CN103536917B - Use of interferon in treatment of tumor, and related product and method - Google Patents

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

Use of interferon in the treatment of tumors and related products and methods

technical field

The invention relates to the field of tumor therapy, in particular to overcoming tumor resistance to antibody therapy. Specifically, the present invention relates to blocking PD-1 (programmed death-1)/PDL (programmed death ligand, PD-1Ligand) pathway by interferon, such as type I interferon and its The combination of the compounds inhibits tumor growth and recurrence and causes tumor regression.

Background technique

It is currently accepted that antibodies targeting oncogenic receptors, such as cetuximab and Herceptin, have antitumor effects because of their ability to block growth signaling induction, cell cycle arrest, and/or through apoptosis or antibody-dependent cell-mediated cytotoxicity (ADCC) to induce cell death. However, most studies on antibody-mediated antitumor mechanisms are based on human tumor experiments from in vitro culture or in vivo xenograft models, which do not include the contribution of adaptive immune responses. More recently, studies using immunocompetent hosts have shown that durable antitumor protection by anti-CD20, anti-Neu and anti-EGFR antibody therapy is indeed dependent on cellular immune responses (Abes et al., 2010; Park et al., 2010 ; Yang et al., 2013). However, it remains undetermined whether and how antibody therapy activates T cell responses.

Currently, most strategies for improving the therapeutic efficacy of antibodies focus on increasing cytotoxicity towards tumor cells. One strategy is to conjugate antibodies to cytotoxic drugs, including chemotherapeutics, to form antibody-drug complexes (ADCs), which achieve better antitumor efficacy than antibodies alone, and compared with This produces fewer side effects compared to cytotoxic chemotherapy (Fayad et al., 2013; Hurvitz et al., 2013; Krop et al., 2012). However, the long-term protective effect of ADCs remains unknown. It is also not clear whether non-specific T cells are also activated during this treatment, increasing the potential for harmful side effects.

Similar to first-generation antibodies, host resistance can develop after long-term use of ADCs. Indeed, although the potential cytotoxic effects of ADCs initially caused regressions, many patients experienced relapse or developed metastases. One possible explanation for this resistance is that antibody-resistant clones arose before or after the initial antibody treatment, causing primary or acquired resistance, respectively.

Therefore, there is an urgent need for a new type of anti-tumor therapy that can effectively cause tumor regression and overcome the resistance of tumor cells to the therapy so as to finally cure the tumor and/or prevent the recurrence of the tumor.

Contents of the invention

Antibody immunotherapy has been used for nearly 16 years to treat cancer by targeting tumor-associated surface markers (Scott et al., 2012). Targeting oncogenic receptors has been shown to be effective, as antibody therapy against these receptors was able to significantly inhibit tumor cell growth in vitro and partially in vivo in human tumor xenograft models in immunodeficient mice Tumor growth (Hynes and Lane, 2005; Li et al., 2005). The primary therapeutic effect of antibody therapy was originally attributed to direct killing of tumor cells through signaling. Recently, however, it has been recognized that Fc receptor (FcR) signaling on immune cells is also important because antibody-mediated antitumor effects are greatly reduced in the absence of FcR signaling in mouse models, in vivo ( Clynes et al., 2000), and FcR polymorphisms are associated with clinical outcome in human breast cancer patients (Musolino et al., 2008). These data raise the possibility that the antitumor efficacy of antibody therapy also depends on antibody-dependent cellular cytotoxicity produced by innate cells, as well as acquired immune cell-dependent immune responses (ADCC).

