WO2010085648A2 - Methods for reducing radiation-induced tissue damage - Google Patents

Methods for reducing radiation-induced tissue damage Download PDF

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WO2010085648A2
WO2010085648A2 PCT/US2010/021809 US2010021809W WO2010085648A2 WO 2010085648 A2 WO2010085648 A2 WO 2010085648A2 US 2010021809 W US2010021809 W US 2010021809W WO 2010085648 A2 WO2010085648 A2 WO 2010085648A2
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tweak
antibody
radiation therapy
pharmaceutical composition
radiation
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WO2010085648A3 (en
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Burkly C. Linda
Taeko Dohi
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Linda Burkly C
Taeko Dohi
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • This invention involves methods for preventing or reducing the severity of tissue damage resulting from radiation therapy.
  • Radiation therapy also known as radiotherapy, X-ray therapy, and irradiation
  • Radiation therapy is the medical use of ionizing radiation in cancer treatment to kill malignant cells and shrink tumors. About 50 to 60 percent of cancer patients receive some type of radiation therapy during the course of their treatment. Radiation therapy is painless, but has several acute side effects, including damage to epithelial surfaces in the area of the body under treatment. In these tissues, both single-cell damage (necrosis or apoptosis) and interstitial damage (edema, fibrosis, vascular alterations, cellular infiltrations) resulting in tissue remodeling can occur. Epithelial surfaces such as oral, pharyngeal, and bowel mucosa, skin, and urothelium are particularly prone to damage.
  • Radiation therapy injures or destroys cells by damaging their DNA, making it impossible for these cells to continue to grow and divide.
  • the damage is caused by a photon, electron, proton, neutron, or ion beam directly or indirectly ionizing the atoms which make up the DNA chain.
  • the radiation is administered either from a machine outside of the body (as in external beam radiotherapy, or teletherapy), or it may be placed inside the body (brachytherapy, or sealed source radiotherapy) or may be emitted from unsealed radioactive materials that go throughout the body (systemic radioisotope therapy, or unsealed source radiotherapy).
  • radiation may be used alone or in combination with other cancer treatments, such as chemotherapy or surgery, and a patient can receive more than one type of radiation therapy.
  • Radiation can be used as palliative treatment, where cure is not possible and the aim is for local disease control or symptomatic relief, or it can be used as therapeutic treatment, where the therapy has survival benefit and it can be curative. It can also be used as prophylactic treatment, with the intent of preventing cancer formation in a specific area that receives the radiation.
  • Radiation therapy may be used to treat almost every type of solid tumor, including cancers of the lung, brain, breast, cervix, prostate larynx, pancreas, skin, stomach, or uterus, and soft tissue sarcomas. Radiation may also be used to treat leukemia and lymphoma.
  • the dosage of radiation depends on a number of factors, including the radiosensitivity of each cancer type and whether there are tissues and organs nearby that may be damaged by radiation. Although radiation damages both cancer cells and normal cells, most normal cells can recover from the effects of radiation and function properly.
  • the goal of radiation therapy is to damage as many cancer cells as possible, while limiting harm to nearby healthy tissue. Hence, it is typically given in many fractions, allowing healthy tissue to recover between fractions.
  • Radiation therapy can be external beam radiotherapy (EBRT or XBRT, also known as teletherapy), brachytherapy (also known as sealed source radiotherapy or endocurietherapy), and systemic radioisotope therapy (also known as unsealed source radiotherapy).
  • EBRT or XBRT external beam radiotherapy
  • brachytherapy also known as sealed source radiotherapy or endocurietherapy
  • systemic radioisotope therapy also known as unsealed source radiotherapy.
  • the differences between types of radiation therapy relate to the position of the radiation source: external is outside the body, brachytherapy uses sealed radioactive sources placed precisely in the area under treatment, and systemic radioisotope therapy uses radioisotopes administered by infusion or oral ingestion.
  • Brachytherapy can use temporary or permanent placement of radioactive sources.
  • Proton therapy is a special case of external beam radiotherapy where the particles are protons.
  • Introperative radiotherapy is a special type of radiotherapy that is delivered immediately after surgical removal of the cancer. This method has been employed
  • Epithelial surfaces such as oral, pharyngeal, and bowel mucosa, skin, and urothelium are particularly prone to damage. If the head and neck area is treated, damage to the lining of the mouth and throat that can create temporary soreness, mucositis (an inflammation of the mucous membranes in the mouth), infection, bleeding, pain, and ulceration. If severe, this can affect swallowing, and the patient may need painkillers and nutritional support. While most of these complications are manageable, they can sometimes become severe enough that treatment must be completely stopped or the dose limited, which may also reduce the therapeutic effect of the radiation against the cancer cell targets.
  • the lining of the esophagus can easily be damaged and become sore if the esophagus receives a dose of collateral radiation during treatment of lung cancer or if it is treated directly.
  • the lower bowel is treated with radiation, as in cases of rectal or anal cancer, or is exposed by radiotherapy to other pelvic structures (bladder, male prostate, female genital tract), epithelial damage frequently produces localized inflammation, nausea, vomiting, cramping abdominal pain, and diarrhea.
  • skin is easily damaged by radiation therapy. In areas receiving radiation, skin typically starts to become pink and sore several weeks into treatment.
  • the reaction may become more severe during the treatment and for up to about one week following the end of radiotherapy, and the skin may break down in a process called moist desquamation, characterized by sloughing of the epidermis and exposure of the dermal layer.
  • moist desquamation characterized by sloughing of the epidermis and exposure of the dermal layer.
  • the rates of onset of damage and recovery from it depend upon the turnover rate of the epithelial cells.
  • Intestinal epithelia have rapid cell turnover rates, accompanied by an equally high rate of apoptosisfor maintaining mucosal homeostasis.
  • Intestinal epithelial cells especially rapidly proliferating cells in the crypts (i.e. mucosal glands), are highly sensitive to both cancer chemotherapy and ⁇ -irradiation. DNA damage caused by exposure to cytotoxic agents or ⁇ -irradiation initiates either a self-repairing process or apoptosis to eliminate injured cells. In the latter situation, cytotoxic treatment results in excessive epithelial apoptosis or dysregulation of apoptosis.
  • the methods of the invention relate, in part, to a discovery that disruption of the activity of TNF (Tumor necrosis factor) family member TWEAK (TNF-like weak inducer of apoptosis) reduces the levels of epithelial cell death and results in increased epithelial cell regeneration in mice following tissue injury by ⁇ -irradiation. Accordingly, the invention provides methods of preventing radiation-induced cell death in a subject using a therapeutically effective amount of a TWEAK antagonist. These methods are useful for treating, preventing, and/or reducing the severity of epithelial cell and tissue damage in subjects receiving radiation therapy for the treatment of cancer and other conditions and in subjects who are otherwise exposed to radiation.
  • TNF Tumor necrosis factor
  • TWEAK TNF-like weak inducer of apoptosis
  • Embodiments of the invention encompass the use of an antagonist of TWEAK for preventing or reducing cell death that occurs as a result of radiation therapy or radiation exposure.
  • the methods comprise administering to a subject who has received, is receiving, or will receive radiation therapy a therapeutically effective amount of a TWEAK antagonist sufficient to prevent or reduce the severity of radiation-induced cell death.
  • the subject has cancer.
  • the cancer is a solid cancer.
  • the cancer is a hematologic cancer.
  • the radiation therapy is palliative radiation therapy, wherein cure is not possible, but radiation therapy can, for example, aid in local disease control or symptomatic relief.
  • the radiation therapy is prophylactic radiation therapy.
  • the radiation therapy is locally administered to a specific area of tissue.
  • the therapy is total body irradiation.
  • the subject receives total body irradiation prior to receiving a bone marrow transplant.
  • the methods of the invention prevent or reduce the severity of radiation-induced cell death of epithelial cells.
  • the epithelial cells are gastrointestinal epithelial cells.
  • the gastrointestinal epithelial cells are cells of the jejunum, ileum, or colon.
  • the TWEAK antagonist is an anti-TWEAK antibody or an anti-TWEAK receptor antibody.
  • the antibody is a full length IgG.
  • the antibody is an antigen- binding fragment of a full length IgG.
  • the antibody can be a monoclonal or humanized antibody.
  • the antibody can contain a human Fc region.
  • the TWEAK antagonist is a soluble TWEAK receptor (Fn 14) polypeptide.
  • Fn 14 soluble TWEAK receptor
  • An example of a soluble form of the TWEAK receptor is an Fc fusion protein that includes at least a portion of the extracellular domain of Fn14 referred to as Fn14-Fc.
  • Fn14-Fc Fn14-Fc
  • Other soluble forms of Fn14, e.g., forms that do not include an Fc domain, may also be used in the methods of the invention.
  • the TWEAK antagonist is administered via an enteric route.
  • the tissue to be targeted with radiation therapy can be gastrointestinal tissue or is proximal to gastrointestinal tissue.
  • the TWEAK antagonist is administered via a parenteral route.
  • the TWEAK antagonist can be delivered to the tissue which will be targeted with radiation therapy.
  • the localized delivery is performed via local injection.
  • the localized delivery is performed via ultrasound-mediated microbubble destruction.
  • FIG. 2 shows the genomic loci for TWEAK WT and mutants, indicating the WT locus, neomycin-containing targeting construct, and neo-deleted locus.
  • the coding exons are shown as black boxes.
  • the arrow indicates the direction of transcription.
  • the targeting construct contains a human CD2 expression cassette (grey box) and a loxP-flanked neomycin cassette (striped box flanked by solid black).
  • the CD2 cassette and one loxP element remain.
  • Bold lines (5' and 3') beneath the WT genomic locus mark the positions of external Southern blot probes.
  • Figure 3 shows Northern blot analysis of tissues from TWEAK KO and WT mice. Fifteen ⁇ g of RNA from the indicated tissues were probed for GAPDH or TWEAK, using the full-length TWEAK cDNA.
  • FIG. 4 shows mRNA expression of TWEAK locus neighbors Senp3 (SMT3IP1), Tnfs13 (APRIL) and Eif4a by gene array analysis of colon tissue from TWEAK KO and WT Balb/c mice. Averages for 3 animals per group are plotted with no significant differences.
  • Figure 5 shows the spleen weight and spleen weight to body weight ratio for 4 or 12 month old TWEAK KO and WT mice (WT open bars, KO filled bars).
  • Figure 6 shows the numbers of cells in CD3, CD4, and CD8 T cell subsets from the spleens of TWEAK KO and WT mice. Twelve-month-old mice were used to measure cell numbers.
  • Figure 7 shows the number of cells in IgM+ B cell, NK cell, and NKT cell subsets from the spleens of TWEAK KO and WT mice.
  • Four-month-old mice were used to measure NK and NKT cells, and 12-month-old mice were used to measure IgM+ B cells.
  • Figure 8 shows the levels of activated/memory cells within the CD4 and CD8 spleen T cell populations from twelve-month old animals. All error bars are SEM, 5-7 mice per group were used for the comparisons.
  • Figures 9A and 9E are photos of isolated colons of WT and TWEAK KO mice, respectively.
  • Figure 9B-9D and 9F-9H show histological features of Balb/c WT and TWEAK KO colons: untreated 9B WT and 9F KO at day 0 of the TNBS colitis protocol; TWEAK WT 9C 1 9D and KO 9G, 9H at day 10 of the colitis protocol.
  • Scale bars in Figures 9B, 9C, 9F, and 9G correspond to 0.1 mm.
  • the images in Figures 9D and 9H are two fold higher magnification.
  • Figure 11 shows mean weight loss +/- SEM in TWEAK KO and WT mice after TNBS treatment on days 0 and 7 (indicated by arrows). The number of WT and KO mice for each time point are in parentheses. Asterisks indicate significant differences (p ⁇ 0.05, unpaired t-test) on days 1 , 3 and 5.
  • Figure 15 shows histological scores for TNBS colitis in TWEAK KO, Fn14 KO and WT mice on the C57BL/6 background.
  • Figure 21 shows the anti-TNP response of sacral lymph node T cells of TNBS-induced colitis mice on day 10 following TNBS administration. Stimulation index was calculated as the level of in vitro 3 H- thymidine incorporation compared to cultures without antigen. WT group is smaller due to the death of 3 mice during the course of the experiment.
  • Figure 21 shows the anti-TNP serum IgG titer on day 10 of TNBS colitis. No significant differences were observed.
  • Figure 22 depicts caspase-3 activation detected by western blot of Balb/c WT and Fn14 KO mouse intestinal epithelial cell samples treated TNF ⁇ .
  • Figure 23 shows representative images of single staining with anti- TWFJAK and epithelial marker EpCAM on serial colon sections from Balb/c WT and TWEAK KO mice, naive or 3 days after TNBS administration to induce colitis.
  • Anti-TWEAK staining of TWEAK KO serves as a control and reflects a low level of nonspecific staining.
  • Figure 24 shows anti-Fn14 immunofluorescent staining (green) of epithelial colon sections from Balb/c WT and Fn14 KO mice, naive (top row) or 3 days after TNBS administration to induce colitis. Edges of the tissue on the lumen side showed nonspecific staining in both WT and Fn 14 KO mice. The bottom image shows immunofluorescent staining for both Fn14 (green) and F4/80 (red) in WT mouse tissue 3 days after TNBS administration.
  • Figure 25 shows representative images of anti-Fn14 double staining with epithelial marker EpCAM or macrophage marker F4/80 on epithelial colon sections from WT and Fn 14 KO mice 3 days after TNBS colitis.
  • Figure 26 shows the Fn14 mRNA relative expression values for primary colon tissue cultures with media or CpG ODN or TNF ⁇ stimuli as shown. Each line represents a culture from an individual mouse.
  • Figure 27 shows a flow cytometry overlay of anti-Fn14 (open profile) vs. control Ig staining (filled profile) of epithelial cell line MODE-K.
  • Figure 28 shows MMP-9 production by MODE-K cells cultured in media alone or in the presence of TWEAK (1.0 ng/ml), TWEAK plus soluble Fn14 decoy protein (10 ⁇ g/ml), of agonistic anti-Fn14 mAb (10 ⁇ g/ml), or control Ig (10 ⁇ g/ml).
  • Figure 29 shows MCP-1 and KC production in MODE-K cells stimulated by agonistic anti-Fn14 mAb versus control Ig. Mean values +/- SD are shown for triplicate cultures. Asterisks indicate significant differences, all p values ⁇ 0.005 by Student's t test.
  • Figure 32 shows the TNBS-induced colitis histology scores of WT mice, TWEAK KO mice, WT mice treated with control Ig, and WT mice treated with anti-TWEAK monoclonal antibody three and ten days after administration of TNBS. Antibodies were injected on day 0, day 3, and day 7 of TNBS-induced colitis.
  • Figure 33 shows representative images of H&E-stained jejunum and colon samples from Balb/c WT and TWEAK-KO mice 24 hours after receiving 3 Gy of whole body irradiation. Apoptotic cells with dense chromatin masses are indicated by arrows.
  • Figure 34 shows a plot of the number of apoptotic cells observed in the jejunum and colon of Balb/c WT and TWEAK-KO mice 24 hours after receiving 3 Gy of whole-body ⁇ -irradiation.
  • Figure 35 shows a plot of the percentage of apoptotic cells at various crypt cell positions along the longitudinal crypt axis in Balb/c WT (dark circles) and TWEAK-KO mice (gray squares) 24 hours after receiving 3 Gy of whole-body ⁇ -irradiation.
  • Figure 36 shows a plot of the percentage of apoptotic cells at various crypt cell positions along the longitudinal crypt axis in C57BL6 WT (dark circles) and Fn-14-KO mice (gray squares) 24 hours after receiving 3 Gy of whole- body ⁇ -irradiation.
  • Figure 37 shows representative images of H&E stained jejunum and ileum samples from Balb/c WT and TWEAK-KO mice 24 hours after receiving 12 Gy of whole body ⁇ -irradiation. Regenerating microcolonies of cells are indicated with arrows.
  • Figure 38 shows a plot of the numbers of regenerating microcolonies of cells in jejunum, ileum, and colon samples from Balb/c WT and TWEAK-KO mice 4 days after receiving 12 Gy of whole body ⁇ -irradiation.
  • Figure 39 shows a plot of the number of BrdU + cells in jejunum samples of Balb/c WT or TWEAK-KO mice or in C57BL6 WT or Fn 14-KO mice 24 hours after receiving 3 Gy of whole-body ⁇ -irradiation.
  • the invention provides methods of preventing tissue damage induced by radiation therapy. Aspects of the invention relate, in part, to the discovery that disrupting the signaling of TNF family member TWEAK (TNF-like weak inducer of apoptosis) through its receptor, Fn14, reduces levels of epithelial cell death and increases epithelial cell regeneration following radiation treatment in a mouse model of tissue injury by ⁇ -irradiation.
  • Embodiments of the invention encompass the use of an antagonist of TWEAK for preventing or reducing cell death that occurs as a result of radiation therapy or radiation exposure.
  • the methods comprise administering to a subject who has received, is receiving, or will receive radiation therapy a therapeutically effective amount of a TWEAK antagonist sufficient to prevent or reduce the severity of radiation-induced cell death.
  • a therapeutically effective amount of a TWEAK antagonist sufficient to prevent or reduce the severity of radiation-induced cell death is meant an amount of a TWEAK antagonist delivered to a subject that reduces the levels of apoptosis (programmed cell dea + h) occurring in an area of tissue treated with radiation therapy or otherwise exposed to radiation to a level that is either 1) not detectably different than that occurring in the tissue prior to administration of the radiation, or 2) that is decreased relative to the level of apoptosis occurring in the tissue receiving radiation when a TWEAK antagonist is not administered to the subject.
  • apoptosis programmed cell dea + h
  • the method of preventing or reducing cell death that occurs as a result of radiation therapy comprises 1) administering a TWEAK antagonist and 2) administering radiation.
  • the TWEAK antagonist is administered a sufficient amount of time prior to administration of radiation to allow the TWEAK antagonist to penetrate into the target tissue.
  • the TWEAK antagonist is administered at least two hours before administration of the radiation.
  • the TWEAK antagonist is administered between two to four hours prior to administration of the radiation.
  • the time between the administration of a TWEAK antagonist and subsequent administration of radiation is four to six hours, six to eight hours, eight to ten hours, ten to twelve hours, twelve to twenty-four hours, twenty-four to forty-eight hours, or more than 48 hours.
  • the TWEAK antagonist is administered prior to multiple rounds of radiation treatment. In other embodiments, the TWEAK antagonist is administered prior to each round of radiation administered over the course of the patient's treatment. In some embodiments, the TWEAK antagonist is administered in repeat doses. In further embodiments, the TWEAK antagonist is administered in repeat doses prior to multiple rounds of radiation treatment. In further embodiments, the TWEAK antagonist is administered in repeat doses for every round of radiotherapy administered over the course of the patients treatment.
  • the TWEAK antagonist is administered to a subject before and after radiation treatment. In further embodiments, the TWEAK antagonist is administered to a subject before, during, and after a course of radiation therapy. In some embodiments, the dosage of the TWEAK antagonist is relatively high just before and after radiation treatment and is then lowered to smaller doses over time following the treatment.
  • the TWEAK antagonist is administered to a subject after exposure to radiation.
  • exposure to radiation includes radiation therapy, accidental radiation exposure, and exposure to individuals at risk for radiation exposure. Accordingly, the methods can benefit individuals who are exposed to radiation inadvertantly or who are exposed to radiation as part of their work, including, for example, individuals working in nuclear power plants or nuclear fuel and/or nuclear waste processing facilities, individuals transporting nuclear material, researchers working with radioactive materials, individuals responsible for handling hazardous materials, members of the military, medical care workers, and the like.
  • the subject is a patient receiving X-rays for purposes of medical monitoring and/or diagnosis.
  • the TWEAK antagonist is administered to a subject following inadvertant exposure to radiation.
  • the TWEAK antagonist can be administered in single or multiple doses within a few hours after exposure to radiation. In some embodiments, the TWEAK antagonist is administered within 24 hours of exposure to radiation. In further embodiments, the antagonist is administered within 48 hours, 72 hours, or 96 hours or more following exposure to radiation. Administration of a TWEAK antagonist following exposure to radiation can be continued for several days, weeks, or months, or as long as the administration may be therapeutically effective. In some embodiments, the dosage of a TWEAK antagonist is lowered from a higher dose immediately following exposure to radiation to a lower dose over the course of several days, weeks, or months.
  • the TWEAK antagonist is administered in a single dose. In some embodiments, the antagonist is administered in multiple doses. In some embodiments, the TWEAK antagonist is administered several times per day, i.e. once every two to three hours, three to six hours, six to nine hours, or nine to twelve or more hours.
  • the methods of the invention are used to prevent or reduce the severity of radiation-induced cell death in a subject with cancer.
  • the cancer can be, for example, a carcinoma, sarcoma, lymphoma, leukemia, myeloma, germ cell tumor, or blastoma.
  • Carcinomas are malignant tumors derived from epithelial cells. Carcinomas are the most common cancers, and include common forms of lung, colon, breast, and prostate cancer.
  • Sarcomas are malignant tumors derived from connective tissue or mesenchymal cells (bone, cartilage, or fat).
  • Lymphomas, leukemias, and myelomas are malignancies derived from hematopoietic (blood-forming) cells, including bone marrow cells and lymphocytes.
  • Germ cell tumors are derived from totipotent cells. In adults, germ cell tumors are most often found in the testicle and ovary. In fetuses, infants, and young children, germ cell tumors are most often found on the body midline, particularly at the tip of the tailbone. Blastomas are tumors that resemble an immature or embryonic tissue, and the majority are typically found in children.
  • the cancer is a solid cancer.
  • the cancer can be, for example, lung cancer, prostate cancer, breast cancer, colon or rectal cancer, bladder cancer, pancreatic cancer, endometrial cancer, ovarian cancer, testicular cancer, melanoma, nonmelanoma skin cancer, cervical cancer, small bowel cancer, stomach cancer, urinary tract cancer, thyroid cancer, kidney (renal cell) cancer, neuroblastoma, rhabdomyosarcoma (arising from muscle), retinoblastoma, osteosarcoma, Ewing's sarcoma, and teratoma.
  • the cancer is a hematological neoplasm.
  • the hematological neoplasm is a leukemia, lymphoma, or multiple myeloma.
  • the subject receiving radiation therapy has a non-malignant condition.
  • non-malignant conditions include trigeminal neuralgia, severe thyroid eye disease, pterygium, pigmented villonodular synovitis, keloid scar growth, and heterotopic ossification.
  • the subject receives total body irradiation (TBI) as part of the preparative regimen for a hematopoietic stem cell (or bone marrow) transplantation due to a disease of the blood, bone marrow, or because of a certain type of cancer.
  • TBI total body irradiation
  • TBI treatment typically is delivered twice daily over 3 days. However, a variety of other approaches may be used depending on the tumor site and the individual protocol.
  • a current multiple myeloma TBI regimen for example, utilizes a single fraction of 2 grays (Gy). In most TBI protocols, however, the total dose is 12 Gy.
  • the radiation therapy is palliative radiation therapy.
  • Palliative radiation therapy is therapy that is not curative, but rather is used to control symptoms associated with localized tumors that cannot be treated by other methods, such as surgery.
  • the radiation therapy is prophylactic radiation therapy.
  • prophylactic radiation may be given to areas that do not have evidence of cancer. This is done to prevent cancer cells from growing in the area receiving the radiation.
  • prophylactic radiation to the contralateral breast can be administered to reduce the rate of subsequent contralateral breast cancer and offer an option for risk reduction in women with BRCA germline mutations.
  • prophylactic radiation can be administered to the central nervous system (whole brain irradiation, also called prophylactic cranial irradiation, or PCI).
  • the methods prevent or reduce the severity of radiation-induced cell death of epithelial cells.
  • Apoptosis or programmed cell death, is an important physiological process in epithelia for eliminating senescent, damaged, redundant, or deleterious cells.
  • Epithelial cells line the cavities and surfaces of structures throughout the body. The outermost layer of skin is composed of dead stratified squamous, keratinized epithelial cells.
  • the tissues that line the inside of the mouth, the esophagus, and part of the rectum are composed of nonkeratinized stratified squamous epithelium.
