CN103536917A

CN103536917A - Use of interferon in treatment of tumor, and related product and method

Info

Publication number: CN103536917A
Application number: CN201310525752.5A
Authority: CN
Inventors: 杨选明; 傅阳心
Original assignee: Suzhou Ding Fu Target Spot Bioisystech Co Ltd
Current assignee: Suzhou Ding Fu Target Spot Bioisystech Co Ltd
Priority date: 2013-10-30
Filing date: 2013-10-30
Publication date: 2014-01-29
Anticipated expiration: 2033-10-30
Also published as: CN103536917B

Abstract

The invention relates to use of interferon in treatment of a tumor, and related products and a method, particularly relates to the field of tumor treatment, and especially aims at overcoming the resistance of the tumor on an antibody therapy. Particularly, the invention relates to application, and related product and method for inhibiting growth and recurrence of the tumor and causing tumor regression by combined utilization of interferon, especially I-type interferon, and a compound for blocking up a programmed death 1 or programmed death ligand (PD-1/PDL) passage.

Description

Interferon is the purposes in tumor and relevant product and method in treatment

Technical field

The present invention relates to therapeutic field of tumor, particularly overcome the resistance of tumor antagonist therapy.Particularly, the present invention relates to by interferon, for example I type interferon with and with the blocking-up PD-1(programmed death factor, Programmed death-1)/PDL(programmed death factor part, PD-1Ligand) combining of the compound of path used and growth and the recurrence of inhibition tumor, and causes tumor regression.

Background technology

Generally acknowledged viewpoint is at present, the antibody of targeting carcinogenecity receptor (for example Cetuximab and He Saiting) has antitumous effect, because they can be blocked growth signals induction, cell cycle be stopped and/or the cytotoxic effect (ADCC) that mediating by apoptosis or antibody dependent cellular carrys out inducing cell death.Yet, about the great majority research of antibody-mediated antitumor mechanism, be the people's tumor experiment based on from heteroplastic transplantation model in In vitro culture or body, they do not comprise the contribution of Acquired immune response.Recently, the research of using immunocompetence host to carry out shows, by anti-CD 20, the lasting antitumor protection that anti-Neu and anti-EGFR-antibodies therapy are brought depends on cellullar immunologic response (people such as Abes, 2010 really; The people such as Park, 2010; The people such as Yang, 2013).Yet, determine yet Antybody therapy whether and how activating T cell reply.

At present, the most of strategies for improvement of the therapeutic effect of antibody focus on the cytotoxicity increasing tumor cell.A kind of strategy is antibody and cytotoxic drug (comprising chemotherapeutant) are puted together mutually and formed antibody-drug complex (ADC); compare this with independent antibody and realized better antitumous effect; and compare with independent cytotoxicity chemotherapy; this has produced less side effect (people such as Fayad, 2013; The people such as Hurvitz, 2013; The people such as Krop, 2012).But the long-term protection effect of ADC is still unknown.Also fail clearly to determine in the process of this treatment, whether also to activate non-specific T cell, thereby increase the potential possibility that produces harmful side effect.

Similar to first generation antibody, after life-time service ADC, can produce host resistance.Really, although the potential cytotoxic effect of ADC originally cause and disappear, many patient experiences recurrence or produced transfer.For this resistance, a kind of possible explanation is that antibody resistance clone produced before or after antibody treatment originally, thereby causes respectively former or acquired resistance.

Therefore, need a kind of novel antitumor therapy badly, it can effectively cause disappearing of tumor and can overcome tumor cell for the resistance of described therapy and final realize the healing of tumor and/or prevent the recurrence of tumor.

Summary of the invention

The antibody mediated immunity treatment surface marker by targeting and Tumor-assaciated is used for the treatment of cancer and has 16 years nearly people such as (, 2012) Scott.Targeting carcinogenecity receptor is shown is effective; because the Antybody therapy for these receptors can partly suppress tumor growth (Hynes and Lane, 2005 in inhibition tumor cell growth and the human tumor xenograft model in immunodeficiency type mice in vitro significantly in vivo; The people such as Li, 2005).The primary treatment effect of antibody therapy is originally by owing to being conducted and directly killed tumor cell by signal.Yet, recently, people recognize that Fc receptor (FcR) the signal conduction on immunocyte is also very important, because in mouse model, in vivo, when lacking the conduction of FcR signal, antibody-mediated antitumous effect reduces the (people such as Clynes greatly, 2000), and FcR polymorphism relevant to the clinical effectiveness in human breast carcinoma patient (people such as Musolino, 2008).These data have proposed following probability: the antitumous effect of antibody therapy also depends on the antibody dependent cellular cytotoxicity being produced by congenital cell, and the immunoreation (ADCC) of acquired immunity cell dependence.

Although, still there are some obstacles for realizing the effective antibody therapy for cancer in the antibody-mediated antitumous effect shown in having above.First, many patients still can produce resistance to main antibody therapy after having experienced long-term and expensive treatment.For example, the colorectal cancer patients of 80 – 90% has resistance (Bardelli and Siena, 2010) to Cetuximab (anti-EGFR), and the patient with breast cancer of 66 – 88% has resistance (people such as Cobleigh, 1999) to He Saiting (anti-HER2).The second, after long-term treatment, originally antagonist therapy has the tumor cell in the patient of response probably to produce natural or acquired antibody resistance clone, and this is the main cause of cancer return.What most research was paid close attention to is the inherent resistance of carcinogenecity signal conduction, for example, by the sudden change in the sudden change in the oncogene of targeting or the gene relevant to the carcinogenic path of facilitating antibody resistance.In following tumor cell, can occur the constitutional of Cetuximab and acquired resistance: the sub-KRAS of mediation that contains EGFR itself or downstream and the tumor cell of the sudden change in BRAF; and the dimerization companion of containing the Her2(EGFR through increasing) tumor cell (Bardelli and Siena, 2010; The people such as Misale, 2012; The people such as Sharma, 2007; The people such as Wheeler, 2008; The people such as Yonesaka, 2011).In order to strengthen by the effect of Antybody therapy killing tumor cell and the resistance that reduces host; developed the treatment of the second filial generation based on antibody; it is comprised of the cytotoxic drug of puting together with some antibody or radioactive substance, kills and wounds (people such as Fayad, 2013 to strengthen directly; The people such as Hurvitz, 2013; The people such as Krop, 2012).The another kind of strategy that improves direct cytotoxic effect relates to the different epi-position of targeting or even relevant receptor people such as (, 2009) Bostrom.At present, the main policies that overcomes the resistance of antagonist in host is through the oncogene of sudden change or through the medicine of the gene relevant to carcinogenic path of sudden change in exploitation target tumor cell.For example, BRAF inhibitor is used to targeting Cetuximab resistance tumor (people such as Yoon, 2011).

In sum, the resistance of antagonist is the main challenge for the cancer therapy based on antibody.And the strategy that overcomes at present the described resistance for antibody focuses on improvement: 1) put together the direct killing to tumor cell of carrying out by antibody and cytotoxic drug, or 2) by non-specific anti-CD3 activation, undertaken to the sub-intensifier target of bispecific T Cell binding of tumor cell to T cell killing.Due to heterogeneous and their residing environment of tumor cell, the strategy of these direct killing can not all tumor cells of targeting, and finally still can cause antibody resistance.

In order to seek more effective strategy, improve antibody mediated immunity therapy, need to differentiate critical molecule, they reply with caused acquired immunity antitumor antibody-mediated antitumor to reply and be associated.

Interferon (IFN) is a kind of broad-spectrum disease resistance toxic agent, and it can suppress virus replication, also can strengthen natural killer cell (NK cell), macrophage and the lymphocytic vigor of T simultaneously, thereby play immunoregulation effect, and strengthen anti-virus ability.Interferon is one group of reactive protein (being mainly glycoprotein) with several functions, is a kind of cytokine being produced by mononuclear cell and lymphocyte.They on allogenic cell, have wide spectrum antiviral, affect Growth of Cells, and differentiation, regulate the multiple biological activitys such as immunologic function.General sorting technique is divided into three types by interferon at present: I type has IFN-α and IFN-β, and wherein IFN-α has more than 20 hypotypes, and IFN-β only has a hypotype.I type interferon have suppress virus replication, parasiticide, inhibition various kinds of cell propagation, immune stimulatory cell killing activity, participate in the effects such as immunomodulating, antitumor; II type: only have IFN-γ, and only have a kind of hypotype, except having antiviral, antiproliferative activity, its main biologic activity is immunoregulation effect; IFN-λ 2(IL-28a) and IFN-λ (IL-28b) III type: i.e. IFN-λ 1(IL-29).IFN-ω belongs to IFN-α family, and its structure and size and other IFN-α be difference slightly, but antigenicity has larger difference.

Recently, the increase of finding interferon (IFN) (such as I type interferon) and anticancer clinical immunne response advantageously relevant people such as (, 2011) Fuertes.In addition, interferon signal conduction at spontaneous tumor, repel and the process of various antitumor therapies in to cause antitumor t cell response be vital (people such as Burnette, 2011; The people such as Diamond, 2011; The people such as Fuertes, 2011; The people such as Stagg, 2011).These data show that interferon is crucial for the antineoplastic specificity t cell response causing for tumor cell.Also reported that interferon activates memory T cell (people such as Kohlmeier, 2010) in the process of viral infection.Yet up to now, interferon is by modestly for clinical and effect is limited (Trinchieri, 2010).Really, people understand very limitedly to the effect of this type cytokines, (the people such as Gonzalez-Navajas because it may work as immune activation agent or immunosuppressant in different disease models, 2012), and potential serious side effects follow the short half-life to limit its application in oncotherapy.

