WO2013189287A1 - Use of interferon in treatment/prevention of tumors resistant to conventional anti-tumor therapy - Google Patents

Abstract

The invention provides for the use of an interferon (IFN) in the treatment and/or prevention of tumors resistant to conventional anti-tumor therapy, such as radiotherapy and chemotherapy, and/or provides for the use of said interferon in combination with other anti-tumor therapies, and provides a related composition and kit.

Description

 Use of interferon in the treatment/prevention of tumors resistant to conventional anti-tumor therapies and related products and methods

 The present invention relates to the treatment and prevention of tumors, in particular cancers, in particular, the use of interferon (IFN) in the treatment and/or prevention of tumors which are resistant to conventional anti-tumor therapies and/or Use in conjunction with other anti-tumor therapies, and related products and methods. Background technique

 The cytotoxicity of many conventionally used cancer therapies for cancer cells is produced by inducing lethal DNA damage. These commonly used cancer therapies are, for example, radiation therapy (RT), certain chemotherapy (e.g., using antibodies against tumor cells), and the like. However, many tumors have been found to be unresponsive or poorly responsive to these conventional anti-tumor therapies. Currently, there is still a lack of effective means to treat/prevent tumors that are resistant to conventional anti-tumor therapies such as radiation therapy, chemotherapy, and the like.

Type I interferons are a family of cytokines that are commonly known for their function in antiviral responses. However, in tumor systems, the function of type I IFN is less studied and characterized. Some evidence suggests that type I IFN may play a role in controlling tumor growth. In particular, early studies using IFN-α/Ρ neutralizing antisera showed that type I IFN can limit the growth of transplantable tumors (Gresser et al., 1983). In addition, complete deletion of type I IFN signaling resulted in faster tumor growth and increased mortality in several synergistic transplantable tumor models, including B16 melanoma (Picaud et al., 2002). These studies have demonstrated the importance of type I IFN. However, no one has studied whether the inhibition of tumor growth by type I IFN is immune-mediated. Recently, the production of endogenous type I IFN has been shown in tumor immunology editing. It plays a key role, and this effect is different from the well-characterized role of type II IFN (IFNy) in the immune response to tumors (Dunn et al, 2005; Dunn et al, 2006). In contrast to the role of type I IFN in mediating "natural" immunity to transplantable tumors, for type I IFN has been established Little is known about the role of the treatment of tumors such as cancer, especially those that have been shown to induce tumor-specific adaptive immune responses (such as localized ablation radiation or certain chemotherapy) (Ma et al., 2010). .

 The Cancer Immunology Editor is a process in which the immune system inhibits tumor growth and shapes tumor immunogenicity. Recent studies have shown that type I IFN is required to elicit an anti-tumor response and that the role of type I IFN is differentiated in time from the effect of type II IFN (i.e., IFN-γ) during cancer immunoediting. In lymphocyte-mediated tumor rejection, dendritic cells (DC), particularly CD8 a (+) DC, are functionally related targets for endogenous type I IFN (Diamond et al., 2011). Spontaneous T cell initiation often occurs in response to growing tumors. However, the intrinsic immune mechanisms that promote natural anti-tumor T cell responses have not been identified. In human metastatic melanoma, there is a correlation between the transcriptional characteristic profile of type I interferon (IFN) and T cell markers in metastatic tumor tissues. In mice, after tumor transplantation, it was produced by CDllc (+) cells, whereas tumor-induced T cell priming was defective in mice lacking IFN-α / P R or Statl. Thus, host type I IFN is critical for intrinsic immune recognition of growing tumors by signaling on CD8 a (+) DC (Fuertes et al., 2011).

 Therefore, it is still unclear to date that for tumors that have been created and established,

Summary of the invention

As discussed above, the cytotoxicity of many conventionally used anti-tumor therapies for cancer cells is produced by inducing lethal DNA damage. The inventors of the present invention have found that an excess of host DNA can induce the production of IFN, which is a cascade of intrinsic immunity and adaptive immunity, thereby causing tumor regression. In tumor cells or tumors (or hosts) that lack IFN or its associated signaling, the immune cascade required to regress tumors cannot be produced, resulting in resistance to conventional anti-tumor immunotherapy. Thus, the present application describes the key role of interferon (eg, type I interferon (IFN)) in local tumor control, particularly when administered in combination with conventional anti-tumor therapies, which can produce synergistic effects that enhance the Conventional anti-tumor therapy Effect or treatment and/or prevention of tumor resistance to said conventional therapy. The inventors of the present invention have also discovered that IFN is required to cross-prime T cells, thereby allowing for frequent t-therapy or chemotherapy by sputum IFN. Resistant tumors resolve. Using adenovirus-mediated expression of IFN-[beta], the inventors have demonstrated that delivery of exogenous type I IFN (e.g., IFN-[R]) to tumor tissue is also sufficient to selectively amplify antigen-specific sputum cells, which results in complete tumors. Regressed. The application further develops an antibody-based system to target the delivery of type I IFN to tumor tissues, causing both innate and adaptive immunity to attack the tumor.

Thus, in one aspect, the key role of type I IFN in tumor growth control is described herein, and in a specific embodiment, this is mediated by antibody-based IFN fusion protein delivery in a sputum cell dependent manner. of. The enhanced cross-initiation ability of tumor invasive DCs is not attributable to the mature state or elevated expression of conventional costimulatory molecules, but rather to the local production of type I IFN. Moreover, in another specific embodiment, local type I IFN delivery using a clinically relevant adenoviral vector encoding Aβ can mediate complete tumor rejection in a CD8 ⁺ T cell dependent manner. The results of the present invention support the positive role of type I IFN in producing a tumor-specific CD8+ T cell response produced by IFN (i.e., Ab-IFN) linked to an anti-tumor antibody.

 One aspect of the invention relates to the use of an interferon, preferably a type I interferon, in particular IFN-β, a fragment thereof or a functional variant thereof for the preparation of a medicament, wherein the medicament

 - for treating and/or preventing tumors that are resistant to conventional anti-tumor therapies (e.g., radiation therapy, chemotherapy); and/or

 - for the treatment and/or prevention of tumors in combination with other anti-tumor therapies (eg radiotherapy, chemotherapy);

 Wherein the conventional therapy induces a tumor-specific adaptive immune response; and wherein the interferon, a fragment thereof or a functional variant thereof is capable of stimulating the production of an anti-tumor cytotoxic sputum lymphocyte.

In a specific embodiment, the conventional anti-tumor therapy is radiation therapy. More specifically, it is X-ray radiation, and the dose of the radiation may be, for example, 1 - 5 Gy (for example, 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy), 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy or higher; The radiation is for 1 day or at least two days, such as 3 days, 4 days, 5 days or more Long.

 In a specific embodiment, the interferon, fragment thereof or a functional variant thereof is contained in a viral vector, for example, an adenovirus; an adeno-associated virus; a retrovirus, such as murine mololo Leukemia virus; mouse Harvey sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; SV40-type virus; polyoma virus; Epstein-Barr virus; papilloma virus; disease virus; vaccinia virus; poliovirus; And an RNA virus such as a retrovirus; preferably, the viral vector is an adenoviral vector.

 In a specific embodiment, the interferon, fragment thereof or a functional variant thereof is linked to a targeting moiety (eg, an antibody) that binds to a tumor associated antigen, wherein the targeting moiety and the interferon, fragment thereof Or a functional variant thereof is directly linked (for example as a fusion protein) or linked by a linker.

 In a specific embodiment, the tumor associated antigen is EGFR, the targeting moiety is an anti-EGFR antibody, and the tumor is a tumor that expresses EGFR.

 In other specific embodiments, the tumor is a malignant tumor, such as a malignant solid tumor, including but not limited to, for example, breast cancer, lung cancer, prostate cancer, colon cancer, skin cancer, head and neck cancer, lymphoma or melanoma.

 In other specific embodiments, the medicament of the invention is for use in combination with at least one other anti-tumor therapy, such as radiation therapy (eg, X-rays as described above) Radiation), chemotherapy, etc. In particular, the chemotherapy may be a therapy for administering an antibody directed against a tumor-associated antigen; the chemotherapy may also be, for example, administration of a chemotherapeutic agent such as, but not limited to: a pit-based reagent , antimetabolites, cytotoxic antibiotics, doxorubicin, actinomycin D, mitogen, magenta, gentamicin, doxorubicin, tamoxifen, taxol, taxotere, vinca Neobase, vinblastine, vinorelbine, etoposide (VP-16), 5-oxouracil (5FU), cytarabine, cyclophosphamide, thiotepa, methotrexate, camptothecin, Actinomycin D, mitomycin C, cisplatin (CDDP), aminopterin, combretastatin, other vinca alkaloids and their derivatives or prodrugs.