  • the methods of the invention prevent or reduce the severity of radiation induced cell death of non-keratinized stratified squamous epithelium. In other embodiments, the methods of the invention prevent or reduce the severity of radiation induced cell death of simple squamous, columnar, or pseud ostratified epithelial cells. In some embodiments, the methods of the invention prevent or reduce the severity of radiation induced cell death of endothelial or mesothelial cells.
  • the methods of the invention prevent or reduce the severity of radiation-induced cell death of gastrointestinal (Gl) epithelial cells.
  • the Gl epithelial cells are cells of the mouth, larynx, or esophagus.
  • the Gl epithelial cells are cells of the stomach, duodenum, jejunum, ileum, cecum, colon, or rectum. TWEAK signaling and TWEAK antagonists
  • TNF superfamily of ligands and receptors are prominent regulators of cell fate decisions including survival, proliferation, and differentiation (Ware et al., Cytokine Growth Factor Rev. 14:181-4 (2003)). They play- essential roles in the organogenesis and homeostasis of multiple systems, including bone, skin appendages such as hair and teeth, and lymphoid tissues. They also play complex immunoregulatory roles, for example in host defense, inflammatory responses, and positive and negative regulation of adaptive immunity.
  • TWEAK a member of the TNF ligand superfamily
  • TNF ligand superfamily is a type II- transmembrane protein that can be cleaved to function as a soluble cytokine and is highly expressed by inflammatory cells (Chicheportiche et al., J. Biol. Chem. 272: 32401-10 (1997)).
  • the TWEAK receptor, Fn14 is a TNF receptor superfamily member expressed by nonlymphoid cell types. TWEAK was first described as a weak inducer of apoptosis (Chicheportiche et al., J. Biol. Chem.
  • Fn14 is expressed by many tissue-resident progenitor cells (Girgenrath et al., EMBO J. 25:5826-39 (2006)); Jakubowski et al., J. Clin. Invest, 115:2330 ⁇ 0 (2005); Perper et al., J. Immunol. 177:2610-20 (2006)).
  • TWEAK is a multifunctional cytokine, similar in this regard to its sibling TNF. TWEAK functions physiologically after acute injury and pathologically in chronic inflammatory disease settings. In contrast to TNF however, TWEAK plays no apparent role in development or homeostasis.
  • TWEAK antagonists used to prevent or reduce the severity of radiation-induced cell death includes, e.g., small organic or inorganic molecules, nucleic acids, proteins, or peptide mimetics that are capable of interrupting the binding of TWEAK to its cognate receptor Fn14 or otherwise interfere with TWEAK receptor signaling.
  • TWEAK receptor signaling refers to all molecular reactions associated with the TWEAK receptor pathway and subsequent molecular reactions which result therefrom.
  • TWEAK antagonists useful in preventing or reducing radiation- induced cell death are known in the art. Such agents include those disclosed in, e.g. International Publication Numbers WO 98/05783, WO 98/35061 , WO 99/19490, WO 00/42073, and WO 01/45730, all of which are incorporated herein by reference in their entirety.
  • the antagonist is a biologic, e.g., a protein having a molecular weight of between 5-300 kDa.
  • a TWEAK antagonist may inhibit binding of TWEAK to a Fn14.
  • a typical TWEAK antagonist can bind to TWEAK or a TWEAK receptor, e.g., Fn14.
  • a TWEAK antagonist that binds to TWEAK or Fn14 may block the binding site on TWEAK or Fn14, alter the conformation of TWEAK or Fn14, or otherwise decrease the affinity of TWEAK for Fn14 or prevent the interaction between TWEAK and Fn14.
  • a TWEAK antagonist may bind to TWEAK or to Fn14 with a K d of less than 10 ⁇ 6 , 10 "7 , 10 '8 , 10 "9 , or 10 "10 M.
  • the antagonist binds to TWEAK with an affinity at least 5, 10, 20, 50, 100, 200, 500, or 1000-fold higher than its affinity for TNF or another TNF superfamily member (other than TWEAK).
  • the antagonist binds to the Fn14 with an affinity at least 5, 10, 20, 50, 100, 200, 500, or 1000-fold better than its affinity for the TNF receptor or a receptor for another TNF superfamily member.
  • Exemplary TWEAK antagonists include antibodies (e.g., monoclonal antibodies) that bind to TWEAK or Fn14 and soluble forms of Fn14 that compete with cell surface Fn14 for binding to TWEAK.
  • Other types of antagonists e.g., small molecules, nucleic acid or nucleic acid-based aptamers, and peptides, can be isolated by screening, e.g., as described in Jhaveri et al. Nat. Biotechnol. 18:1293-1297 (2000) and U.S. Patent No. 5,223,409.
  • the TWEAK antagonists used in the methods of the invention include antibodies that bind to TWEAK and/or Fn14.
  • Antibodies directed to TWEAK or a TWEAK receptor that are useful in the invention are described herein and are known in the art. Exemplary antibodies are described, e.g., in International Patent Publication No. WO 2006/130374 and U.S. Patent Publication No. 2008/0279853, which are incorporated herein by reference in their entirety.
  • antibody refers to a protein that includes at least one immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or an immunoglobulin variable domain sequence.
  • Antigen-binding fragments of antibodies include, e.g., single chain antibodies, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, and dAb fragments.
  • Complete antibodies include, e.g., intact and/or full length immunoglobulins of types IgA, IgG (e.g., IgGI, lgG2, lgG3, lgG4), IgE, IgD, IgM (as well as subtypes thereof).
  • the light chains of the immunoglobulin may be of types kappa or lambda.
  • An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity, or may be non-functional for one or both of these activities.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), referenced above, interspersed with regions that are more conserved, termed "framework regions" (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • the extent of the FR's and CDR's has been precisely defined (see, Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.
  • Each VH and VL is typically composed of three CDR's and four FR 1 s, arranged from amino-terminus to carboxyl-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • an "immunoglobulin domain” refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two beta- sheets formed of about seven beta-strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay, Ann. Rev Immunol. 6:381-405 (1988)).
  • An "immunoglobulin variable domain sequence” refers to an amino acid sequence that can form a structure sufficient to position CDR sequences in a conformation suitable for antigen binding. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain.
  • the sequence may omit one, two or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other alterations.
  • a polypeptide that includes an immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form a target binding structure (or "antigen binding site"), e.g., a structure that interacts with TWEAK or Fn 14. - ⁇ -
  • the VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively.
  • the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains.
  • the heavy and light immunoglobulin chains can be connected by disulfide bonds.
  • the heavy chain constant region typically includes three constant domains, CH1 , CH2, and CH3.
  • the light chain constant region typically includes a CL domain.
  • the variable region of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • One or more regions of an antibody can be human, effectively human, or humanized, as described below.
  • one or more of the variable regions can be human or effectively human.
  • An "effectively human" immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human.
  • An "effectively human” antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.
  • one or more of the CDRs can be human.
  • Each of the light chain CDRs can be human.
  • HC CDR3 can be human.
  • One or more of the framework regions can be human, e.g., FRI, FR2, FR3, and FR4 of the HC or LC. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non- hematopoietic cell.
  • the human sequences are germline sequences, e.g., encoded by a germline nucleic acid.
  • One or more of the constant regions can be human, effectively human, or humanized.
  • at least 70, 75, 80, 85, 90, 92, 95, or 98% of the framework regions (e.g., FR1 , FR2, and FR3, collectively, or FR1 , FR2, FR3, and FR4, collectively) or the entire antibody can be human, effectively human, or humanized.
  • FR1 , FR2, and FR3 collectively can be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical, or completely identical, to a human sequence encoded by a human germline segment.
  • a "humanized" immunoglobulin variable region is an immunoglobulin variable region that is modified such that the modified form elicits less of an immune response in a human than does the non-modified form, e.g., is modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response or elicits a reduced immunogenic response in a normal human as compared to a non-humanized antibody.
  • humanized immunoglobulins include, for example, U.S. Patent Numbers 6,407,213 and 5,693,762.
  • humanized immunoglobulins can include a non-human amino acid at one or more framework amino acid positions.
  • Exemplary humanized antibodies that bind to TWEAK are described below as huP2D10-1 and huP2D10-2.
  • the antibodies can be conjugated to a moiety, e.g., can be conjugated to poly(ethylene glycol) (e.g., PEGylated), e.g., to reduce the immunogenicity and/or increase the circulating half-lives of antibodies.
  • the antibody inhibits the interaction between TWEAK and Fn14, e.g., by physically blocking the interaction, decreasing the affinity of TWEAK and/or Fn14 for its counterpart, reducing the signaling activity of Fn14, disrupting or destabilizing TWEAK/Fn14 complexes, sequestering TWEAK or Fn14, or targeting TWEAK or Fn14 for degradation.
  • the antibody can bind to TWEAK or Fn14 at one or more amino acid residues that participate in the TWEAK/ Fn14 binding interface. Such amino acid residues can be identified, e.g., by alanine scanning.
  • the antibody can bind to residues that do not participate in the TWEAK/ Fn14 binding.
  • the antibody can alter a conformation of TWEAK or Fn 14 and thereby reduce binding affinity, or the antibody may sterically hinder TWEAK/ Fn14 binding.
  • the antibody can prevent activation of a TWEAK/ Fn14 mediated event or activity (e.g., NF- ⁇ B activation).
  • the TWEAK antagonist used in the methods of the invention is an anti-TWEAK antibody.
  • the TWEAK antagonist is an anti- Fn 14 antibody.
  • the sequence of an exemplary human TWEAK to which anti-TWEAK antibodies may be directed is shown in SEQ ID NO:1.
  • the sequence of a mouse TWEAK is shown in SEQ ID NO:2.
  • International Patent Application No. WO 2006/130374 discloses the sequences of specific examples of anti-TWEAK antibodies that are useful in the methods of the invention, such as P2D10, huP2D10-1 , and huP2D10-2.
  • amino acid sequence of the IgG heavy chain in each of mature huP2D10-1 and huP2D10-2 antibodies is as follows (SEQ ID NO:3):
  • amino acid sequence of the light chain of huP2D10-1 is as follows (SEQ ID NO:4): 1 DWMTQSPLS LPVTPGEPAS ISCRSSQSLV SSKGNTYLHW YLQKPGQSPQ 51 FLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YFCSQSTHFP 101 RTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASWCL LNNFYPREAK 151 VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE 201 VTHQGLSSPV TKSFNRGEC
  • amino acid sequence of the light chain of huP2D10-2 is as follows (SEQ ID NO:5):
  • Suitable antibodies for use in the methods of the invention include an antibody that can bind to an epitope on TWEAK which includes at least one, two, three or four amino acid residues from an epitope on TWEAK recognized by P2D10, to a peptide from TWEAK that is bound by P2D10 (e.g., a peptide less than 25, 20, or 15 amino acids in length) or to a region of TWEAK recognized by P2D10.
  • the antibody may specifically bind to an epitope, e.g., a linear or a conformational epitope, of TWEAK, in particular human TWEAK, e.g., the soluble region of TWEAK.
  • the antibody may compete with P2D10 for binding to TWEAK, e.g., to human TWEAK.
  • the antibody may competitively inhibit binding of P2D10 to TWEAK, e.g., human TWEAK.
  • the antibody may bind to an epitope which overlaps with that of P2D10, e.g., includes at least one, two, three or four amino acids in common with the P2D10 epitope, or an epitope which, when bound, sterically prevents TWEAK interaction with P2D10.
  • the antibody specifically binds to at least a part of the interaction site on TWEAK that contacts human Fn 14.
  • the antibody may compete with Fn14 for binding to TWEAK, e.g., to human TWEAK.
  • the antibody may competitively inhibit binding of Fn 14 to TWEAK.
  • the antibody may interact with an epitope on TWEAK which, when bound, sterically prevents interaction between TWEAK and Fn14 (e.g., between human TWEAK and human Fn14).
  • the protein includes at least one, two and preferably three CDRs from the light or heavy chain variable region of P2D10.
  • CDRs refer to CDRs as defined by Chothia's hypervariable loops.
  • the protein includes, in the heavy chain variable domain sequence, at least one, two, or three of the following sequences within a CDR region:
  • VLYYDYDGDRIEVMDY CDR3 (SEQ ID NO:8), or a CDR having an amino acid sequence that differs by no more than 4, 3, 2.5, 2, 1.5, 1 , or 0.5 alterations (e.g., substitutions, insertions or deletions) for every 10 amino acids (e.g., the number of differences being proportional to the CDR length) relative to a sequence listed above, e.g., at least one alteration but not more than two, three, or four per CDR.
  • the heavy chain variable domain sequence may include these CDR sequences particularly in CDR3, or in at least two CDRs, e.g., CDR1 and CDR3, CDR2 and CDR3, or in all three CDRs.
  • the protein can include, in the heavy chain variable domain sequence, at least one, two, or three of the following sequences within a CDR region (amino acids in parentheses represent alternatives for the particular position):
  • the antibody can include, in the light chain variable domain sequence, at least one, two, or three of the following sequences within a CDR region:
  • SQSTHFPRT SEQ IDNO:14
  • CDR3 SQSTHFPRT (SEQ IDNO:14); (CDR3), or a CDR having an amino acid sequence that differs by no more than 4, 3, 2.5, 2, 1.5, 1 , or 0.5 alterations (e.g., substitutions, insertions or deletions) for every 10 amino acids (e.g., the number of differences being proportional to the CDR length) relative to a sequence listed above, e.g., at least one alteration but not more than two, three, or four per CDR.
  • the light chain variable domain sequence may include these CDR sequences particularly in CDR3, or in at least two CDRs, e.g., CDR1 and CDR3, CDR2 and CDR3, or in all three CDRs.
  • the antibody can include, in the light chain variable domain sequence, at least one, two, or three of the following sequences within a CDR region (amino acids in parentheses represent alternatives for the particular position): (i) (RK)-S-S-Q-S-(LI)-(KV)-S-S-(KR)-G-N-(TN)-Y-L-(EHDNQY) (SEQ ID NO: 15), or (RK)-S-S-Q-S-(LI)-V-S-S-(KR)-G-N-(TN)-Y-L-H (SEQ ID NO: 16);
  • the antibody includes all six CDRs from P2D10 or closely related CDRs, e.g., CDRs which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions), or other CDR described herein.
  • the antibody includes at least one, two, or three CDRs that have the same canonical structures and the corresponding Chothia CDRs of P2D10, e.g., the same canonical structures as at least CDR1 and/or CDR2 of the heavy and/or light chain variable domains of P2D10.
  • antibodies such as these, can be made, for example, by preparing and expressing synthetic genes that encode the recited amino acid sequences or by mutating human germline genes to provide a gene that encodes the recited amino acid sequences.
  • Antibodies that bind to TWEAK or Fn14 may also be generated by a variety of other means. (See, for example, "Antibodies: A Laboratory Manual,” ed. by Harlow and Lane, Cold Spring Harbor press: 1988).
  • One exemplary method includes screening protein expression libraries, e.g., phage or ribosome display libraries.
  • Phage display is described, for example, U.S. 5,223,409; Smith, Science 228:1315-1317 (1985); WO 92/18619; WO 91/17271 ; WO 92/20791 ; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809.
  • the display of Fab's on phage is described, e.g., in U.S. Pat. Nos. 5,658,727; 5,667,988; and 5,885,793.
  • TWEAK- or Fn14-binding antibody In addition to the use of display libraries, other methods can be used to obtain a TWEAK- or Fn14-binding antibody.
  • all or part of TWEAK or Fn14 may be used as an immunogen or as a target for selection.
  • the immunized animal contains immunoglobulin-producing cells with natural, human, or partially human immunoglobulin loci.
  • the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected.
  • Non-human antibodies to TWEAK or Fn14 can also be produced, for example, in a rodent.
  • the non-human antibody can be humanized, e.g., as described in EP 239 400; U.S. Pat. Nos. 6,602,503; 5,693,761 ; and 6,407,213, deimmunized, or otherwise modified to make it effectively human.
  • EP 239 400 (Winter et al.) describes altering antibodies by substitution (within a given variable region) of their complementarity determining regions (CDRs) for one species with those from another.
  • CDRs of a non-human (e.g., murine) antibody are substituted into the corresponding regions in a human antibody, e.g., from germline immunoglobulin genes, by using recombinant nucleic acid technology to produce sequences encoding the desired substituted antibody.
  • Human constant region gene segments of the desired isotype usually gamma I for CH and kappa for CL
  • the humanized heavy and light chain genes can be co-expressed in mammalian cells to produce soluble humanized antibody.
  • a non-human TWEAK- or Fn14 binding antibody may be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy and light chain variable regions of an antibody can be analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317).
  • peptide threading For detection of potential T-cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class Il binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class Il DR allotypes, and thus constitute potential T cell epitopes.
  • Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable regions, or preferably, by single amino acid substitutions. As far as possible, conservative substitutions are made. Often, but not exclusively, an amino acid common to a position in human germline antibody sequences may be used.
  • nucleic acids encoding VH and VL can be constructed by mutagenesis or other synthetic methods (e.g., de novo synthesis, cassette replacement, and so forth).
  • a mutagenized variable sequence can, optionally, be fused to a human constant region, e.g., human IgGI or kappa constant regions.
  • a potential T cell epitope will include residues which are known or predicted to be important for antibody function. For example, potential T cell epitopes are usually biased towards the CDRs. In addition, potential T cell epitopes can occur in framework residues important for antibody structure and binding. Changes to eliminate these potential epitopes will in some cases require more scrutiny, e.g., by making and testing chains with and without the change. Where possible, potential T cell epitopes that overlap the CDRs can be eliminated by substitutions outside the CDRs. In some cases, an alteration within a CDR is the only option, and thus variants with and without this substitution can be tested.
  • the substitution required to remove a potential T cell epitope is at a residue position within the framework that might be critical for antibody binding, hi these cases, variants with and without this substitution are tested.
  • variants with and without this substitution are tested.
  • several variant deimmunized heavy and light chain variable regions are designed and various heavy/light chain combinations are tested to identify the optimal deimmunized antibody.
  • the choice of the final deimmunized antibody can then be made by considering the binding affinity of the different variants in conjunction with the extent of deimmunization, particularly, the number of potential T cell epitopes remaining in the variable region.
  • Deimmunization can be used to modify any antibody, e.g., an antibody that includes a non-human sequence, e.g., a synthetic antibody, a murine antibody other non-human monoclonal antibody, or an antibody isolated from a display library.
  • a non-human sequence e.g., a synthetic antibody, a murine antibody other non-human monoclonal antibody, or an antibody isolated from a display library.
  • the antibody can include a human Fc region, e.g., a wild-type Fc region or an Fc region that includes one or more alterations.
  • the constant region is altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).
  • the human IgGI constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237.
  • Antibodies may have mutations in the CH2 region of the heavy chain that reduce or alter effector function, e.g., Fc receptor binding and complement activation.
  • antibodies may have mutations such as those described in U.S. Patent Nos. 5,624,821 and 5,648,260.
  • Antibodies may also have mutations that stabilize the disulfide bond between the two heavy chains of an immunoglobulin, such as mutations in the hinge region of lgG4, as disclosed in the art (e.g., Angal et al., MoI. Immunol. 30:105-08 (1993)). See also, e.g., U.S. 2005-0037000.
  • Fully human monoclonal antibodies that bind to a TWEAK receptor can be produced, e.g., using in wYro-primed human splenocytes, as described by Boerner et al., J.
  • phage display libraries may also be used to isolate high affinity antibodies that can be developed as human therapeutics using standard phage technology (see, for example, Hoogenboom et al., Immunotechnology, 4:1-20 (1998); Hoogenboom et al., Immunol Today, 2: 371-378 (2000); and U.S. Patent Publication No. 2003/0232333).
  • An anti-TWEAK or anti-Fn14 antibody may be modified, e.g., by mutagenesis, to provide a pool of modified antibodies for use in the methods of the invention.
  • the modified antibodies are evaluated to identify one or more antibodies which have altered functional properties (e.g., improved binding, improved stability, reduced antigenicity, or increased stability in vivo).
  • display library technology is used to select or screen the pool of modified antibodies. Higher affinity antibodies are then identified from the second library, e.g., by using higher stringency or more competitive binding and washing conditions. Other screening techniques can also be used.
  • the mutagenesis is targeted to regions known or likely to be at the binding interface. If, for example, the identified binding proteins are antibodies, then mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs, e.g., framework regions, particularly within 10, 5, or 3 amino acids of a CDR junction. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make step-wise improvements.
  • mutagenesis is used to make an antibody more similar to one or more germline sequences.
  • One exemplary germlining method can include: identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Then mutations (at the amino acid level) can be made in the isolated antibody, either incrementally, in combination, or both. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In one embodiment, as many germline residues are introduced into an isolated antibody as possible.
  • mutagenesis is used to substitute or insert one or more germline residues into a CDR region.
  • the germline CDR residue can be from a germline sequence that is similar (e.g., most similar) to the variable region being modified.
  • activity e.g., binding or other functional activity
  • Similar mutagenesis can be performed in the framework regions.
  • a germline sequence can be selected if it meets a predetermined criteria for selectivity or similarity, e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity, relative to the donor non-human antibody.
  • the selection can be performed using at least 2, 3, 5, or 10 germline sequences.
  • identifying a similar germline sequence can include selecting one such sequence.
  • identifying a similar germline sequence can include selecting one such sequence, but may include using two germline sequences that separately contribute to the amino-terminal portion and the carboxy- terminal portion. In other implementations, more than one or two germline sequences are used, e.g., to form a consensus sequence.
  • the antibody may be modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern).
  • altered means having one or more carbohydrate moieties deleted, and/or having one or more glycosylation sites added to the original antibody. Addition of glycosylation sites to the presently disclosed antibodies may be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences; such techniques are well known in the art. Another means of increasing the number of carbohydrate moieties on the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody.
  • an antibody has CDR sequences that differ only insubstantially from those of P2D10. Insubstantial differences include minor amino acid changes, such as substitutions of 1 or 2 out of any of typically 5-7 amino acids in the sequence of a CDR, e.g., a Chothia or Kabat CDR. Typically an amino acid is substituted by a related amino acid having similar charge, hydrophobic, or stereochemical characteristics. Such substitutions would be within the ordinary skills of an artisan. Unlike in CDRs, more substantial changes in structure framework regions (FRs) can be made without adversely affecting the binding properties of an antibody.
  • FRs structure framework regions
  • Changes to FRs include, but are not limited to, humanizing a nonhuman-derived framework or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, e.g., changing the class or subclass of the constant region, changing specific amino acid residues which might alter an effector function such as Fc receptor binding (Lund et al., J. Immun. 147:2657-62 (1991); Morgan et al., Immunology 86:319-24 (1995)), or changing the species from which the constant region is derived.
  • humanizing a nonhuman-derived framework or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site e.g., changing the class or subclass of the constant region, changing specific amino acid residues which might alter an effector function such as Fc receptor binding (Lund et al., J. Immun. 147:2657-62 (1991); Morgan et al., Immunology 86:319-24 (1995)), or changing the species from which the constant region is
  • the anti-TWEAK or anti-Fn14 antibodies used in the methods of the invention can be in the form of full length antibodies, or in the form of fragments of antibodies, e.g., Fab, F(ab')2, Fd, dAb, and scFv fragments. Additional forms include a protein that includes a single variable domain, e.g., a camel or camelized domain. See, e.g., U.S. 2005-0079574 and Davies et al., Protein Eng. 9(6):531-7 (1996). Antibodies can further be linked to one or more heterologous polypeptides.
  • an anti-Tweak antibody is linked to one or more antigen binding domains of the same or a different antibody.
  • an anti-Tweak antibody can be linked to one or more scFvs, e.g,. at the C- terminus of the Fc portions.
  • the TWEAK antagonist is a soluble Fn14 polypeptide.
  • the sequence of an exemplary human Fn14 is shown in SEQ ID NO:21.
  • the sequence of an exemplary mouse Fn14 is shown in SEQ ID NO:22.
  • An exemplary soluble form of the Fn14 protein includes a region of the Fn14 protein that binds to TWEAK, e.g., about amino acids 32-75, 31-75, 31-78, or 28- 79 of SEQ ID NO:21.
  • the TWEAK antagonist is an Fn 14 fusion protein.
  • the term "fusion protein" refers to a chimeric protein comprising amino acid sequences of two or more different proteins.
  • the Fn14 fusion protein includes, in addition toFn14, one or more polypeptide portions that enhance one or more of in vivo stability, in vivo half-life, uptake/administration, tissue localization or distribution, formation of protein complexes, and/or purification. Fusion proteins may be generated recombinantly using molecular cloning techniques well known in the art.