On the other hand, in antibody response tumor model, in ADCC process or by stress the immune activation molecule that discharges of antibodies tumor cell effectively active antigen be delivery cell (APC), strengthen the ability of its inducing cytotoxic t cell response.Yet this immunne response is weak with instantaneous, particularly when tumor is immediately built.In antibody resistance tumor model, the APC or the T cell that need other strategy to come reactivation to be suppressed by inhibitive ability of immunity tumor microenvironment.Current inventor has observed recently CpG and Cetuximab coupling has greatly been strengthened to its therapeutic effect, infers that this is dendritic cell in tumor microenvironment and excite Acquired immune response subsequently people such as (, 2013) Yang by activation.The method that these researchs have shown to be designed for the activation that promotes acquired immunity for the cancer therapy based on antibody long-term be successfully important, even can be crucial.

As the support to this idea, nearest clinical experiment has used antibody to carry out the co-suppression signal on blocking t cell, and this comprises CTLA-4, PD-1 and PD-L1.There are many inhibition signal paths that can weaken t cell response and promote T cell tolerance in B7-CD28 family, the PD-1/PD-L path of wherein in recent years finding is noticeable due to unique inhibit feature, be considered to affect the key factor of autoimmune and infectious disease chronicity, this path comprises two part: PD-L1 and the PD-L2 of PD-1 and it.PD-1(programmed cell death1), also claim CD279, contactin member, the cytoplasmic region of containing two tyrosine signal motifs, one forms immunity receptor tyrosine and suppresses motif (immunoreceptor tyrosine-based inhibition motif, ITIM), another is immunity receptor tyrosine exchange motif (Immunoreceptor tyrosine-based switch motif, ITSM).ITSM raises phosphatase SHP-1 and SHP-2, the effect signal generation dephosphorylation that TCR or BCR are transmitted, and meanwhile, PD-1 signal reduces the signal of CD28 mediation, suppresses the expression of Akt tyrosine phosphorylation, carbohydrate metabolism and Bcl-XL.It is comparatively extensive that PD-1 expresses, and thymus development stage PD-1 is mainly expressed on jack to jack adapter sexual cell, and the periphery CD4+ after activation, CD8+T cell, B cell and mononuclear cell can abduction deliverings, and on NK-T cell, expression is lower.

PD-1 has PD-L1 and two kinds of parts of PD-L2, and PD-L1 is extensive compared with the expression of PD-L2, and the affinity of PD-L2 is 2～6 times of PD-L1.PD-L2(also claims B7-DC or CD273) can abduction delivering on the bone marrow mastocyte of dendritic cell (dendritic cells, DC), macrophage and cultivation; And PD-L1(also claims B7-H1 or CD274) the bone marrow mastocyte constructive expression of muroid T cell, B cell, dendritic cell, macrophage, mescenchymal stem cell and cultivation, after activation, express and strengthen, constructive expression's level of human PD-L 1 is low compared with muroid.PD-L1 is also expressed on the cell of many non-hemopoietic types, in immunity, exempts position, and as eye and Placenta Hominis also have expression, PD-L1 may regulate autoreactive T cell or B cell and inflammatory response in these tissues.

Nearest studies show that out that it is the advantageous manner that improves therapeutic effect that reverse T cell suppresses (for example, by blocking-up PD-1/PDL), but lamentedly, and this has also brought serious side effect (people such as Brahmer, 2012; The people such as Topalian, 2012; Weber, 2007).And, clinical data demonstrates, response rate for PD-1 blocking-up remains relatively low (15-25%) in selected patient storehouse, and this replying depended on the expression of PD-L1 in tumor tissues and lymphocytic infiltration originally (people such as Topalian, 2012) to a great extent.Therefore, the critical problem of immunotherapy for cancer is how further to reverse immunosuppressant in described microenvironment to reactivate specific antitumor t cell response, thereby realizes the higher response rate of the immunotherapy based on antibody and long-term effectiveness.

So the medicine based on antibody of being badly in need of a new generation is with target tumor tissue more effectively and reduce systematic side effect.

The not direct target tumor cell of antibody therapy (for example antibody-interferon beta) that has shown associating interferon (for example I type interferon) in the present invention, because not response of the interferon receptors-adequacy tumor antagonist-interferon beta in interferon receptors-defective mice.In addition, bone marrow transplantation (BMT) experiment demonstrates, and need on the cell of derived from bone marrow, express interferon receptors, and this shows wherein not relate to the angiogenesis inhibitor effect of interferon.In order to determine the Relative Contribution of ADCC and Acquired immune response, inventor has processed immunodefiiciency Rag-1KO mice with antibody-interferon beta, and observed in the situation that lacking acquired immunity cell, antibody-interferon beta can not suppress tumor growth completely.Although T cell and dendritic cell all can be in response to interferon betas, inventor has further determined that dendritic cell are cells of essential expression interferon receptors, because the mice with I type interferon receptors-defective dendritic cell is for the not response completely of antibody-interferon beta, and the mice with I type interferon receptors-defective T cell only demonstrates the tumor suppression slightly reducing.In addition, the dendritic cell of the mice that the antibody interferes with of hanging oneself element β processes can direct activation tumour-specific T cell, and this antibody therapy (for example antibody-interferon beta) that has shown to combine interferon (as I type interferon) is processed and increased dendritic cell antigen presentation and T cell activation.Therefore, dendritic cell probably correspond directly to combined interferon antibody therapy (for example antibody-interferon beta) to increase antigen presentation and T cell activation, and described antigen presentation and T cell activation reactivate T cell.

In the present invention, inventor has also shown that antibody treatment itself can produce by inducing interferon (for example I type interferon), and it is facilitated dendritic cell activation and produces subsequently antitumor T cellular immunity.This has also shown that the main SC type in tumor microenvironment is dendritic cell and non-T cell, for example, because when I type interferon receptors selectivity when disappearance in dendritic cell, the therapeutic effect of having combined the antibody therapy (antibody-interferon beta fusion rotein) of I type interferon has just disappeared.Therefore, except targeting T-cells, go back the effect that targeting dendritic cells can further increase antibody therapy.

For the immune-mediated damage of balance and ongoing immunne response, after immune activation, host immune is induced the signal conduction of inhibition conventionally.Among this; it is the most noticeable as a kind of protective mechanism in cancer immunotherapy process that PD-L1 in tumor tissues raises, and inventor has observed this phenomenon after processing with the antibody therapy of associating interferon (as I type interferon).Really, blocking-up PD-L1/PD-1 path has further strengthened antitumous effect and has removed tumor.This is specific for PD-L1/PD-1, because when by the antibody therapy of associating I type interferon and blocking-up from T cell on two kinds of other antibody of signal conduction of important inhibition molecule (CTLA-4 or BTLA) when combined, do not observe synergy.This inhibitory pathway that has clearly illustrated that targeting is induced specifically by active immunotherapy can instruct following clinical treatment.

Analysis from the above can recognize, the antibody (Ab) of preferential targeting carcinogenecity receptor is used to treatment of cancer more and more, but after long-term and expensive treatment, tumor usually develops the resistance to these antibody.Current inventor for example, with interferon (I type interferon, as interferon beta) equip with arms antibody, as biological preparation of new generation, it is superior to the antibody therapy of the first generation far away and is independent of various resistance mechanisms and is used to control the tumor that antagonist therapy has resistance.This new strategy is by the resistance reactivating and the repressed congenital and acquired immunity of bridge joint is controlled antagonist again.From mechanism, the dendritic cell in the main target tumor of use in conjunction of antibody and I type interferon, it reactivates cytotoxic T cell by the intersection induction increasing in Tumor-assaciated microenvironment.The signal conduction of the PD-1/PD-L1 path that in addition, blocking-up is induced by the use in conjunction of antibody and I type interferon in tumor has further overcome the resistance of obstruction treatment and has removed the larger tumor of having set up completely.Therefore, the present invention has set up the immunotherapy of a new generation based on antibody, and it can remove the tumor that antagonist has resistance.

Therefore, the Antybody therapy of a new generation of the present invention based on immune can be replied the problem that overcomes use antibody therapy mentioned above by the specific T-cells that reactivates the congenital and acquired immunity cell in tumor and produce a plurality of tumors of targeting sudden change antigen.Therefore, this new strategy may can be removed the directly tumor cell of targeting of antibody.

Aspect other, PD-1/PD-L path not only plays a significant role in regulating self tolerance, also has pivotal role in anti-microbial infection immunity.Manyly cause that chronically infected microorganism may utilize PD-1/PD-L path to weaken anti-infectious immunity mostly, and promote persistent infection.Powerful effect is different with Memorability CD8+T cell from producing after actute infection or vaccination, and when chronic viral infection, the function of virus-specific CD8+T cell all can weaken conventionally.In chronic infection model, as mice LCMV infects or primates SIV infection, or the viral infection such as HIV, HBV, HCV, conventionally can observe non-functional virus-specific CD8+T cell sustainable existence, this phenomenon is called exhaustion (exhaustion), and T cell is exhausted and represented a series of immunodeficiency.With the rising of virus load, the function of virus specific t cell is impaired more serious, and propagation and the ability i.e. disappearance in early days that produces IL-2, produce responsiveness cytokine and cytolytic function and disappear in the later stage.

Meanwhile, interferon (IFN), as a kind of broad-spectrum disease resistance toxic agent, is also shown and can suppresses copying of virus; Also can strengthen natural killer cell (NK cell), macrophage and the lymphocytic vigor of T simultaneously, thereby play immunoregulation effect, and strengthen anti-virus ability.Thereby, according to the present invention, can also recognize, combine and use interferon (I type interferon for example, as interferon beta), the reagent (for example anti-PDL1 antibody) of the interferon of (for example merge, particularly the form with fusion rotein exists) and the signal conduction of blocking-up PD-1/PD-L path of particularly combining with antibody also can be used to treat infected by microbes, viral infection and/or other infectious disease.

Therefore, one aspect of the present invention relates to interferon, and as I type interferon, for example IFN β or IFN α 5 are for the preparation of the purposes of medicine, and described medicine is for overcoming tumor for the resistance of antibody therapy.