In a specific embodiment, the conventional anti-tumor therapy is radiation therapy, and the tumor or host carrying the tumor is defective in one or more of the following: 1) interferon (eg, type I interferon, preferably interferon alpha or beta) expression and/or function, particularly interferon expressed by CD45+ hematopoietic cells; 2) expression and/or function of an interferon receptor, wherein the interferon is The body is, for example, an IFNa receptor and/or an IFNP receptor.

 Another aspect of the invention relates to a composition (e.g., a pharmaceutical composition) comprising: - an interferon, a fragment thereof, or a functional variant thereof, the interferon, a fragment thereof, or a functional variant thereof, and a tumor-associated antigen Targeting moieties (eg, antibodies) linked, wherein the targeting moiety is directly linked (eg, as a fusion protein) to the interferon, a fragment thereof, or a functional variant thereof, or joined by a linker;

 - optionally a pharmaceutically acceptable carrier.

 In a specific embodiment of the invention, the tumor associated antigen is EGFR, the targeting moiety is an anti-EGFR antibody, and preferably, the tumor is a tumor expressing EGFR. In a specific embodiment, the targeting moiety (e. g., an anti-EGFR antibody) forms a fusion protein (e.g., by direct ligation) with the interferon, a fragment thereof, or a functional variant thereof (e.g., Aβ).

 In a further aspect, the invention relates to the use of a composition of the invention as defined above for the preparation of a medicament, wherein the medicament is for the treatment and/or prevention of a tumor, such as a malignant tumor (especially a malignant solid tumor) It includes, for example, breast cancer, lung cancer, prostate cancer, colon cancer, skin cancer, head and neck cancer, lymphoma or melanoma. In a specific embodiment, the tumor is melanoma.

 In a further aspect, the invention relates to a kit comprising:

 a) an interferon, a fragment thereof or a functional variant thereof as defined above; or a composition of the invention as defined above;

 b) instructions for use in which the kit is used to treat and/or prevent tumors that are resistant to conventional anti-tumor therapies (eg, radiation therapy, chemotherapy); or for use with other anti-tumor therapies (eg, radiation) Therapy, chemotherapy, combined with the treatment and / or prevention of tumors.

In a further aspect, the invention relates to a method of preventing and/or treating a tumor, the method comprising administering to a patient a therapeutically and/or prophylactically effective amount of an interferon, a fragment thereof or a functional change thereof as defined above Or a composition of the invention as defined above. In a specific embodiment, the patient does not respond to conventional anti-tumor therapies (e.g., radiation therapy). In other embodiments, the patient is also defective in one or more of the following: 1) expression and/or function of an interferon (eg, type I interferon, preferably interferon alpha or beta), particularly CD45+ An interferon expressed by a hematopoietic cell; 2) an expression and/or function of an interferon receptor, such as an IFNa receptor and/or an IFN receptor.

 In additional embodiments, the prophylactic and/or therapeutic method further comprises administering to the patient simultaneously, sequentially (in any order) or separately at least one other anti-tumor therapy (eg, radiation therapy, administration of antibody chemistry) Therapy, administration of other chemotherapeutic agents, etc.). DRAWINGS

 Figure 1. Radiation therapy increases IFN-& in the tumor microenvironment.

 a) Real-time PCR analysis of IFN-β mRNA levels in all tumor RNA of established B16F10 tumors (16-20 days), wherein the tumors were untreated or subjected to a local RT treatment of 20 Gy. The data shown was from RNA extracted 6 hours after RT (*p=0. 0123).

 b). Using whole tumor homogenate, IFNP protein levels were detected by ELISA for 48 hours after RT (*p=0. 0375).

 c). Time course analysis of IFN-β mRNA levels by RT-PCR at the indicated time points. CD45+ and CD45- cells were sorted from established B16F10 tumors at the indicated time points (the tumors were treated with 20 Gy local RT), then RNA was extracted and RT-PCR was performed, and the data shown were similar. Representative data for the results of the three experiments.

 Figure 2. The therapeutic response to RT depends on the host's response to type I IFN. a) Tumor growth curve of WT and IFNaRl K0 mice bearing established B16F10 tumors that were either untreated or subjected to topical RT (15 Gy, continuous treatment for three days). Tumor growth was plotted starting from the starting dose of RT (n = 6-9 mice, pooled data) (**p = 0. 0012, on day 14).

b) . Lethal injection of π mice and π or IFNaRl 0 bone marrow (BM) Refactoring. After reconstitution, chimeric mice were stimulated with B16F10 and tumors were allowed to establish for 14 days or the mean volume of tumors was allowed to reach 100 ^mm3 . The established tumors received local ablation RT (15 Gy) on days 14, 15 and 16 (n= 7-8/group, **p=0. 0011, for RT group ψΤ→ΤΓ vs. IFNAR 0→WT) , after treatment for 20 days).

 c). Same as b) except for the following: IFNctRl K0 mice were also used as recipients of WT BM. (***p<0. 0001, for both WT→WT and WT→IFNAR K0, on day 21 after RT). The data shown is representative of two experiments with similar results.

E). B6/RAG1+ mice were reconstituted with purified polyclonal T cells from WT or IFNaR1 K0 mice according to the procedure detailed in the Materials and Methods section. T cells were allowed to reconstitute for 7 days, and then tumor-stimulated by subcutaneously using 5x10 ⁵ B16-SIY (in which SIY is an abbreviation for SIYRYYGL-K (b) recognized by 2C T cells). On day 7 after tumor stimulation, tumors were treated with a total RT of 25 Gy, at which time the volume of the tumor was approximately 80-100 «M ³ . Tumor growth was monitored and the volume of the tumor was calculated according to the procedure shown in Materials and Methods. n = 4 mice per group, and the data represents one of two experiments with similar results.

 Figure 3. Local ablation RT confers T cell stimulation to TIDCs in WT mice but does not produce functional TIDCs in IFNAR K0 mice;

a) C57B1/6 mice or IFNRK0 mice were inoculated with 5xl 0 ⁵ B16-SIY tumor cells. Tumors established for 15 days received 25 Gy of local RT radiation or were untreated. CDllc ⁺ cells were isolated from tumors (a) and draining lymph nodes (d) of 3 mice per group and pooled for analysis. The isolated CD1 lc ⁺ cells were co-cultured with naive 2C TCR transgenic cells under different conditions and T cell proliferation was assessed by incorporation of sputum-containing thymidine. Error bars represent the standard deviation between triplicate wells (*P=0. 0167).

 b, c) The supernatant of the in vitro culture from TIDC (c, d) and lymph node DC (f) was isolated at 72 hr, which was subjected to multiple doubling just prior to the addition of sputum-thymidine Cytokine Bead Array (CBA) was used to measure cytokine concentrations. The levels of IFNy (b) and TNFa (c) from the culture supernatant are shown (*ρ=0·0192). The data shown is representative of two experiments with similar results. (Unless otherwise indicated, ***ρ<0. 001, **ρ<0. 01, *ρ<0. 05; as shown above, a separate ρ value can be obtained).

Figure 4. The anti-tumor effect of ad-IFN-[beta] is immune mediated and depends on sputum cells. a) . The established B16-SIY tumor was treated by intratumoral injection of 2χ10 ¹⁽⁾ νρ ad-nul l or ad- ¹ FN-β, and tumor growth was monitored (η=4, **ρ=0. 0064 ) .

 b). Transferring the Rag K0 host with WT peripheral T cells or PBS followed by stimulation of the host with B16-SIY.

 c) Treatment of established tumors by intratumoral injection with 2 10 1> ad-nul l or ad-IFN-β. The ad-IFN-β-treated mouse subgroup also received anti-CD4 or anti-CD8 depletion antibodies (η=4, **ρ=0. 0069, vs ad-IFN-β treated vs ad-IFN-β and Anti-CD8 treated mice, on day 32). The data shown is representative of three experiments with similar results.

 Figure 5. Ad-IFN-β promotes preferential amplification of tumor antigen-specific cells.