  • the Fn14 fusion protein contains amino acids 28-79 of SEQ ID NO.21.
  • This region of Fn14 can be physically associated, e.g., fused to another amino acid sequence, e.g., an Fc domain, at its N- or C- terminus.
  • the Fn14 polypeptide can be from the heterologous amino acid sequence spaced by a linker.
  • the Fn14 fusion protein includes a purification subsequence, such as an epitope tag, a FLAG tag, a 6xHis sequence, or GST polypeptide.
  • U.S. Patent No. 6,824,773 describes an exemplary Fn14 fusion protein.
  • the Fn14 fusion protein may contain a mutated form of Fn14 as described above.
  • conservative substitutions of one or more amino acids present in a native Fn 14 polypeptide can be made without adversely effecting the activity of the polypeptide.
  • conservative substitutions include substitution of amino acids outside of regions of Fn 14 that are conserved between species (such as between human and mouse Fn14), and substitution of amino acids that do not alter the secondary and/or tertiary structure of Fn14.
  • substitution of one aliphatic residue for another such as lie, VaI, Leu, or Ala for one another, or substitution of one polar residue for another, such as between Lys and Arg; GIu and Asp; or GIn and Asn, or substitution of one aromatic residue for another, such as Phe, Trp, or Tyr for one another.
  • Other conservative substitutions such as substitutions of entire regions having similar hydrophobicity characteristics, are known in the art and are contemplated in the methods of the invention.
  • Methods of generating mutated forms of Fn14 are well known in the art of molecular biology, and include altering DNA molecules by random mutagenesis, site directed mutagenesis, deletions, and truncations.
  • PCR polymerase chain reaction
  • saturation i.e. chemical or radiation
  • mutagenesis saturation (i.e. chemical or radiation) mutagenesis
  • chemical DNA synthesis alanine scanning mutagenesis
  • oligonucleotide-mediated mutagenesis hybridization to a DNA template in vitro followed by enzymatic elongation
  • cassette recombinant
  • combinatorial mutagenesis introduction of random degenerate sequences into the TWEAK DNA.
  • the methods of the invention employ an Fn14 fusion protein that includes the Fc domain of an immunoglobulin such as, e.g., IgGI , lgG2, lgG3, lgG4), IgE, IgD, IgM (Fn14-Fc).
  • an immunoglobulin such as, e.g., IgGI , lgG2, lgG3, lgG4), IgE, IgD, IgM (Fn14-Fc).
  • the Fc portion of an immunoglobulin has the meaning commonly given to the term in the field of immunology. Specifically, this term refers to an antibody fragment which does not contain the two antigen binding regions (the Fab fragments) from the antibody.
  • the Fc portion consists of the constant region of an antibody from both heavy chains, which associate through non-covalent interactions and disulfide bonds.
  • the Fc portion can include the hinge regions and extend through the CH2 and CH3 domains to the C-terminus of the antibody.
  • the Fc portion can further include one or more glycosylation sites.
  • the immunoglobulin Fc portion of the fusion protein contains mutations designed to remove unwanted effector functions and/or reduce the risk of inducing an immune response after repeated and prolonged administration, as described in U.S. Patent No. 7,452,966.
  • Fn14 has been characterized in J. Biol. Chem., 274: 33166-76 (1999) and International Publication Number WO 02/022166, the entire disclosure of which is incorporated herein by reference.
  • the human and mouse amino acid sequences for this type I transmembrane protein are provided herein as SEQ ID NO:21 and SEQ ID NO:22, respectively.
  • Antibodies and other proteins described herein can be produced in prokaryotic and eukaryotic cells.
  • the antibodies are expressed in a yeast cell such as Pichia (see, for example, Powers et al., J. Immunol. Methods, 251 : 123-35 (2001)), Hanseula, or Saccharomyces.
  • Antibodies particularly full length antibodies, for example, IgG's, and soluble Fn14 proteins may be expressed in mammalian cells.
  • exemplary mammalian host cells for recombinant expression include Chinese Hamster Ovary (CHO cells) (including dhfr CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. U. SA, 77: 4216-4220 (1980), in which recombinant constructs include a DHFR selectable marker, as described in Kaufman and Sharp, MoI.
  • lymphocytic cell lines including NSO myeloma cells and SP2 cells, COS cells, K562 cells, and cells from a transgenic animal, such as a transgenic mammal.
  • Antibodies may be expressed in mammary epithelial cells.
  • the recombinant expression vectors may carry additional nucleic acid sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017).
  • Exemplary selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr CHO cells by calcium phosphate-mediated transfection.
  • the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes.
  • enhancer/promoter regulatory elements e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element
  • the recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification.
  • the selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, to transfect the host cells, to select for transformants, to culture the host cells, and to recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G.
  • Antibodies may also include modifications, including, for example, modifications that alter Fc function. Such modifications include changes that decrease or remove interaction with an Fc receptor or with C1q, or both.
  • the human IgGI constant region can be mutated at one or more residues, including, for example, one or more of residues 234 and 237, according to the numbering in U.S. Pat. No. 5,648,260.
  • Other exemplary modifications include those described in U.S. Pat. No. 5,648,260.
  • the protein/antibody production system may be designed to synthesize the fusion protein or antibody proteins in which the Fc region is glycosylated.
  • the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain.
  • the Fc domain can also include other eukaryotic post-translational modifications.
  • the protein is produced in a form that is not glycosylated.
  • Antibodies and other proteins can also be produced by a transgenic animal.
  • U.S. Pat. No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal.
  • a transgene is constructed that includes a milk-specific promoter and nucleic acid sequences encoding the antibody of interest, e.g., an antibody described herein, and a signal sequence for secretion.
  • the milk produced by females of such transgenic mammals includes, secreted-therein, the protein of interest, e.g., an antibody or Fc fusion protein.
  • the protein can be purified from the milk, or for some applications, used directly.
  • the methods of this invention include the administration of an effective dose of a TWEAK antagonist to a subject to prevent or reduce cell death induced by radiation therapy. Determination of a preferred pharmaceutical formulation and a therapeutically efficient dose regiment for a given subject is well within the skill of the art taking into consideration, for example, the condition and weight of the patient, the extent of desired treatment and the tolerance of the patient for the treatment. In some embodiments, the TWEAK antagonist is administered locally to the region of tissue to be treated with radiation therapy. [0131]
  • the TWEAK antagonist can be administered by any route of administration which is compatible with the antagonist, and may be formulated with any pharmaceutically acceptable carrier appropriate to the route of administration. Such carriers are well known to those skilled in the art.
  • Administration can be performed, for example, intravenously, intraperitoneally, orally, via implant, transmucosally, transdermally, intramuscularly, and subcutaneously.
  • Preferred routes of administration are parenteral and, in particular, intravenous, intraperitoneal, and intracapsular.
  • Administration can be performed over an extended period on an outpatient basis, similar in this regard to radiation treatment.
  • Daily dosages of a TWEAK antagonist are expected to be in the range of about 0.01 to 1000 ⁇ g/kg body weight, and more preferably about 10 to 300 ⁇ g/kg body weight, although precise dosages will vary depending upon the agent employed and the particular subject's medical condition and history.
  • the TWEAK antagonist is administered via an injectable drug delivery system.
  • injectable drug delivery system can include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (including, for example, ethanol, propylene glycol and sucrose) and polymers (including, for example, polycaprylactones and polylactic-co-glycolic acids (PLGA's)).
  • solubility-altering agents including, for example, ethanol, propylene glycol and sucrose
  • polymers including, for example, polycaprylactones and polylactic-co-glycolic acids (PLGA's)
  • the TWEAK antagonist is delivered to via ultrasound-mediated microbubble destruction. Drugs can be i n corporated into microbubbles in a number of different ways, including binding of the drug to the microbubble shell and attachment of site-specific ligands.
  • microbubble delivery is known in the art, and has been demonstrated to be effective in targeted delivery of biologies (i.e. proteins having a molecular weight of between 5-300 kDa; see, for example, Mukherjee, D., et al. J. Am. Coll. Cardiol. 35:1678-1686 (2000)).
  • biologies i.e. proteins having a molecular weight of between 5-300 kDa; see, for example, Mukherjee, D., et al. J. Am. Coll. Cardiol. 35:1678-1686 (2000)).
  • Oral delivery systems for the TWEAK antagonist include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropyl- methylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc).
  • excipients such as binders (e.g., hydroxypropyl- methylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials
  • Transmucosal delivery systems for the TWEAK antagonist include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).
  • solubilizers and enhancers e.g., propylene glycol, bile salts and amino acids
  • other vehicles e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid.
  • Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone).
  • solubilizers e.g., permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone).
  • permeation enhancers e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids
  • hydrophilic polymers e.
  • the TWEAK antagonist is administered via an implantable system.
  • Implantable systems can include rods and discs, and can contain excipients such as PLGA and polycaprylactone.
  • Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).
  • suspending agents e.g., gums, zanthans, cellulosics and sugars
  • humectants e.g., sorbitol
  • solubilizers e.g., ethanol, water, PEG and propylene glycol
  • TWEAK antagonist may, for example, be placed into sterile, isotonic formulations with or without cofactors which stimulate uptake or stability.
  • the formulation is preferably liquid, or may be lyophilized powder.
  • the TWEAK antagonist may be diluted with a formulation comprising 5.0 mg/ml citric acid monohydrate, 2.7 mg/ml trisodium citrate, 41 mg/ml mannitol, 1 mg/ml glycine and 1 mg/ml polysorbate 20.
  • This solution can be lyophilized, stored under refrigeration and reconstituted prior to administration with sterile Water-For- Injection (U.S.P).
  • the TWEAK antagonists of this invention may also be administered using microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in, near, or otherwise in communication with affected tissues or the bloodstream.
  • sustained release carriers include semipermeably polymer matrices in the form of shaped articles such as suppositories or microcapsules.
  • Implantable or microcapsular sustained release matrices include polylactides (U.S. Pat. No.
  • the methods, formulations, and dosages of the invention may be evaluated in a known model of irradiation-induced tissue injury. These models include animal models such as the mouse model described in the following examples. When testing the compositions of the invention in animal models, the biological therapeutic agent should have activity in the animal. For example, a mouse anti-TWEAK antibody may be used in lieu of a human antibody if the human antibody does not cross-react with mouse TWEAK.
  • Example 1 The TWEAK/Fn14 pathway has a nonredundant role in intestinal damage in mice through a TWEAK/intestinal epithelial cell axis
  • Fn14 expression in the gastrointestinal tract are limited, with moderate mRNA levels observed in normal human tissue mRNA arrays (Wiley et al., Cytokine Growth Factor Rev 14:241-9 (2003)).
  • expression of Fn 14 is well documented in cells of epithelial origin such as bronchial, mammary and bile duct epithelium and human keratinocytes (Jakubowski et al., J CHn Invest 115:2330-40 (2005); Jin et al., J Invest Dermatol 122:1175-9 (2004); Michaelson et al., Oncogene 24:2613-24 (2005); Xu et al., Biochem Biophys Res Commun 318:422-7 (2004)).
  • TWEAK is proinflammatory (Burkly et al., Cytokine 40:1-16 (2007)); or (3) TWEAK represses innate immunity (Maecker et al., Cell 123:931- 44 (2005)).
  • TNBS-induced colitis is a model of human IBD in which intrarectal instillation of TNBS with ethanol injures the epithelial barrier followed by a T cell me ⁇ iated immune response against haptenated colonic proteins, leading to mucosal inflammation and epithelial damage involving both innate and adaptive immune components.
  • the ⁇ - irradiation injury model (Example 2) induces much more limited damage directed at the rapidly dividing progenitor cells of the intestinal crypts without a significant inflammatory component (Booth et al., lnt J Cancer 86:53-9 (2000); Leedham et al., J Cell MoI Med 9:11-24 (2005)).
  • TWEAK/Fn14 pathway a novel pathogenic mediator in models of intestinal injury, promoting pathology through its effects on epithelial cell inflammatory responses and turnover. TWEAK deficiency limits local intestinal pathology without impairing systemic adaptive immune responses.
  • TWEAK or Fn14 knockout mice were backcrossed onto Balb/c or C57BL/6 backgrounds for 5-6 generations under SPF conditions at Biogenrez or the International Medical Center of Japan (IMCJ). All experimental protocols were approved by the institutional animal care and use committees. Generation of the TWEAK- and Fn14-deficient mice
  • TWEAK KO mice were generated as described (Campbell et al., J Immunol 176:1889-98 (2006)).
  • a 8.12-kb Hind ⁇ subclone containing 3.36 kb of upstream sequence and the first five exons of the TWEAK gene was obtained from a BAC clone of 129 sv/J genomic DNA (Genome Systems).
  • the targeting vector was constructed from this subclone using the bacterial recombination method (Zhang, Y., et al. Nat. Genet. 20: 123-128 (1998)).
  • TWEAK-deficient mice were generated by standard procedures using homologous recombination in embryonic stem cells (Bronson, S. K., et al. Proc. Natl. Acad. Sci. USA 93: 9067-9072 (1996)).
  • the neomycin expression cassette was excised from the locus by mating TWEAK heterozygous mice with MSX2-Cre recombinase-expressing mice (Lewandoski, M., et al. Nat.
  • mice Homozygous TWEAK-deficient, neomycin-deleted mice were generated by breeding heterozygous mice. Absence of TWEAK mRNA expression in knockout mice was confirmed by RT-PCR and Northern blot using the full length TWEAK cDNA, and GAPDH probed as a control. TWEAK knockout (KO) mice were backcrossed onto Balb/c backgrounds for 5-6 generations under specific pathogen free (SPF) conditions. Expression of genes neighboring the TWEAK locus was unaltered.
  • SPF pathogen free
  • Fn14 KO mice generation of Fn14 KO mice was described (Jakubowski et al., J CHn Invest 115:2330-40 (2005)).
  • a 10-kb Kpn1 genomic DNA fragment containing the full murine Fn14 gene was isolated, and a targeting vector was designed to delete the first 2 exons, which contained the entire extracellular ligand-binding domain of Fn14.
  • the target vector was transfected into the J1 129 ES cell line and selected with G418.
  • ES cell clones were screened for homologous recombination using Southern blot, and the correct clones were injected into C57BL/6 blastocysts to generate chimeras.
  • mice heterozygous for targeted Fn14 alleles were obtained through further breeding and identified using Southern blot or PCR.
  • the null mutation was confirmed by both Northern blot and RT-PCR. Mice were bred to homozygosity on the 129 background.
  • the Fn14 mutation was backcrossed 5 times onto the C57BL/6 background under SPF conditions.
  • TNBS colitis was induced by intrarectal administration of a 2 % solution of TNBS in PBS:ethanol (1 :1) as described in Dohi et al., Gastroenterology 119:724-33 (2000) and Dohi et al., J Exp Med 189:1169-80 (1999).
  • 60 ⁇ g/g body weight of TNBS was given on day 0 and animals sacrificed on day 3.
  • 36 ⁇ g/g body weight of TNBS was administered on days 0 and 7 with sacrifice on day 10.
  • mice were treated with murine anti-TWEAK mAb P2D10 (Campbell et al., J Immunol 176:1889-98 (2006)) or isotype-matched control Pl 17 (ATCC, Manassas, VA), 300 ⁇ g on days 3 and 7 with sacrifice on day 10, or as otherwise specified.
  • the colons of surviving mice were cut into proximal, middle and distal segments, formalin-fixed, paraffin-embedded and 4- ⁇ m sections stained with H&E. Each colon segment was scored individually and these scores were summed to reach a total score for the entire colon. Histological scores were assigned to each segment as follows: 0 - normal, 1 - ulcer or cell infiltration limited to the mucosa, 2 - ulcer or limited cell infiltration in the submucosa, 3 - focal ulcer involving all layers of the colon, 4 - multiple lesions involving all layer of the colon or necrotizing ulcer larger than 3mm in length. Thus the total possible histological score is 12.
  • the SV40 immortalized, nontransformed cell lines MODE-K (Vidal et al., J Immunol Methods 166:63-73 (1993)) and MCE301 (Tabuchi et al., Cell Struct Fund 25:297-307 (2000)) were cultured with media alone, or with specified concentrations of murine TWEAK, soluble Fn 14 plus TWEAK, an agonistic anti- Fn14 mAb P2.D3 or isotype-matched control Ig, MOPC-21 , for 24 hours.
  • Culture supernatants were analyzed by SearchLight multiplex ELISA (Pierce Boston Technology Center, Woburn, MA) for chemokines, cytokines and MMPs. Cell subsets analysis for WT and TWEAK KO mice
  • TWEAK KO and WT splenocytes were stained using antibodies (BD Biosciences) for: CD3, CD4, CD8, IgM, NK1.1 , DX5, CD44 and CD62L. lmmunohistochemistry for fluorescently-stained samples
  • Fn14 detection was carried out as described above with double staining for PE-labeled EpCAM mAb or PE-labeled anti-F4/80 (Becton, Dickinson and Company, 1 :200 dilution) for 2 hours at room temperature.
  • PE-labeled EpCAM mAb or PE-labeled anti-F4/80 Becton, Dickinson and Company, 1 :200 dilution
  • 1 mg bromodeoxyuridine (BrdU) was injected i.p.
  • jejunum was collected at 24 hours post irradiation, and paraffin- embedded sections were deparaffinized, treated with 4N HCI, blocked with 1% BSA and stained with rat anti-BrdU antibody (Oxford Biotech, Ltd, United Kingdom) followed by FITC-labeled anti-rat IgG (Southern Biotechnology Associates, Inc., Birmingham, AL). Numbers of BrdU + cells were counted in 20 separate crypts in 3 or more fields for each animal.
  • Fn14 expression was evaluated as relative expression to the mean value for cultures with media alone.
  • Fn14 mRNA increased in TNBS-treated colons of Balb/c mice
  • a gene profiling study was conducted with Balb/c colons collected 3 days after intrarectal TNBS/EtOH to determine acute changes in gene expression.
  • animals were given a second dose of TNBS on day seven and colons collected three days later (day 10) to examine the later phase of the pathology.
  • Three members of the TNFR superfamily showed significant up- regulation in colons of TNBS-treated mice in comparison to untreated controls in both the acute and late stages of colitis (Figure 1).
  • TNFR1 and 2 were increased, however expression of their ligand TNF ⁇ was not detected, consistent with the prior report of no effect of TNF blockade in TNBS-induced colitis in the Balb/c strain ⁇ Dohi et al., J Immunol 167:2781-90 (2001)).
  • TWEAK KO mice have normal immune system/intestinal architecture
  • TWEAK deficient animals were healthy and normal.
  • Comprehensive necropsy revealed no abnormalities, and extensive immune compartment analysis showed normal spleen weights in both young and aged mice, and normal percentages and absolute numbers of splenic B, T, NK, and NKT cell subsets and activated/memory cells within the CD4 and CD8 T cell populations ( Figures 5, 6, 7, 8).
  • Baseline levels of Ig subclasses were also comparable to those of WT mice (data not shown).
  • TWEAK KO mice protected from TNBS-induced colitis/ulceration
  • TWEAK KO mice exhibited a significantly lower histological score than WT controls (Figure 13) reflecting two prominent differences on both days 3 and 10: reduction in frequency and extent of epithelial ulcers and reduction in leukocyte infiltration into the submucosa. The overall magnitude of inflammatory cell infiltration was also reduced in TWEAK KO as compared on WT colons at both the early and late disease stages ( Figure 14).
  • TNBS colitis was also induced in TWEAK or Fn14 deficient mice on the C57BL/6 background. A similar protective effect was seen in this strain in the absence of either TWEAK or Fn14 ( Figure 15). Additionally, WT Balb/c mice were treated with an anti-TWEAK blocking antibody starting either on day 0 ( Figures 16, 17) or starting 3 days after TNBS administration and diminished clinical score and histological damage to the colon likewise observed ( Figures 18, 19). Taken together, these results indicate that TWEAK/Fn14 pathway contributes to both the onset and progression of TNBSinduced colitis.
  • TWEAK KO mice exhibited a significantly reduced level of peroxidase activity (Figure 20 A&B), and staining for neutrophil marker Gr-1 ( Figure 20 C&D) was likewise diminished. Monocyte infiltration was also reduced in TWEAK KO colons ( Figure 20 E&F).
  • TNBS colitis is also associated with a robust adaptive immune response to trinitrophenyl (TNP)-modified proteins.
  • TWEAK deficiency did not alter generation of the TNP-specific T cell response in draining sacral lymph nodes ( Figure 21 left graph) or serum levels of anti-TNP antibodies ( Figure 21 , right graph). These findings suggest that TWEAK deficiency ameliorates local pathogenic events rather than the systemic adaptive immune component.
  • chemokines/chemokine receptors were markedly reduced in TWEAK KO colons. Most of the chemokines affected were those involved in neutrophil and monocyte chemotaxis. Some of these chemokines are known to be secreted by colon epithelial cells (CXCL1 , CXCL5, CCL2), while others are produced by activated macrophages (CXCL2, CCL3) and serve to recruit additional cells into the tissue (Papadakis Curr Allergy Asthma Rep 4:83-9 (2004)).
  • MMPs matrix metalloproteinases
  • TWEAK induces epithelial cell production of inflammatory mediators
  • TWEAK TWEAK and its receptor, Fn 14, were identified.
  • TWEAK was expressed by intestinal epithelial cells in WT tissue ( Figure 23), whereas expression by other cell types was equivocal, warranting further investigation. TWEAK expression was similar before and 3 days after disease induction. In contrast, there was little if any Fn14 in normal WT colon ( Figure 24, upper left panel) whereas Fn 14 expression by colon epithelial cells was markedly increased in WT mice three days after TNBS administration ( Figure 24, middle left panel).
  • TWEAK stimulation of proinflammatory mediator production was also observed with the MCE301 cell line ( Figures 30, 31). These results suggest that TWEAK acts locally through Fn 14 expressing epithelial cells to promote pathogenic tissue inflammation and matrix remodeling in TNBS colitis. Anti-TWEAK antibody protects WT Balb/c mice from colitis
  • FIG. 32 shows the TNBS-induced colitis histology scores of WT mice, TWEAK KO mice, WT mice treated with control Ig, and WT mice treated with anti-TWEAK monoclonal antibody three and ten days after administration of TNBS. Antibodies were injected on day 0, day 3, and day 7 of TNBS-induced colitis.
  • TWEAK/Fn14 pathway As a nonredundant pathogenic mediator in contexts of intestinal injury and inflammatory disease.
  • Significant amelioration of TNBS-induced colitis is observed in TWEAK or Fn14 deficient mice, and in WT mice treated with TWEAK blocking mAbs prophylactically or therapeutically.
  • TWEAK deficient animals show an improved clinical course, reduced colon epithelial ulcers and less infiltration by granulocytes and macrophages.
  • TWEAK does not regulate the systemic adaptive response to TNP.
  • blocking TWEAK can be an intervention point for inhibiting local immune effector mechanisms, as well as controlling epithelial injury, in contexts of intestinal injury and inflammatory disease. Blocking TWEAK can be a favorable intervention point, since it orchestrates events specific to the disease target tissue without systemically impairing host immunity. .
  • the TWEAK KO mice of the present study were extensively characterized, with no differences observed between KO and WT. Since full length TWEAK cDNA was used as a probe in the Northern blot analysis, it is extremely unlikely that TWEAK is expressed in the test animals.
  • Intestinal epithelial cells markedly upregulate Fn14 after TNBS administration. Although other local Fn14 expressing cells cannot be ruled out, colon infiltrating macrophages do not appear to express this receptor. Intestinal epithelium is a well established player in the innate defense of the intestine (Abreu et al., J Immunol 174:4453-60 (2005)). The present in vitro studies suggest that Fn14 can be induced by exposure to bacterial DNA after injury to the epithelial barrier. Although TNF ⁇ is not expressed in the Balb/c strain after TNBS administration, it might may also induce Fn14 in contexts of intestinal injury on other genetic backgrounds.
  • Intestinal epithelial cells also respond to TWEAK, with increased production of chemokines involved in monocyte and neutrophil recruitment and MMP-9. Conversely, expression of these gene signatures was significantly reduced locally in TWEAK KO as compared to WT colons. While the pathogenic role of inflammatory cytokines, chemokines and MMPs in models of colitis is well established (Papadakis Curr Allergy Asthma Rep 4:83-9 (2004); Naito et al., MoI Aspects Med 26:379-90 (2005)), the present studies identify TWEAK as an important instigator of their production in TNBS induced colitis apparently through stimulation of intestinal epithelial cells.