In one embodiment, described interferon (as I type interferon) for example, is connected (for example forming fusion rotein) with the targeting moiety (antibody) in conjunction with tumor associated antigen, and wherein said targeting moiety is directly connected or is connected by connexon with described interferon.In a specific embodiment, described targeting moiety is positioned at the N end of described interferon.In another specific embodiment, described targeting moiety is positioned at the C end of described interferon.In a specific embodiment, described interferon can be connected with two or more described targeting moieties, thereby forms polyspecific (for example bispecific) molecule (as polyspecific fusion rotein, as bispecific fusion protein).

At described interferon, by connexon, be connected and form in the situation of fusion rotein with described targeting moiety, described connexon can comprise the aminoacid sequence of any suitable length, for example, described connexon can comprise 1-10,1-20,1-30 or more aminoacid (or consisting of), for example described connexon can comprise 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31 or more aminoacid, or is comprised of described aminoacid.

In specific embodiment, described targeting moiety is antibody, for example anti-egfr antibodies or anti-Neu antibody.

In other specific embodiments, described tumor is malignant tumor, pernicious solid tumor for example, and it comprises for example breast carcinoma, pulmonary carcinoma, carcinoma of prostate, colon cancer, skin carcinoma, head and neck cancer, lymphoma or melanoma.

In yet another aspect, the present invention relates to interferon (I type interferon for example, as IFN-β or IFN α 5) with the compound of blocking-up PD-1/PDL signal transduction pathway (antibody for example, as the antagonistic antibodies of anti-PD1 or PD-L1) jointly for example, for the preparation of the purposes of medicine (pharmaceutical kit), wherein said medicine is used for the treatment of tumor (pernicious solid tumor for example, breast carcinoma particularly, pulmonary carcinoma, carcinoma of prostate, colon cancer, skin carcinoma, head and neck cancer, lymphoma or melanoma), for example, for overcoming tumor for the resistance of antibody therapy.Wherein, described interferon exists independently of one another with the form that the compound of described blocking-up PD-1/PDL signal transduction pathway can mutually not mix in described medicine, or mixes and exist not affect each other the mode of effect.

In a specific embodiment, the above-mentioned host who has the tumor of resistance or carry described tumor for antibody therapy is at following one or more middle defectiveness: 1) interferon (as I type interferon), for example expression of interferon-ALPHA 5 or interferon beta and/or function; 2) expression and/or the function of the interferon receptors in expression and/or the function, particularly dendritic cell of interferon receptors (as I type interferon receptors); 3) intersection of dendritic cell is presented and consequent antitumor cell toxicity T cell.

Wherein, in specific embodiment, described interferon (as I type interferon) can for example, be connected (for example forming fusion rotein) with the targeting moiety (antibody) in conjunction with tumor associated antigen, and wherein said targeting moiety is directly connected or is connected by connexon with described interferon.In a specific embodiment, described targeting moiety is positioned at the N end of described interferon.In another specific embodiment, described targeting moiety is positioned at the C end of described interferon.In a specific embodiment, described interferon can be connected with two or more described targeting moieties, thereby forms polyspecific (for example bispecific) molecule (as polyspecific fusion rotein, as bispecific fusion protein).

Aspect other, the present invention relates to a kind of test kit, it contains:

A) interferon, I type interferon for example, as IFN β or IFN α 5; With

B) compound of blocking-up PD-1/PDL signal transduction pathway, antibody for example, as anti-PDL1 antibody.

Wherein when using described test kit, described interferon (for example I type interferon, as IFN β or IFN α 5) with as described in the compound of blocking-up PD-1/PDL signal transduction pathway can be by simultaneously, (with any order) or respectively to there being the experimenter who needs to use in turn.And in described test kit, described interferon (for example I type interferon, as IFN β or IFN α 5) with as described in the form that can not mix mutually each other of the compound of blocking-up PD-1/PDL signal transduction pathway there is (for example in the independent space of each leisure, existing); Or exist not affect the form mixing of effect each other.

Wherein, in specific embodiment, described interferon in described test kit (as I type interferon) can for example, be connected (for example forming fusion rotein) with the targeting moiety (antibody) in conjunction with tumor associated antigen, and wherein said targeting moiety is directly connected or is connected by connexon with described interferon.In a specific embodiment, described targeting moiety is positioned at the N end of described interferon.In another specific embodiment, described targeting moiety is positioned at the C end of described interferon.In a specific embodiment, described interferon can be connected with two or more described targeting moieties, thereby forms polyspecific (for example bispecific) molecule (as polyspecific fusion rotein, as bispecific fusion protein).

In another embodiment, described targeting moiety is antibody, for example anti-egfr antibodies or anti-Neu antibody.

In other specific embodiments, described tumor is malignant tumor, pernicious solid tumor for example, and it comprises for example breast carcinoma, pulmonary carcinoma, carcinoma of prostate, colon cancer, skin carcinoma, head and neck cancer, lymphoma or melanoma.Especially, described tumor can be breast tumor or melanoma.

In a specific embodiments, described tumor or the host that carries described tumor are at following one or more middle defectiveness: 1) interferon (as I type interferon), for example expression of interferon-ALPHA 5 or interferon beta and/or function; 2) expression and/or the function of the described interferon receptors in expression and/or the function, particularly dendritic cell of interferon receptors (as I type interferon receptors); 3) intersection of dendritic cell is presented and consequent antitumor cell toxicity T cell.

Aspect other, the invention still further relates to a kind of test kit, it contains:

A) as the interferon defined in above; With

B) operation instructions, have wherein recorded described test kit for overcoming tumor for the resistance of antibody therapy.

Aspect other, the present invention relates to a kind ofly (for example treat tumor, for overcoming tumor for the resistance of antibody therapy) method, described method comprise to patient's administering therapeutic effective dose as the interferon defined in above or as above defined in test kit of the present invention.

In specific embodiment, described patient has resistance for antibody therapy.In other embodiment, described patient is at following one or more middle defectiveness: interferon, and I type interferon for example, as the expression of interferon-ALPHA 5 or interferon beta and/or function; 2) expression and/or the function of the interferon receptors in the expression of interferon receptors and/or function, particularly dendritic cell; 3) intersection of dendritic cell is presented and consequent antitumor cell toxicity T cell.

In other embodiments, described in prevent and/or treat that method also comprises simultaneously, (with any order) or to described patient, use respectively in turn: 1) as the interferon defined in above; With 2) compound of blocking-up PD-1/PDL signal transduction pathway, antibody for example, as anti-PDL1 antibody.

In yet another aspect, the present invention relates to as interferon defined above or test kit of the present invention, it is for overcoming tumor for the resistance of antibody therapy.

Aspect other, I type interferon of the present invention, test kit or method can be used to and antineoplastic antibody therapy or other antitumor therapy (such as chemotherapy, radiation therapy etc.) co-administered (for example or use in turn with any order) simultaneously.

In addition, optionally, in reagent of the present invention and/or test kit, can further comprise pharmaceutically acceptable carrier.

In a word, the present invention has brought some important impacts to cancer immunotherapy field.First, it has been set up a kind of approach and has produced the treatment (antibody therapy of for example combining I type interferon, for example antibody-interferon beta fusion rotein) of a new generation based on antibody, and it makes Acquired immune response can more effectively answer antagonist resistance and recurrence.Then, enhancing cytotoxic T cell is replied then can be killed more tumor cell to produce following positive feedback loop: the death of neoplastic cells of cytotoxic T cell mediation causes dangerous/congenital signal conduction of endogenous, it further produces the congenital and acquired immunity for tumor, and finally causes tumor regression more completely.Second, the invention provides evidence shows, interferon (as I type interferon) (it is associated congenital and acquired antineoplastic immune) is crucial factor for antibody-mediated tumor regression, and therefore provides the important target for immunotherapy for cancer.The 3rd, it is tolerance cell types main in tumor that the present invention has disclosed dendritic cell, shows that they play a major role in definite inhibitive ability of immunity tumor microenvironment.Therefore, targeting dendritic cells will be another important strategy for improvement of the effect of cancer immunotherapy.The 4th, the present invention proposes acquired resistance that antagonism induced by immunization therapy and will make the therapeutic effect of immunization therapy maximize and the final host's of healing tumor.In a word, the strategy adopting in the present invention has been pointed out some new directions for optimizing targeting immunization therapy, and it can greatly affect discovery and the clinical cancer treatment of antitumor drug.

Accompanying drawing explanation

Fig. 1. in antibody-mediated tumor regression process, induced interferon to produce and it is necessary for described tumor regression process.A) to WT BALB/c mouse (n=4/ group) subcutaneous injection 5 * 10 ⁵tUBO cell was used the anti-Neu (left hurdle) of 100 μ g or contrasts IgG in the time of the 14th day.To WT B6 mice (n=4/ group) subcutaneous injection 7 * 10 ⁵b16-EGFR cell, and in the time of the 14th day, used the anti-EGFR (right hurdle) of 100 μ g or contrasted IgG.After four days, tumor is digested and isolate the CD45 in tumor tissues ⁺and CD45 ^-group.Carried out PCR in real time to detect the expression of the mRNA level of I type interferon.B) to WT BALB/c mouse (n=5/ group) subcutaneous injection 5 * 10 ⁵tUBO-EGFR cell, has used the anti-Neu of 100 μ g at the 14th day and 21 days.In tumor, using the anti-I type interferon receptors of 200 μ g or contrasting Ig on the same day.Measure the growth of tumor and compare for twice weekly.C) to WT B6 mice (n=5/ group) subcutaneous injection 5 * 10 ⁵b16-EGFR cell, has used 2 * 10 at the 14th day and 21 days in tumor ⁹adenovirus-interferon beta or contrast virus.Measure tumor growth and compare for twice weekly.*, compare P<0.05 with matched group.Shown one group of representative experimental results in three groups of experiments.