WT mice bearing 12-day established B16-SIY tumors were subsequently transferred with a mixture of CFSE-labeled 2C Tg T cells and OTI/Thyr Tg T cells. In the 13th and 15th the tumor treated with IFN- β ad-nul l or the ad- 3xl0 ^1Q vp. DL and spleen were collected after three days.

 a). Representative FACS plot showing the frequency of antigen-specific 2C cells relative to non-specific 0T-1 TCR Tg T cells.

 b) . Pooled data showing a significant increase in the frequency of 2C cells relative to 0Τ-1 TCR Tg T cells after ad-IFN-β treatment (η=5-7, **ρ=0. 0075) c) . Pooled data showing that the ratio of 2C to OT-1 TCR Tg T cells was approximately equal when treated with control ad- nul l, but significantly increased after ad-IFN-β treatment (η) =5-7) , (*ρ=0. 0128).

 d) . Representative histogram, which shows dilution of CFSE by proliferation.

 e) Mice carry a 12-day established B16-SIY tumor treated with ad- nul l or ad-IFN-β, which is 3 - 5 days after the last intratumoral adenovirus injection, Target cells loaded with SI Υ peptide were received by intravenous injection. Specific lysis of target cells (**ρ=0. 0015) was assessed in the spleen 18-24 hours after transfer. The data shown is representative of two experiments with similar results.

Figure 6. Targeting tumors with adenovirus expressing IFN reduces tumor growth. Balb/C mice were inoculated with 5*10 ⁵ TUB0-EGFR cells. Two weeks after the injection, through the 笫14, Mice were treated with intraperitoneal injection at 17 and 20 days with PBS, Ad-nul or Ad-IFNP (1*10 ¹⁰ vp). Tumor growth was measured twice a week.

Figure 7. Anti-EGFR-fusion proteins are effective in controlling primary tumor growth. WT Balb/C mice were inoculated with 5*10 ⁵ TUB0-EGFR cells. Two weeks after the injection, 25 g of anti-EGFR or anti-antigen was administered by intratumoral injection on days 14, 17 and 20

EGFR-treated mice. Tumor growth was measured twice a week.

 Figure 8. Anti-EGFR-IFN beta is effective in controlling EGFR-B16 tumor growth.

FT Β6 mice were inoculated with 5*10 ⁵ B16-EGFR-SIY cells. Ten days after the injection, the mice were treated with 254 g of hlg, anti-EGFR or anti-EGFR-IFNβ by intratumoral injection on days 10, 13 and 16. Tumor growth was measured twice a week.

Figure 9. CD8+ T cells are required for anti-tumor effects against EGFR-IFNp. Use 5*10 ⁵

B16-EGFR-SIY cells were seeded with WT B6 mice. Ten days after the injection, the mice were treated with 25 g of hlg, anti-EGFR or anti-EGFR-IFNβ by intratumoral injection on days 10, 13 and 16. Depletion of antibody anti-CD4 and anti-CD8 by intraperitoneal administration on days 9, 14, and 19

(200μ ₈ )„ Measurement of tumor growth twice a week.

 Terms and definitions

 Unless otherwise stated, the terms and definitions used in this application are used conventionally in the art and are known to those skilled in the art.

 As used in this application, the term "subsequent transfer" refers to the transfer of sputum cells to a recipient.

 As used in this application, the term "tumor site" refers to an in vivo or ex vivo location that contains or is suspected of containing tumor cells. The tumor site includes a solid tumor and a location near or adjacent to where the tumor is growing.

As used in this application, the term "administering" refers to systemic and/or topical administration. The term "systemic administration" refers to administration non-locally such that the substance being administered may affect several organs or tissues throughout the body; or such that the administered substance may cross several organs or tissues throughout the body to reach the target site point. For example, to the subject's circulatory system Administration can cause the therapeutic product to be expressed from the administered vector in more than one tissue or organ, or can cause the therapeutic product to be expressed at the specific site by the administered vector, for example, due to natural tropism. Or due to an operably linked to a tissue-specific promoter element. One of ordinary skill in the art will appreciate that such systemic administration encompasses various forms of administration including, but not limited to, parenteral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, oral administration, and the like.

 The term "topical administration" refers to administration at or around a specific site. One skilled in the art will appreciate that topical administration encompasses various forms of administration, such as direct injection to a particular site or injection into it (e.g., intratumoral administration).

 As used herein, the term "therapeutically and/or prophylactically effective amount" refers to the requirement to achieve a disease or condition for the treatment and/or prophylaxis (eg, a tumor/cancer, eg, for resolving a tumor or reducing the size of a tumor). The interferon of the invention, a fragment or functional variant thereof, or the amount of the composition of the invention. The effective amount can be determined for a specific purpose by practice, in a conventional manner. In particular, the therapeutically effective amount can be an amount required to achieve a reduction in the number of cancer cells; a reduction in tumor size; inhibition (ie, slowing or halting) infiltration of cancer cells into peripheral organs; inhibition (ie, slowing down) Or stop) tumor metastasis; inhibit tumor growth; and/or alleviate one or more symptoms associated with cancer.

 The term "antibody" encompasses, for example, monoclonal antibodies, polyclonal antibodies, single chain antibodies, antibody fragments (which exhibit the desired biological or immunological activity). In the present application, the term "immunoglobulin" (Ig) is used interchangeably with an antibody. The antibody can specifically target a tumor antigen, such as a surface tumor antigen, such as EGFR, CD4, CD8, and the like.

 The term "fragment" (eg, a fragment of an interferon) refers to a portion of a biological molecule (eg, a protein, such as an interferon or its encoding nucleic acid) that is capable of effecting a desired biological function, such as inducing expansion of a tumor-specific T cell. increase.

 The term "functional variant" means that it is modified (eg, mutated, inserted, deleted, fused, conjugated, cross-linked, etc.) to be different from the parent molecule (eg, interferon), but retains its desired biological activity. Variant.

Interferons, fragments thereof, or functional variants thereof can be ligated to the targeting moiety by a variety of conventional methods known in the art. The connection can be direct or indirect (eg As in the case of direct ligation, in the case of direct ligation, it can be achieved by the formation of fusion proteins, conjugation or chemical ligation. When the ligation forms a fusion protein, it can be achieved, for example, by recombinant techniques or peptide synthesis techniques. In certain embodiments, the fusion protein may also comprise a linker that does not disrupt the desired properties of the formed product (eg, induces expansion of tumor-specific tau cells).

 A person skilled in the art can select an appropriate dosage form according to a specific condition, type of disease (e.g., tumor type, stage of development of the tumor, etc.), severity, patient's body, other therapies that may be administered in combination, previously administered therapies, and the like. Mode of administration.

 The term "cancer" refers to a condition that is generally characterized by unregulated cell growth (e.g., in a mammal, such as a human). Such cancers include, but are not limited to, breast cancer, lung cancer, prostate cancer, colon cancer, cutaneous cancer, head and neck cancer, lymphoma or melanoma, and the like.

 The term "conventional anti-tumor therapy" refers to a therapy that has hitherto been used in the art to treat and/or prevent the production, progression, metastasis, etc. of a tumor, including, but not limited to, radiation therapy (eg, by using X-ray radiation, Radioisotopes, etc.), use of chemotherapeutic agents, use of tumor-specific antibodies, etc.

 The term "radiation therapy resistance" (or RT resistance) means that a tumor or tumor cell does not respond to radioactive treatment of a conventional dose and/or a lethal dose (eg, 15 Gy of X-ray radiation for 3 consecutive days), ie, for example with Compared with tumors of the same shape that were not subjected to the radioactive treatment, the size of the tumor subjected to the radioactive treatment was not significantly reduced, the number of tumor cells was not significantly decreased, the tendency of tumor recurrence was not inhibited, and tumor metastasis was not obtained. Control, etc.

 "Type I IFN" refers to a Type I interferon which includes, for example, IFNα, IFNp, IFN w, IFN t , IFN d , IFNk and the like.

In the present application, the term "viral vector" refers to any suitable viral vector for delivery of a protein of interest (eg, an interferon) into a target cell or tissue, including but not limited to, for example, an adenoviral vector, an adeno-associated viral vector. , retroviral vector, mouse Harvey sarcoma virus vector, mouse mammary tumor virus vector, Rous sarcoma virus vector, SV40-type virus vector, polyoma virus vector, EB virus vector, papilloma virus vector, herpes virus Vector, vaccinia virus vector, sputum scutellaria virus vector and RNA viral vector; preferably, the disease The virulence vector is an adenoviral vector.

 In the present application, the term "tumor-associated antigen" includes, for example, tumor surface antigens, including but not limited to members such as the epidermal growth factor receptor family (EGFR), including EGFR, HER1, HER2, HER4 and HER8, etc. (Nam, NH , & Parang, Κ · (2003), Current targets for anti cancer drug discovery. Current Drug Targets, 4 (2) , 159-179) , STEAP (six-transmembrane epithelial antigen of the prostate; Hubert et al, STEAP : a prostate-specific cel l-surface ant igen highly expressed in human prostate tumors. , Proc Natl Acad Sci US A. 1999; 96 (25) : 14523-8. ) , CD55 (Hsu et al, Generation and characterizat ion of Monobolic antibodies directed agains t the surface antigens of cervical cancer cel ls. , Hybrid Hybridomics. 2004; 23 (2) : 121-5).