Despite the antibody-mediated anti-tumor effects shown above, several obstacles remain to achieve effective antibody therapy against cancer. First, many patients become resistant to primary antibody therapies after long and expensive treatments. For example, 80–90% of colon cancer patients are resistant to cetuximab (anti-EGFR) (Bardelli and Siena, 2010), and 66–88% of breast cancer patients are resistant to Herceptin (anti-HER2). Sex (Cobleigh et al., 1999). Second, after long-term treatment, tumor cells in patients who initially respond to antibody therapy are likely to develop natural or acquired antibody-resistant clones, which are a major cause of cancer recurrence. Most studies have focused on intrinsic resistance to oncogenic signaling, such as mutations in targeted oncogenes or genes associated with oncogenic pathways that contribute to antibody resistance. Primary and acquired resistance to cetuximab can occur in tumor cells containing mutations in EGFR itself, or in the downstream mediators KRAS and BRAF, and in tumor cells containing amplified Her2 (Dimerization partner of EGFR) in tumor cells (Bardelli and Siena, 2010; Misale et al., 2012; Sharma et al., 2007; Wheeler et al., 2008; Yonesaka et al., 2011). In order to enhance the efficacy of killing tumor cells by antibody therapy and reduce host resistance, second-generation antibody-based therapy has been developed, which consists of cytotoxic drugs or radioactive substances conjugated to certain antibodies to enhance direct killing (Fayad et al., 2013; Hurvitz et al., 2013; Krop et al., 2012). Another strategy to improve direct cytotoxic effects involves targeting different epitopes or even related receptors (Bostrom et al., 2009). Currently, the main strategy to overcome resistance to antibodies in the host is to develop drugs targeting mutated oncogenes or mutated genes associated with oncogenic pathways in tumor cells. For example, BRAF inhibitors have been used to target cetuximab-resistant tumors (Yoon et al., 2011).

Taken together, resistance to antibodies is a major challenge for antibody-based cancer therapies. Current strategies to overcome such resistance to antibodies focus on improving either: 1) direct tumor cell killing by antibody and cytotoxic drug conjugation, or 2) tumor cell killing by nonspecific anti-CD3 activation. Bispecific T cell binders enhance targeted T cell killing. Due to the heterogeneity of tumor cells and their environment, these direct killing strategies cannot target all tumor cells and still eventually cause antibody resistance.

In order to find more effective strategies to improve antibody immunotherapy, it is necessary to identify the key molecules that link the antibody-mediated anti-tumor response to the elicited adaptive immune anti-tumor response.

Interferon (IFN) is a broad-spectrum antiviral agent, which can inhibit virus replication, and at the same time enhance the activity of natural killer cells (NK cells), macrophages and T lymphocytes, thereby playing an immunomodulatory role, and Enhance antiviral ability. Interferon is a group of active proteins (mainly glycoproteins) with multiple functions, and is a cytokine produced by monocytes and lymphocytes. They have broad-spectrum anti-virus, affect cell growth, differentiation, regulation of immune function and other biological activities on the same kind of cells. The current general classification method divides interferon into three types: Type I includes IFN-α and IFN-β, of which IFN-α has more than 20 subtypes, and IFN-β has only one subtype. Type I interferon has the functions of inhibiting virus replication, anti-parasite, inhibiting the proliferation of various cells, stimulating the killing activity of immune cells, participating in immune regulation, and anti-tumor; type II: only IFN-γ, and only one subtype, In addition to antiviral and antiproliferative activities, its main biological activity is immune regulation; Type III: IFN-λ1 (IL-29), IFN-λ2 (IL-28a) and IFN-λ (IL-28b) . IFN-ω belongs to the IFN-α family, and its structure and size are slightly different from other IFN-α, but the antigenicity is quite different.

Recently, an increase in interferons (IFNs), such as type I interferons, was found to correlate favorably with clinical immune responses against cancer (Fuertes et al., 2011). Furthermore, interferon signaling is critical for eliciting antitumor T cell responses during spontaneous tumor rejection and various antitumor therapies (Burnette et al., 2011; Diamond et al., 2011; Fuertes et al., 2011 ; Stagg et al., 2011). These data suggest that interferons are critical for eliciting antitumor-specific T cell responses against tumor cells. It has also been reported that interferon activates memory T cells during viral infection (Kohlmeier et al., 2010). However, to date, interferon has been used cautiously and with limited efficacy in the clinic (Tnchieri, 2010). Indeed, the role of this class of cytokines is very poorly understood, as they may act as immune activators or immunosuppressants in different disease models (Gonzalez-Navajas et al., 2012), and potentially serious side effects accompany The short half-life limits its application in tumor therapy.

On the other hand, in antibody-responsive tumor models, immune-activating molecules released during ADCC or by stressed antibody-bound tumor cells can effectively activate antigen-presenting cells (APCs) and enhance their induction of cytotoxic T-cell responses. Ability. However, this immune response is weak and transient, especially when tumors are established. In antibody-resistant tumor models, additional strategies are needed to reactivate APCs or T cells suppressed by the immunosuppressive tumor microenvironment. The present inventors have recently observed that conjugating CpG to cetuximab greatly enhances its therapeutic effect, presumably by activating dendritic cells within the tumor microenvironment and eliciting a subsequent adaptive immune response (Yang et al. , 2013). These studies suggest that approaches designed to drive activation of adaptive immunity are important, and may even be critical, for the long-term success of antibody-based cancer therapies.