Fig. 2 .Ab-IFN β has greatly improved the antitumous effect of antibody.A) to WT BALB/c mouse (n=5/ group) subcutaneous injection 5 * 10 ⁵tUBO-EGFR the 14th, anti-EGFR-interferon beta or control antibodies with 25 μ g in the time of 18 and 22 days are processed.Measure tumor growth and compare for twice weekly.B) to WT B6 mice (n=5/ group) subcutaneous injection 1 * 10 ⁶b16-EGFR the 14th, anti-EGFR-interferon beta or control antibodies with 25 μ g in the time of 18 and 22 days are processed.Measured weekly tumor growth for twice and compared.C) to Rag1KO mice (n=5/ group) subcutaneous injection 3 * 10 ⁶h460 cell, and adoptive transfer 2 * 10 in the time of the 13rd day ⁶oTI LN cell.The 14th, in the time of 18 and 22 days, used anti-EGFR-interferon beta or the control antibodies of 25 μ g.Measured weekly tumor growth for twice and compared.D) to Neu ^oTI/OTII-Tg female mice (n=5/ group) subcutaneous injection 1 * 10 ⁶nOP23 the 14th, anti-Neu-interferon beta or control antibodies with 25 μ g in the time of 18 and 22 days are processed.Measured weekly tumor growth for twice and compared.*, compare P<0.05 with matched group.Shown three groups experiment in representational one group.

Fig. 3. anti-EGFR-interferon beta can be replied by inducing antitumor specific cytotoxic t lymphocytes, and it facilitates tumor regression.A) to WT and Rag1KO B6 mice (n=5/ group) subcutaneous injection 5 * 10 ⁵b16-EGFR-SIY cell, and the 14th, anti-EGFR-interferon beta or control antibodies with 25 μ g in the time of 18 and 22 days are processed.Monitor tumor growth twice weekly.B) process the last time afterwards the 7th day, collected dLN cell and carried out interferon gamma ELISPOT and measured.C) to WT B6 mice (n=5/ group) subcutaneous injection 5 * 10 ⁵b16-EGFR-SIY the 14th, anti-EGFR-interferon beta or control antibodies with 25 μ g in the time of 18 and 22 days are processed.Use on the same day CD8-with anti-EGFR-interferon beta and exhausted antibody (200 μ g/ mice).Measured weekly tumor growth for twice and compared.D) the 7th day after processing the last time, has collected dLN cell and has carried out interferon gamma ELISPOT and measured.*, than matched group P<0.05.Shown three groups experiment in representational one group.

Fig. 4. the antitumous effect of anti-EGFR-interferon beta requires to express I type interferon receptors on host's hematopoietic cell.A) to WT and I type interferon receptors 1KO B6 mice (n=5/ group) subcutaneous injection 5 * 10 ⁵b16-EGFR-SIY cell and the 14th, anti-EGFR-interferon beta or control antibodies with 25 μ g in the time of 18 and 22 days are processed.Monitor tumor growth twice weekly.B) during the 7th day after processing the last time, collect dLN, spleen and measure and detected the interferon in supernatant and produce through tumor-infiltrated cell and by CBA.C) shown in bone marrow chimera reconstruct after 30 days, to mouse subcutaneous injection 5 * 10 ⁵b16-EGFR-SIY the 14th, anti-EGFR-interferon beta or contrast Ig with 25 μ g in the time of 18 and 22 days process.Measured weekly tumor growth for twice and compared.*, than matched group P<0.05.One of two groups of representative experiments have been shown.

Fig. 5. anti-EGFR-interferon beta has recovered the intersection of dendritic cell and has presented ability.A) to WTB6 mice (n=5/ group) subcutaneous injection 5 * 10 ⁵b16-EGFR-SIY and the 14th with process by anti-EGFR-interferon beta or the control antibodies of 25 μ g 18 days time.Process the last time afterwards the 7th day, collected dLN cell and carried out interferon gamma ELISPOT and measured.B) to WT B6 mice (n=5/ group) subcutaneous injection 5 * 10 ⁵b16-EGFR-SIY the 14th with process by anti-EGFR-interferon beta or the control antibodies of 25 μ g 18 days time.After processing for the first time the 8th day, passes through CD11c ⁺positive selected purification from the dendritic cell of dLN and hatched with purified inmature 2C T cell.After 2 days, measured interferon generation.C) by flow cytometry, measured dendritic cell activation marker.D) shown in the reconstruct of bone marrow chimerism thing after 30 days, to mouse subcutaneous injection 5 * 10 ⁵b16-EGFR-SIY the 14th, anti-EGFR-interferon beta or contrast Ig with 25 μ g in the time of 18 and 22 days process.DT or PBS have been used on the same day with described processing.Measured weekly tumor growth for twice and compared.*, compared to matched group P<0.05.Shown two groups experiment in representational one group.

Fig. 6. dendritic cell are to correspond directly to the main cell type that anti-EGFR-interferon beta is processed.A) to WT and CD11c-Cre I type interferon receptors 1 ^flox/floxmice (n=5/ group) subcutaneous injection 5 * 10 ⁵b16-EGFR-SIY the 14th, anti-EGFR-interferon beta or control antibodies with 25 μ g in the time of 18 and 22 days are processed.Measure tumor growth and compare for twice weekly.B) to WT and CD4-Cre I type interferon receptors 1 ^flox/floxmice (n=5/ group) subcutaneous injection 5 * 10 ⁵b16-EGFR-SIY the 14th, anti-EGFR-interferon beta or control antibodies with 25 μ g in the time of 18 and 22 days are processed.Measure tumor growth and compare for twice weekly.*, compared to matched group P<0.05.Shown two groups experiment in representational one group.

Fig. 7. antagonism has realized by the PD-L1 expression of anti-EGFR-interferon beta induction the result that tumor disappears completely.A) to WT B6 mouse subcutaneous injection 5 * 10 ⁵b16-EGFR-SIY cell was also processed by anti-EGFR-interferon beta or the control antibodies of 25 μ g in the time of the 14th day.After two days, collect tumor cell and by flow cytometry, analyze PD-L1 and express.Red line represents the group of processing through contrast Ig, and blue line represents the group of processing through anti-EGFR-interferon beta.B) B16-EGFR cell, processes with anti-EGFR-interferon beta of 0.02 μ g/ml in cell cultivation process in vitro.After one day, collected tumor cell and by flow cytometry, PD-L1 expressed and analyzed.Red line represents the cell stimulating through contrast Ig, and blue line represents the cell stimulating through anti-EGFR-interferon beta.C) to WT B6 mice (n=5/ group) subcutaneous injection 5 * 10 ⁵b16-EGFR-SIY cell, and the 14th, anti-EGFR-interferon beta or control antibodies with 25 μ g in the time of 18 and 22 days are processed.Used on the same day PD-L1 blocking antibody (400 μ g/ mice) with anti-EGFR-interferon beta.Measure tumor growth and compare for twice weekly.D) the 14th day after processing the last time, collects splenocyte and has also carried out interferon gamma ELISPOT and measure.*, than matched group P<0.05.Shown three groups experiment in representational one group.

Fig. 8. the model how the I type interferon of proposition is associated congenital and acquired antineoplastic immune.After antibody therapy, from antibody response tumor and the DAMP that non-antibody resistance tumor discharges will induce the generation of I type interferon.Ab-interferon produces at antibody resistance tumor situ Simulation with I type interferon.I type interferon increases dendritic cell intersection simultaneously to be presented, and to produce better cytotoxic T cell, replys, and its direct activated T cell and induction PD-L1 express to weaken antineoplastic immune.Ab-interferon combines anti-tumor immune response is maximized with PD-L1 blocking-up.

Fig. 9. anti-EGFR-IFN β can be delivered to IFN β EGFR+ cell.A) structure of anti-EGFR-IFN beta fusion proteins.B) with hIg, anti-EGFR or anti-EGFR-IFN β and Anti-Human IgG Fc γ-PE dye to A431 cell.C) use 5x10 ⁵b16-EGFR cell skin hemostasis WT B6 mice (n=5/ group) was also processed with anti-EGFR-IFN β of 25 μ g at the 14th day.At different time points, by hIg ELISA, measure the concentration of anti-EGFR-IFN β in different tissues.Shown a group in three groups of representative experiments.

Figure 10. the side effect of anti-EGFR-IFN β.Anti-EGFR-IFN β intravenous injection B6 mice by various dose.Shown in time point, by BD CBA test kit, measure the concentration of cytokine shown in serum.

Figure 11. for the beta induced tumor regression of anti-EGFR-IFN, do not need CD20 ⁺, NK1.1 ⁺and Ly-6G ⁺cell.Use 5x10 ⁵b16-EGFR subcutaneous injection WT B6 mice (n=5/ group) the 14th, anti-EGFR-IFN β or contrast Ab with 25 μ g in the time of 18 and 22 days process.The Ab of deletion specific cells group shown in having used on the same day with anti-EGFR-IFN β (200 μ g/ mice).Measured weekly tumor growth for twice and compared.

Figure 12. by anti-EGFR-IFN β direct activation DC and T cell in vitro.Anti-EGFR-IFN the β that has cultivated in vitro concentration shown in also using from the splenocyte of WT B6 mice processes.After one day, collected cell for by the expression of CD86 on CD69 on Flow Cytometry Analysis T cell and dendritic cell.

Figure 13. between anti-EGFR-IFN β and anti-CTLA 4 and anti-BTLA, there is no concertedness antitumous effect.Use 5x10 ⁵b16-EGFR cell skin hemostasis WT B6 mice (n=5/ group) was also processed by anti-EGFR-IFN β or the control antibodies of 25 μ g at the 14th, 18 and 22 days.Use on the same day CTLA4 or BTLA blocking antibody (400 μ g/ mice) with anti-EGFR-IFN β.Measured weekly tumor growth for twice and compared.