 Other suitable antibodies that can be used in the present invention include rituximab (RituxanTM, a chimeric anti-CD20 antibody), Campath-1H (anti-CD52 antibody), and antibodies to any cancer-specific cell surface antigen. The following are exemplarily listed for antibodies of a particular cancer type suitable for use in conjunction with interferons for the purposes of the present invention: alemtuzumab (CampathTM) for chronic leukemia; bevacizumab (AvastinTM) For colon and lung cancer; cetuximab (ErbituxTM) for colon and head and neck cancer; jituzumab (MylotargTM) for acute sputum leukemia; Ibritumomab (Zeval inTM) for non-Hodgkin's lymph Tumor; Parumimide (VectibixTM) for colon cancer; RituxanTM non-Hodgkin's lymphoma; Tosimizumab (BexxarTM) for non-Hodgkin's lymphoma; and trastuzum HerceptinTM is used in breast cancer.

In the present invention, "pharmaceutically acceptable carrier" means a carrier which does not cause an allergic reaction or other unpleasant effects in the administered cells or subjects, and which does not affect the activity of the drug. Suitable pharmaceutically acceptable carriers include, but are not limited to, for example, one or more of water, physiological saline, phosphate buffer, levulose, glycerol, ethanol, and the like, as well as combinations of the foregoing. The pharmaceutically acceptable carrier may further comprise minor auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or utility of the nucleic acid, polypeptide, viral particle or cell. In the present application, "defective" in interferon and/or its receptor means that the expression of the interferon or interferon receptor does not reach the level required to achieve its biological function, or the interferon expressed Or the interferon receptor is unable to exert the desired biological function (eg, in the form of a mutation), or the interferon (or interferon receptor) is unable to interact with its receptor (ligand) to cause downstream signaling. The following examples are merely illustrative of the invention and are not intended to limit the invention in any way. Example

 Materials and Method

 Mouse

 C57BL/6 mice, rats, B6/0TI TCR transgenic mice, Ly5.1 mice and B6/Rag-1 K0 mice were purchased from Jackson Laboratory and were 6-7 weeks old. 2C TCR-transgenic mice were supplied by Jianzhu Chen, MIT, Cambridge, MA and were deposited in the No Specific Pathogen (SPF) facility at the University of Chicago. The B6/IFNA1R K0 mouse was generously provided by Anita Chong of the University of Chicago. For all experiments, mice were 6-16 weeks old, mice were incubated under SPF and mice were used according to animal care and using the Animal Experimental Guide (IACUC) set by the Commission. Cell line

 Bl 6-F10 mouse melanoma cells were obtained from the American Type Culture Collection.

B16-SIY melanoma cells and anti-2C TCR (1B2) antibodies were obtained from Tom Gajewski (University of Chicago). The cells were cultured in RPMI 1640 medium containing L-glutamate at 37 Ό and 53⁄4 C0 ₂ supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, in the RPMI 1640, lniM sodium pyruvate, 0. ImM non-essential amino acids and HEPES. The B16-SIY cell line was maintained in medium containing G418 (1 mg/ml).

RNA purification and gene array analysis B16F1 tumors were irradiated (20 Gy) or not treated. Five hours after irradiation, the tumors were excised, snap frozen in liquid nitrogen and stored at -80 Torr until further processing. The frozen tumors were cut into pieces of about 5 mm ³ in size and soaked overnight in RNA a ICE solution (Applied Biosystems-Ambion). The samples were centrifuged, washed in RLT buffer (QIAGEN), and homogenized on ice using a glass-Teflon homogenizer set to 3000 rpm. Further purification was performed using a combination of RNeasy spin columns and TRIzol reagents as previously described (Khodarev et al., 2004). Gel electrophoresis (1.8% agarose) and spectrophotometry were used to assess the quality of the samples. RNA samples were normalized to a concentration of 1 mg/ml, samples from at least three tumors/group were pooled in equal amounts, and the pooled samples were transferred to a functional genomics facility at the University of Chicago. For labeling and hybridization with the mouse genome ⁴ 30 2. 0 GeneChips® array (Af fymetrix). Selection and analysis of genes differentially expressed in irradiated vs. untreated tumors is based on the process detailed above. (Khodarev et al., 2004; Kimchi et al., 2005; Pitroda et al., 2009). Briefly, each array was hybridized to pooled total RNA samples. After obtaining the data, the "integral median normalization" (Kimchi et al., 2005) was used to re-adjust the data throughout the data set and filter the data as described (Khodarev et al., 2004). Subsequent analysis was based on a pairwise comparison of replicate arrays using a saliency analysis of the microarray (Tusher et al., 2001) version 3. 02, setting the false discovery rate to <1%. Gene-annotate the selected probe set ID and perform a functional analysis using the Netaffx Analysis Center (https://www.afffymetrix.com/analysis/netaffx/index.affx) to generate bone marrow chimeras

Lethal radiation was administered to wild-type (WT) or IFNAR K0 mice with a single dose of 1000 rad. On the next day, 2 - ³ x 106 bone marrow (BM) cells were intravenously transferred to irradiated mice. After reconstitution, the mice were maintained on antibiotics diluted with sulfamethoxazole and trimethoprim (complex sulfamethoxazole) in drinking water for 4 weeks. Tumor cells were injected into mice 5-6 weeks after reconstitution. Subsequent transfer of T cells

Lymph node (LN) cells and spleen cells were isolated from 2C or OTI Tg mice. Then, a total of 2 X 10 ⁶ 2C or 0T1 T cells labeled with carboxyfluorescein amber Bt imidate (CFSE) were intravenously transferred to C57BL/6 mice bearing B16-SIY tumors. Cells were isolated from draining lymph nodes (DLN), spleen or tumor at the indicated time points. CFSE dilution was evaluated as previously described (Yu et al, 2004; Yu et al, 2007). For reconstitution of RAG K0 recipients, splenocytes and lymph node cells were collected from ^^ mice, and Pan T cell isolation kit and automated magnetic cells ^ (autoMACSTM Miltenyi Biotec) were used to control T cells. Flow cytometry analysis

 Tumors, DLN and spleen (SP) were excised from the mice, minced and digested with 1.5 mg/ml collagenase, lU/mL dispase and 0.4 mg/ffll DNase I for 40 min at 37 ,, then added EDTA was brought to a final concentration of 6 mM to inactivate the enzyme. The single cell suspension of the cells was incubated with anti-CD16/32 (anti-FCTI II/II receptor, clone 2. 4G2) for 20 min at room temperature, followed by staining with the following conjugated antibody: anti-CD45. 2 ( Clone 104), anti-CD90. 1 (anti-Thy- 1. 1, clone OX-7), anti-CD8a (lon 53-6.7), anti-CDllc (^long HL3), anti-CDllb (clone M1/70), Anti-Ly6C (clone AL-21), anti-I-A/I-E (clone M5/114. 15. 2), anti-CD80 (B7-1, clone 16-10A1), anti-CD86 (B7-2, clone GL1 ), anti-CCR7 (^long 4B12) or anti-CD4 (^long GK1. 5). All purified and fluorescently labeled monoclonal antibodies were purchased from BD Pharmingen. Samples were analyzed on a FACSCanto flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar, Inc.). Tumor growth and treatment

The cultured cancer cells were trypsinized, washed with a medium, and subcutaneously injected into the corresponding mice on the back. Tumor size was determined at 3-3-4 day intervals. Tumor volume was measured along three orthogonal axes (a, 6 and c) and the tumor volume was calculated to be equal to a^/2. Tumor nodules were inoculated in vivo with the indicated amounts of Ad-IFN-P or Ad-null virus tumors. Through and Biogen Idee collaborated to obtain Ad-IFN-β. For antibody-mediated cell depletion, 200/mouse of anti-CD4 or anti-CD8 was administered intraperitoneally to mice on day 9, day 11 and day 13 after inoculation of the original tumor (YTS. 169. 4. 2) Antibodies. Mice were subjected to local X-ray radiation at the doses indicated in each experiment using a GE Maxitron x-ray generator. Each mouse was protected with a lead cap to expose the tumor, allowing for local IR (ionizing radiation) radiation. Real-time PCR

Tumors were collected at the indicated time points after topical RT at 20 Gy. Real-time PCR was performed using cDNA prepared from DNase I-treated RNA, which was extracted from the entire tumor or was sorted by the BD FACSAria cell sorter into CD45. 2 ^CD and CD45. 2_ populations. Single cell suspension extracted. The primers and probes used are as follows. For IFN β: forward 5' - ATG AGT GGT GGT TGC AGG C-3' (SEQ ID NO: 1), reverse 5' - TGA CCT TTC AAA TGC AGT AGA TTC Α-3' (SEQ ID NO: 2 ). For GAPDH: forward 5'-TTC ACC ACC ATG GAG AAG GC-3' (SEQ ID NO: 3), reverse 5'-GGC ATG GAC TGT GGT CAT GA-3' (SEQ ID NO: 4). Real-time PCR reactions were performed on ABI/Prism 7300 (Appl ied Biosystems) with a final volume of 25 μl containing 2. 5 μΜ of forward and reverse primers, 2x Taqman Master Mix (Applied Biosystems) containing Ampl iTaq Gold polymerase ). The cycling conditions were: a single denaturation step, 15 min; followed by 45 cycles, each cycle at 94 for 15 s and at 60 Torr for 1 min. Use the standard curve to analyze /? /; ^ and gene expression, and then normalize the sample relative to ί3⁄4 ^. The standard curve has an R ² value > 0.99.