  • TWEAK was previously shown to significantly contribute to joint inflammation and damage in mouse collagen-induced arthritis, likely through its effect on mesenchymal joint cell types, without affecting systemic collagen-specific T cell antibody responses (Kamata et al., J Immunol 177:6433-6439 (2006); Perper et al., J Immunol 177:2610-20 (2006)). These studies underscore the important disease-promoting contribution of TWEAK-epithelial and TWEAK-stromal cell interactions locally in the disease target tissue.
  • MMPs enzymes important for degradation of extracellular matrix proteins and cell migration are significantly increased after TNBS administration in WT mice and may contribute to disease pathogenesis in multiple ways.
  • MMP-9 was shown to be abundantly associated with inflamed areas in ulcer bases in human IBD tissue (Arihiro et al., Histopathology 39:50-9 (2001)) and MMP-3 shown to produce severe tissue injury in gut explant cultures (Pender et al., J Immunol 160:4098-103 (1998)).
  • proteolysis by MMPs may contribute to ulcer formation and severity
  • reduced MMPs including MMP-3 and MMP-9 in TWEAK KO animals may contribute to reduced ulceration.
  • MMPs are also involved in accumulation of inflammatory cells into the intestine in TNBS colitis as evidenced by reduced neutrophil accumulation in MMP inhibitor treated animals (Di Sebastiano et al., Digestion 63:234-9 (2001); Sykes et al., Aliment Pharmacol Ther 13:1535-42 (1999)).
  • reduced MMP expression in TWEAK KO mice may contribute to decreased inflammation.
  • TWEAK has been demonstrated to induce MMP-9 dependent proliferation and branching of mammary gland epithelial cells in vitro (Michaelson et al., Oncogene 24:2613-24 (2005)). Decreased anchorage of epithelial cells as a consequence of matrix degradation may trigger cell proliferation and branching.
  • TWEAK-induced MMP activity may reduce epithelial anchoring and thereby promote cell proliferation, crypt deformity and branching, features of abnormal repair observed 10 days after TNBS insult. These features are reduced in the TWEAK KO mice.
  • TWEAK produced by adaptive and innate cell types acts on epithelial cells to enhance the local production of well known inflammatory mediators, tissue remodeling enzymes, and regulate epithelial turnover.
  • Anti-TNF- ⁇ antibody treatment was a milestone in the therapy of Crohn's disease (Targan et al., N Engl J Med 337:1029-35 (1997)) and now in ulcerative colitis (Rutgeerts et al., N Engl J Med 353:2462-76 (2005). However, 30-40% of patients are refractory to this treatment. Thus new therapeutics are sought to address this unmet need.
  • Example 2 TWEAK or Fn14 deficiency reduces intestinal epithelial cell death following ⁇ -irradiation
  • TWEAK- or Fn14-KO mice received 3 Gy of whole-body irradiation using the ⁇ -irradiation apparatus MBR-1520-R (Hitachi Medical Corporation, Tokyo, JAPAN). Twenty-four hours post-irradiation, the mice were sacrificed, and small intestine and colon were collected. Paraffin-embedded sections were prepared from a Swiss roll of whole colon or a 7 cm length from the oral end of the small intestine for analysis of jejunum. Numbers of apoptotic cells per crypt, detected in H&E stained sections by chromatin condensation, were counted in 20 separate crypts in 3 or more fields for each animal.
  • Balb/c TWEAK-KO mice showed a significantly lower number of apoptotic cells than WT in both jejunum and colon after 3 Gy of whole-body irradiation.
  • Representative H&E stained images of crypts from Balb/c WT and TWEAK-KO jejunum and colon samples 24 hours after irradiation are shown in Figure 33. Apoptotic cells in a single representative crypt are indicated by arrowheads.
  • the number of apoptotic cells in the jejunum or colon crypts in Balb/c WT or TWEAK-KO mice is shown in Figure 34. Asterisks indicate p ⁇ 0.01.
  • Example 3 TWEAK deficiency increases the appearance of regenerating epithelial cells after irradiation
  • mice [0185] The effect of TWEAK deficiency on cell survival was also assessed by measuring cell regeneration in intestinal epithelial cells of WT and TWEAK- deficient mice receiving radiation treatment.
  • Balb/c WT and Balb/c TWEAK-KO mice received 12 Gy of whole-body irradiation using the ⁇ -irradiation apparatus MBR-1520-R (Hitachi Medical Corporation, Tokyo, JAPAN).
  • MBR-1520-R Haitachi Medical Corporation, Tokyo, JAPAN.
  • mice were sacrificed, and small intestine was collected. Paraffin- embedded sections of both the jejunum and ileum were prepared for H&E staining and analysis.
  • Example 4 Enhanced survival of intestinal epithelial cells in
  • TWEAK deficiency was also assessed by measuring cell proliferation in intestinal epithelial cells of WT and TWEAK- deficient mice receiving radiation treatment.
  • the number of proliferating cells in crypts of Balb/c WT and TWEAK-KO mice and in C57BL/6 WT and Fn 14-KO mice after irradiation was determined.
  • 1 mg 5-bromo-2'-deoxy-uridine (BrdU; Sigma-Aldrich) was injected intraperitoneally into the mice 1 hour prior to sacrifice.
  • Jejunum was collected at 24 hours post irradiation, and paraffin-embedded sections were deparaffinized, treated with 4N HCl, blocked with 1% BSA and stained with rat anti-BrdU antibody (Oxford Biotech, Ltd, United Kingdom) followed by FITC-labeled anti-rat IgG (Southern Biotechnology Associates, Inc., Birmingham, AL). Numbers of BrdU + cells were counted in 20 separate crypts in 3 or more fields for each animal.
  • TWEAK participates in TNF ⁇ -induced apoptosis pathway

Abstract

Methods of preventing tissue damage induced by radiation therapy are provided. Disrupting the signaling of TNF family member TWEAK (TNF-like weak inducer of apoptosis) through its receptor Fn14 reduces levels of epithelial cell death and increases epithelial cell regeneration following radiation treatment in a mouse model of tissue injury by gamma-irradiation. Applications include use of an antagonist of TWEAK for preventing or reducing cell death that occurs as a result of radiation therapy.

Description

METHODS FOR REDUCING RADIATION-INDUCED TISSUE DAMAGE
[001] This invention involves methods for preventing or reducing the severity of tissue damage resulting from radiation therapy.
[002] Radiation therapy (also known as radiotherapy, X-ray therapy, and irradiation) is the medical use of ionizing radiation in cancer treatment to kill malignant cells and shrink tumors. About 50 to 60 percent of cancer patients receive some type of radiation therapy during the course of their treatment. Radiation therapy is painless, but has several acute side effects, including damage to epithelial surfaces in the area of the body under treatment. In these tissues, both single-cell damage (necrosis or apoptosis) and interstitial damage (edema, fibrosis, vascular alterations, cellular infiltrations) resulting in tissue remodeling can occur. Epithelial surfaces such as oral, pharyngeal, and bowel mucosa, skin, and urothelium are particularly prone to damage.
[003] Radiation therapy injures or destroys cells by damaging their DNA, making it impossible for these cells to continue to grow and divide. The damage is caused by a photon, electron, proton, neutron, or ion beam directly or indirectly ionizing the atoms which make up the DNA chain. The radiation is administered either from a machine outside of the body (as in external beam radiotherapy, or teletherapy), or it may be placed inside the body (brachytherapy, or sealed source radiotherapy) or may be emitted from unsealed radioactive materials that go throughout the body (systemic radioisotope therapy, or unsealed source radiotherapy).
[004] For cancer patients that receive radiation therapy, radiation may be used alone or in combination with other cancer treatments, such as chemotherapy or surgery, and a patient can receive more than one type of radiation therapy. Radiation can be used as palliative treatment, where cure is not possible and the aim is for local disease control or symptomatic relief, or it can be used as therapeutic treatment, where the therapy has survival benefit and it can be curative. It can also be used as prophylactic treatment, with the intent of preventing cancer formation in a specific area that receives the radiation.
[005] Radiation therapy may be used to treat almost every type of solid tumor, including cancers of the lung, brain, breast, cervix, prostate larynx, pancreas, skin, stomach, or uterus, and soft tissue sarcomas. Radiation may also be used to treat leukemia and lymphoma. The dosage of radiation depends on a number of factors, including the radiosensitivity of each cancer type and whether there are tissues and organs nearby that may be damaged by radiation. Although radiation damages both cancer cells and normal cells, most normal cells can recover from the effects of radiation and function properly. The goal of radiation therapy is to damage as many cancer cells as possible, while limiting harm to nearby healthy tissue. Hence, it is typically given in many fractions, allowing healthy tissue to recover between fractions.
[006] Radiation therapy can be external beam radiotherapy (EBRT or XBRT, also known as teletherapy), brachytherapy (also known as sealed source radiotherapy or endocurietherapy), and systemic radioisotope therapy (also known as unsealed source radiotherapy). The differences between types of radiation therapy relate to the position of the radiation source: external is outside the body, brachytherapy uses sealed radioactive sources placed precisely in the area under treatment, and systemic radioisotope therapy uses radioisotopes administered by infusion or oral ingestion. Brachytherapy can use temporary or permanent placement of radioactive sources. Proton therapy is a special case of external beam radiotherapy where the particles are protons. Introperative radiotherapy is a special type of radiotherapy that is delivered immediately after surgical removal of the cancer. This method has been employed in breast cancer (targeted intraoperative radiotherapy), brain tumors and rectal cancers.
[007] While radiation therapy is in itself painless, it has several acute side effects, including damage to epithelial surfaces in the area of the body under treatment. In these tissues, both single-cell damage (necrosis or apoptosis) and interstitial damage (edema, fibrosis, vascular alterations, cellular infiltrations) resulting in tissue remodeling can occur.
[008] Epithelial surfaces such as oral, pharyngeal, and bowel mucosa, skin, and urothelium are particularly prone to damage. If the head and neck area is treated, damage to the lining of the mouth and throat that can create temporary soreness, mucositis (an inflammation of the mucous membranes in the mouth), infection, bleeding, pain, and ulceration. If severe, this can affect swallowing, and the patient may need painkillers and nutritional support. While most of these complications are manageable, they can sometimes become severe enough that treatment must be completely stopped or the dose limited, which may also reduce the therapeutic effect of the radiation against the cancer cell targets. Similarly, the lining of the esophagus can easily be damaged and become sore if the esophagus receives a dose of collateral radiation during treatment of lung cancer or if it is treated directly. If the lower bowel is treated with radiation, as in cases of rectal or anal cancer, or is exposed by radiotherapy to other pelvic structures (bladder, male prostate, female genital tract), epithelial damage frequently produces localized inflammation, nausea, vomiting, cramping abdominal pain, and diarrhea. Likewise, skin is easily damaged by radiation therapy. In areas receiving radiation, skin typically starts to become pink and sore several weeks into treatment. The reaction may become more severe during the treatment and for up to about one week following the end of radiotherapy, and the skin may break down in a process called moist desquamation, characterized by sloughing of the epidermis and exposure of the dermal layer. The rates of onset of damage and recovery from it depend upon the turnover rate of the epithelial cells.
[009] Intestinal epithelia have rapid cell turnover rates, accompanied by an equally high rate of apoptosisfor maintaining mucosal homeostasis. Intestinal epithelial cells, especially rapidly proliferating cells in the crypts (i.e. mucosal glands), are highly sensitive to both cancer chemotherapy and γ-irradiation. DNA damage caused by exposure to cytotoxic agents or γ-irradiation initiates either a self-repairing process or apoptosis to eliminate injured cells. In the latter situation, cytotoxic treatment results in excessive epithelial apoptosis or dysregulation of apoptosis. Studies have demonstrated that excessive apoptosis is one of the main etiological factors that contributes to the Gl syndrome after use of cytotoxic agents and/or γ-irradiation (Ijiri, K., and Potten, CS. BrJ Cancer 47: 175-185 (1983); Keefe, D.M., et al. Gut 47: 632-637 (2000); Potten C.S., et al. IntJ Radiat Biol 65: 71-78 (1994)). This Gl syndrome includes diarrhea, general malabsorption, and infection. These severe complications inflict not only intestinal discomfort on patients undergoing chemoradiotherapy, but can also limit the dose of therapeutic agents. SUMMARY OF THE INVENTION
[010] The methods of the invention relate, in part, to a discovery that disruption of the activity of TNF (Tumor necrosis factor) family member TWEAK (TNF-like weak inducer of apoptosis) reduces the levels of epithelial cell death and results in increased epithelial cell regeneration in mice following tissue injury by γ-irradiation. Accordingly, the invention provides methods of preventing radiation-induced cell death in a subject using a therapeutically effective amount of a TWEAK antagonist. These methods are useful for treating, preventing, and/or reducing the severity of epithelial cell and tissue damage in subjects receiving radiation therapy for the treatment of cancer and other conditions and in subjects who are otherwise exposed to radiation.
[011] Embodiments of the invention encompass the use of an antagonist of TWEAK for preventing or reducing cell death that occurs as a result of radiation therapy or radiation exposure. In exemplary embodiments, the methods comprise administering to a subject who has received, is receiving, or will receive radiation therapy a therapeutically effective amount of a TWEAK antagonist sufficient to prevent or reduce the severity of radiation-induced cell death.
[012] In some embodiments, the subject has cancer. In some embodiments, the cancer is a solid cancer. In further embodiments, the cancer is a hematologic cancer.
[013] In some embodiments of the invention, the radiation therapy is palliative radiation therapy, wherein cure is not possible, but radiation therapy can, for example, aid in local disease control or symptomatic relief. In some embodiments, the radiation therapy is prophylactic radiation therapy. In some embodiments, the radiation therapy is locally administered to a specific area of tissue. In further embodiments, the therapy is total body irradiation. In some exemplary embodiments, the subject receives total body irradiation prior to receiving a bone marrow transplant.
[014] In some embodiments, the methods of the invention prevent or reduce the severity of radiation-induced cell death of epithelial cells. In some embodiments, the epithelial cells are gastrointestinal epithelial cells. In some embodiments, the gastrointestinal epithelial cells are cells of the jejunum, ileum, or colon.
[015] In some embodiments, the TWEAK antagonist is an anti-TWEAK antibody or an anti-TWEAK receptor antibody. In some embodiments, the antibody is a full length IgG. In further embodiments, the antibody is an antigen- binding fragment of a full length IgG.. The antibody can be a monoclonal or humanized antibody. In some embodiments, the antibody can contain a human Fc region.
[016] In further embodiments, the TWEAK antagonist is a soluble TWEAK receptor (Fn 14) polypeptide. An example of a soluble form of the TWEAK receptor is an Fc fusion protein that includes at least a portion of the extracellular domain of Fn14 referred to as Fn14-Fc. Other soluble forms of Fn14, e.g., forms that do not include an Fc domain, may also be used in the methods of the invention.
[017] In some embodiments, the TWEAK antagonist is administered via an enteric route. In exemplary embodiments, the tissue to be targeted with radiation therapy can be gastrointestinal tissue or is proximal to gastrointestinal tissue. In further embodiments, the TWEAK antagonist is administered via a parenteral route. [018] The TWEAK antagonist can be delivered to the tissue which will be targeted with radiation therapy. In some embodiments, the localized delivery is performed via local injection. In further embodiments, the localized delivery is performed via ultrasound-mediated microbubble destruction.
[019] Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF DRAWINGS
[020] Figure 1 shows the fold-change in mean intensity of expression of TNFR family members in TNBS-induced colitis mice compared to untreated control Balb/c mice. n=3 for untreated, 6 animals for day 3 (white bars) and 5 animals for day 10 (grey bars) TNBS treated mice. p<0.01 for all treated vs. untreated comparisons, error bars are SEM.
[021] Figure 2 shows the genomic loci for TWEAK WT and mutants, indicating the WT locus, neomycin-containing targeting construct, and neo-deleted locus. The coding exons are shown as black boxes. The arrow indicates the direction of transcription. The targeting construct contains a human CD2 expression cassette (grey box) and a loxP-flanked neomycin cassette (striped box flanked by solid black). In the neo-deleted TWEAK KO locus, the CD2 cassette and one loxP element remain. Bold lines (5' and 3') beneath the WT genomic locus mark the positions of external Southern blot probes. [022] Figure 3 shows Northern blot analysis of tissues from TWEAK KO and WT mice. Fifteen μg of RNA from the indicated tissues were probed for GAPDH or TWEAK, using the full-length TWEAK cDNA.
[023] Figure 4 shows mRNA expression of TWEAK locus neighbors Senp3 (SMT3IP1), Tnfs13 (APRIL) and Eif4a by gene array analysis of colon tissue from TWEAK KO and WT Balb/c mice. Averages for 3 animals per group are plotted with no significant differences.
[024] Figure 5 shows the spleen weight and spleen weight to body weight ratio for 4 or 12 month old TWEAK KO and WT mice (WT open bars, KO filled bars).
[025] Figure 6 shows the numbers of cells in CD3, CD4, and CD8 T cell subsets from the spleens of TWEAK KO and WT mice. Twelve-month-old mice were used to measure cell numbers.
[026] Figure 7 shows the number of cells in IgM+ B cell, NK cell, and NKT cell subsets from the spleens of TWEAK KO and WT mice. Four-month-old mice were used to measure NK and NKT cells, and 12-month-old mice were used to measure IgM+ B cells.
[027] Figure 8 shows the levels of activated/memory cells within the CD4 and CD8 spleen T cell populations from twelve-month old animals. All error bars are SEM, 5-7 mice per group were used for the comparisons.
[028] Figures 9A and 9E are photos of isolated colons of WT and TWEAK KO mice, respectively. Figure 9B-9D and 9F-9H show histological features of Balb/c WT and TWEAK KO colons: untreated 9B WT and 9F KO at day 0 of the TNBS colitis protocol; TWEAK WT 9C1 9D and KO 9G, 9H at day 10 of the colitis protocol. Scale bars in Figures 9B, 9C, 9F, and 9G correspond to 0.1 mm. The images in Figures 9D and 9H are two fold higher magnification.
[029] Figure 10 shows survival curves for TWEAK KO (dotted line, n=15) and WT mice (solid line, n=16) after TNBS treatment on days 0 and 7, Kaplan- Maier analysis p=0.086.
[030] Figure 11 shows mean weight loss +/- SEM in TWEAK KO and WT mice after TNBS treatment on days 0 and 7 (indicated by arrows). The number of WT and KO mice for each time point are in parentheses. Asterisks indicate significant differences (p<0.05, unpaired t-test) on days 1 , 3 and 5.
[031] Figure 12 shows mean clinical score of TNBS-induced colitis in TWEAK KO (n=16) and WT (n=15) mice. Difference between the groups was significant, two-way Anova p value < 0.0001.
[032] Figure 13 shows mean histological scores of TNBS-induced colitis mice +/- SEM for surviving mice on day 3 (n=7 per group) or day 10 for (n=8 WT, n=12 TWEAK KO). Asterisks indicate p<0.01 by Mann Whitney test.
[033] Figure 14 shows inflammatory cell infiltration scores for TNBS colitis in Balb/c WT and TWEAK KO mice on day 3 (n=7/group) and day 10 (WT n=8, KO n=11). Mean values (horizontal lines) and p values by Mann-Whitney test are shown.
[034] Figure 15 shows histological scores for TNBS colitis in TWEAK KO, Fn14 KO and WT mice on the C57BL/6 background. (Top graph) Scores on day 3 of the colitis protocol. (Bottom graph) Scores on day 10 of the colitis protocol. All differences are significant, p<0.05 by the Mann-Whitney test.
[035] Figure 16 (left graph) shows mean TNBS-induced colitis clinical scores in Balb/c mice treated with anti-TWEAK mAb or control Ig (n=8/group) starting on day O. Mice sacrificed on day 3 received 300 μg of Ab i.p. on day 0 and mice sacrificed on day 10 received 300 μg on days 0, 3 and 7. Difference between the groups was statistically significant by two way Anova, p value = 0.049. Figure 16 (right graph) shows mean histological scores +/- SEM for colons of surviving mice treated with anti-TWEAK antibody or control Ig starting on day 0 relative to TNBS administration and sacrificed on day 3 (n=12 anti-TWEAK, n=15 control Ig treated) or day 10 (n=7 anti-TWEAK, n=6 control Ig treated). Asterisks indicate significant p-values by Mann-Whitney test, p=0.0025 for day 3, p=0.0184 for day 10.
[036] Figure 17 shows inflammatory cell infiltrations score on day 3 and day 10 (n=6-7/group) after TNBS administration. Mean values (horizontal lines) and p values by Mann-Whitney test are shown.
[037] Figure 18 shows mean clinical scores in Balb/c mice treated with anti-TWEAK mAb (n=14) or control Ig (n=13) starting on day 3 after TNBS administration. Difference between the groups was significant, two-way Anova p value =0.0014.
[038] Figure 19 shows mean histology scores +/- SEM for colons of surviving mice treated with anti-TWEAK antibody or control Ig starting on day 3 after TNBS administration and sacrificed on day 10 (anti-TWEAK n=8, control Ig treated n=5). Asterisks indicate significant p-values by Mann-Whitney test, p=0.0062.
[039] Figures 20 shows WT (20A, 2OC, and 20E) and TWEAK KO (2OB, 2OD, and 20F) colon sections on day 10 of the colitis protocol stained for peroxidase activity (2OA and 20B), neutrophil marker Gr-1 (2OC and 20D), or macrophage marker F4/80 (2OE and 20F). Images are representative of results obtained in n=8 mice/group.
[040] Figure 21 (left graph) shows the anti-TNP response of sacral lymph node T cells of TNBS-induced colitis mice on day 10 following TNBS administration. Stimulation index was calculated as the level of in vitro 3H- thymidine incorporation compared to cultures without antigen. WT group is smaller due to the death of 3 mice during the course of the experiment. Figure 21 (right graph) shows the anti-TNP serum IgG titer on day 10 of TNBS colitis. No significant differences were observed.
[041] Figure 22 depicts caspase-3 activation detected by western blot of Balb/c WT and Fn14 KO mouse intestinal epithelial cell samples treated TNFα.
[042] Figure 23 shows representative images of single staining with anti- TWFJAK and epithelial marker EpCAM on serial colon sections from Balb/c WT and TWEAK KO mice, naive or 3 days after TNBS administration to induce colitis. Anti-TWEAK staining of TWEAK KO serves as a control and reflects a low level of nonspecific staining.
[043] Figure 24 shows anti-Fn14 immunofluorescent staining (green) of epithelial colon sections from Balb/c WT and Fn14 KO mice, naive (top row) or 3 days after TNBS administration to induce colitis. Edges of the tissue on the lumen side showed nonspecific staining in both WT and Fn 14 KO mice. The bottom image shows immunofluorescent staining for both Fn14 (green) and F4/80 (red) in WT mouse tissue 3 days after TNBS administration.
[044] Figure 25 shows representative images of anti-Fn14 double staining with epithelial marker EpCAM or macrophage marker F4/80 on epithelial colon sections from WT and Fn 14 KO mice 3 days after TNBS colitis. [045] Figure 26 shows the Fn14 mRNA relative expression values for primary colon tissue cultures with media or CpG ODN or TNFα stimuli as shown. Each line represents a culture from an individual mouse.
[046] Figure 27 shows a flow cytometry overlay of anti-Fn14 (open profile) vs. control Ig staining (filled profile) of epithelial cell line MODE-K.
[047] Figure 28 shows MMP-9 production by MODE-K cells cultured in media alone or in the presence of TWEAK (1.0 ng/ml), TWEAK plus soluble Fn14 decoy protein (10 μg/ml), of agonistic anti-Fn14 mAb (10 μg/ml), or control Ig (10 μg/ml).
[048] Figure 29 shows MCP-1 and KC production in MODE-K cells stimulated by agonistic anti-Fn14 mAb versus control Ig. Mean values +/- SD are shown for triplicate cultures. Asterisks indicate significant differences, all p values <0.005 by Student's t test.