Figure 14. in the embodiment of the present invention, the antibody therapy of associating interferon there is is the summary of the tumor model of response.

Specific embodiments

term and definition

Unless stated otherwise, the term and definition using in the application is all usual implication and dawns known to those skilled in the art of using in this area.

As used in this application, term " tumor sites " refer to contain or the body that contains tumor cell under a cloud in or in vitro position.Described tumor sites comprises the position at solid tumor and approaching or contiguous tumor growth place.

As used in this application, term administering " refer to general and/or local application.Term " general is used " refers to non locally to be used, thereby the material of using may affect the some organ or tissues in whole health; Thereby or the material of using may pass through the several organ or tissues in whole health and arrive target site.For example, to experimenter's blood circulation use can cause therapeutic product in more than one tissue or organ from used vector expression, or can cause therapeutic product in specificity site by used vector expression, for example, this be due to natural tropism or due to being operatively connected of tissue-specific promoter's element.It will be understood by those skilled in the art that described general is used contains various forms of using, and this includes but not limited to: parenteral administration, intravenous are used, intramuscular administration, subcutaneous administration, applied dermally, oral etc.

Term " local application " refers in specificity site or its and around uses.It will be understood by those skilled in the art that local application contains various forms of using, for example, be injected directly into specific site place or be expelled to (for example in tumor, using) around it.

As used herein, term " treatment effective dose " refers to and reaches therapeutic purposes disease or the patient's condition (lesion/cancer disease for example, for example, for making tumor regression or reducing the size of tumor) required interferon of the present invention, or the amount of component in test kit of the present invention.Can about specific object, determine described effective dose by putting into practice, coming in a conventional manner.Especially, described treatment effective dose can be to reach the required amount of following object: the number that reduces cancerous cell; Reduce tumor size; Suppress (slow down or stop) cancer cell infiltration in peripheral organ; Suppress (slow down or stop) neoplasm metastasis; Suppress tumor growth; And/or alleviation one or more symptoms relevant to cancer.

Term " antibody " is for example contained, monoclonal antibody, polyclonal antibody, single-chain antibody, antibody fragment (it demonstrates required biology or immunologic competence).In this application, term " immunoglobulin " (Ig) is used interchangeably with antibody.Described antibody is target tumor antigen specifically, surface tumours antigen for example, EGFR for example, CD4, CD8, Neu etc.

Term " interferon " comprises complete interferon molecule (for example human interferon, mouse interferon, for example people or mice I type interferon, as interferon-ALPHA or β), its functional fragment, derivant, equivalent or any functional variant, it can realize required biological function, and for example antitumor cell toxicity T cell is presented and produced thus to the intersection of the amplification of inducing tumor-specific T cell, particularly dendritic cell.In situation of the present invention, described interferon can be selected from I type, II type and/or type iii interferon, such as IFN-α, IFN-β, IFN-γ, IFN-λ 1(IL-29), IFN-λ 2(IL-28a), IFN-λ (IL-28b) and IFN-ω etc.

Term " functional variant " refers to, through modifying, (for example suddenlys change, inserts, deletes.Merge, put together, be cross-linked etc.) and for example, from parent's molecule (interferon) different, but retained the variant of its required biologic activity.

Can make by the method for various routines known in the art interferon, its fragment or its functional variant with described targeting moiety to being connected.Described connection can be direct or indirect (for example passing through connexon), for example, and can be by forming fusion rotein, put together or chemistry connects and realizes.When described, while being connected to form fusion rotein, it can be realized by for example recombinant technique or peptide synthetic technology.In certain embodiments, described fusion rotein also can comprise connexon, and described connexon does not destroy the object characteristic (for example generation of inducing antitumor cytotoxic T cell) of formed product.For example, described targeting moiety can be positioned at N end or the C end of described interferon.In a specific embodiment, described interferon can be connected with two or more described targeting moieties, thereby forms polyspecific (for example bispecific) molecule (as polyspecific fusion rotein, as bispecific fusion protein).At described interferon, by connexon, be connected and form in the situation of fusion rotein with described targeting moiety, described connexon can comprise the aminoacid sequence of any suitable length, for example, described connexon can comprise 1-10,1-20,1-30 or more aminoacid (or consisting of), for example described connexon can comprise 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31 or more aminoacid, or is comprised of described aminoacid

Those skilled in the art can select suitable dosage form and method of application according to the concrete patient's condition, disease type (such as the developmental stage of tumor type, tumor etc.), the order of severity, patient's body constitution, other therapies that may be co-administered, the therapy of once using before etc.

Term " cancer " refers to the patient's condition (for example, in mammal, for example people) that is conventionally characterized as being not modulated Growth of Cells.Described cancer includes but not limited to, breast carcinoma for example, pulmonary carcinoma, carcinoma of prostate, colon cancer, skin carcinoma, head and neck cancer, lymphoma or melanoma etc.

Term " antibody therapy " refers to the method by treating with the antibody in selectively targeted object tissue or site.In situation of the present invention, it refers to the method for the treatment of tumor (for example cancer) by the specific antibody of target tumor cell especially.

In situation of the present invention, " resistance of antagonist therapy " refers to, after using specific antibody preparation to treat, response is not (for example for described treatment for tumor (for example cancer) or the patient that carries described tumor, tumor cell is not killed, tumor does not disappear), and/or described therapy can not cause disappearing, reducing of described tumor or suppress transfer or the further growth of tumor.

In this application; term " tumor associated antigen " comprises for example TSA; this includes but not limited to for example member of Epidermal Growth Factor Receptor Family (EGFR); this comprises EGFR; HER1; HER2; (the Nam such as HER4 and HER8; N.H., & Parang, K. (2003); Current targets for anti cancer dr μ g discovery.Current Dr μ g Targets; 4 (2), 159-179), STEAP (six-transmembrane epithelial antigen of the prostate; The people such as Hubert, STEAP:a prostate-specific cell-surface antigen highly expressed in human prostate tumors., Proc Natl Acad Sci U S is A.1999; 96 (25): 14523-8.); CD55 (the people such as Hsu; Generation and characterization of monoclonal antibodies directed against the surface antigens of cervical cancer cells., Hybrid Hybridomics.2004; 23 (2): 121-5).

Can comprise Rituximab (RituxanTM, chimeric anti-CD 20 antibodies), Campath-1H (anti-CD 52 antibody), and the antibody of any cancer specific cell surface antigen for other suitable antibody of the present invention.Following exemplarily listed for particular cancers type, be suitable for being combined with interferon and for the antibody of object of the present invention: A Lun pearl monoclonal antibody (Campath ^tM) for chronic leukemia; Avastin (Avastin ^tM) for colon cancer and pulmonary carcinoma; Cetuximab (Erbitux ^tM) for colon cancer and head and neck cancer; Lucky trastuzumab (Mylotarg ^tM) for acute myeloid leukaemia; Ibritumomab (Zevalin ^tM) for non-Hodgkin lymphoma; Handkerchief wood monoclonal antibody (Vectibix ^tM) for colon cancer; Rituximab (Rituxan ^tM) for non-Hodgkin lymphoma; Tositumomab (Bexxar ^tM) for non-Hodgkin lymphoma; And Herceptin (He Saiting ^tM) for breast carcinoma.

In the present invention, " pharmaceutically acceptable carrier " refers to and can in used cell or experimenter, not cause anaphylaxis or other uncomfortable impacts, and can not affect the carrier of pharmaceutically active.Suitable pharmaceutically suitable carrier includes but not limited to, for example, and one or more water, normal saline, phosphate buffer, levulose, glycerol, ethanol and other analog, and the combination of above-mentioned substance.Pharmaceutically acceptable carrier also can further comprise can improve the pot-life of nucleic acid, polypeptide, virion or cell or the micro-auxiliary substance of effectiveness, for example wetting agent or emulsifying agent, antiseptic or buffer.

In this application, in interferon and/or its receptor, " defectiveness " refers to that the expression of described interferon or interferon receptors can not reach the required level of its biological function that realizes, for example, or expressed interferon or interferon receptors can not be brought into play required biological function (form with sudden change exists), or interferon (or interferon receptors) can not interact with its receptor (part) and cause the signal conduction in downstream.

The following examples are only used to explain better the present invention, and are not intended to limit by any way the present invention.

embodiment

Materials and methods

Mice

C57BL/6J and BALB/c mouse are purchased from Harlan.Rag1 ^-/-, 2C CD8 ⁺tCR transgenic mice, CD11c-DTR transgenic mice, CD11c-Cre and CD4-Cre transgenic mice are purchased from JAX.IFN AR1 ^-/-anita doctor Chong of mice Shi You Chicago University provides.IFN AR1 ^fl/flmice is (Kamphuis, the people such as Junt 2006) that Ulrich doctor Kalinke by Hanoverian, Germany experimental infection institute provides.Neu ^oT-I/OT-IItransgenic mice (B6 background) is by Canadian Trev & Joyce Deeley Research Centre, (Wall, the people such as Milne 2007) that Brad doctor H.Nelson of British Columbia provides.All mices are all raised under specific pathogen-free domestic condition.Animal care and use are to carry out according to studying scheme and the guidance of NIH in one's power, and all research has all obtained Chicago University's animal care and the approval of using committee.