ELISA

After local X-ray irradiation, the tumors were collected and weighed at the indicated time points, and were performed on water in IX phosphate buffer (PBS) and IX Hal t protein preparation mixture (Thermo Fisher Scientific). Qualitative. Supernatants were collected and IFN-[beta] was measured using a VeriKine mouse IFN-P ELISA kit (PBL IFN Source) according to the manufacturer's instructions. In vivo specific lysis assay

Mice were injected subcutaneously with 0.5×x0 ⁶ B16-SIY cells. Mice were treated with axltT ad-IFN-P or ad-nul 1 tumors on day 12 and day 14 after injection. An equal number of CFSE-labeled splenocytes were intravenously transferred to mice supplemented with SIY peptide (lyg/ml) or 0T-1 peptide (3). Lyg/ml). Cells supplemented with SIY were labeled with CFSE* (ΙΟμΜ), while cells filled with 0Τ-1 were labeled with CFSE (1. ΟμΜ). Splenocytes loaded with the peptide were also transferred to naive, non-tumor-bearing mice as controls. After 18-24 hours, the spleens of recipient mice were collected and analyzed by FACS. The specific lysis was calculated as follows:

 % specific lysis = [ (%CFSE low X A - %CFSE high) / (%CFSE low x A) ] x 100

 Wherein A = % CFSE * (specific peptide - SI Y) divided by % CFSE low (non-specific peptide - 0T-1) (in naive control mice, the results for multiple controls were averaged).

DC cross-priming assay and cytokine assay

⁵ ×10 ⁵ B16-SIY tumor cells were subcutaneously injected into the lower back of C57BL/6 mice. After the tumor was established, the mice received local RT (20 Gy) at the tumor and tumors were collected for DC purification after 3 days. The tumor was finely shredded with scissors and a solution containing 1.5 mg/mL j^, drunk (Sigma), lU/mL drunk (BD Biosciences) and 0.4 mg/mL DNase I (Sigma) was used at 37°. C, digest for 40 minutes in a rotary incubator set to low speed. Live cells from the resulting single cell suspension were subjected to Ficoll-Paque Plus (GE Healthcare) centrifugation, and the isolated cells were used for DC purification using CDllc+ magnetic bead kit and automated magnetic cell sorting (autoMACS) TM Mi lteayi Biotec). For in vitro culture, 1x10 ⁵ naive 2C cells were plated with lxlO ⁵ DCs with or without exogenous SIY peptide (g/ml), and supernatants were collected 3 days later for cell flow according to the manufacturer's instructions. Bead array (CBA) (BD Biosciences) for analysis. Cell proliferation was assessed according to conditions similar to those in vitro, by the following procedure: supplementation of ³ H-thymidine at 72 hours, harvesting of cells at 96 hours for analysis, and on a liquid scintillation counter^ board. Production of adenovirus expressing IFN β (Ad- IFN β )

 To construct the recombinant adenovirus "mIFN beta, the mouse IFN beta cDNA was amplified by PCR and cloned into the Notl/EcoRV site of pAdenoVator-CMV5 (CuO) under the control of the CMV5 promoter. The pAdenoVator-mIFN P was linearized and electroporated into the electrocompetent cell BJ5183 at 2. 5 kV for recombination with the backbone vector containing the adenoviral genome. Selection of hybrids on kanamycin LB agar plates Cosmid. Pad digestion was used to further identify recombinant cosmids containing the insert mlFN P. Ad-mIFN β DNA was linearized by Pa digestion and the Pa-digested mixture was transfected into 293 cells for further purification without further purification. Recombinant adenovirus. Adenovirus-mIFN P is called Ad- IFN β. Statistical analysis

Statistical analysis was performed using unpaired two-tailed t-test. Error bars represent standard deviations. Unless otherwise stated, the materials and methods used in the following examples were carried out in accordance with the teachings in the "Materials and Methods" section above. Example 1 Radiation therapy increases intratumoral IFN-P production Since DC maturation is not measurably affected by local RT and individual tumor antigen cross-presentation does not explain increased functionality in DCs from tumors receiving local RT, the inventors propose Local RT may enhance the local tumor microenvironment to respond to DC function through changes in the local cytokine environment. Looking at the obtained gene array data for significant differences in cytokine gene expression, few interesting candidates (data representations) that met the detection threshold were obtained. However, the invention AJ « saw a small, but consistent increase in IFNP expression in treated tumors. RT-PCR and ELISA of tumor samples confirmed that IFNP was indeed upregulated after local RT, both at RNA (Fig. la) and protein levels (Fig. lb). Further analysis by cell sorting revealed that type I IFN was mainly produced by CD45+ hematopoietic cells infiltrating tumors (Fig. lc). In order to confirm that a small amount of IFN-β produced in the CD45- fraction is not derived from tumor cells, the inventors tested whether radiation can be directly induced from B16 in vitro. IFN-β of tumor cells. After X-ray irradiation of tumor cells in vitro, no IFN-β transcript was detected at the protein and mRNA levels, suggesting that a small amount of IFN-β signal in the CD45 fraction may be derived from non-hematopoietic stromal cells (data not shown) These data indicate that after local RT, Aβ is up-regulated in tumors and these data also support that hematopoietic cells are the main source of IFN-β in irradiated tumors. Example 1 IFN-α/β response is critical for the therapeutic effect of RT in order to first test whether type I IFN is critical for RT-mediated tumor reduction, Applicants in wild-type (WT) and IFN receptors Parental B16F10 tumors were established in both alpha knockout (IFNaRl K0) mice. These mice lack the cc subunit of the IFN-[alpha]/[beta] receptor, so they do not respond to the Al ll type IFN (Muller et al, 1994). Tumor-bearing mice (15 Gy χ3) were treated daily with 15 Gy (local) RT and tumor growth was monitored for three consecutive days. The inventors previously established that this treatment regimen was able to successfully control tumor growth in wild-type mice stimulated with B16F10 (Lee, 2009). Untreated tumors developed rapidly in the FT and IFNotRl K0 groups (Fig. 2a). T mice receiving 15 Gy X 3 showed strong tumor growth inhibition compared to tumors in untreated mice. . In contrast, tumors in IFNaR10 mice grew with kinetics similar to untreated tumors and showed almost complete resistance to even high dose RT. Therefore, the response to RT does depend on the host's response to type I IFN.

Type I IFN exerts a pleiotropic effect including, for example, antiviral, antiproliferative, immunomodulatory and antiangiogenic responses. Common heterozygous dimeric IFN-[alpha]/[beta] receptors are ubiquitously expressed, but the effects of IFN receptor action may vary depending on cell type (Uddin and Platanias, 2004; van Boxel-Dezaire et al, 2006). Due to the IFN-[alpha]/[beta] receptor Puda, tumor cells are potential targets for type I IFN, where direct signaling on tumor cells can mediate anti-proliferative effects (Figure 2). Thus, the lack of response to RT in the IFNaRI KO host may be due to receptor defects in non-hematopoietic tumor-associated mesenchymal cells, immune cells, or a combination thereof. To test the need for a type I IFN response in these populations, the inventors prepared a mutual bone marrow (BM) chimera. The 1000 rad (ie lethal dose) pair or IFNaRl K0 host was irradiated and reconstituted with FT or IFNaRl K0 osteophytes. Treated with a partial burning candle RT (15 Gy x3) Tumor-bearing mice, as expected, tumors gradually grew in both untreated groups

(Figure 2b). Tumors in mice reconstituted with WT bone marrow responded in a similar manner to wild-type mice. However, in IFNaRl KO BM recipients, although in all other tissues, including tumor cells themselves, IFN-α was present. /β sensitivity, in mice lacking IFNaRl only on BM cells, tumors no longer respond to RT (Fig. 2b). Whether type I IFN signaling was sufficient on cells of the source osteophytes was tested by reconstitution of WT or IFNaRl KO recipients with osteophytes. As expected, tumor response in WT recipients of WT BM to RT was wild type Animals are similar (Figure 2). Interestingly, IFNaR1 K0 mice responded to RT once the hematopoietic cells were restored to IFN-α/β (Fig. 2c). Therefore, in non-tumor cells, an IFN-α/β response in the hematopoietic system is required to achieve the therapeutic effect of RT.