[049] Figure 30 shows production of inflammatory mediators IL-6 and MMP-9 by the colon epithelial cell line MCE301 cultured in the presence of 100 ng/ml Fc-TWEAK, control Ig, or without stimulation (media). Differences between Fc-TWEAK and media are significant for each mediator, p< 0.01. Asterisks indicate that differences between Fc-TWEAK and control Ig are significant for MMP-9 (p=0.008), MCP-1 (p=0.001), KC (p= 0.03).
[050] Figure 31 shows production of inflammatory mediators MCP-1 and KC by the colon epithelial cell line MCE301 cultured in the presence of 100 ng/ml Fc-TWEAK1 control Ig, or without stimulation (media). Differences between Fc- TWEAK and media are significant for each mediator, p< 0.01. Asterisks indicate that differences between Fc-TWEAK and control Ig are significant for MMP-9 (p=0.008), MCP-1 (p=0.001), KC (p= 0.03). [051] Figure 32 shows the TNBS-induced colitis histology scores of WT mice, TWEAK KO mice, WT mice treated with control Ig, and WT mice treated with anti-TWEAK monoclonal antibody three and ten days after administration of TNBS. Antibodies were injected on day 0, day 3, and day 7 of TNBS-induced colitis.
[052] Figure 33 shows representative images of H&E-stained jejunum and colon samples from Balb/c WT and TWEAK-KO mice 24 hours after receiving 3 Gy of whole body irradiation. Apoptotic cells with dense chromatin masses are indicated by arrows.
[053] Figure 34 shows a plot of the number of apoptotic cells observed in the jejunum and colon of Balb/c WT and TWEAK-KO mice 24 hours after receiving 3 Gy of whole-body γ-irradiation.
[054] Figure 35 shows a plot of the percentage of apoptotic cells at various crypt cell positions along the longitudinal crypt axis in Balb/c WT (dark circles) and TWEAK-KO mice (gray squares) 24 hours after receiving 3 Gy of whole-body γ-irradiation.
[055] Figure 36 shows a plot of the percentage of apoptotic cells at various crypt cell positions along the longitudinal crypt axis in C57BL6 WT (dark circles) and Fn-14-KO mice (gray squares) 24 hours after receiving 3 Gy of whole- body γ-irradiation.
[056] Figure 37 shows representative images of H&E stained jejunum and ileum samples from Balb/c WT and TWEAK-KO mice 24 hours after receiving 12 Gy of whole body γ-irradiation. Regenerating microcolonies of cells are indicated with arrows. [057] Figure 38 shows a plot of the numbers of regenerating microcolonies of cells in jejunum, ileum, and colon samples from Balb/c WT and TWEAK-KO mice 4 days after receiving 12 Gy of whole body γ-irradiation.
[058] Figure 39 shows a plot of the number of BrdU+ cells in jejunum samples of Balb/c WT or TWEAK-KO mice or in C57BL6 WT or Fn 14-KO mice 24 hours after receiving 3 Gy of whole-body γ-irradiation.
DETAILED DESCRIPTION
[059] The invention provides methods of preventing tissue damage induced by radiation therapy. Aspects of the invention relate, in part, to the discovery that disrupting the signaling of TNF family member TWEAK (TNF-like weak inducer of apoptosis) through its receptor, Fn14, reduces levels of epithelial cell death and increases epithelial cell regeneration following radiation treatment in a mouse model of tissue injury by γ-irradiation. Embodiments of the invention encompass the use of an antagonist of TWEAK for preventing or reducing cell death that occurs as a result of radiation therapy or radiation exposure. In exemplary embodiments, the methods comprise administering to a subject who has received, is receiving, or will receive radiation therapy a therapeutically effective amount of a TWEAK antagonist sufficient to prevent or reduce the severity of radiation-induced cell death.
[060] By "a therapeutically effective amount of a TWEAK antagonist sufficient to prevent or reduce the severity of radiation-induced cell death" is meant an amount of a TWEAK antagonist delivered to a subject that reduces the levels of apoptosis (programmed cell dea+h) occurring in an area of tissue treated with radiation therapy or otherwise exposed to radiation to a level that is either 1) not detectably different than that occurring in the tissue prior to administration of the radiation, or 2) that is decreased relative to the level of apoptosis occurring in the tissue receiving radiation when a TWEAK antagonist is not administered to the subject.
[061] In some embodiments, the method of preventing or reducing cell death that occurs as a result of radiation therapy comprises 1) administering a TWEAK antagonist and 2) administering radiation. In exemplary embodiments, the TWEAK antagonist is administered a sufficient amount of time prior to administration of radiation to allow the TWEAK antagonist to penetrate into the target tissue. In some embodiments, the TWEAK antagonist is administered at least two hours before administration of the radiation. In some embodiments, the TWEAK antagonist is administered between two to four hours prior to administration of the radiation. In further embodiments, the time between the administration of a TWEAK antagonist and subsequent administration of radiation is four to six hours, six to eight hours, eight to ten hours, ten to twelve hours, twelve to twenty-four hours, twenty-four to forty-eight hours, or more than 48 hours. In some embodiments, the TWEAK antagonist is administered prior to multiple rounds of radiation treatment. In other embodiments, the TWEAK antagonist is administered prior to each round of radiation administered over the course of the patient's treatment. In some embodiments, the TWEAK antagonist is administered in repeat doses. In further embodiments, the TWEAK antagonist is administered in repeat doses prior to multiple rounds of radiation treatment. In further embodiments, the TWEAK antagonist is administered in repeat doses for every round of radiotherapy administered over the course of the patients treatment.
[062] In some embodiments, the TWEAK antagonist is administered to a subject before and after radiation treatment. In further embodiments, the TWEAK antagonist is administered to a subject before, during, and after a course of radiation therapy. In some embodiments, the dosage of the TWEAK antagonist is relatively high just before and after radiation treatment and is then lowered to smaller doses over time following the treatment.
[063] In some embodiments, the TWEAK antagonist is administered to a subject after exposure to radiation. The term "exposure to radiation" includes radiation therapy, accidental radiation exposure, and exposure to individuals at risk for radiation exposure. Accordingly, the methods can benefit individuals who are exposed to radiation inadvertantly or who are exposed to radiation as part of their work, including, for example, individuals working in nuclear power plants or nuclear fuel and/or nuclear waste processing facilities, individuals transporting nuclear material, researchers working with radioactive materials, individuals responsible for handling hazardous materials, members of the military, medical care workers, and the like. In some embodiments, the subject is a patient receiving X-rays for purposes of medical monitoring and/or diagnosis. In some embodiments, the TWEAK antagonist is administered to a subject following inadvertant exposure to radiation. The TWEAK antagonist can be administered in single or multiple doses within a few hours after exposure to radiation. In some embodiments, the TWEAK antagonist is administered within 24 hours of exposure to radiation. In further embodiments, the antagonist is administered within 48 hours, 72 hours, or 96 hours or more following exposure to radiation. Administration of a TWEAK antagonist following exposure to radiation can be continued for several days, weeks, or months, or as long as the administration may be therapeutically effective. In some embodiments, the dosage of a TWEAK antagonist is lowered from a higher dose immediately following exposure to radiation to a lower dose over the course of several days, weeks, or months.
[064] In some embodiments, the TWEAK antagonist is administered in a single dose. In some embodiments, the antagonist is administered in multiple doses. In some embodiments, the TWEAK antagonist is administered several times per day, i.e. once every two to three hours, three to six hours, six to nine hours, or nine to twelve or more hours.
[065] In exemplary embodiments, the methods of the invention are used to prevent or reduce the severity of radiation-induced cell death in a subject with cancer. The cancer can be, for example, a carcinoma, sarcoma, lymphoma, leukemia, myeloma, germ cell tumor, or blastoma. Carcinomas are malignant tumors derived from epithelial cells. Carcinomas are the most common cancers, and include common forms of lung, colon, breast, and prostate cancer. Sarcomas are malignant tumors derived from connective tissue or mesenchymal cells (bone, cartilage, or fat). Lymphomas, leukemias, and myelomas are malignancies derived from hematopoietic (blood-forming) cells, including bone marrow cells and lymphocytes. Germ cell tumors are derived from totipotent cells. In adults, germ cell tumors are most often found in the testicle and ovary. In fetuses, infants, and young children, germ cell tumors are most often found on the body midline, particularly at the tip of the tailbone. Blastomas are tumors that resemble an immature or embryonic tissue, and the majority are typically found in children. [066] In some embodiments, the cancer is a solid cancer. The cancer can be, for example, lung cancer, prostate cancer, breast cancer, colon or rectal cancer, bladder cancer, pancreatic cancer, endometrial cancer, ovarian cancer, testicular cancer, melanoma, nonmelanoma skin cancer, cervical cancer, small bowel cancer, stomach cancer, urinary tract cancer, thyroid cancer, kidney (renal cell) cancer, neuroblastoma, rhabdomyosarcoma (arising from muscle), retinoblastoma, osteosarcoma, Ewing's sarcoma, and teratoma. In some embodiments, the cancer is a hematological neoplasm. In some embodiments, the hematological neoplasm is a leukemia, lymphoma, or multiple myeloma.
[067] In some embodiments, the subject receiving radiation therapy has a non-malignant condition. Exemplary non-malignant conditions include trigeminal neuralgia, severe thyroid eye disease, pterygium, pigmented villonodular synovitis, keloid scar growth, and heterotopic ossification. In some embodiments, the subject receives total body irradiation (TBI) as part of the preparative regimen for a hematopoietic stem cell (or bone marrow) transplantation due to a disease of the blood, bone marrow, or because of a certain type of cancer. In adult and pediatric patients undergoing stem cell transplantation for leukemias, lymphomas, and other hematologic conditions, total body irradiation is typically used in conjunction with high dose chemotherapy. TBI treatment typically is delivered twice daily over 3 days. However, a variety of other approaches may be used depending on the tumor site and the individual protocol. A current multiple myeloma TBI regimen, for example, utilizes a single fraction of 2 grays (Gy). In most TBI protocols, however, the total dose is 12 Gy.
[068] In some embodiments, the radiation therapy is palliative radiation therapy. Palliative radiation therapy is therapy that is not curative, but rather is used to control symptoms associated with localized tumors that cannot be treated by other methods, such as surgery.
[069] In some embodiments, the radiation therapy is prophylactic radiation therapy. For some types of cancer, prophylactic radiation may be given to areas that do not have evidence of cancer. This is done to prevent cancer cells from growing in the area receiving the radiation. For example, prophylactic radiation to the contralateral breast can be administered to reduce the rate of subsequent contralateral breast cancer and offer an option for risk reduction in women with BRCA germline mutations. As another example, in patients with small-cell lung cancers, prophylactic radiation can be administered to the central nervous system (whole brain irradiation, also called prophylactic cranial irradiation, or PCI). Individuals with small-cell lung cancer may have chest tumors disappear completely following chemotherapy and/or radiotherapy, but many later develop metastatic brain cancer, because the chemotherapy did not penetrate the blood- brain barrier. Thus, for small-cell lung cancer patients who are free of central nervous system symptoms or in which tumors are not detected in brain imaging, prophylactic cranial irradiation is often administered to extend symptom-free quality of life, if not actually increasing survival. Epithelial cells
[070] In some embodiments, the methods prevent or reduce the severity of radiation-induced cell death of epithelial cells. Apoptosis, or programmed cell death, is an important physiological process in epithelia for eliminating senescent, damaged, redundant, or deleterious cells. Epithelial cells line the cavities and surfaces of structures throughout the body. The outermost layer of skin is composed of dead stratified squamous, keratinized epithelial cells. The tissues that line the inside of the mouth, the esophagus, and part of the rectum are composed of nonkeratinized stratified squamous epithelium. Other sufaces that separate body cavities from the outside environment are lined by simple squamous, columnar, or pseud ostratified epithelial cells. Other epithelial cells line the insides of the lungs, the gastrointestinal tract, the reproductive and urinary tracts, and make up the exocrine and endocrine glands. The outer surface of the cornea is covered with fast-growing, easily-regenerated epithelial cells. Endothelium (the inner lining of blood vessels, the heart, and lymphatic vessels) is a specialized form of epithelium. Another type, mesothelium, forms the walls of the pericardium, pleurae, and peritoneum.
[071] Thus, in some embodiments, the methods of the invention prevent or reduce the severity of radiation induced cell death of non-keratinized stratified squamous epithelium. In other embodiments, the methods of the invention prevent or reduce the severity of radiation induced cell death of simple squamous, columnar, or pseud ostratified epithelial cells. In some embodiments, the methods of the invention prevent or reduce the severity of radiation induced cell death of endothelial or mesothelial cells.
[072] In some embodiments, the methods of the invention prevent or reduce the severity of radiation-induced cell death of gastrointestinal (Gl) epithelial cells. In some embodiments, the Gl epithelial cells are cells of the mouth, larynx, or esophagus. In some embodiments, the Gl epithelial cells are cells of the stomach, duodenum, jejunum, ileum, cecum, colon, or rectum. TWEAK signaling and TWEAK antagonists
[073] The TNF superfamily of ligands and receptors are prominent regulators of cell fate decisions including survival, proliferation, and differentiation (Ware et al., Cytokine Growth Factor Rev. 14:181-4 (2003)). They play- essential roles in the organogenesis and homeostasis of multiple systems, including bone, skin appendages such as hair and teeth, and lymphoid tissues. They also play complex immunoregulatory roles, for example in host defense, inflammatory responses, and positive and negative regulation of adaptive immunity.
[074] TWEAK, a member of the TNF ligand superfamily, is a type II- transmembrane protein that can be cleaved to function as a soluble cytokine and is highly expressed by inflammatory cells (Chicheportiche et al., J. Biol. Chem. 272: 32401-10 (1997)). The TWEAK receptor, Fn14, is a TNF receptor superfamily member expressed by nonlymphoid cell types. TWEAK was first described as a weak inducer of apoptosis (Chicheportiche et al., J. Biol. Chem. 272:32401-10 (1997)), and has been found to trigger multiple cellular responses ranging from proliferation to cell death through its cognate receptor Fn 14. Fn 14 expression is highly inducible (Meighan-Mantha et al., J. Biol. Chem. 274:33166- 76 (1999); Feng et al., Am. J. Pathol. 156:1253-61 (2000)). A comprehensive survey of TWEAK and Fn14 expression in normal versus injured and diseased tissues in both mouse and human recently demonstrated that in both species, expression of TWEAK, and in particular Fn14, is relatively low in normal tissues but undergoes dramatic upregulation in settings of tissue injury and diseases. The studies also showed that Fn14 is expressed by many tissue-resident progenitor cells (Girgenrath et al., EMBO J. 25:5826-39 (2006)); Jakubowski et al., J. Clin. Invest, 115:2330^0 (2005); Perper et al., J. Immunol. 177:2610-20 (2006)).
[075] After acute injury, inflammatory cytokines induce the influx of scavenger cells to remove dying cells and tissue debris, and promote wound closure and healing through controlled proliferation, migration of cells, and extracellular matrix turnover. More recently, the concept that inflammatory cells also regulate tissue regeneration via their effects on tissue progenitor cells has been suggested (Duffield et al., Clin. Sci. (Lond) 104:27-38 (2003)). However, in settings of chronic disease, these multifaceted activities are often dysregulated and pathogenic. It is now evident that TWEAK is a multifunctional cytokine, similar in this regard to its sibling TNF. TWEAK functions physiologically after acute injury and pathologically in chronic inflammatory disease settings. In contrast to TNF however, TWEAK plays no apparent role in development or homeostasis.
[076] TWEAK antagonists used to prevent or reduce the severity of radiation-induced cell death-includes, e.g., small organic or inorganic molecules, nucleic acids, proteins, or peptide mimetics that are capable of interrupting the binding of TWEAK to its cognate receptor Fn14 or otherwise interfere with TWEAK receptor signaling. "TWEAK receptor signaling" refers to all molecular reactions associated with the TWEAK receptor pathway and subsequent molecular reactions which result therefrom.
[077] Many TWEAK antagonists useful in preventing or reducing radiation- induced cell death are known in the art. Such agents include those disclosed in, e.g. International Publication Numbers WO 98/05783, WO 98/35061 , WO 99/19490, WO 00/42073, and WO 01/45730, all of which are incorporated herein by reference in their entirety.
[078] In one embodiment, the antagonist is a biologic, e.g., a protein having a molecular weight of between 5-300 kDa. For example, a TWEAK antagonist may inhibit binding of TWEAK to a Fn14. A typical TWEAK antagonist can bind to TWEAK or a TWEAK receptor, e.g., Fn14. A TWEAK antagonist that binds to TWEAK or Fn14 may block the binding site on TWEAK or Fn14, alter the conformation of TWEAK or Fn14, or otherwise decrease the affinity of TWEAK for Fn14 or prevent the interaction between TWEAK and Fn14. A TWEAK antagonist (e.g., an antibody) may bind to TWEAK or to Fn14 with a Kd of less than 10~6, 10"7, 10'8, 10"9, or 10"10 M. In some embodiments, the antagonist binds to TWEAK with an affinity at least 5, 10, 20, 50, 100, 200, 500, or 1000-fold higher than its affinity for TNF or another TNF superfamily member (other than TWEAK). In some embodiments, the antagonist binds to the Fn14 with an affinity at least 5, 10, 20, 50, 100, 200, 500, or 1000-fold better than its affinity for the TNF receptor or a receptor for another TNF superfamily member.
[079] Exemplary TWEAK antagonists include antibodies (e.g., monoclonal antibodies) that bind to TWEAK or Fn14 and soluble forms of Fn14 that compete with cell surface Fn14 for binding to TWEAK. Other types of antagonists, e.g., small molecules, nucleic acid or nucleic acid-based aptamers, and peptides, can be isolated by screening, e.g., as described in Jhaveri et al. Nat. Biotechnol. 18:1293-1297 (2000) and U.S. Patent No. 5,223,409. Exemplary assays for determining if an agent binds to TWEAK or Fn14 and for determining if an agent modulates a TWEAK/Fn14 interaction are described, e.g., in U.S. 2004-0033225. Antibodies
[080] In some embodiments, the TWEAK antagonists used in the methods of the invention include antibodies that bind to TWEAK and/or Fn14. Antibodies directed to TWEAK or a TWEAK receptor that are useful in the invention are described herein and are known in the art. Exemplary antibodies are described, e.g., in International Patent Publication No. WO 2006/130374 and U.S. Patent Publication No. 2008/0279853, which are incorporated herein by reference in their entirety.
[081] The term "antibody" refers to a protein that includes at least one immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or an immunoglobulin variable domain sequence. Antigen-binding fragments of antibodies include, e.g., single chain antibodies, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, and dAb fragments. Complete antibodies include, e.g., intact and/or full length immunoglobulins of types IgA, IgG (e.g., IgGI, lgG2, lgG3, lgG4), IgE, IgD, IgM (as well as subtypes thereof). The light chains of the immunoglobulin may be of types kappa or lambda. An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity, or may be non-functional for one or both of these activities. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), referenced above, interspersed with regions that are more conserved, termed "framework regions" (FR). The extent of the FR's and CDR's has been precisely defined (see, Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991); and Chothia, C. et al., J. MoI. Biol. 196:901-917 (1987)). Kabat definitions and numbering are used herein. Each VH and VL is typically composed of three CDR's and four FR1 s, arranged from amino-terminus to carboxyl-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
[082] An "immunoglobulin domain" refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two beta- sheets formed of about seven beta-strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay, Ann. Rev Immunol. 6:381-405 (1988)). An "immunoglobulin variable domain sequence" refers to an amino acid sequence that can form a structure sufficient to position CDR sequences in a conformation suitable for antigen binding. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may omit one, two or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other alterations. In one embodiment, a polypeptide that includes an immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form a target binding structure (or "antigen binding site"), e.g., a structure that interacts with TWEAK or Fn 14. - ~ -
[083] The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains. The heavy and light immunoglobulin chains can be connected by disulfide bonds. The heavy chain constant region typically includes three constant domains, CH1 , CH2, and CH3. The light chain constant region typically includes a CL domain. The variable region of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. [084] One or more regions of an antibody can be human, effectively human, or humanized, as described below. For example, one or more of the variable regions can be human or effectively human. An "effectively human" immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. An "effectively human" antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.
For example, one or more of the CDRs, e.g., HC CDR1 , HC CDR2, HC CDR3, LC CDR1 , LC CDR2, and LC CDR3, can be human. Each of the light chain CDRs can be human. HC CDR3 can be human. One or more of the framework regions can be human, e.g., FRI, FR2, FR3, and FR4 of the HC or LC. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non- hematopoietic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. One or more of the constant regions can be human, effectively human, or humanized. In another embodiment, at least 70, 75, 80, 85, 90, 92, 95, or 98% of the framework regions (e.g., FR1 , FR2, and FR3, collectively, or FR1 , FR2, FR3, and FR4, collectively) or the entire antibody can be human, effectively human, or humanized. For example, FR1 , FR2, and FR3 collectively can be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical, or completely identical, to a human sequence encoded by a human germline segment. [085] A "humanized" immunoglobulin variable region is an immunoglobulin variable region that is modified such that the modified form elicits less of an immune response in a human than does the non-modified form, e.g., is modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response or elicits a reduced immunogenic response in a normal human as compared to a non-humanized antibody. Descriptions of "humanized" immunoglobulins include, for example, U.S. Patent Numbers 6,407,213 and 5,693,762. In some cases, humanized immunoglobulins can include a non-human amino acid at one or more framework amino acid positions. Exemplary humanized antibodies that bind to TWEAK are described below as huP2D10-1 and huP2D10-2.The antibodies can be conjugated to a moiety, e.g., can be conjugated to poly(ethylene glycol) (e.g., PEGylated), e.g., to reduce the immunogenicity and/or increase the circulating half-lives of antibodies.
[086] In some embodiments, the antibody inhibits the interaction between TWEAK and Fn14, e.g., by physically blocking the interaction, decreasing the affinity of TWEAK and/or Fn14 for its counterpart, reducing the signaling activity of Fn14, disrupting or destabilizing TWEAK/Fn14 complexes, sequestering TWEAK or Fn14, or targeting TWEAK or Fn14 for degradation. In some embodiments, the antibody can bind to TWEAK or Fn14 at one or more amino acid residues that participate in the TWEAK/ Fn14 binding interface. Such amino acid residues can be identified, e.g., by alanine scanning. In other embodiments, the antibody can bind to residues that do not participate in the TWEAK/ Fn14 binding. For example, the antibody can alter a conformation of TWEAK or Fn 14 and thereby reduce binding affinity, or the antibody may sterically hinder TWEAK/ Fn14 binding. In one embodiment, the antibody can prevent activation of a TWEAK/ Fn14 mediated event or activity (e.g., NF-κB activation).
[087] In some embodiments, the TWEAK antagonist used in the methods of the invention is an anti-TWEAK antibody. In other embodiments, the TWEAK antagonist is an anti- Fn 14 antibody. The sequence of an exemplary human TWEAK to which anti-TWEAK antibodies may be directed is shown in SEQ ID NO:1. The sequence of a mouse TWEAK is shown in SEQ ID NO:2. International Patent Application No. WO 2006/130374, incorporated herein by reference in its entirety, discloses the sequences of specific examples of anti-TWEAK antibodies that are useful in the methods of the invention, such as P2D10, huP2D10-1 , and huP2D10-2.
[088] The amino acid sequence of the IgG heavy chain in each of mature huP2D10-1 and huP2D10-2 antibodies is as follows (SEQ ID NO:3):
1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYAMSWVRQA PGKGLEWVAE 51 ISSGGSYPYY PDTVTGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVL 101 YYDYDGDRIE VMDYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL 151 GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSWTVPSSS 201 LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAP ELLGGPSVFL 251 FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VKFNWYVDGV EVHNAKTKPR 301 EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ 351 PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK 401 TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS 451 LSPG
[089] The amino acid sequence of the light chain of huP2D10-1 is as follows (SEQ ID NO:4): 1 DWMTQSPLS LPVTPGEPAS ISCRSSQSLV SSKGNTYLHW YLQKPGQSPQ 51 FLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YFCSQSTHFP 101 RTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASWCL LNNFYPREAK 151 VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE 201 VTHQGLSSPV TKSFNRGEC
[090] The amino acid sequence of the light chain of huP2D10-2 is as follows (SEQ ID NO:5):
1 DWMTQSPLS LPVTPGEPAS ISCRSSQSLV SSKGNTYLHW YLQKPGQSPQ
51 LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCSQSTHFP
101 RTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASWCL LNNFYPREAK
151 VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE 201 VTHQGLSSPV TKSFNRGEC
[091] Suitable antibodies for use in the methods of the invention include an antibody that can bind to an epitope on TWEAK which includes at least one, two, three or four amino acid residues from an epitope on TWEAK recognized by P2D10, to a peptide from TWEAK that is bound by P2D10 (e.g., a peptide less than 25, 20, or 15 amino acids in length) or to a region of TWEAK recognized by P2D10. For example, the antibody may specifically bind to an epitope, e.g., a linear or a conformational epitope, of TWEAK, in particular human TWEAK, e.g., the soluble region of TWEAK. The antibody may compete with P2D10 for binding to TWEAK, e.g., to human TWEAK. The antibody may competitively inhibit binding of P2D10 to TWEAK, e.g., human TWEAK. In one embodiment, the antibody may bind to an epitope which overlaps with that of P2D10, e.g., includes at least one, two, three or four amino acids in common with the P2D10 epitope, or an epitope which, when bound, sterically prevents TWEAK interaction with P2D10.