Cell line and reagent

H460 is purchased from ATCC.TUBO is that the spontaneous breast tumor from BALB/c Neu-transgenic mice is cloned.TUBO-EGFR is carrying out transfection or is carrying out selecting after transfection with the plasmid that contains 2 μ g/mL blasticidin Ss (InvivoGen) with pSEB-EGFR.After transduceing with the slow virus of expressing Human epidermal growth factor receptor or EGFR-SIY, select the monoclonal of B16-EGFR and B16-EGFR-SIY.NOP23 is from B6Neu ^oT-I/OT-IIspontaneous breast tumor clone's in transgenic mice, and be that Brad doctor H.Nelson by Canadian Trev & Joyce Deeley Research Centre provides.At 5%CO ₂condition under cultivate H460, TUBO, B16 and their derivant, and they are maintained in vitro and have supplemented 10% heat-inactivated hyclone (Sigma), 2mmol/L L-glutaminate, in the DMEM of 0.1mmol/L MEM non essential amino acid ，100 unit/mL penicillin and 100 μ g/mL streptomycins.Anti-EGFR mAb Cetuximab is purchased from Imclone.Anti-CD8 (YTS169.4.2), anti-NK1.1 (PK136), anti-Neu (7.16.4) antibody is the inner manufacture of laboratory.Anti-PD-L1 (10F.9G2) and anti-Ly-6G (1A8) antibody are purchased from BioXcell.Anti-CD 20 (5D2) antibody is to be provided by Ouyang Wenjun (Genentech, San Franscisco).Anti-I type interferon receptors (MAR-5A3) antibody is to be provided by Robert doctor Schreiber (University of Washington, St. Louis).The adenovirus of expressing Mus interferon beta is people such as (, 2011) Burnette preparing as previously described.

The generation of Ab-IFN beta fusion proteins

From anti-EGFR (LA22) hybridoma (ATCC), cloned the V region of anti-EGFR.The V region of heavy chain and light chain is cloned into respectively to (people such as Smith, 2009) in Abvec-IgG1 and Abvec-kappa.Then, the C-terminal that mouse interferon β is inserted to heavy chain is as the fusion rotein that contains SGGGGSGGGGSGGGGSGGGG (SEQ ID NO:1) connexon.The whole heavy chain that contains interferon beta and light chain are cloned into respectively in pEE6.4 and pEE12.4 (Lonza).Then with NotI and BamHI, digest this two carriers.The complete hCMV-MIE-heavy chain/SV40 transcriptional units of pEE6.4 plasmid of digestion of hanging oneself is in the future connected with the large NotI-BamHI fragment (containing light chain expression box) of the pEE12.4 plasmid that digests of hanging oneself.By containing the two plasmid transfection of heavy chain and light chain, in Chinese hamster ovary celI and according to Guide Book (Lonza), set up stable clone.The supernatant that contains anti-EGFR-interferon beta by protein A column purification according to Guide Book (Repligen Corporation).Produced in an identical manner anti-Neu-interferon beta, difference is that the cDNA in V region is from anti-Neu (7.16.4) hybridoma.

Tumor growth and processing

By about 6 * 10 ⁵tUBO, TUBO-EGFR, B16-EGFR, B16-EGFR-SIY or NOP23 cell are subcutaneously injected in 6 to 8 weeks large mices at abdomen place, right side.Along three normal axis (a, b and c), having measured gross tumor volume and calculated gross tumor volume is abc/2.After tumor inoculation the 14th, 18 and 22 days, by anti-EGFR-interferon beta antibody or the control antibodies of three swollen intratumor injection 25 μ g, process mice.For CD8, exhaust experiment, process simultaneously with described anti-EGFR-interferon beta, the anti-CD8 antibody of intraperitoneal ground injection 200 μ g.By about 6 * 10 ⁶h460 cell is subcutaneously injected in 6 to 8 weeks large Rag1KO mices at abdomen place, right side.After tumor is set up (approximately 14 days), by intravenous injection by from 2 * 10 of OTI TCR transgenic mice ⁶lN cell continues being subject to property and transfers in mice.After one day, by anti-EGFR-interferon beta antibody or the control antibodies of three swollen intratumor injection 25 μ g, process mice.

The expression of I type interferon mRNA after antibody treatment

By about 6 * 10 ⁵tUBO, TUBO-EGFR or B16-EGFR cell are subcutaneously injected in 6 to 8 weeks large mices at right side abdomen.After tumor inoculation the 14th and 16 days, by the anti-Neu of twice swollen intratumor injection 100 μ g, anti-EGFR or control antibodies are processed mice.With the Collagenase V III (Sigma) of 1mg/ml and the DNA enzyme I (Sigma) of 200 μ g/ml, at 37 ℃, digest tumor 30 minutes.The CD45 living by FACS AriaII (BD Bioscience) sorting ⁺and CD45 ^-cell mass.The RNA separated total by the mini test kit of RNeasy (Qiagen) is also cDNA by Sensiscript RT test kit (Qiagen) by its reverse transcription.By quantitative PCR in real time, analyze the expression of I type interferon mRNA.Primer for described mensuration is as follows:

Mouse interferon α 5,5'-ATGAAGTCCATCAGCAGCTC(SEQ ID NO:2), 5'-AGGGGCTGTGTTTCTTCTCT(SEQ ID NO:3);

Beta-actin, 5'-ACACCCGCCACCAGTTCGC(SEQ ID NO:4), 5'-ATGGGGTACTTCAGGGTCAGGATA(SEQ ID NO:5).

By ELISPOT, measure to measure the T cell of secretion interferon gamma

By ELISPOT, measure and measure SIY reactive polypeptide T cell.Spleen or lymph-node cell are resuspended in and have supplemented 10%FCS, 2mmol/L L-glutaminate ，100 unit/mL penicillin, and in the RPMI1640 of 100 μ g/mL streptomycins.Altogether by 1-4 * 10 ⁵individual spleen or lymph-node cell are for this mensuration.Concentration by SIY peptide with 5 μ g/mL adds.After the hatching of 48h, according to Guide Book, by interferon gamma ELISPOT test kit (BD Bioscience) or CBA, measure the generation that (BD Bioscience) determined interferon gamma.With ImmunoSpot analyser (cytotoxic T cell), counted seen cytokine point.

In vitro dendritic cell intersect presents mensuration

At the 14th day and 17 days, with anti-EGFR or the swollen intratumor injection of anti-EGFR-interferon beta of 25 μ g, process the mice of carrying B16-EGFR-SIY.After four days, with Collagenase V III (Sigma) and the 200 μ g/ml DNA enzyme I (Sigma) of 1mg/ml, at 37 ℃, draining lymph node is digested 15 minutes.By positive selective reagent box (Stemcell) purification of CD11c dendritic cell.By about 1 * 10 ⁵dendritic cell and purified 2 * 10 ⁵2C T mixing with cells (containing or do not contain the SIY peptide of 5 μ g/mL) is again to stimulate T cell.After two days, collected supernatant, and by CBA, measured (BD Bioscience) and measured interferon gamma.

In vitro T cell activation is measured

The 14th with 17 days time, by the anti-EGFR with 25 μ g or the anti-EGFR-interferon beta intratumor injection that swells, process the mice that carries B16-EGFR-SIY.After four days, by negative selective reagent box (Stemcell) purification of T cell dLN T cell.By from about 1 * 10 of inmature mice ⁵purified dendritic cell and 2 * 10 ⁵t mixing with cells (contains or does not contain the SIY peptide of 5 μ g/mL) to stimulate T cell together again.After two days, collect supernatant and by CBA, measure (BD Bioscience) and carry out measurements interference element γ.

The generation of bone marrow chimera

Single dose Lethal irradiation WT mice with 1000rads.Next day, by 2-3x10 ⁶wT, IFN AR1 ^-/-or CD11c-DTR Tg donor bone marrow cell passes through intravenous adoptive transfer to the described mice through radiation.After reconstruct, mice is remained in Sulfamethoxazole Compound (Bactrim) antibiotic to (in drinking water, diluting) 4 weeks.In 5-6 week after reconstruct, use tumor cell injection mice.

Detect the endotoxin in mAb and fusion rotein prepared product

By LAL, measure (Cambrex inc.MD) and measured endotoxin.For all mAb prepared products, having measured endotoxic amount is <0.2E.U./mg mAb.

Flow Cytometry Analysis

With anti-CD16/32 (anti-Fc γ III/II receptor, clone 2.4G2), hatch single-cell suspension liquid 10 minutes, then with the antibody of puting together, dye.All through fluorescently-labeled monoclonal antibody all purchased from Biolegend or eBioscience.At the automatic flow cytometry of FACSC (BD Biosciences), above sample is analyzed, and analyzed data with FlowJo software (TreeStar, Inc.).

Statistical analysis

Use and not match the two tail t tests of Student and compared meansigma methods.Error bars represents SD.Statistically significant difference p<0.05 and p<0.01 are marked respectively with * and * *.

Embodiment 1

Need I type interferon in body for effective tumor response of antibody therapy

Shown I type interferon as potential key danger signal spontaneous tumor repel and various antitumor therapy in cause the current inventor of antineoplastic t cell response and supposed that carcinogenesis receptor antibody induces the generation of I type interferon in tumor tissues, and bridge joint is congenital and acquired immunity.In order to test in model as herein described, the sensitivity of tumor antagonism carcinogenecity receptor antibody whether with process after the Horizontal correlation of I type interferon, first inventor has prepared two different tumor cell lines, i.e. antibody response cell line and antibody resistant cell line.TUBO breast tumor cell is derived from Her2/neu Tg mice, and wherein Neu is the main signal of Growth of Cells, and TUBO cell is used as anti-neu antibody response tumor cell line.Through the B16 of EGFR transfection K-1735 (wherein EGFR can not send growth signals), be used as resisting completely the tumor cell line that anti-EGFR processes.Inventor by corresponding antibody treatment carry the mice of these tumors and assessed process after the generation of I type interferon in tumor.Inventor has found that being created in antibody response tumor model of interferon-ALPHA 5 and interferon beta increased, but in antibody resistance tumor model, there is no to increase (Figure 1A and the data that do not show), this shows that the generation of the I type interferon that increases is that carcinogenecity receptor blocking and stress by antibody induction causes.In order whether to need I type interferon by antibody-mediated antitumous effect in test body, in the process that inventor processes in antibody response TUBO mice with tumor model, at anti-Neu, with anti-I type interferon receptors blocking antibody, processed mice.Inventor has found, blocks the therapeutic effect (Figure 1B) that the conduction of I type interferon signal has damaged anti-Neu antibody, and this shows the really critical cytokine of I type interferon for antibody-mediated tumor regression.Therefore, this has proposed following probability: impaired I type interferon may limit with the immunne response in the host of antibody resistance tumor.In order further testing, other I type interferon to be directly delivered in tumor to whether be enough to control tumor growth (even in antibody resistance tumor), with the adenovirus (adenovirus-interferon beta) of coded interference element β, to have processed two groups of mices of carrying tumor.As shown in Fig. 1 C, the processing of Ad-interferon beta itself is just enough to control the growth (data do not show) of antibody resistance tumor and antibody response tumor.In sum, these data show, I type interferon is targeted to and in tumor, may be enough to overcome tumour immunity and avoids.