 In order to specifically study the response of T cells to type I IFN, the inventors utilized a mode of subsequent transfer. Mice knocked out with the recombinase activating gene (RAG) were reconstituted with total T cells purified from WT or IFNaR10 mice, and these T cell chimeric mice were inoculated with tumor cells one week after T cell transfer. At the time of inoculation, steady-state expansion of the transferred T cells had stopped (data not shown). Tumors established in T cell chimeric mice were treated with local ablation RT and tumor growth was monitored. As expected, treated mice that received sputum cells were able to control tumor growth (Fig. 2e). Surprisingly, mice that received IFNaRl KO T cells were still able to mediate equal tumor control after local RT. Therefore, direct T cell responses to type I IFN are not required to achieve RT-mediated tumor control. Example 3 Local RT restored the ability of tumor infiltrating DCs to elicit T cells in an IFN-dependent manner.

The inventors' data highlight the critical role of type I IFN response of hematopoietic cells in the therapeutic efficacy of local RT, however, the inventors wanted to know whether there is a relationship between type I IFN signaling and DC function in the system. Connected. The inventors wished to determine whether the response to type I interferons was able to unravel the functional differences between the DCs they observed, the untreated tumors and the DCs of the locally irradiated tumors. When analyzing the function of tumor infiltrating dendritic cells from untreated B16-SIY tumors in IFNa R1 K0 mice, they were found to stimulate naive 2C T The ability of cell proliferation was severely reduced (Fig. 3a), which was similarly observed when DCs from untreated tumors were used (Fig. 3a). Interestingly, local irradiation of tumors in IFNa R1 K0 mice did not restore the ability of TIDCs (ie, tumor infiltrating dendritic cells) to stimulate T cells, which is associated with an increase in π DC isolated from tumors that received local RT. The ability to form a sharp contrast (Figure 3a). In addition, by? Γ TIDC-driven T cell proliferation induced robust IF production in culture supernatants, and clear T cell stimulation led to a polarized effector T cell phenotype (Fig. 3b). IFNa R1 K0 DC purified from irradiated tumors does not stimulate the ability of T cells to proliferate, which cannot be restored by the addition of exogenous SIY peptides, and is co-cultured in vitro with the provision of exogenous SIY peptides. Stimulation of TLR with bacterial lipopolysaccharide (LPS) peptides failed to restore the ability of TIDCs to stimulate T cell proliferation (Fig. 3a), and these results further confirmed the results previously obtained by the inventors in wild type mice. In addition, the maturation of IFNa Rl KO DC was assessed by surface staining with MHC class I, MHC class II, B7-1, B7-2 and CCR7, showing expression equivalent to FT TIDC, which was obtained from treated and not No significant differences were observed between DCs of treated tumors (data not shown). Furthermore, because TNFa production in response to LPS is equivalent (if not increased) compared to WT TIDCs from irradiated tumors, IFNa Rl KO TIDC is unlikely to be fully functionally compromised (Fig. 3c). In order to confirm that the function of DC was not completely impaired in IFNa Rl KO mice, the inventors analyzed DCs isolated from tumor-draining lymph nodes of IFNa Rl KO mice. They drive T cell proliferation and effector cytokine production. In terms of ability, lymph node DCs between WT and IFNa Rl KO mice were functionally indistinguishable (Fig. 3d and data not shown). DCs from the lymph nodes of WT and IFNa Rl KO mice also showed an equivalent response to LPS as measured by TNFa production (data not shown). Therefore, RT-directed type I IFN is required for obtaining DC cross-inducing ability in the microenvironment of tumors. Example 4: The tumor reduction caused by local delivery of type I IFN to the source is dependent on CD8+

 T cell

The inventors' results show that type I IFN is a key downstream mediator of local RT, however, local RT has the potential to induce many factors that affect tumor control and rejection. In order to study whether type I IFN can play a sufficient role without RT, the inventors tested Whether local delivery of type I IFN to a tumor by recombinant adenoviral vector (ad-IFNp) can cause tumor rejection. The inventors treated established B16-SIY tumors with ad-null (blank vector control) or ad-IFN-P and monitored tumor growth. Surprisingly, even for this aggressive tumor, ad-IFN-p also showed a very strong anti-tumor effect (Fig. 4a).

 Since ad-IFN-β may have multiple inhibitory effects on B16-SIY tumor cells, in order to test for the inhibition of sputum cells, the inventors used B6/RAG K0 mice (these mice are defective in lymphocytes). And compared to untransferred hosts and those reconstituted with sputum cells collected from wild-type donors. Interestingly, a tumor response to ad-IFN-β was not detected in Rag-1+ mice, but it was restored by transferring peripheral sputum cells (Fig. 4b). Therefore, the treatment with ad-IFN-P is immune-mediated and dependent on T cells, as is the case with RT treatment.

Recognizing that RAG-1 K0 mice may have other defects that may affect the therapeutic response, and in order to further differentiate the function of CD4+ and CD8+ T cell subsets, the inventors utilized WT mice and antibody-mediated CD8 ⁺ Depletion of T cells or CD4 ⁺ T cells. Surprisingly, depletion of CD4 ⁺ T cells had little effect on ad-IFN-P treatment, as tumors responded equally with and without CD4 depletion (Fig. 4c). However, depletion of CD8+ T cells severely reduced therapeutic efficacy and tumors were fast: il Ol (Fig. 4c). NK cells appear to be not critical for tumor regression (data not shown). Therefore, CD8 ⁺ T cells are critical for the anti-tumor effect of ad-IFN-β. Example 5 IFN-Ρ causes preferred amplification of antigen-specific sputum cells. Since the inventors determined that the therapeutic effect of ad-IFN- Ρ depends on CD8 ⁺ T cells (Fig. 4b, c), the inventors hypothesized that it is like radiation therapy. , ad-IFN-Ρ can induce antigen-specific T cell initiation and expansion. To test this, the inventors transferred naive 2C transgenic T cells recognizing the SIY antigen into mice bearing B16-SIY tumors. After treatment with ad-IFN-P, the inventors collected draining lymph nodes (DLN) and used clonal antibody 1B2 (anti-2C TCR) to quantify antigen-specific CD8+ T cell expansion. Treatment with ad- resulted in an approximately 6-fold increase in antigen-specific cells compared to ad-null (data not shown). This significant increase is likely due to antigen-driven proliferation, however, the following possibilities exist: ad-IFN- Beta can generally promote non-specific amplification of cells by reversing the inhibitory cytokine environment or by creating space by IFN-[alpha]/[beta]-mediated apoptosis of sputum cells. Type I IFN has also been shown to be involved in mediating bystander cell proliferation (Tough et al., 1996). Type I IFN is critical for viral infection and has been shown to be decisive for infection by some bacteria (eg, Listeria monocytogenes) (Auerbuch et al., 2004; Carrero et al., 2004; O' Connel l et al., 2004). Therefore, it is still unclear what effect IFNP released from tumor tissue after treatment will have on antigen-specific T cell immune responses. To test whether this amplification is limited to antigen-specific cells and/or increased proliferation, the inventors labeled 2C (antigen-specific) and 0T-1 (non-specific) cells with carboxyfluorescein succinimidyl ester (CFSE). They were then transferred to mice bearing B16-SIY tumors. To help identify the transferred cell population, the inventors utilized 0T-1 cells containing a congenic marker (Thyl. 1). Five days after treatment with ad-nul l or ad-IFN-P, the degree of CFSE dilution of 1B2+CD8+ (antigen-specific cells) in DLN and spleen relative to Thyl.l ⁺ CD8 ⁺ (non-specific) was determined. Ad-IFN-P induced a preferred amplification of antigen-specific 2C cells compared to non-specific OT-1 cells (Fig. 5a-c). When the ad- nul l control group was used, the ratio of 2C to OT-1 TCR transgenic cells was approximately equal, confirming that a similar number of transgenic Ts were transferred to the recipient, but the therapeutic effect of ad-IFN-P was Significantly increased (Figure 5c). In addition, 2C cells showed vigorous proliferation, which was confirmed by almost complete CFSE dilution (Fig. 5d). In contrast, non-specific cells were unable to proliferate (data not shown). Taken together, these data indicate that ad-IFN-P-induced antigen-specific CD8+ T cell expansion is relatively selective and mediated by increased proliferation. Example 6 Increased antigen-specific cytolytic activity in vivo Although proliferation is a good indicator of an effective immune response, it does not always translate into potent effector functions. Therefore, the inventors investigated whether ad-therapy causes increased in vivo specific lysis. Tumor-bearing mice were treated with ad- nul l or ad-IFN-P, and then target cells loaded with CFSE-labeled peptides were transferred thereto. As a negative control, target cells loaded with CFSE-labeled peptides were also transferred to naive mice, and as expected, these mice showed normal specific lysis. By comparison, it is found that it is processed with ad-nul l Compared to mice, ad-IFN-P treated mice showed significantly higher specific lysis (Fig. 5e). These data indicate that amplification of antigen-specific cells observed in ad-IFNP-treated mice caused potent effector CTL activity,