[092] In one embodiment, the antibody specifically binds to at least a part of the interaction site on TWEAK that contacts human Fn 14. The antibody may compete with Fn14 for binding to TWEAK, e.g., to human TWEAK. The antibody may competitively inhibit binding of Fn 14 to TWEAK. The antibody may interact with an epitope on TWEAK which, when bound, sterically prevents interaction between TWEAK and Fn14 (e.g., between human TWEAK and human Fn14).
[093] In one embodiment, the protein includes at least one, two and preferably three CDRs from the light or heavy chain variable region of P2D10. In this context, CDRs refer to CDRs as defined by Chothia's hypervariable loops. For example, the protein includes, in the heavy chain variable domain sequence, at least one, two, or three of the following sequences within a CDR region:
GFTFSRYAMS (CDR1) (SEQ ID NO:6),
EISSGGSYPYYPDTVTG (CDR2) (SEQ ID NO:7),
VLYYDYDGDRIEVMDY (CDR3) (SEQ ID NO:8), or a CDR having an amino acid sequence that differs by no more than 4, 3, 2.5, 2, 1.5, 1 , or 0.5 alterations (e.g., substitutions, insertions or deletions) for every 10 amino acids (e.g., the number of differences being proportional to the CDR length) relative to a sequence listed above, e.g., at least one alteration but not more than two, three, or four per CDR. The heavy chain variable domain sequence may include these CDR sequences particularly in CDR3, or in at least two CDRs, e.g., CDR1 and CDR3, CDR2 and CDR3, or in all three CDRs.
[094] The protein can include, in the heavy chain variable domain sequence, at least one, two, or three of the following sequences within a CDR region (amino acids in parentheses represent alternatives for the particular position):
(i) G-(YF)-(NT)-F-(STDN)-(RY)-Y-A-(MIL)-(HS) (SEQ ID NO:9); or
(ii) Y-Y-(PV)-D-(TS)-V-(TK)-G (SEQ ID NO: 10); and
(iii) (VL)-(IL)-(YF)-(YF)-D-(YF)-D; or (DE)-(RK)-(ILVM)- (EQD)-(VAL)-M- (DE) (SEQ ID NO:11).
[095] The antibody can include, in the light chain variable domain sequence, at least one, two, or three of the following sequences within a CDR region:
(i) RSSQSLVSSKGNTYLH (SEQ ID NO:12); (CDR1),
(ii) KVSNRFS (SEQ ID NO:13); (CDR2), and
(iii) SQSTHFPRT (SEQ IDNO:14); (CDR3), or a CDR having an amino acid sequence that differs by no more than 4, 3, 2.5, 2, 1.5, 1 , or 0.5 alterations (e.g., substitutions, insertions or deletions) for every 10 amino acids (e.g., the number of differences being proportional to the CDR length) relative to a sequence listed above, e.g., at least one alteration but not more than two, three, or four per CDR. The light chain variable domain sequence may include these CDR sequences particularly in CDR3, or in at least two CDRs, e.g., CDR1 and CDR3, CDR2 and CDR3, or in all three CDRs.
[096] The antibody can include, in the light chain variable domain sequence, at least one, two, or three of the following sequences within a CDR region (amino acids in parentheses represent alternatives for the particular position): (i) (RK)-S-S-Q-S-(LI)-(KV)-S-S-(KR)-G-N-(TN)-Y-L-(EHDNQY) (SEQ ID NO: 15), or (RK)-S-S-Q-S-(LI)-V-S-S-(KR)-G-N-(TN)-Y-L-H (SEQ ID NO: 16);
(ii) (KE)-(LVI)-S-(NYS)-(RW)-(FAD)-S (SEQ ID NO: 17), or K(LVI)-S-(NYS)- R-(FAD)-S (SEQ ID NO: 18); and
(iii) (SM)-Q-(GSA)-(ST)-(HEQ)-(FWL)-P (SEQ ID NO: 19), or S-Q-(GSA)- (SIT)-(HEQ)-F-P (SEQ ID NO:20).
[097] In one preferred embodiment, the antibody includes all six CDRs from P2D10 or closely related CDRs, e.g., CDRs which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions), or other CDR described herein.
[098] In still another embodiment, the antibody includes at least one, two, or three CDRs that have the same canonical structures and the corresponding Chothia CDRs of P2D10, e.g., the same canonical structures as at least CDR1 and/or CDR2 of the heavy and/or light chain variable domains of P2D10.
[099] Particular antibodies, such as these, can be made, for example, by preparing and expressing synthetic genes that encode the recited amino acid sequences or by mutating human germline genes to provide a gene that encodes the recited amino acid sequences. Antibodies that bind to TWEAK or Fn14 may also be generated by a variety of other means. (See, for example, "Antibodies: A Laboratory Manual," ed. by Harlow and Lane, Cold Spring Harbor press: 1988).
[0100] One exemplary method includes screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, U.S. 5,223,409; Smith, Science 228:1315-1317 (1985); WO 92/18619; WO 91/17271 ; WO 92/20791 ; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809. The display of Fab's on phage is described, e.g., in U.S. Pat. Nos. 5,658,727; 5,667,988; and 5,885,793.
[0101] In addition to the use of display libraries, other methods can be used to obtain a TWEAK- or Fn14-binding antibody. For example, all or part of TWEAK or Fn14 may be used as an immunogen or as a target for selection. In one embodiment, the immunized animal contains immunoglobulin-producing cells with natural, human, or partially human immunoglobulin loci. In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected. See, for example, XENOMOU.S.E™, Green et al., Nat. Gen., 7: 13-21 (1994); U.S. Patent Publication No. 2003/0070185; U.S. Patent No. 5,789,650; and International Patent Publication No. WO 96/34096.
[0102] Non-human antibodies to TWEAK or Fn14 can also be produced, for example, in a rodent. The non-human antibody can be humanized, e.g., as described in EP 239 400; U.S. Pat. Nos. 6,602,503; 5,693,761 ; and 6,407,213, deimmunized, or otherwise modified to make it effectively human.
[0103] EP 239 400 (Winter et al.) describes altering antibodies by substitution (within a given variable region) of their complementarity determining regions (CDRs) for one species with those from another. Typically, CDRs of a non-human (e.g., murine) antibody are substituted into the corresponding regions in a human antibody, e.g., from germline immunoglobulin genes, by using recombinant nucleic acid technology to produce sequences encoding the desired substituted antibody. Human constant region gene segments of the desired isotype (usually gamma I for CH and kappa for CL) can be added and the humanized heavy and light chain genes can be co-expressed in mammalian cells to produce soluble humanized antibody. Other methods for humanizing antibodies can also be used. For example, other methods can account for the three dimensional structure of the antibody, framework positions that are in three- dimensional proximity to binding determinants, and immunogenic peptide sequences. See, for example, International Application No. WO 90/07861 ; U.S. Patent No's. 5,693,762; 5,693,761 ; 5,585,089; 5,530,101 ; and 6,407,213; and Tempest et al., Biotechnology 9: 266-271 (1991).
[0104] A non-human TWEAK- or Fn14 binding antibody may be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy and light chain variable regions of an antibody can be analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317). For detection of potential T-cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class Il binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class Il DR allotypes, and thus constitute potential T cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable regions, or preferably, by single amino acid substitutions. As far as possible, conservative substitutions are made. Often, but not exclusively, an amino acid common to a position in human germline antibody sequences may be used. After the deimmunizing changes , are identified, nucleic acids encoding VH and VL can be constructed by mutagenesis or other synthetic methods (e.g., de novo synthesis, cassette replacement, and so forth). A mutagenized variable sequence can, optionally, be fused to a human constant region, e.g., human IgGI or kappa constant regions.
[0105] In some cases, a potential T cell epitope will include residues which are known or predicted to be important for antibody function. For example, potential T cell epitopes are usually biased towards the CDRs. In addition, potential T cell epitopes can occur in framework residues important for antibody structure and binding. Changes to eliminate these potential epitopes will in some cases require more scrutiny, e.g., by making and testing chains with and without the change. Where possible, potential T cell epitopes that overlap the CDRs can be eliminated by substitutions outside the CDRs. In some cases, an alteration within a CDR is the only option, and thus variants with and without this substitution can be tested. In other cases, the substitution required to remove a potential T cell epitope is at a residue position within the framework that might be critical for antibody binding, hi these cases, variants with and without this substitution are tested. Thus, in some cases several variant deimmunized heavy and light chain variable regions are designed and various heavy/light chain combinations are tested to identify the optimal deimmunized antibody. The choice of the final deimmunized antibody can then be made by considering the binding affinity of the different variants in conjunction with the extent of deimmunization, particularly, the number of potential T cell epitopes remaining in the variable region. Deimmunization can be used to modify any antibody, e.g., an antibody that includes a non-human sequence, e.g., a synthetic antibody, a murine antibody other non-human monoclonal antibody, or an antibody isolated from a display library.
[0106] Other methods for humanizing antibodies can also be used. For example, other methods can account for the three dimensional structure of the antibody, framework positions that are in three dimensional proximity to binding determinants, and immunogenic peptide sequences. See, e.g., WO 90/07861 ; U.S. Pat. Nos. 5,693,762; 5,693,761 ; 5,585,089; 5,530,101 ; and 6,407,213; Tempest et al., Biotechnology 9:266-271 (1991). Still another method is termed "humaneering" and is described, for example, in U.S. 2005-008625.
[0107] The antibody can include a human Fc region, e.g., a wild-type Fc region or an Fc region that includes one or more alterations. In one embodiment, the constant region is altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function). For example, the human IgGI constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237. Antibodies may have mutations in the CH2 region of the heavy chain that reduce or alter effector function, e.g., Fc receptor binding and complement activation. For example, antibodies may have mutations such as those described in U.S. Patent Nos. 5,624,821 and 5,648,260. Antibodies may also have mutations that stabilize the disulfide bond between the two heavy chains of an immunoglobulin, such as mutations in the hinge region of lgG4, as disclosed in the art (e.g., Angal et al., MoI. Immunol. 30:105-08 (1993)). See also, e.g., U.S. 2005-0037000. [0108] Fully human monoclonal antibodies that bind to a TWEAK receptor can be produced, e.g., using in wYro-primed human splenocytes, as described by Boerner et al., J. Immunol., 147:86-95 (1991). They may be prepared by repertoire cloning as described by Persson et al., Proc. Nat. Acad. Sci. U. SA, 88: 2432-2436 (1991), or by Huang and Stollar, J. Immunol. Methods, 141 : 227-236 (1991); or as described in U.S. Patent. No. 5,798,230. Large nonimmunized human phage display libraries may also be used to isolate high affinity antibodies that can be developed as human therapeutics using standard phage technology (see, for example, Hoogenboom et al., Immunotechnology, 4:1-20 (1998); Hoogenboom et al., Immunol Today, 2: 371-378 (2000); and U.S. Patent Publication No. 2003/0232333).
[0109] An anti-TWEAK or anti-Fn14 antibody may be modified, e.g., by mutagenesis, to provide a pool of modified antibodies for use in the methods of the invention. The modified antibodies are evaluated to identify one or more antibodies which have altered functional properties (e.g., improved binding, improved stability, reduced antigenicity, or increased stability in vivo). In one implementation, display library technology is used to select or screen the pool of modified antibodies. Higher affinity antibodies are then identified from the second library, e.g., by using higher stringency or more competitive binding and washing conditions. Other screening techniques can also be used.
[0110] In some implementations, the mutagenesis is targeted to regions known or likely to be at the binding interface. If, for example, the identified binding proteins are antibodies, then mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs, e.g., framework regions, particularly within 10, 5, or 3 amino acids of a CDR junction. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make step-wise improvements.
[0111] In one embodiment, mutagenesis is used to make an antibody more similar to one or more germline sequences. One exemplary germlining method can include: identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Then mutations (at the amino acid level) can be made in the isolated antibody, either incrementally, in combination, or both. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In one embodiment, as many germline residues are introduced into an isolated antibody as possible.
[0112] In one embodiment, mutagenesis is used to substitute or insert one or more germline residues into a CDR region. For example, the germline CDR residue can be from a germline sequence that is similar (e.g., most similar) to the variable region being modified. After mutagenesis, activity (e.g., binding or other functional activity) of the antibody can be evaluated to determine if the germline residue or residues are tolerated. Similar mutagenesis can be performed in the framework regions.
[0113] Human germline sequences, for example, are disclosed in Tomlinson, LA. et al., J. MoI. Biol. 227:776-798 (1992); Cook, G. P. et al., Immunol. Today 16: 237- 242 (1995); Chothia, D. et al., J. MoI. Bio. 227:799-817 (1992); and Tomlinson et al., EMBO J. 14:4628-4638 (1995). The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al. MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequence, e.g., for framework regions and CDRs. Consensus human framework regions can also be used, e.g., as described in U.S. Pat. No. 6,300,064.
[0114] Selecting a germline sequence can be performed in different ways. For example, a germline sequence can be selected if it meets a predetermined criteria for selectivity or similarity, e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity, relative to the donor non-human antibody. The selection can be performed using at least 2, 3, 5, or 10 germline sequences. In the case of CDR1 and CDR2, identifying a similar germline sequence can include selecting one such sequence. In the case of CDR3, identifying a similar germline sequence can include selecting one such sequence, but may include using two germline sequences that separately contribute to the amino-terminal portion and the carboxy- terminal portion. In other implementations, more than one or two germline sequences are used, e.g., to form a consensus sequence.
[0115] In other embodiments, the antibody may be modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern). As used in this context, "altered" means having one or more carbohydrate moieties deleted, and/or having one or more glycosylation sites added to the original antibody. Addition of glycosylation sites to the presently disclosed antibodies may be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences; such techniques are well known in the art. Another means of increasing the number of carbohydrate moieties on the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody. These methods are described in, e.g., WO 87/05330, and Aplin and Wriston,. CRC Crit. Rev. Biochem. 22:259-306 (1981). Removal of any carbohydrate moieties present on the antibodies may be accomplished chemically or enzymatically as described in the art (Hakimuddin et al., Arch. Biochem. Biophys. 259:52 (1987); Edge et al., Anal. Biochem. 118:131 (1981); and Thotakura et al., Meth. Enzymol. 138:350 (1987)). See, e.g., U.S. Pat. No. 5,869,046 for a modification that increases in vivo half life by providing a salvage receptor binding epitope.
[0116] In one embodiment, an antibody has CDR sequences that differ only insubstantially from those of P2D10. Insubstantial differences include minor amino acid changes, such as substitutions of 1 or 2 out of any of typically 5-7 amino acids in the sequence of a CDR, e.g., a Chothia or Kabat CDR. Typically an amino acid is substituted by a related amino acid having similar charge, hydrophobic, or stereochemical characteristics. Such substitutions would be within the ordinary skills of an artisan. Unlike in CDRs, more substantial changes in structure framework regions (FRs) can be made without adversely affecting the binding properties of an antibody. Changes to FRs include, but are not limited to, humanizing a nonhuman-derived framework or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, e.g., changing the class or subclass of the constant region, changing specific amino acid residues which might alter an effector function such as Fc receptor binding (Lund et al., J. Immun. 147:2657-62 (1991); Morgan et al., Immunology 86:319-24 (1995)), or changing the species from which the constant region is derived.
[0117] The anti-TWEAK or anti-Fn14 antibodies used in the methods of the invention can be in the form of full length antibodies, or in the form of fragments of antibodies, e.g., Fab, F(ab')2, Fd, dAb, and scFv fragments. Additional forms include a protein that includes a single variable domain, e.g., a camel or camelized domain. See, e.g., U.S. 2005-0079574 and Davies et al., Protein Eng. 9(6):531-7 (1996). Antibodies can further be linked to one or more heterologous polypeptides. In one embodiment, an anti-Tweak antibody is linked to one or more antigen binding domains of the same or a different antibody. For example, an anti-Tweak antibody can be linked to one or more scFvs, e.g,. at the C- terminus of the Fc portions.
Soluble Fn 14 polypeptides
[0118] In some embodiments, the TWEAK antagonist is a soluble Fn14 polypeptide. The sequence of an exemplary human Fn14 is shown in SEQ ID NO:21. The sequence of an exemplary mouse Fn14 is shown in SEQ ID NO:22. An exemplary soluble form of the Fn14 protein includes a region of the Fn14 protein that binds to TWEAK, e.g., about amino acids 32-75, 31-75, 31-78, or 28- 79 of SEQ ID NO:21.
[0119] In certain exemplary embodiments, the TWEAK antagonist is an Fn 14 fusion protein. The term "fusion protein" refers to a chimeric protein comprising amino acid sequences of two or more different proteins. In some embodiments, the Fn14 fusion protein includes, in addition toFn14, one or more polypeptide portions that enhance one or more of in vivo stability, in vivo half-life, uptake/administration, tissue localization or distribution, formation of protein complexes, and/or purification. Fusion proteins may be generated recombinantly using molecular cloning techniques well known in the art. In some embodiments, the Fn14 fusion protein contains amino acids 28-79 of SEQ ID NO.21. This region of Fn14 can be physically associated, e.g., fused to another amino acid sequence, e.g., an Fc domain, at its N- or C- terminus. The Fn14 polypeptide can be from the heterologous amino acid sequence spaced by a linker. In other exemplary embodiments, the Fn14 fusion protein includes a purification subsequence, such as an epitope tag, a FLAG tag, a 6xHis sequence, or GST polypeptide. U.S. Patent No. 6,824,773 describes an exemplary Fn14 fusion protein.
[0120] In certain embodiments, the Fn14 fusion protein may contain a mutated form of Fn14 as described above. Generally, conservative substitutions of one or more amino acids present in a native Fn 14 polypeptide can be made without adversely effecting the activity of the polypeptide. Examples of conservative substitutions include substitution of amino acids outside of regions of Fn 14 that are conserved between species (such as between human and mouse Fn14), and substitution of amino acids that do not alter the secondary and/or tertiary structure of Fn14. Specific examples include substitution of one aliphatic residue for another, such as lie, VaI, Leu, or Ala for one another, or substitution of one polar residue for another, such as between Lys and Arg; GIu and Asp; or GIn and Asn, or substitution of one aromatic residue for another, such as Phe, Trp, or Tyr for one another. Other conservative substitutions, such as substitutions of entire regions having similar hydrophobicity characteristics, are known in the art and are contemplated in the methods of the invention. Methods of generating mutated forms of Fn14 are well known in the art of molecular biology, and include altering DNA molecules by random mutagenesis, site directed mutagenesis, deletions, and truncations. Specific techniques include polymerase chain reaction (PCR) mutagenesis, saturation (i.e. chemical or radiation) mutagenesis, chemical DNA synthesis, alanine scanning mutagenesis, oligonucleotide-mediated mutagenesis (hybridization to a DNA template in vitro followed by enzymatic elongation), cassette (recombinant) mutagenesis, and combinatorial mutagenesis (introduction of random degenerate sequences into the TWEAK DNA).
[0121] In certain exemplary embodiments, the methods of the invention employ an Fn14 fusion protein that includes the Fc domain of an immunoglobulin such as, e.g., IgGI , lgG2, lgG3, lgG4), IgE, IgD, IgM (Fn14-Fc). As used herein, the Fc portion of an immunoglobulin has the meaning commonly given to the term in the field of immunology. Specifically, this term refers to an antibody fragment which does not contain the two antigen binding regions (the Fab fragments) from the antibody. The Fc portion consists of the constant region of an antibody from both heavy chains, which associate through non-covalent interactions and disulfide bonds. The Fc portion can include the hinge regions and extend through the CH2 and CH3 domains to the C-terminus of the antibody. The Fc portion can further include one or more glycosylation sites. In some embodiments, the immunoglobulin Fc portion of the fusion protein contains mutations designed to remove unwanted effector functions and/or reduce the risk of inducing an immune response after repeated and prolonged administration, as described in U.S. Patent No. 7,452,966.
[0122] Fn14 has been characterized in J. Biol. Chem., 274: 33166-76 (1999) and International Publication Number WO 02/022166, the entire disclosure of which is incorporated herein by reference. The human and mouse amino acid sequences for this type I transmembrane protein are provided herein as SEQ ID NO:21 and SEQ ID NO:22, respectively. Antibody and Protein Production
[0123] Antibodies and other proteins described herein can be produced in prokaryotic and eukaryotic cells. In one embodiment, the antibodies are expressed in a yeast cell such as Pichia (see, for example, Powers et al., J. Immunol. Methods, 251 : 123-35 (2001)), Hanseula, or Saccharomyces.
[0124] Antibodies, particularly full length antibodies, for example, IgG's, and soluble Fn14 proteins may be expressed in mammalian cells. Exemplary mammalian host cells for recombinant expression include Chinese Hamster Ovary (CHO cells) (including dhfr CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. U. SA, 77: 4216-4220 (1980), in which recombinant constructs include a DHFR selectable marker, as described in Kaufman and Sharp, MoI. Biol., 159: 601-621 (1982)), lymphocytic cell lines including NSO myeloma cells and SP2 cells, COS cells, K562 cells, and cells from a transgenic animal, such as a transgenic mammal. Antibodies may be expressed in mammary epithelial cells.
[0125] In addition to the nucleic acid sequence encoding the immunoglobulin domain, the recombinant expression vectors may carry additional nucleic acid sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). Exemplary selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). [0126] In an exemplary system for recombinant expression of an antibody or antibody fragment, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, to transfect the host cells, to select for transformants, to culture the host cells, and to recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G.
[0127] Antibodies (and Fc fusions) may also include modifications, including, for example, modifications that alter Fc function. Such modifications include changes that decrease or remove interaction with an Fc receptor or with C1q, or both. For example, the human IgGI constant region can be mutated at one or more residues, including, for example, one or more of residues 234 and 237, according to the numbering in U.S. Pat. No. 5,648,260. Other exemplary modifications include those described in U.S. Pat. No. 5,648,260. [0128] For some Fn14 fusion proteins that include an Fc domain and antibodies, the protein/antibody production system may be designed to synthesize the fusion protein or antibody proteins in which the Fc region is glycosylated. For example, the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain. The Fc domain can also include other eukaryotic post-translational modifications. In other cases, the protein is produced in a form that is not glycosylated.
[0129] Antibodies and other proteins can also be produced by a transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acid sequences encoding the antibody of interest, e.g., an antibody described herein, and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the protein of interest, e.g., an antibody or Fc fusion protein. The protein can be purified from the milk, or for some applications, used directly. Formulations and routes of administration
[0130] The methods of this invention include the administration of an effective dose of a TWEAK antagonist to a subject to prevent or reduce cell death induced by radiation therapy. Determination of a preferred pharmaceutical formulation and a therapeutically efficient dose regiment for a given subject is well within the skill of the art taking into consideration, for example, the condition and weight of the patient, the extent of desired treatment and the tolerance of the patient for the treatment. In some embodiments, the TWEAK antagonist is administered locally to the region of tissue to be treated with radiation therapy. [0131] The TWEAK antagonist can be administered by any route of administration which is compatible with the antagonist, and may be formulated with any pharmaceutically acceptable carrier appropriate to the route of administration. Such carriers are well known to those skilled in the art. Administration can be performed, for example, intravenously, intraperitoneally, orally, via implant, transmucosally, transdermally, intramuscularly, and subcutaneously. Preferred routes of administration are parenteral and, in particular, intravenous, intraperitoneal, and intracapsular. Administration can be performed over an extended period on an outpatient basis, similar in this regard to radiation treatment. Daily dosages of a TWEAK antagonist are expected to be in the range of about 0.01 to 1000 μg/kg body weight, and more preferably about 10 to 300 μg/kg body weight, although precise dosages will vary depending upon the agent employed and the particular subject's medical condition and history.