Embodiment 2

The targeted delivery of interferon beta strengthens antibody-mediated therapeutic effect

Data in embodiment 1 show I type interferon to be targeted in tumor and to have potential immunization therapy effect, particularly can be used for overcoming tumor for the resistance of antibody therapy.For further this conclusion of checking and further improve therapeutic effect, current inventor prepared anti-EGFR-interferon beta (Ab-IFN β) fusion rotein with targeting interferon beta is directly delivered in the tumor tissues of expressing EGFR (Fig. 9 A).First current inventor has checked in this fusion rotein, whether the anti-tumor function of anti-EGFR and interferon beta still remains intact.Inventor's discovery, described fusion rotein can be in conjunction with EGFR ⁺cell (Fig. 9 B) and can activate I type interferon receptors signal path (data do not show), therefore known, in fusion rotein, the anti-tumor function of anti-EGFR and interferon beta has all obtained good maintenance.Whether then inventor has detected anti-EGFR-interferon beta can be delivered to EGFR by interferon beta in vivo specifically ⁺tumor sites.Result confirmed, after originally injection, the concentration of anti-EGFR-interferon beta is almost all keeping higher level (Fig. 9 C) in whole one week in tumor tissues, and its after originally injection less than the time of one week in just reduction significantly in other tissue.In addition, inventor has detected the side effect of anti-EGFR-interferon beta by measuring serum cytokines, ALT and AST level.The cytokine detecting inventor comprises TNF, IL-12, and interferon gamma, MCP-1, IL-6 and IL-10, slightly increase (Figure 10) at the expression of latter 6 hours of injection and 1 o'clock interferon gamma and MCP-1.Inventor has also observed after process processing recited above, and the level of ALT and AST does not increase (data do not show).

For the antineoplaston effect between anti-EGFR-interferon beta fusion rotein more of new generation and first generation anti-EGFR-antibodies (Cetuximab), inventor has treated and has carried the EFGR having set up with each reagent ⁺the host of tumor.Surprisingly, inventor observes in having the tumor model of partial resistance, compares with independent use anti-EGFR-antibodies, and anti-EGFR-interferon beta has much effective therapeutic effect (Fig. 2 A) in lower dosage and shorter persistent period.In mammary gland, in fatty Partial tumors injection model, observed similarly the antitumous effect (data do not show) improving.In order further to test Ab-IFN β, whether can in having the tumor model of complete resistance, control tumor growth, inventor has tested the effect of anti-EGFR-interferon beta in B16-EGFR model.As predict, independent anti-EGFR is processed can not suppress B16-EGFR tumor growth in vivo, and anti-EGFR-interferon beta is processed and again can effectively be controlled tumor growth (Fig. 2 B).Reported that KRAS sudden change is to facilitate whether the key factor of clinical middle anti-EGFR resistance is effectively in order to test anti-EGFR-interferon beta in the antibody resistance tumor model of KRAS sudden change induction, inventor has processed H460 people's tumor of KRAS sudden change in the acquired immunity reconstruct Rag-1KO mice building before with anti-EGFR-interferon beta.Even in this model, than independent use anti-EGFR, anti-EGFR-interferon beta has also demonstrated superior therapeutic effect (Fig. 2 C).In order to test anti-EGFR-interferon beta, whether can control neoplasm metastasis, current inventor has injected B16-EGFR with simulation neoplasm metastasis in WT mouse vein.Inventor has found to compare with independent use anti-EGFR, anti-EGFR-interferon beta better controls metastasis tumor and can extend the survival (data do not show) of mice.

The antineoplastic immune of being led by anti-Neu antibody greatly reduces in neu Tg mice, this is to make life stage in early days because of the character due to transgene expression, and the high neu in all mammary gland expresses (as oneself and tumor associated antigen) tolerance host immune cell.In order to test Ab-IFN β, whether can break this toleration and induce neu in neu Tg mice ⁺disappearing of tumor set up neu in neu Tg host ⁺tumor is that NOP23(produces from neu Tg mice at first) surprisingly, to compare with the anti-Neu antibody of independent use, anti-Neu-interferon beta has suppressed tumor growth (Fig. 2 D) more significantly.In a word, these data show, for controlling tumor (or even in host of antibody resistance tumor model and tolerance), to compare with first generation antibody therapy, Ab-IFN beta fusion proteins therapy can be with low dosage, reach at short notice more advantageous effect (Figure 14).

Embodiment 3

The therapeutic effect of anti-EGFR-interferon beta fusion rotein depends on acquired immunity

I type interferon has multiple potential effect for the growth of tumor, and this comprises and suppresses propagation, suppresses angiogenesis, activates innate immune cells, bridge joint is congenital and acquired immunity and directly activate Acquired immune response.In order to be evaluated in numerous other anti-EGFR-interferon beta antitumous effects, the Relative Contribution of acquired immunity, has processed the B6 Rag1 KO mice of carrying B16-EGFR with anti-EGFR-interferon beta.Contrary with the therapeutic effect of observing in WT mice, the anti-EGFR-interferon beta of similar dosage can not suppress the tumor (Fig. 3 A) in the Rag1 KO mice of these immunocompromised hosts.Therefore, these Data supports following conclusion: the therapeutic effect of anti-EGFR-interferon beta mediation needs acquired immunity to a great extent.

CD8 ⁺t lymphocyte is the main cell group who participates in the growth of the many tumors of control.In order to determine whether they also participate in the antitumous effect of anti-EGFR-interferon beta mediation, and inventor has followed the trail of antitumor t cell response in initiating stage.Inventor's model B16-EGFR-SIY tumor cell line, wherein SIY peptide (SIYRYYGL) by ectopic expression in Human epidermal growth factor receptor molecule; This SIY peptide is to indicate as an alternative, and it can be by endogenous or 2C Tg CD8 ⁺the identification of T cell-specific ground.After processing with anti-EGFR-interferon beta, separated draining lymph node (dLN) lymphocyte from carry the mice of tumor, and stimulate with SIY peptide, and measured the generation of interferon gamma, as the effector-functional parameter of the T cell being activated.As shown in Figure 3 B, process and compare with independent anti-EGFR, anti-EGFR-interferon beta is processed the interferon gamma having increased from specific for tumour antigen T cell and is produced.In order to understand CD8 ⁺whether cell is crucial for the therapeutic effect of anti-EGFR-interferon beta, has used CD8 and has exhausted antibody, and measured tumor growth in the process that inventor processes in carrying the WT B6 mice of B16-EGFR, at anti-EGFR-interferon beta.CD8 ⁺cell is exhausted the therapeutic effect (Fig. 3 C) reduced widely anti-EGFR-interferon beta, and this shows that the acquired immunne response of needs controls tumor growth.Consistent with this discovery, inventor has found that external t cell response reduces (Fig. 3 D) greatly after exhausting with anti-CD8.The exhaustion of other cell does not affect (comprising NK and B cell) antitumous effect (Figure 11) of anti-EGFR-interferon beta.Inventor is supposition therefore, and anti-EGFR-interferon beta has been induced the more effective initiation to tumor-specific cytotoxicity T cell, causes the inhibition to tumor growth.In a word, these data show that the processing of anti-EGFR-interferon beta can increase T cell and cause, and this has facilitated the antitumous effect of anti-EGFR-interferon beta.

Embodiment 4

The therapeutic effect of anti-EGFR-interferon beta need to be expressed I type interferon receptors on host's hematopoietic cell

I type interferon receptors wide expression all in nearly all cell type, therefore normal cell and tumor cell are all potential anti-EGFR-interferon beta targets.Reported that therefore the effect of anti-CD 20-interferon alpha fusion protein need to express I type interferon receptors on tumor cell, inventor infers for the therapeutic effect of anti-EGFR-interferon beta, may also need to be by the I type interferon receptors of tumor cells expression.For this reason, inventor has compared the I type interferon receptors that carries tumor and has knocked out the antitumous effect in (KO) mice, and described mice lacks the expression of I type interferon receptors on host cell, but on tumor cell, expresses I type interferon receptors.Surprisingly, in I type interferon receptors KO mice, the therapeutic effect of anti-EGFR-interferon beta has been destroyed (Fig. 4 A) completely, and the cytotoxic T cell of not observing increase is replied (Fig. 4 B).These results show, the activation that the therapeutic effect of anti-EGFR-interferon beta need to be mediated by I type interferon receptors in host cell, but do not need in tumor cell.Because all host tissues are all expressed I type interferon receptors, whether inventor has built I type interferon receptors bone marrow chimerism (BMC) mice, further to analyze, need hematopoietic cell or the host matrix cell of expressing I type interferon receptors to realize antitumous effect.Inventor has found, need on hematopoietic cell, express I type interferon receptors because in the mice of I type interferon receptors KO BM reconstruct the antitumous effect of anti-EGFR-interferon beta be badly damaged (Fig. 4 C).These data show that anti-EGFR-interferon beta is not by the growth of direct inhibition tumor cell but mediates its Graft Versus Tumor by activation host's hematopoietic cell (then it change tumor microenvironment).