 To determine whether targeting tumors can cause tumor regression, TUB0-EGFR cells were inoculated and IFN-expressing adenoviruses were injected intratumorly on day 14, 17 and 20 days. Ad- nul l has a mild effect on tumor growth, while ad- causes tumor regression (Figure 6). This suggests that local delivery of ad-IFN beta on TUB0 can also induce a strong response, causing tumor regression.

To test whether the actual fusion prepared by CH0 cells can induce rapid tumor regression, encode the antibody-IFN (for anti-EGFR-ΙΡΝβ, connect the 3' end of Cetuximab to IFNp and then introduce it into the vector pcDNA In the middle, the subclones are all carried out using standard molecular biology techniques. Briefly, 1) HC-Fc-IFN is cloned into the vector Abvec-hlgGl; 2) the light chain of the antibody is cloned into the vector Abvec-lamda; The HC-Fc-IFN is cleaved from the product obtained in 1) and cloned into the lonza vector pEE6.4 by blunt-end ligation; 4) the light chain is cleaved from the product obtained in step 2) And cloning it into lonza vector PEE12.4 by blunt-end ligation; 5) excising the expression cassette from the product of step 4) and cloning it into the product of step 3), thereby obtaining the final plasmid pEEl ^{2 4} - anti-EGFR-IFNP) plasmid was transfected into CH0 cells and cells expressing the fusion protein were selected in large amounts. The supernatant of CH0 containing this plasmid was collected and the fusion protein was purified by Protein A column, the protein A The column binds the Fc portion of the fusion protein with high affinity. The antibody or the antibody-IFN was used to treat mice bearing TUB0-EGFR. Only the fusion protein caused tumor tachycardia regression (Fig. 7). Similar results were obtained from mice bearing B16-EGFR tumors ( Figure 8) Thus, this fusion protein can also be used to better eliminate tumors that express EGFR in tumors compared to antibodies alone.

To determine if this significant effect was caused by T cell immunity, mice bearing EGFR-TUB0 tumors were treated with low doses of the fusion protein and T cell depleting antibodies. Although the growth of EGFR-TUB0 was significantly delayed, depletion of CD8+ T cells caused rapid recurrence, whereas depletion of CD4+ T cells did not cause tumors (JL) (Fig. 9). references:

 Anderson, U., Wang, H., Palmblad, K., Aveberger, AC, Bloom, 0. , Erlandsson-Harr is, H., Janson, A., okkola, R., Zhang, M., Yang, H . and Tracey, . J. (2000). High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. The Journal of experimental medicine 192, 565-570.

 Apetoh, L., Ghiringhelli, F., Tesniere, A., Obeid, M., Ortiz, C., Criollo, A., Mignot, G., Maiuri, MC, Ullrich, E., Saulnier, P., et Al. (2007). Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 13, 1050-10059.

 Auerbuch, V., Brockstedt, DG, Meyer-Morse, N., O'Riordan, M., and Portnoy, DA (2004) . Mice lacking the type I interferon receptor are resistant to Listeria monocytogenes. The Journal of experimental medicine 200 , 527-533.

 Bianchi, M. E., and Manfred i, A. A. (2007). High-mobility group box 1 (HMGB1) protein at the crossroads between innate and adaptive immunity. Immunol Rev 220, 35-46.

 Carrero, J. A, Calderon, B., and Unanue, E. R. (2004). Type I interferon sensitizes lymphocytes to apoptosis and reduces resistance to Listeria infection. The Journal of experimental medicine 200, 535-540.

 Curtsinger, JM, Valenzuela, J. 0., Agarwal, P., Litis, D., and Mescher, MF (2005). Type I IFNs provide a third signal to CD8 T cells to stimulate clonal expansion and differentiation. J Immunol 174 , 4465-4469.

Diamond, MS, Kinder, M., Matsushita, H., Masha ekhi, M., Dunn, GP, Archambault, JM, Lee, H., Arthur, CD, White, JM, Kal inke, U., et al. (2011). Type I interferon is selectively required by dendritic cel ls for immune rejection of tumors. The Journal of exper imental medicine 208, 1989-2003.

 Dunn, G. P., Bruce, A. T., Sheehan, K. C., Shankaran, V., Uppaluri, R., Bui, J. D., Diamond, M. S., Koebel, C. M., Arthur,

C., White, J. M., and Schreiber, R. D. (2005) . A cri tical function for type I interferons in cancer immunoediting. Nat Immunol 6, 722-729.

 Dunn, G. P., Koebel, C. M., and Schreiber, R. D. (2006) . Interferons, immunity and cancer immunoediting. Nat Rev Immunol 6, 836-848.

 Fuertes, M. B., Kacha, A. K., Kl ine, J., Woo, S. R., Kranz,

DM , Murphy , . M. , and Gajewski , TF (2011) . Host type II FN signals are required for anti tumor CD8+ T cel l responses through CD8 {alpha} + dendritic cel ls. The Journal of experimental medicine 208, 2005— 2016.

 Gresser, I, , Belardell i, F., Maury, C., Maunoury, MT, and Tovey, MG (1983) . Injection of mice with antibody to interferon enhances the growth of transplantable murine tumors. The Journal of experimental medicine 158, 2095-2107.

 Jiang, W., and Pisetsky, DS (2006) . The role of IFN-alpha and nitric oxide in the release of HMGBl by RAW 264. 7 cel ls st imulated with polyinosinic-polycytidyl ic acid or l ipopolysacchar ide. J Immunol 177 , 3337-3343.

Khodarev, NN, Beckett, M., Labay, E., Darga, T., Roizman, B., and Weichselbaum, RR (2004) . STATl is overexpressed in tumors selected for radioresitance and confers protect ion from radiation in transduced sensi tive Cel ls. Proc Natl Acad Sci USA 101, 1714-1719. Khodarev, NN, Roach, P., Pitroda, SP, Golden, DW, Bhayani, M., Shao, MY, Darga, TE, Beveridge, MG, Sood, RF, Sutton, HG, et al. (2009) . STATl Pathway mediates ampl if ication of metastatic potential and resistance to therapy. PLoS One 4, e5821.

 Kim, JH, Kim, SJ, Lee, IS, Lee, MS, Uematsu, S., Akira, S., and Oh, KI (2009) . Bacterial endotoxin induces the release of high mobi li ty group box 1 via the IFN -beta signal ing pathway. J Immunol 182, 2458-2466.

 Kimchi, ET, Posner, MC, Park, J. 0. , Darga, TE, Kocherginsky, M., Karrison, T., Hart, J., Smith, KD, Mezhir, JJ, Weichselbaum, RR, and Khodarev, NN (2005). Progression of Barrett's metaplasia to adenocarcinoma is associated with the suppression of the transcriptional programs of epidermal differentiation. Cancer Res 65, 3146-3154.

 Kolumam, GA, Thomas, S., Thompson, LJ, Sprent, J., and Mural i- Krishna, . (2005) . Type I interferons act directly on CD8 T cel ls to al low clonal expansion and memory formation in response to Viral infection. The Journal of experimental medicine 202, 637-650.

 Le Bon, A. , Durand, V. , Kamphuis, E. , Thompson, C. , Bulf one-Paus, S. , Rossmann, C. , Kal inke, U., and Tough, DF (2006) . Direct stimulation Of T cells by type II FN enhances the CD 8+ T cel l response during cross-priming. J Immunol 176, 4682-4689.