[0132] The following delivery systems, which employ a number of routinely used carriers, are only representative of the many embodiments envisioned for administering the TWEAK antagonist according to the methods of the invention.
[0133] In some embodiments, the TWEAK antagonist is administered via an injectable drug delivery system. Such systems can include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (including, for example, ethanol, propylene glycol and sucrose) and polymers (including, for example, polycaprylactones and polylactic-co-glycolic acids (PLGA's)). In some embodiments, the TWEAK antagonist is delivered to via ultrasound-mediated microbubble destruction. Drugs can be incorporated into microbubbles in a number of different ways, including binding of the drug to the microbubble shell and attachment of site-specific ligands. As perfluorocarbon-filled microbubbles are sufficiently stable for circulating in the vasculature as blood pool agents, they act as carriers of these agents until the site of interest is reached. Ultrasound applied over the skin surface can then be used to burst the microbubbles at this site, causing localized release of the drug. Microbubble delivery is known in the art, and has been demonstrated to be effective in targeted delivery of biologies (i.e. proteins having a molecular weight of between 5-300 kDa; see, for example, Mukherjee, D., et al. J. Am. Coll. Cardiol. 35:1678-1686 (2000)).
[0134] Oral delivery systems for the TWEAK antagonist include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropyl- methylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc).
[0135] Transmucosal delivery systems for the TWEAK antagonist include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).
[0136] Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone).
[0137] In some embodiments, the TWEAK antagonist is administered via an implantable system. Implantable systems can include rods and discs, and can contain excipients such as PLGA and polycaprylactone.
[0138] Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).
[0139] TWEAK antagonist may, for example, be placed into sterile, isotonic formulations with or without cofactors which stimulate uptake or stability. The formulation is preferably liquid, or may be lyophilized powder. For example, the TWEAK antagonist may be diluted with a formulation comprising 5.0 mg/ml citric acid monohydrate, 2.7 mg/ml trisodium citrate, 41 mg/ml mannitol, 1 mg/ml glycine and 1 mg/ml polysorbate 20. This solution can be lyophilized, stored under refrigeration and reconstituted prior to administration with sterile Water-For- Injection (U.S.P).
[0140] The TWEAK antagonists of this invention may also be administered using microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in, near, or otherwise in communication with affected tissues or the bloodstream. Suitable examples of sustained release carriers include semipermeably polymer matrices in the form of shaped articles such as suppositories or microcapsules. Implantable or microcapsular sustained release matrices include polylactides (U.S. Pat. No. 3,773,319; EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-56 (1985)); poly(2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem. Tech, 12: 98-105 (1982)).
[0141] The methods, formulations, and dosages of the invention may be evaluated in a known model of irradiation-induced tissue injury. These models include animal models such as the mouse model described in the following examples. When testing the compositions of the invention in animal models, the biological therapeutic agent should have activity in the animal. For example, a mouse anti-TWEAK antibody may be used in lieu of a human antibody if the human antibody does not cross-react with mouse TWEAK.
[0142] The following examples provide illustrative embodiments of the invention. The skilled artisan will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the present invention. Such modifications and variations are encompassed within the scope of the invention. The examples do not in any way limit the invention.
EXAMPLES
Example 1 -- The TWEAK/Fn14 pathway has a nonredundant role in intestinal damage in mice through a TWEAK/intestinal epithelial cell axis
TWEAK/Fn14 pathway in gastrointestinal cells
[0143] Reports of Fn14 expression in the gastrointestinal tract are limited, with moderate mRNA levels observed in normal human tissue mRNA arrays (Wiley et al., Cytokine Growth Factor Rev 14:241-9 (2003)). However, expression of Fn 14 is well documented in cells of epithelial origin such as bronchial, mammary and bile duct epithelium and human keratinocytes (Jakubowski et al., J CHn Invest 115:2330-40 (2005); Jin et al., J Invest Dermatol 122:1175-9 (2004); Michaelson et al., Oncogene 24:2613-24 (2005); Xu et al., Biochem Biophys Res Commun 318:422-7 (2004)). Several colon carcinoma cell lines constitutively express Fn14 (Kawakita et al., lnt J Oncol 26:87-93 (2005); Nakayama et al., J Immunol 170:341-8 (2003)). Experiments were conducted to investigate Fn14 expression in the Gl tract and potential involvement of TWEAK/Fn14 in models of intestinal epithelial damage and repair and to determine whether (1) TWEAK regulates proliferation vs. death of mature epithelium or crypt progenitors, thereby impacting repair; (2) TWEAK is proinflammatory (Burkly et al., Cytokine 40:1-16 (2007)); or (3) TWEAK represses innate immunity (Maecker et al., Cell 123:931- 44 (2005)).
[0144] The role of the TWEAK/Fn14 pathway was investigated in two murine models of intestinal damage. TNBS-induced colitis (Example 1) is a model of human IBD in which intrarectal instillation of TNBS with ethanol injures the epithelial barrier followed by a T cell meαiated immune response against haptenated colonic proteins, leading to mucosal inflammation and epithelial damage involving both innate and adaptive immune components. The γ- irradiation injury model (Example 2) induces much more limited damage directed at the rapidly dividing progenitor cells of the intestinal crypts without a significant inflammatory component (Booth et al., lnt J Cancer 86:53-9 (2000); Leedham et al., J Cell MoI Med 9:11-24 (2005)).
[0145] These studies elucidate the TWEAK/Fn14 pathway as a novel pathogenic mediator in models of intestinal injury, promoting pathology through its effects on epithelial cell inflammatory responses and turnover. TWEAK deficiency limits local intestinal pathology without impairing systemic adaptive immune responses.
Fn 14 Knockout Mice
[0146] TWEAK or Fn14 knockout (KO) mice were backcrossed onto Balb/c or C57BL/6 backgrounds for 5-6 generations under SPF conditions at Biogen Idee or the International Medical Center of Japan (IMCJ). All experimental protocols were approved by the institutional animal care and use committees. Generation of the TWEAK- and Fn14-deficient mice
[0147] TWEAK KO mice were generated as described (Campbell et al., J Immunol 176:1889-98 (2006)). In brief, a 8.12-kb Hind\\\ subclone containing 3.36 kb of upstream sequence and the first five exons of the TWEAK gene was obtained from a BAC clone of 129 sv/J genomic DNA (Genome Systems). The targeting vector was constructed from this subclone using the bacterial recombination method (Zhang, Y., et al. Nat. Genet. 20: 123-128 (1998)). Construction of the targeting vector introduced an in-frame human CD2 cDNA upstream of a loxP-flanked neomycin gene in place of the TWEAK sequences spanning the translational start site through exon 3, thereby removing the first three TWEAK exons. TWEAK-deficient mice were generated by standard procedures using homologous recombination in embryonic stem cells (Bronson, S. K., et al. Proc. Natl. Acad. Sci. USA 93: 9067-9072 (1996)). The neomycin expression cassette was excised from the locus by mating TWEAK heterozygous mice with MSX2-Cre recombinase-expressing mice (Lewandoski, M., et al. Nat. Genet. 17: 223-225 (1997)). Homozygous TWEAK-deficient, neomycin-deleted mice were generated by breeding heterozygous mice. Absence of TWEAK mRNA expression in knockout mice was confirmed by RT-PCR and Northern blot using the full length TWEAK cDNA, and GAPDH probed as a control. TWEAK knockout (KO) mice were backcrossed onto Balb/c backgrounds for 5-6 generations under specific pathogen free (SPF) conditions. Expression of genes neighboring the TWEAK locus was unaltered.
[0148] Generation of Fn14 KO mice was described (Jakubowski et al., J CHn Invest 115:2330-40 (2005)). In brief, a 10-kb Kpn1 genomic DNA fragment containing the full murine Fn14 gene was isolated, and a targeting vector was designed to delete the first 2 exons, which contained the entire extracellular ligand-binding domain of Fn14. The target vector was transfected into the J1 129 ES cell line and selected with G418. ES cell clones were screened for homologous recombination using Southern blot, and the correct clones were injected into C57BL/6 blastocysts to generate chimeras. Mice heterozygous for targeted Fn14 alleles were obtained through further breeding and identified using Southern blot or PCR. The null mutation was confirmed by both Northern blot and RT-PCR. Mice were bred to homozygosity on the 129 background. The Fn14 mutation was backcrossed 5 times onto the C57BL/6 background under SPF conditions.
Induction and evaluation of colitis
[0149] TNBS colitis was induced by intrarectal administration of a 2 % solution of TNBS in PBS:ethanol (1 :1) as described in Dohi et al., Gastroenterology 119:724-33 (2000) and Dohi et al., J Exp Med 189:1169-80 (1999). For acute inflammatory responses, 60 μg/g body weight of TNBS was given on day 0 and animals sacrificed on day 3. For late phase responses, 36 μg/g body weight of TNBS was administered on days 0 and 7 with sacrifice on day 10. Clinical severity was scored as follows: normal stool = 0, soft stool =1 , diarrhea or proctitis (macroscopically visible perianal erosions and ulcers) = 2, anal bleeding = 3, found dead or sacrificed due to moribund condition = 4. In some studies mice were treated with murine anti-TWEAK mAb P2D10 (Campbell et al., J Immunol 176:1889-98 (2006)) or isotype-matched control Pl 17 (ATCC, Manassas, VA), 300 μg on days 3 and 7 with sacrifice on day 10, or as otherwise specified.
Histological evaluation
[0150] The colons of surviving mice were cut into proximal, middle and distal segments, formalin-fixed, paraffin-embedded and 4-μm sections stained with H&E. Each colon segment was scored individually and these scores were summed to reach a total score for the entire colon. Histological scores were assigned to each segment as follows: 0 - normal, 1 - ulcer or cell infiltration limited to the mucosa, 2 - ulcer or limited cell infiltration in the submucosa, 3 - focal ulcer involving all layers of the colon, 4 - multiple lesions involving all layer of the colon or necrotizing ulcer larger than 3mm in length. Thus the total possible histological score is 12.
Immunohistochemistrv
[0151] Four μm-thick frozen colon sections were cut from a Swiss roll of the whole colon, fixed with cold acetone for 20 min, treated with BlockAce (Dainippon Pharmaceutical Co., Ltd. Osaka Japan) for 20 min at room temperature, the Avidin-biotin blocking kit (Vector Labs), and then biotinylated anti-Fn14 mAb (P3D8) (1 μg/ml in 0.2% BlockAce) (Tran et al., Cancer Res 66:9535-42 (2006)) for 2 h at room temperature, and detected with Alexa488-labeled streptavidin (1 :500 dilution) for 30 min. Images were captured with a fluorescence microscope (BX50/BXFLA; Olympus) with a CCD camera and merged using Adobe Photoshop CS2. Fixed frozen sections were stained with biotinylated anti-Gr-1 mAb or anti-F4/80 followed by PE-streptavidin (BD Biosciences), and directly incubated with substrate 3,3'- deaminobenzidine tetrahydrochloride in the presence of H2O2 to detect myeloperoxidase activity. Sacral lymph node T cell responses
[0152] Cells isolated from sacral lymph nodes were treated with 0.3 mg/ml TNBS in RPMI 1640 for 15 min at room temperature, extensively washed and cultured in complete medium for 3 days at a density of 2x105cell/well. 0.5 μCi of [3H] thymidine per well was added 18 h prior to harvesting and scintillation counting.
Serum anti-TNP titer
[0153] Sera were collected on day 10, assayed on microtiter plates coated with TNP-coupled ovalbumin, with detected by peroxidase-labeled secondary antibodies against mouse Ig isotypes (Southern Biotechnology Associates Inc., Birmingham, AL) were used. Endpoint titers were expressed as the reciprocal Iog2 of the last dilution, which gave an optical density of 0.1 greater than the pooled serum obtained from naive mice of the same strain. Total RNA isolation for gene profiling
[0154] Colon middle and distal segments were resuspended in TRIzol® (Invitrogen Life Technologies, Carlsbad, CA), homogenized and further purified using an RNeasy Mini column (QIAGEN, Valencia, CA). Gene expression profiling and analysis
[0155] Samples were profiled using the Mouse Genome Mouse 430 2.0 GeneChip® probe array according to the manufacturer's protocol (Affymetrix, Santa Clara, CA) and supplemental information. Colon epithelial in vitro cultures -
[0156] The SV40 immortalized, nontransformed cell lines MODE-K (Vidal et al., J Immunol Methods 166:63-73 (1993)) and MCE301 (Tabuchi et al., Cell Struct Fund 25:297-307 (2000)) were cultured with media alone, or with specified concentrations of murine TWEAK, soluble Fn 14 plus TWEAK, an agonistic anti- Fn14 mAb P2.D3 or isotype-matched control Ig, MOPC-21 , for 24 hours. Culture supernatants were analyzed by SearchLight multiplex ELISA (Pierce Boston Technology Center, Woburn, MA) for chemokines, cytokines and MMPs. Cell subsets analysis for WT and TWEAK KO mice
[0157] TWEAK KO and WT splenocytes were stained using antibodies (BD Biosciences) for: CD3, CD4, CD8, IgM, NK1.1 , DX5, CD44 and CD62L. lmmunohistochemistry for fluorescently-stained samples
[0158] Fixed frozen colon sections were blocked with human IgG (10 μg/ml) for 15 minutes, treated with BlockAce for 30 min. at room temperature, blocked with Avidin-biotin blocking kit and then incubated with biotinylated anti-TWEAK mAb (huP2D10) (1.5 μg/ml in 0.2% BlockAce) for 1 hr at room temperature, followed by Streptavidin-Alexa488 (1:500 dilution, 30 min). Serial sections were stained with PE-labeled anti-EpCAM mAb (eBiosicence, 1 :3000). Fn14 detection was carried out as described above with double staining for PE-labeled EpCAM mAb or PE-labeled anti-F4/80 (Becton, Dickinson and Company, 1 :200 dilution) for 2 hours at room temperature. To visualize proliferating cells after 3-Gy γ- irradiation, 1 mg bromodeoxyuridine (BrdU) was injected i.p. 1 hour prior to sacrifice, jejunum was collected at 24 hours post irradiation, and paraffin- embedded sections were deparaffinized, treated with 4N HCI, blocked with 1% BSA and stained with rat anti-BrdU antibody (Oxford Biotech, Ltd, United Kingdom) followed by FITC-labeled anti-rat IgG (Southern Biotechnology Associates, Inc., Birmingham, AL). Numbers of BrdU+ cells were counted in 20 separate crypts in 3 or more fields for each animal.
Histological scoring system for cell infiltration
[0159] The colons of surviving mice were cut into proximal, middle and distal segments, formalin-fixed, and paraffin-embedded; 4μm sections were stained with H&E. Each colon segment was scored individually and these scores were summed to reach a total score for the entire colon. The overall magnitude of inflammatory cell infiltration was scored for each colon segment as follows: no infiltration=0, mild infiltration=1 , severe infiltration =2. Thus the total possible cell infiltration score is 6.
Gene expression profiling and analysis
[0160] Sample labeling, hybridization, and scanning were carried out according to the Eukaryotic Target Preparation protocol in the Affymetrix Technical Manual for Genechip® Expression Analysis (Affymetrix, Santa Clara, CA). The cRNA product was purified, fragmented, and applied to a Mouse Genome Mouse 430 2.0 GeneChip® probe array representing over 45,000 transcripts and transcript variants. The percent of genes called present, an indicator of sample quality, was determined using Microarray Suite 5.0 probe reduction algorithms. All sample hybridization data, in the form of CEL files, was imported into BRB-Array Tools (Gautier et al., Bioinformatics 20:307-15 (2004); Gentleman et al., Genome Bio. 5:R80 (2004)). Comparisons between groups used a random-variance t-test to generate p-values (Wright et al., Bioinformatics 19:2448-55 (2003)). False positives were controlled for by limiting the lists based on the multivariate permutation test (Korn et al., J. of Statistical Planning and Inference 124:379-398 (2004)). A 90% confidence rate was specified such that the false discovery rate within each list is less than 10%. Fold changes were generated using the Iog2 average intensity for each group of samples.
Stimulation of primary colon tissue cultures [0161] Balb/c colons were taken and 1 cm length opened segments cultured as previously described (Kawashima et al., Gastroenterology 131 :130-41 (2006)) for 6 hours with media alone or media plus a synthetic oligodeoxynucleotide containing CpG motifs, CpG ODN 1688 (Invivogen, San Diego, CA) which mimics bacterial DNA, or TNFα (R&D Systems), with segments from 3 individual mice per treatment group. Fn14 RNA levels were quantified by real-time PCR and normalized to GAPDH RNA levels as described (Jakubowski et al., J Clin Invest 115:2330-40 (2005)). Fn14 expression was evaluated as relative expression to the mean value for cultures with media alone. Fn14 mRNA increased in TNBS-treated colons of Balb/c mice [0162] To explore the role of the TNF superfamily in murine TNBS colitis, a gene profiling study was conducted with Balb/c colons collected 3 days after intrarectal TNBS/EtOH to determine acute changes in gene expression. Alternatively, animals were given a second dose of TNBS on day seven and colons collected three days later (day 10) to examine the later phase of the pathology. Three members of the TNFR superfamily showed significant up- regulation in colons of TNBS-treated mice in comparison to untreated controls in both the acute and late stages of colitis (Figure 1). Expression of TNFR1 and 2 was increased, however expression of their ligand TNFα was not detected, consistent with the prior report of no effect of TNF blockade in TNBS-induced colitis in the Balb/c strain {Dohi et al., J Immunol 167:2781-90 (2001)). A similar upregulation of Fn 14, the TWEAK receptor, was also observed, and mRNA of TWEAK was expressed in both the normal and inflamed colon, suggesting a role for the TWEAK/Fn14 pathway in TNBS-induced colitis.
TWEAK KO mice have normal immune system/intestinal architecture
[0163] To study the role of the TWEAK/Fn14 pathway in TNBS colitis, TWEAK KO mice were used (Campbell et al., J Immunol 176:1889-98 (2006)) (Figure 2). Lack of TWEAK expression was confirmed by Northern blot (Figure 3). Expression of genes neighboring the TWEAK locus was unaltered (Figure 4).
[0164] TWEAK deficient animals were healthy and normal. Comprehensive necropsy revealed no abnormalities, and extensive immune compartment analysis showed normal spleen weights in both young and aged mice, and normal percentages and absolute numbers of splenic B, T, NK, and NKT cell subsets and activated/memory cells within the CD4 and CD8 T cell populations (Figures 5, 6, 7, 8). Baseline levels of Ig subclasses were also comparable to those of WT mice (data not shown). Intestines of TWEAK KO and previously described Fn14 KO mice (Jakubowski et al., J CHn Invest 115:2330-40 (2005); Girgenrath et al., Embo J (2006)) were normal in gross morphology and histology (Figure 9 C&H). WT and TWEAK KO colons also showed comparable gene expression profiles with no statistically significant differences, suggesting that this pathway does not play a major role in the normal homeostasis of the gastrointestinal tract.
TWEAK KO mice protected from TNBS-induced colitis/ulceration
[0165] After colonic TNBS administration, WT animals displayed wasting disease accompanied by bloody diarrhea and in some animals rectal obstruction. More TWEAK KO animals survived (12 of 15) than WT control animals (8 of 16)- (Figure 10). TWEAK KO animals exhibited a significant reduction in weight loss (Figure 11) along with a significantly reduced clinical score (Figure 12). These results indicate a significant protective effect of TWEAK deficiency in the TNBS- induced colitis model.
[0166] WT Balb/c animals treated with TNBS displayed typical colonic macroscopic and histological features associated with TNBS colitis, including colon wall thickening and focal ulcers in the mucosa of the distal half of the colon (Figure 9 C&D) (Dohi et al., J Exp Med 189:1169-80 (1999)). Loss of goblet cells, mononuclear cell infiltration and crypt distortion were also observed. TWEAK KO mice treated with TNBS displayed reduced epithelial ulceration and infiltrate levels and corresponding reductions in crypt deformity as compared to WT on day 10 (Figure 9 G&H). TWEAK KO mice exhibited a significantly lower histological score than WT controls (Figure 13) reflecting two prominent differences on both days 3 and 10: reduction in frequency and extent of epithelial ulcers and reduction in leukocyte infiltration into the submucosa. The overall magnitude of inflammatory cell infiltration was also reduced in TWEAK KO as compared on WT colons at both the early and late disease stages (Figure 14).
[0167] TNBS colitis was also induced in TWEAK or Fn14 deficient mice on the C57BL/6 background. A similar protective effect was seen in this strain in the absence of either TWEAK or Fn14 (Figure 15). Additionally, WT Balb/c mice were treated with an anti-TWEAK blocking antibody starting either on day 0 (Figures 16, 17) or starting 3 days after TNBS administration and diminished clinical score and histological damage to the colon likewise observed (Figures 18, 19). Taken together, these results indicate that TWEAK/Fn14 pathway contributes to both the onset and progression of TNBSinduced colitis.
Adaptive immune response not altered in TWEAK KO mice
[0168] The mucosal damage induced by TNBS/EtOH enema leads to extensive neutrophil and macrophage infiltrates that persist and secrete proinflammatory mediators and reactive oxygen species that contribute to tissue damage. TWEAK KO mice exhibited a significantly reduced level of peroxidase activity (Figure 20 A&B), and staining for neutrophil marker Gr-1 (Figure 20 C&D) was likewise diminished. Monocyte infiltration was also reduced in TWEAK KO colons (Figure 20 E&F).
[0169] TNBS colitis is also associated with a robust adaptive immune response to trinitrophenyl (TNP)-modified proteins. TWEAK deficiency did not alter generation of the TNP-specific T cell response in draining sacral lymph nodes (Figure 21 left graph) or serum levels of anti-TNP antibodies (Figure 21 , right graph). These findings suggest that TWEAK deficiency ameliorates local pathogenic events rather than the systemic adaptive immune component.
Reduced Expression of inflammation genes in TWEAK KO mice [0170] To investigate the basis for protection from colitis in TWEAK KO mice at a molecular level, gene profiling was conducted with colons from acute (day 3) or late stage (day 10) colitis or untreated control animals. No significant differences in gene expression were observed between untreated KO and WT colons. Overall, 638 genes exhibited a statistically significant change in expression of greater than 2-fold in WT colons on day 3 as compared to only 239 gene changes in TWEAK KO. A similar trend was observed at day 10. A group of genes expressed by neutrophils and macrophages was present at a greatly reduced level in TWEAK deficient colons on both days 3 and 10 (Table 1), consistent with the reduced number of infiltrating cells (Figures 20, 21).
Table 1. Fold change in gene expression in WT and TWEAK KO colons treated with TNBS relative to untreated animals.
Figure imgf000064_0001
Genes listed are those for which a significant difference is observed between TNBS treated KO and WT in acute colitis samples (day 3). BColons from animals euthanized 3 days after TNBS treatment, relative to colons of untreated animals. cColons from animals treated with TNBS on days 0 and 7 and euthanized on day 10, relative to colons of untreated animals, ns = no significant difference from untreated control colon. Italicized fold-changes have a random variance f-test p-value between 0.01 and 0.05. p-values are less than 0.01 for all others. [0171] Profiling revealed differences in the molecular signatures of WT and KO colons that were not shown by histology (Figure 22). Expression of 12 genes encoding chemokines/chemokine receptors was markedly reduced in TWEAK KO colons. Most of the chemokines affected were those involved in neutrophil and monocyte chemotaxis. Some of these chemokines are known to be secreted by colon epithelial cells (CXCL1 , CXCL5, CCL2), while others are produced by activated macrophages (CXCL2, CCL3) and serve to recruit additional cells into the tissue (Papadakis Curr Allergy Asthma Rep 4:83-9 (2004)). In addition, two acute phase cytokines IL-6 and IL-1 β, previously associated with colonic inflammation (Kucharzik et al., lnflamm Bowel Dis 12:1068-83 (2006)), were greatly decreased in TWEAK KO mice. Also reduced were matrix metalloproteinases (MMPs) and their inhibitors, enzymes important for turnover of extracellular matrix proteins and cell migration, whose expression is associated with inflammation and subsequent tissue repair (Naito et al., MoI Aspects Med 26:379-90 (2005)).