Embodiment 5

The dendritic cell that improve intersect presents the antitumous effect of facilitating anti-EGFR-interferon beta

In view of CD8 ⁺cell is critical for the antitumous effect of anti-EGFR-interferon beta treatment, and inventor has further explored anti-EGFR-interferon beta and how to have strengthened the basic mechanism that cytotoxic T cell is replied.Inventor observes, and even without the stimulation of exogenous SIY peptide, also has interferon gamma generation property CD8 after anti-EGFR-interferon beta is processed ⁺the remarkable increase of cell, this shows that the intersection of APC is presented has increased (Fig. 5 A).Because intersect present be activating cytotoxic T cell for the main induced mechanism inventor of antineoplastic immune infer increase that described intersection presents for recover Dendritic Cell Function with reactivate in tumor and draining lymph node in cytotoxic T cell may be critical.For the anti-EGFR-interferon beta that begins one's study can increase the probability that the intersection of APC is presented, inventor has used the antigenic specificity system in body to follow the trail of initiation and the activation of tumor-T cells with antigenic specificity.Therefore, the dendritic cell of having assessed the dLN of the mice of carrying B16-EGFR-SIY that the anti-EGFR of hanging oneself-interferon beta processes, detect by hatch them with the reactive 2C T of SIY-cell in isolated measuring the ability that its enhancing specificity antineoplastic cytotoxic T cell is replied.Really, process and compare with anti-EGFR, the dendritic cell of the mice that the anti-EGFR-interferon beta of hanging oneself is processed have been induced more interferon gammas (approximately 33 times) that produced by 2C T cell, when there is no the stimulating again of exogenous SIY peptide, are also even so (Fig. 5 B).Comprehensive, these data show, intersect initiation function strengthened CD8 through the dendritic cell of anti-EGFR-interferon beta activation by increases ⁺t cell activation.In addition, these results show, are that the interferon beta component of described fusion rotein has caused the activation that dendritic cell intersection is presented path, because independent anti-EGFR is not induced strong dendritic cell activation.In order to test anti-EGFR-interferon beta, whether also increased direct initiation function, the antigen of external source generation property peptide (SIY) is added in culture medium, it has further increased SIY-specificity 2C t cell response.After external source SIY-peptide stimulates again, to process and compare with anti-EGFR, the dendritic cell induction 2C T cell of the mice that the anti-EGFR of hanging oneself-interferon beta is processed produces approximately 2 times of more interferon gammas (Fig. 5 B).These results show, compare with the dendritic cell of mice from processing through anti-EGFR separately, and the dendritic cell of the host's that the anti-EGFR of hanging oneself-interferon beta is processed dLN are probably being activated to a greater extent.Really, these dendritic cell have the Activation marker high expressed of (comprising CD86), as (Fig. 5 C) that assessed by flow cytometry.For whether the dendritic cell activation that further parsing is induced by anti-EGFR-interferon beta has facilitated the T cell activation strengthening, in the process of processing at anti-EGFR-interferon beta, with DT processing, carry the CD11c-DTR BMC mice of tumor with exhaustion dendritic cell.Inventor's discovery, when not there are not dendritic cell, the described therapeutic effect significantly impaired (Fig. 5 D) by the mediation of anti-EGFR-interferon beta.In a word, these data show that the dendritic cell that increase intersect and present the CD8 that causes improvement ⁺cytotoxic T cell causes and function, and this may be the main essence mechanism of the therapeutic effect of anti-EGFR-interferon beta.

Embodiment 6

The direct targeting dendritic cells of anti-EGFR-interferon beta with the tumor microenvironment that reverses tolerance for improvement of antitumous effect

The application's data show, for processing by anti-EGFR-interferon beta, to bring improved antitumous effect be important in the intersection induction of increase.Yet, how by anti-EGFR-interferon beta, process that to activate the mechanism of dendritic cell still unclear.Especially, need to identify the cell of the expression I type interferon receptors of the therapeutic effect of directly facilitating anti-EGFR-interferon beta.In order to address this problem, by I type interferon receptors ^fl/flmice and various Cre-Tg mice together feed.When I type interferon receptors is at CD11c-Cre-I type interferon receptors ^fl/flcD11c in mice ⁺when in cell, selectivity lacks, the antitumous effect of anti-EGFR-interferon beta significantly reduces (Fig. 6 A), and this shows, by anti-EGFR-interferon beta direct activation dendritic cell, may be the factor of main contributions that its therapeutic effect is made.When I type interferon receptors is at CD4-Cre-I type interferon receptors ^fl/flwhen in the T cell of mice, selectivity lacks, the antitumous effect of anti-EGFR-interferon beta is slightly impaired but not significantly impaired (Fig. 6 B), this shows to make the direct targeting T-cells of I type interferon receptors can make the T cell having been activated by dendritic cell further be activated, thereby produces improved antitumous effect.In order further to test this idea, inventor has assessed anti-EGFR-interferon beta in measuring in vitro stimulate the effect in dendritic cell and T cell activation; Really, anti-EGFR-interferon beta has increased the activation (Figure 12) of purified dendritic cell and T cell.These data combine and have shown, the direct activation of I type interferon receptors expressivity dendritic cell plays a major role in the therapeutic effect of anti-EGFR-interferon beta mediation, and has further strengthened this effect by express I type interferon receptors on T cell.

Embodiment 7

The PD-L1 of antagonism anti-EGFR-interferon beta induction expresses and has realized tumor free effect

Although compare with independent anti-EGFR-antibodies, anti-EGFR-interferon beta merges has realized more effective antitumous effect, and residual tumor finally can recur.Therefore inventor wants to understand, and anti-EGFR-interferon beta is processed the expression of possibility induction inhibition molecule to prevent tissue injury, and this will be along with the time is weakened antitumous effect.Inventor has assessed the expression of PD-L1 after anti-EGFR-interferon beta is processed, and expresses (Fig. 7 A and 7B) with the external PD-L1 increasing on tumor cell that all clearly observed in vivo.Then inventor has tested anti-PD-L1 and the anti-EGFR-interferon beta of combination and has processed the long-term effect that whether can further strengthen anti-EGFR-interferon beta.Surprisingly, when combining while using anti-PD-L1 with the processing of anti-EGFR-interferon beta, long-term treatment effect is significantly improved; After described processing, mice keeps stimulating without tumor at least 60 days and to tumor has resistance (Fig. 7 C, and the data that do not show).In addition, the interaction by anti-PD-L1 processing blocking-up PD-1 and PD-L1 has further improved specific antitumor t cell response (Fig. 7 D).On the contrary, when by the anti-CTLA-4 of antibody of anti-EGFR-interferon beta and two other main Inhibitory molecules of expressing, anti-BTLA combined administration, do not observe any similar synergy (Figure 13) on T cell.In a word, inventor's data show on antagonism tumor cell that the PD-L1 by the induction of anti-EGFR-interferon beta expresses can make the antitumous effect of anti-EGFR-interferon beta maximize and obtain impressive without tumor result, for antibody resistance tumor, is even also like this.The integral body that this strategy based on combination probably increases antibody resistance host is replied and cure rate, even in the host that can not respond anti-PD-1 antibody (PD-1 that its direct blocking t cell is expressed), also can realize.

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Claims

1. the compound of interferon and blocking-up PD-1/PDL signal transduction pathway (antibody for example, as anti-PDL1 antibody) jointly for example, for the preparation of the purposes of medicine (pharmaceutical kit), wherein said medicine is used for the treatment of tumor, for example pernicious solid tumor, particularly breast carcinoma, pulmonary carcinoma, carcinoma of prostate, colon cancer, skin carcinoma, head and neck cancer, lymphoma or melanoma.

2. the compound of interferon and blocking-up PD-1/PDL signal transduction pathway (antibody for example, as anti-PDL1 antibody) jointly for example, for the preparation of the purposes of medicine (pharmaceutical kit), wherein said medicine is used for overcoming tumor (pernicious solid tumor for example, breast carcinoma particularly, pulmonary carcinoma, carcinoma of prostate, colon cancer, skin carcinoma, head and neck cancer, lymphoma or melanoma) for the resistance of antibody therapy.

3. according to the purposes of claim 1 or 2, wherein said interferon is I type interferon, preferably interferon-ALPHA and/or interferon beta.

4. according to the purposes of any one in claim 1-3, wherein said interferon with for example, in conjunction with the targeting moiety of tumor associated antigen (antibody, as anti-egfr antibodies or anti-Neu antibody) be connected (for example forming fusion rotein), wherein said targeting moiety is directly connected or is connected by connexon with described interferon.

5. according to the purposes of any one in claim 2-4, the wherein said host who has the tumor of resistance or carry described tumor for antibody therapy is at following one or more middle defectiveness:

1) interferon, I type interferon for example, as the expression of interferon-ALPHA 5 and/or interferon beta and/or function;

2) interferon receptors, as expression and/or the function of the described interferon receptors in expression and/or the function, particularly dendritic cell of I type interferon receptors; And/or

3) intersection of dendritic cell is presented and consequent antitumor cell toxicity T cell.

6. test kit, it contains:

A) interferon, I type interferon for example, as IFN β and/or IFN α 5; With

7. the test kit of claim 6, wherein said interferon for example, is connected (for example forming fusion rotein) with the targeting moiety (antibody) in conjunction with tumor associated antigen, and wherein said targeting moiety is directly connected or is connected by connexon with described interferon.

8. according to the test kit of claim 7, wherein said targeting moiety is antibody, for example anti-egfr antibodies or anti-Neu antibody.

9. according to the test kit of any one in claim 6-8, wherein said tumor is malignant tumor, pernicious solid tumor for example, and it comprises for example breast carcinoma, pulmonary carcinoma, carcinoma of prostate, colon cancer, skin carcinoma, head and neck cancer, lymphoma or melanoma.

10. according to the test kit of any one in claim 6-8, wherein said tumor or the host that carries described tumor are at following one or more middle defectiveness:

3) antitumor cell toxicity T cell is presented and produced thus to the intersection of dendritic cell.