Le Bon, A. , Etchart, N. , Rossmann, C. , Ashton, M. , Hou, S. , Gewert, D. , Borrow, P. , and Tough, DF (2003) . Cross-priming of CD8+ T Cel ls stimulated by virus-induced type I interferon. Nat Immunol 4, 1009-1015. Le Bon, A. , and Tough, DF (2002) . Links between innate and adaptive immunity via type I interferon. Curr Opin Immunol 14, 432-436.

 Lee, Y., Sogyong L. Auh, Yugang Wang, Byron Burnette, Yang fang, Yuru Meng, Michael Beckett, Rohit Sharma, Robert Chin, Tony Tu, Ralph R. Weichselbaum, and Yang-Xin Fu (2009) . Therapeutic effects Of ablat ive radiation on local tumor require CD8+ T cells: changing strategies for cancer treatment Blood.

 Ma, Y. , epp, 0. , Ghiringhel li, F. , Apetoh, L. , Aymeric, L. , Locher, C. , Tesniere, A. , Martins, I. , Ly, A. , Haynes, NM , Et al. (2010) . Chemotherapy and radiotherapy: cryptic anticancer vaccines. Semin Immunol 22, 113-124.

 Marrack, P., Kappler, J., and Mitchel l, T. (1999) . Type I interferons keep activated T cel ls al ive. The Journal of experimental medicine 189, 521-530.

 McBride, ? H. , Chiang, CS, Olson, JL, Wang, CC, Hong, JH, Pajonk, F., Dougherty, GJ, Iwamoto, KS, Pervan M., and Liao, YP (2004) . A sense of danger from Radiation. Radiat Res 162, 1-19.

 Muller, U., Steinhoff, U., Reis, LF, Hemmi, S., Pavlovic, J., Zinkernagel, RM, and Ague t, M. (1994) . Functional role of type I and type II interferons in antiviral Defense. Science 264, 1918-1921.

0' Co. 11, RM, Saha, SK, Vaidya, SA, Bruhn, KW, Miranda, GA, Zarnegar, B., Perry, AE, Nguyen, B. 0. , Lane, TF, Taniguchi, T. , et Al. (2004) . Type I interferon production enhances susceptibility to Listeria monocytogenes infection. The Journal of experimental medicine 200, 437-445. Picaud, S., Bardot, B., De Maeyer, E., and Seif, I. (2002) . Enhanced tumor development in mice lacking a functional type I interferon receptor. J Interferon Cytokine Res 22, 457-462.

 Pi troda, SP, fakim, BT, Sood, RF, Beveridge, MG, Beckett, MA, MacDermed, DM, Weichselbaum, RR, and Khodarev, NN (2009) . STAT 1 -dependent expression of energy metabol ic pathways l inks tumour Growth and radioresi stance to the Warburg effect. BMC Med 7, 68.

 Santini, SM, Di Pucchio, T., Lapenta, C., Par lato, S. , Logozzi, M. , and Belardel li, F. (2002) . The natural al l i between between type I interferon and dendritic cel ls and Its role in l inking innate and adaptive iniffluni ty. J Interferon Cytokine Res 22, 1071-1080.

 Tough, D. F., Borrow, P., and Sprent, J. (1996) . Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 272, 1947-1950.

 Tusher, V. G., Tibshirani, R., and Chu, G. (2001). Signif icance analysis of microarrays ap l ied to the ionizing radiation response. Proc Natl Acad Sci U S A 98, 5116-5121.

 Uddin, S., and Platanias, L. C. (2004) . Mechanisms of type — I interferon s ignal transduction. J Biochem Mol Biol 37, 635-641.

 Van Boxel-Dezaire, A. H., Rani, M. R., and Stark, G. R. (2006) . Complex modulation of cel l type-specif ic signal ing in response to type I interferons. Immunity 25, 361-372.

 Wang, H. , Bloom, 0. , Zhang, M., Vishnubhakat, JM, Ombrel l ino, M. , Che, J. , Frazier, A. , Yang, H. , Ivanova, S. , Borovikova, L. , et al. (1999) . HMG-1 as a late mediator of endotoxin lethality in mice. Science 285, 248-251.

Weichselbauffl, RR, Ishwaran, H., Yoon, T., Nuyten, DS, Baker, S. . , hodarev, N. , Su, AW , Shaikh, AY, Roach, P. , Kreike, B. , et al. (2008) . An inter fer on-related gene signature for DNA damage res istance is a predictive marker for chemotherapy and radiation for breast cancer. Proc Natl Acad Sci USA 105, 18490-18495.

 Yu, P. , Lee, Y. , Liu, f. , Chin, RK, Wang, J. , Wang, Y. , Schietinger, A. , Phi l ip, M. , Schreiber, H. , and Fu, YX (2004) . Pr iming of naive T cel ls inside tumors leads to eradication of establ ished tumors. Nat Immunol 5, 141-149.

 Yu, P. , Lee, Y., Wang, Y. , Liu, X., Auh, S., Gajewski, TF, Schreiber, H., You, Z. , Kaynor, C., fang, X. , and Fu, YX (2007) . Targeting the primary tumor to generate CTL for the effective eradication of spontaneous metastases. J Immunol 179, 1960-1968.

Claims

1 . Use of an interferon (preferably a type I interferon, in particular IFN-β), a fragment thereof or a functional variant thereof for the preparation of a medicament, wherein the medicament

 - for treating and/or preventing tumors that are resistant to conventional anti-tumor therapies (e.g., radiation therapy, chemotherapy); and/or

 - for the treatment and/or prevention of tumors in combination with other anti-tumor therapies (eg radiotherapy, chemotherapy);

 Wherein the conventional therapy induces a tumor-specific adaptive immune response; and wherein the interferon, a fragment thereof or a functional variant thereof is capable of stimulating the production of an anti-tumor cytotoxic sputum lymphocyte.

2. The use according to claim 1, wherein the interferon, a fragment thereof or a functional variant thereof is contained in a viral vector which is, for example, an adenovirus; an adeno-associated virus; a retrovirus, for example Mouse Moloney leukemia virus; mouse Harvey sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; SV40-type virus; polyoma virus; prion; papilloma virus; disease virus; vaccinia virus; Shield inflammatory virus; and an RNA virus such as a retrovirus; preferably, the viral vector is an adenovirus vector.

3. The use according to claim 1 or 2, wherein the interferon, a fragment thereof or a functional variant thereof is linked to a targeting moiety (e.g., an antibody) that binds to a tumor associated antigen, wherein the targeting moiety and the interference are The merins, fragments thereof or functional variants thereof are directly linked (for example as a fusion protein) or linked by a linker.

The use according to claim 3, wherein the tumor-associated antigen is EGFR, the targeting moiety is, for example, an anti-EGFR antibody, and the tumor is, for example, a tumor expressing EGFR.

The use according to any one of claims 1 to 4, wherein the tumor is a malignant tumor, such as a malignant solid tumor, which includes, for example, breast cancer, lung cancer, prostate cancer, knot Intestinal cancer, cutaneous cancer, head and neck cancer, lymphoma or melanoma.

The use according to any one of claims 1 to 5, wherein the medicament is for administration in combination with at least one other anti-tumor therapy, such as radiation therapy, chemotherapy or the like.

The use according to any one of claims 1 to 6, wherein the conventional anti-tumor therapy is radiation therapy, and the tumor or host carrying the tumor is defective in one or more of the following: 1) Expression and/or function of an interferon (eg, a type I interferon, preferably interferon alpha or beta), particularly an interferon expressed by a CD45+ hematopoietic cell; 2) expression and/or function of an interferon receptor, wherein the interference The receptor is, for example, an IFNa receptor and/or a ΙΡΝβ receptor.

8. A composition comprising an interferon as defined in claim 3 or 4, a fragment thereof or a functional variant thereof, and optionally a pharmaceutically acceptable carrier.

9. A kit containing:

 a) an interferon, a fragment thereof or a functional variant thereof as defined in any one of claims 1 to 4; or a composition according to claim 8;

 b) instructions for use in the treatment and/or prevention of tumors that are resistant to conventional anti-tumor therapies (eg, radiation therapy, chemotherapy); or for use with other anti-tumor therapies (eg, radiation therapy) , chemotherapy) combined with the treatment and / or prevention of tumors.

10. Use of a composition according to claim 8 for the preparation of a medicament, wherein the medicament is for the treatment and/or prevention of a tumor, such as a malignant tumor, such as a malignant solid tumor, including, for example, breast cancer, lung cancer, prostate cancer, colon Cancer, skin cancer, head and neck cancer, lymphoma or melanoma.