TWEAK induces epithelial cell production of inflammatory mediators
[0172] To further define the pathogenic mechanism of TWEAK in TNBS colitis, cells expressing TWEAK and its receptor, Fn 14, were identified. TWEAK was expressed by intestinal epithelial cells in WT tissue (Figure 23), whereas expression by other cell types was equivocal, warranting further investigation. TWEAK expression was similar before and 3 days after disease induction. In contrast, there was little if any Fn14 in normal WT colon (Figure 24, upper left panel) whereas Fn 14 expression by colon epithelial cells was markedly increased in WT mice three days after TNBS administration (Figure 24, middle left panel). Double staining for Fn14 and EpCAM confirmed the epithelial localization, and for Fn 14 and F4/80 indicated that Fn 14 was not expressed by the colon infiltrating macrophages (Figure 25). The potential mechanism for upregulation of Fn 14 was explored using colon tissue cultures stimulated with inflammatory cytokines or bacterial products. These results suggest that Fn14 expression can be induced by exposure to an oligodeoxynucleotide containing CpG motifs (CpG ODN) (Figure 26).
[0173] To directly demonstrate whether TWEAK can stimulate intestinal epithelial cells to produce proinflammatory mediators, cell lines that express Fn 14 (Figure 27) were employed. Culture of MODE-K cells with TWEAK significantly increased MMP-9 production. This stimulatory effect of TWEAK is mediated by Fn14, as supported by the ability of soluble Fn14 to block it, as well as by the direct stimulatory effect of ligating the cell surface Fn14 receptor with an Fn14- specific agonist mAb (Figure 28). This direct approach was further employed to demonstrate Fn14-mediated induction of KC and MCP-1 (Figure 29). There was a tendency to induce IL-6; an array of other mediators, including 1L-1 β and TNFα, were unchanged (not shown). TWEAK stimulation of proinflammatory mediator production was also observed with the MCE301 cell line (Figures 30, 31). These results suggest that TWEAK acts locally through Fn 14 expressing epithelial cells to promote pathogenic tissue inflammation and matrix remodeling in TNBS colitis. Anti-TWEAK antibody protects WT Balb/c mice from colitis
[0174] The effectiveness of an anti-TWEAK antibody in ameliorating TNBS- induced colitis in WT mice was compared with the effect of deleting the TWEAK gene in TNBS-induced colitis mice. Figure 32 shows the TNBS-induced colitis histology scores of WT mice, TWEAK KO mice, WT mice treated with control Ig, and WT mice treated with anti-TWEAK monoclonal antibody three and ten days after administration of TNBS. Antibodies were injected on day 0, day 3, and day 7 of TNBS-induced colitis. The effect of administering an anti-TWEAK antibody was comparable to the effect of TWEAK gene deletion, indicating that inhibition of the TWEAK signaling pathway by a blocking antibody interferes with the role of TWEAK in colitis in a similar manner as inhibiting the pathway by TWEAK deletion.
[0175] This study identifies the TWEAK/Fn14 pathway as a nonredundant pathogenic mediator in contexts of intestinal injury and inflammatory disease. Significant amelioration of TNBS-induced colitis is observed in TWEAK or Fn14 deficient mice, and in WT mice treated with TWEAK blocking mAbs prophylactically or therapeutically. TWEAK deficient animals show an improved clinical course, reduced colon epithelial ulcers and less infiltration by granulocytes and macrophages. Data indicates that TWEAK acts locally on intestinal epithelial cells which upregulate Fn14 after TNBS induction, stimulating production of inflammatory and tissue remodeling enzymes, thereby contributing both acutely and during disease progression to intestinal tissue damage and abnormal tissue repair. In contrast to its contribution to end-organ pathology, TWEAK does not regulate the systemic adaptive response to TNP. These data indicate that blocking TWEAK can be an intervention point for inhibiting local immune effector mechanisms, as well as controlling epithelial injury, in contexts of intestinal injury and inflammatory disease. Blocking TWEAK can be a favorable intervention point, since it orchestrates events specific to the disease target tissue without systemically impairing host immunity. . [0176] The TWEAK KO mice of the present study were extensively characterized, with no differences observed between KO and WT. Since full length TWEAK cDNA was used as a probe in the Northern blot analysis, it is extremely unlikely that TWEAK is expressed in the test animals. However, prior studies in another TWEAK KO strain showed augmented innate immunity and :.- ceased numbers of immune cell subsets, including NK and T cells (Maecker et al., Cell 123:931-44 (2005)). The differences between the two TWEAK deficient strains might be explained by different housing conditions and/or distinct targeting strategies for the TWEAK locus. The results in the TWEAK KO mice of the present study are independently validated in Fn 14 KO and WT mice treated with TWEAK blocking mAb.
[0177] Intestinal epithelial cells markedly upregulate Fn14 after TNBS administration. Although other local Fn14 expressing cells cannot be ruled out, colon infiltrating macrophages do not appear to express this receptor. Intestinal epithelium is a well established player in the innate defense of the intestine (Abreu et al., J Immunol 174:4453-60 (2005)). The present in vitro studies suggest that Fn14 can be induced by exposure to bacterial DNA after injury to the epithelial barrier. Although TNFα is not expressed in the Balb/c strain after TNBS administration, it might may also induce Fn14 in contexts of intestinal injury on other genetic backgrounds. Intestinal epithelial cells also respond to TWEAK, with increased production of chemokines involved in monocyte and neutrophil recruitment and MMP-9. Conversely, expression of these gene signatures was significantly reduced locally in TWEAK KO as compared to WT colons. While the pathogenic role of inflammatory cytokines, chemokines and MMPs in models of colitis is well established (Papadakis Curr Allergy Asthma Rep 4:83-9 (2004); Naito et al., MoI Aspects Med 26:379-90 (2005)), the present studies identify TWEAK as an important instigator of their production in TNBS induced colitis apparently through stimulation of intestinal epithelial cells. Likewise, TWEAK was previously shown to significantly contribute to joint inflammation and damage in mouse collagen-induced arthritis, likely through its effect on mesenchymal joint cell types, without affecting systemic collagen-specific T cell antibody responses (Kamata et al., J Immunol 177:6433-6439 (2006); Perper et al., J Immunol 177:2610-20 (2006)). These studies underscore the important disease-promoting contribution of TWEAK-epithelial and TWEAK-stromal cell interactions locally in the disease target tissue.
[0178] MMPs, enzymes important for degradation of extracellular matrix proteins and cell migration are significantly increased after TNBS administration in WT mice and may contribute to disease pathogenesis in multiple ways. Previously, MMP-9 was shown to be abundantly associated with inflamed areas in ulcer bases in human IBD tissue (Arihiro et al., Histopathology 39:50-9 (2001)) and MMP-3 shown to produce severe tissue injury in gut explant cultures (Pender et al., J Immunol 160:4098-103 (1998)). Thus proteolysis by MMPs may contribute to ulcer formation and severity, and reduced MMPs including MMP-3 and MMP-9 in TWEAK KO animals may contribute to reduced ulceration. MMPs are also involved in accumulation of inflammatory cells into the intestine in TNBS colitis as evidenced by reduced neutrophil accumulation in MMP inhibitor treated animals (Di Sebastiano et al., Digestion 63:234-9 (2001); Sykes et al., Aliment Pharmacol Ther 13:1535-42 (1999)). Thus reduced MMP expression in TWEAK KO mice may contribute to decreased inflammation. It is notable that TWEAK has been demonstrated to induce MMP-9 dependent proliferation and branching of mammary gland epithelial cells in vitro (Michaelson et al., Oncogene 24:2613-24 (2005)). Decreased anchorage of epithelial cells as a consequence of matrix degradation may trigger cell proliferation and branching. Likewise, in the case of intestinal epithelium, TWEAK-induced MMP activity may reduce epithelial anchoring and thereby promote cell proliferation, crypt deformity and branching, features of abnormal repair observed 10 days after TNBS insult. These features are reduced in the TWEAK KO mice.
[0179] In summary, these studies demonstrate that TWEAK produced by adaptive and innate cell types acts on epithelial cells to enhance the local production of well known inflammatory mediators, tissue remodeling enzymes, and regulate epithelial turnover. Anti-TNF-α antibody treatment was a milestone in the therapy of Crohn's disease (Targan et al., N Engl J Med 337:1029-35 (1997)) and now in ulcerative colitis (Rutgeerts et al., N Engl J Med 353:2462-76 (2005). However, 30-40% of patients are refractory to this treatment. Thus new therapeutics are sought to address this unmet need.
Example 2 -- TWEAK or Fn14 deficiency reduces intestinal epithelial cell death following γ-irradiation
[0180] The decrease in radiation-induced epithelial apoptosis in the absence of TWEAK activity was demonstrated in mice deficient for either TWEAK or the TWEAK receptor Fn 14, which are described in Example 1.
[0181] TWEAK- or Fn14-KO mice, along with corresponding WT controls for each, received 3 Gy of whole-body irradiation using the γ-irradiation apparatus MBR-1520-R (Hitachi Medical Corporation, Tokyo, JAPAN). Twenty-four hours post-irradiation, the mice were sacrificed, and small intestine and colon were collected. Paraffin-embedded sections were prepared from a Swiss roll of whole colon or a 7 cm length from the oral end of the small intestine for analysis of jejunum. Numbers of apoptotic cells per crypt, detected in H&E stained sections by chromatin condensation, were counted in 20 separate crypts in 3 or more fields for each animal.
[0182] Balb/c TWEAK-KO mice showed a significantly lower number of apoptotic cells than WT in both jejunum and colon after 3 Gy of whole-body irradiation. Representative H&E stained images of crypts from Balb/c WT and TWEAK-KO jejunum and colon samples 24 hours after irradiation are shown in Figure 33. Apoptotic cells in a single representative crypt are indicated by arrowheads. The number of apoptotic cells in the jejunum or colon crypts in Balb/c WT or TWEAK-KO mice is shown in Figure 34. Asterisks indicate p<0.01.
[0183] The percentage of apoptotic cells at various crypt positions in the irradiated mice was also measured. In WT mice, a relatively high frequency of apoptotic cells was found in the lower crypt positions on the longitudinal crypt axis (Figure 35; dark circles) where small intestine crypt progenitor cells are localized (Leedham, S.J., et al. J Cell MoI Med 9:11-24 (2005)), although increased apoptosis was also observed at higher crypt positions. TWEAK-KO mice displayed a marked reduction in the percentage of apoptotic cells at both lower and higher crypt positions (Figure 35, gray squares). Values shown in Figure 35 are mean ± SEM based on analysis of 10-20 crypts for each of the 5 animals per group. All positions except 1 , 2, 4, and 13 showed significant difference between WT and KO mice, p<0.05 by two-tailed t test.
[0184] The reduction in apoptosis after γ-irradiation injury was also observed in Fn14-KO mice. Like TWEAK-KO mice, Fn-14 mice had a reduced percentage of apoptotic cells relative to WT mice at both lower and higher crypt positions (Figure 36; C57BL/6 WT in black circles; Fn14-KO in gray squares). As for Figure 35, values shown in Figure 36 are mean ± SEM based on analysis of 5 animals per group. Significant differences by two-tailed t test (p<0.05) between WT and KO mice were observed at positions 3, 4, and 5 in the lower crypt.
Example 3 -- TWEAK deficiency increases the appearance of regenerating epithelial cells after irradiation
[0185] The effect of TWEAK deficiency on cell survival was also assessed by measuring cell regeneration in intestinal epithelial cells of WT and TWEAK- deficient mice receiving radiation treatment. Balb/c WT and Balb/c TWEAK-KO mice received 12 Gy of whole-body irradiation using the γ-irradiation apparatus MBR-1520-R (Hitachi Medical Corporation, Tokyo, JAPAN). Four days post- irradiation, the mice were sacrificed, and small intestine was collected. Paraffin- embedded sections of both the jejunum and ileum were prepared for H&E staining and analysis.
[0186] Microcolonies of regenerating cells were visible in both the jejunum and ileum samples from TWEAK-KO mice, whereas none were visible in WT mice (Figure 37, quantitated in Figure 38), indicating that TWEAK disruption enhances regenerative cell growth in mice gastrointestinal epithelial tissue damaged by radiation, thereby contributing to epithelial cell turnover.
Example 4 -- Enhanced survival of intestinal epithelial cells in
TWEAK-deficient mice following radiation treatment
[0187] The effect of TWEAK deficiency on cell survival was also assessed by measuring cell proliferation in intestinal epithelial cells of WT and TWEAK- deficient mice receiving radiation treatment. The number of proliferating cells in crypts of Balb/c WT and TWEAK-KO mice and in C57BL/6 WT and Fn 14-KO mice after irradiation was determined. To visualize proliferating cells after 3 Gy γ-irradiation, 1 mg 5-bromo-2'-deoxy-uridine (BrdU; Sigma-Aldrich) was injected intraperitoneally into the mice 1 hour prior to sacrifice. Jejunum was collected at 24 hours post irradiation, and paraffin-embedded sections were deparaffinized, treated with 4N HCl, blocked with 1% BSA and stained with rat anti-BrdU antibody (Oxford Biotech, Ltd, United Kingdom) followed by FITC-labeled anti-rat IgG (Southern Biotechnology Associates, Inc., Birmingham, AL). Numbers of BrdU+ cells were counted in 20 separate crypts in 3 or more fields for each animal. The number of BrdlT cells was reduced in WT mice relative to either TWEAK-KO or Fn14-KO by a significant difference (Figure 39; p<0.05 between WT and either TWEAK-KO or Fn14-KO), indicating that the proliferation of intestinal crypt cells after irradiation is greater in the absence of TWEAK. Thus, in the γ-irradiation injury model, TWEAK and Fn 14 deficiency result in greater survival of intestinal epithelial cells.
2 xample 5 -- TWEAK participates in TNFα-induced apoptosis pathway
[0188] Primary cultures of mouse intestinal epithelial cells from wild type
(Balb/c) and Fn 14 KO mice were treated with TN Fa for either 15 or 40 minutes. The activation of apoptotic mediator caspase-3 was then detected in the TNFα- treated cells by western blot analysis. of homogenized cell samples. As Figure 40 demonstrates, active caspase-3 (CASP3) was present in wild type cells treated with TNFα after 15 minutes (early phase of caspase-3 activation) and after 40 minutes (late phase of caspase-3 activation). In Fn14 KO cells treated with TNFα, active caspase-3 was present after 40 minutes, but was absent after 15 minutes, demonstrating that Fn14 is required for the early phase of TNFα-induced caspase-3 activation.
[0189] The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.
[0190] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification, including claims, are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method of preventing or reducing the severity of radiation-induced cell death in a subject, comprising administering a sufficient amount of a TWEAK antagonist to a subject who has received, is receiving, or will receive radiation therapy or radiation exposure to prevent or reduce the severity of radiation- induced cell death.
2. The method of claim 1 , wherein the method comprises administering a TWEAK antagonist to a subject before the subject receives radiation therapy.
3. The method of claim 1 , wherein the subject has cancer.
4. The method of claim 3, wherein the cancer is a solid cancer.
5. The method of claim 3, wherein the radiation therapy is palliative radiation therapy.
6. The method of claim 1 , wherein the radiation therapy is prophylactic radiation therapy.
7. The method of claim 1 , wherein radiation therapy is locally administered to a specific area of tissue.
8. The method of claim 1 , wherein the radiation therapy is total body irradiation.
9. The method of claim 8, wherein the subject receives total body irradiation prior to receiving a bone marrow transplant.
10. The method of claim 1 , wherein the method prevents radiation- induced cell death of epithelial cells.
11. The method of claim 10, wherein the epithelial cells are gastrointestinal epithelial cells.
12. The method of claim 11 , wherein the gastrointestinal cells are cells of the jejunum, ileum, or colon.
13. The method of claim 1 , wherein the TWEAK antagonist is an anti- TWEAK antibody, an anti-TWEAK receptor antibody or an antigen binding fragment thereof.
14. The method of claim 13, wherein the antibody is a full length IgG.
15. The method of claim 13, wherein the antibody is an antigen-binding fragment of a full length IgG.
16. The method of claim 13, wherein the antibody is a monoclonal antibody.
17. The method of claim 13, wherein the antibody comprises a human Fc region.
18. The method of claim 13, wherein the antibody is a humanized antibody or antigen-binding fragment thereof.
19. The method of claim 1 , wherein the TWEAK antagonist is a soluble TWEAK receptor polypeptide.
20. The method of claim 19, wherein the soluble TWEAK receptor polypeptide is fused with an antibody Fc region.
21. The method of claim 19, wherein the soluble TWEAK receptor polypeptide comprises an amino acid sequence that is at least 90% identical to amino acids 28-79 of SEQ ID NO:3.
22. The method of claim 21 , wherein the soluble TWEAK receptor polypeptide comprises an amino acid sequence that is at least 95% identical to amino acids 28-79 of SEQ ID NO:21.
23. The method of claim 22, wherein the soluble TWEAK receptor polypeptide comprises amino acids 28-79 of SEQ ID NO:21.
24. The method of claim 23, wherein the soluble TWEAK receptor polypeptide comprises human Fn 14-Fc.
25. The method of claim 1 , wherein the TWEAK antagonist is administered via an enteric route.
26. The method of claim 25, wherein the tissue to be targeted with radiation therapy is gastrointestinal tissue or is proximal to gastrointestinal tissue.
27. The method of claim 1 , wherein the TWEAK antagonist is administered via a parenteral route.
28. The method of claim 27, wherein the TWEAK antagonist is administered via localized delivery to the tissue to be targeted with radiation therapy.
29. The method of claim 28, wherein the localized delivery is performed via local injection.
30. The method of claim 28, wherein the localized delivery is performed via ultrasound-mediated microbubble destruction.
31. A pharmaceutical composition comprising a TWEAK antagonist for preventing or reducing the severity of radiation-induced cell death in a subject who has received, is receiving, or will receive radiation therapy or radiation exposure,.
32. The pharmaceutical composition of claim 31 administered to a subject before the subject receives radiation therapy.
33. The of pharmaceutical composition of claim 31 , wherein the subject has cancer.
34. The pharmaceutical composition of claim 33, wherein the cancer is a solid cancer.
35. The pharmaceutical composition of claim 33, wherein the radiation therapy is palliative radiation therapy.
36. The pharmaceutical composition of claim 31 , wherein the radiation therapy is prophylactic radiation therapy.
37. The pharmaceutical composition of claim 31 , wherein radiation therapy is locally administered to a specific area of tissue.
38. The pharmaceutical composition of claim 31 , wherein the radiation therapy is total body irradiation.
39. The pharmaceutical composition of claim 38, wherein the subject receives total body irradiation prior to receiving a bone marrow transplant.
40. The pharmaceutical composition of claim 31 for preventing radiation- induced cell death of epithelial cells.
41. The pharmaceutical composition of claim 40, wherein the epithelial cells are gastrointestinal epithelial cells.
42. The pharmaceutical composition of claim 41 , wherein the gastrointestinal cells are cells of the jejunum, ileum, or colon.
43. The pharmaceutical composition of claim 31 , wherein the TWEAK antagonist is an anti-TWEAK antibody, an anti-TWEAK receptor antibody, or an antigen binding fragment thereof.
44. The pharmaceutical composition of claim 43, wherein the antibody is a full length IgG.
45. The pharmaceutical composition of claim 43, wherein the antibody is an antigen-binding fragment of a full length IgG.
46. The pharmaceutical composition of claim 43, wherein the antibody is a monoclonal antibody.
47. The pharmaceutical composition of claim 43, wherein the antibody comprises a human Fc region.
48. The pharmaceutical composition of claim 43, wherein the antibody is a humanized antibody or antigen-binding fragment thereof.
49. The pharmaceutical composition of claim 31 , wherein the TWEAK antagonist is a soluble TWEAK receptor polypeptide.
50. The pharmaceutical composition of claim 49, wherein the soluble TWEAK receptor polypeptide is fused with an antibody Fc region.
51. The pharmaceutical composition of claim 49, wherein the soluble TWEAK receptor polypeptide comprises an amino acid sequence that is at least 90% identical to amino acids 28-79 of SEQ ID NO:21.
52. The pharmaceutical composition of claim 51 , wherein the soluble TWEAK receptor polypeptide comprises an amino acid sequence that is at least 95% identical to amino acids 28-79 of SEQ ID NO:21.
53. The pharmaceutical composition of claim 52, wherein the soluble TWEAK receptor polypeptide comprises amino acids 28-79 of SEQ ID NO:21.
54. The pharmaceutical composition of claim 53, wherein the soluble TWEAK receptor polypeptide comprises human Fn14-Fc.
55. The pharmaceutical composition of claim 31 formulated for administration via an enteric route.
56. The pharmaceutical composition of claim 55, wherein the tissue to be targeted with radiation therapy is gastrointestinal tissue or is proximal to gastrointestinal tissue.
57. The pharmaceutical composition of claim 31 formulated for administration via a parenteral route.
58. The pharmaceutical composition of claim 57 formulated for administration via localized delivery to the tissue to be targeted with radiation therapy.
59. The pharmaceutical composition of claim 58, wherein the localized delivery is performed via local injection.
60. The pharmaceutical composition of claim 58, wherein the localized delivery is performed via ultrasound-mediated microbubble destruction.
61. Use of a TWEAK antagonist in the formulation of a medicament for preventing or reducing the severity of radiation-induced cell death in a subject who has received, is receiving, or will receive radiation therapy or radiation.
62. The use of claim 61 , wherein the medicament is administered to a subject before the subject receives radiation therapy.
63. The use of claim 61 , wherein the subject has cancer.
64. The use of claim 63, wherein the cancer is a solid cancer.
65. The use of claim 63, wherein the radiation therapy is palliative radiation therapy.
66. The use of claim 61 , wherein the radiation therapy is prophylactic radiation therapy.
67. The use of claim 61 , wherein radiation therapy is locally administered to a specific area of tissue.
68. The use of claim 61 , wherein the radiation therapy is total body irradiation.
69. The use of claim 68, wherein the subject receives total body irradiation prior to receiving a bone marrow transplant.
70. The use of claim 61 , wherein the medicament prevents radiation- induced cell death of epithelial cells.
71. The use of claim 70, wherein the epithelial cells are gastrointestinal epithelial cells.
72. The use of claim 71 , wherein the gastrointestinal cells are ceils of the jejunum, ileum, or colon.
73. The use of claim 61 , wherein the medicament comprising a TWEAK antagonist is an anti-TWEAK antibody, an anti-TWEAK receptor antibody or an antigen binding fragment thereof.
74. The use of claim 73, wherein the antibody is a full length IgG.
75. The use of claim 73, wherein the antibody is an antigen-binding fragment of a full length IgG.
76. The use of claim 73, wherein the antibody is a monoclonal antibody.
77. The use of claim 73, wherein the antibody comprises a human Fc region.
78. The use of claim 73, wherein the antibody is a humanized antibody or antigen-binding fragment thereof.
79. The use of claim 61 , wherein the medicament comprising a TWEAK antagonist is a soluble TWEAK receptor polypeptide.
80. The use of claim 79, wherein the soluble TWEAK receptor polypeptide is fused with an antibody Fc region.
81. The use of claim 79, wherein the soluble TWEAK receptor polypeptide comprises an amino acid sequence that is at least 90% identical to amino acids 28-79 of SEQ ID NO:21.
82. The use of claim 81 , wherein the soluble TWEAK receptor polypeptide comprises an amino acid sequence that is at least 95% identical to amino acids 28-79 of SEQ ID NO:21.
83. The use of claim 82, wherein the soluble TWEAK receptor polypeptide comprises amino acids 28-79 of SEQ ID NO:21.
84. The use of claim 83, wherein the soluble TWEAK receptor polypeptide comprises human Fn14-Fc.
85. The use of claim 61 , wherein the medicament comprising a TWEAK antagonist is administered via an enteric route.
86. The use of claim 85, wherein the tissue to be targeted with radiation therapy is gastrointestinal tissue or is proximal to gastrointestinal tissue.
87. The use of claim 61 , wherein the medicament comprising a TWEAK antagonist is administered via a parenteral route.
88. The use of claim 87, wherein the medicament comprising a TWEAK antagonist is administered via localized delivery to the tissue to be targeted with radiation therapy.
89. The use of claim 88, wherein the localized delivery is performed via local injection.
90. The use of claim 88, wherein the localized delivery is performed via ultrasound-mediated microbubble destruction.
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