In support of this idea, recent clinical trials have used antibodies to block co-inhibitory signals on T cells, including CTLA-4, PD-1, and PD-L1. The B7-CD28 family has many inhibitory signaling pathways that can weaken T cell responses and promote T cell tolerance. Among them, the PD-1/PD-L pathway discovered in recent years has attracted attention due to its unique inhibitory function, and is considered to affect the immune system. An important factor in the chronicity of immune and infectious diseases, this pathway includes PD-1 and its two ligands: PD-L1 and PD-L2. PD-1 (programmed cell death1), also known as CD279, is a member of the immunoglobulin superfamily and contains two cytoplasmic regions of tyrosine signaling motifs, one of which forms the immunoreceptor tyrosine inhibitory motif (immunoreceptor tyrosine -basedinhibition motif, ITIM), and the other is Immunoreceptor tyrosine-based switch motif (ITSM). ITSM recruits phosphatases SHP-1 and SHP-2 to dephosphorylate the effector signals transmitted by TCR or BCR. At the same time, PD-1 signals reduce CD28-mediated signals, inhibiting Akt kinase phosphorylation, glucose metabolism and Bcl-XL expression. The expression of PD-1 is relatively extensive, and PD-1 is mainly expressed on double-negative cells in the developmental stage of the thymus, and can be induced to express in peripheral CD4+, CD8+ T cells, B cells and monocytes after activation, and the expression level in NK-T cells is relatively low. Low.

PD-1 has two ligands, PD-L1 and PD-L2. PD-L1 is more widely expressed than PD-L2, and the affinity of PD-L2 is 2-6 times that of PD-L1. PD-L2 (also known as B7-DC or CD273) can be induced to express on dendritic cells (dendritic cells, DC), macrophages and cultured bone marrow-derived mast cells; while PD-L1 (also known as B7-H1 or CD274) is constitutively expressed in murine T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells and cultured bone marrow-derived mast cells, and the expression is enhanced after activation. The constitutive expression level of human PD-L1 is higher than that of mouse class low. PD-L1 is also expressed on many non-hematopoietic cell types, and is also expressed in immune privileged sites, such as the eye and placenta, where PD-L1 may regulate autoreactive T cells or B cells and inflammatory responses.

Recent studies have shown that reversing T cell suppression (e.g., by blocking PD-1/PDL) is a favorable way to improve therapeutic efficacy, but unfortunately, this also comes with serious side effects (Brahmer et al., 2012; Topalian et al., 2012; Weber, 2007). Moreover, clinical data show that the response rate to PD-1 blockade is still relatively low (15-25%) in the selected patient pool, and this response is largely dependent on the presence of PD-L1 in Expression in tumor tissue and initial lymphocyte infiltration (Topalian et al., 2012). Therefore, the key issue of cancer immunotherapy is how to further reverse the immunosuppression in the microenvironment to reactivate the specific anti-tumor T cell response, so as to achieve a higher response rate and long-term efficacy of antibody-based immunotherapy. effectiveness.

Therefore, a new generation of antibody-based drugs is urgently needed to more effectively target tumor tissues and reduce systemic side effects.

It is shown in the present invention that antibody therapy (such as antibody-interferon beta) combined with interferon (such as type I interferon) does not directly target tumor cells because the interferon receptor-interferon receptor in deficient mice- Adequate tumors do not respond to antibody-interferon beta. Furthermore, bone marrow transplantation (BMT) experiments have shown that interferon receptors are required to be expressed on bone marrow-derived cells, suggesting that the anti-angiogenic effects of interferon are not involved. To determine the relative contribution of ADCC to the adaptive immune response, the inventors treated immunodeficient Rag-1 KO mice with antibody-interferon β and observed that in the absence of adaptive immune cells, antibody-interferon β completely Cannot inhibit tumor growth. Although both T cells and dendritic cells are capable of responding to interferon beta, the inventors further determined that dendritic cells are essential interferon receptor-expressing cells because small cells with type I interferon receptor-deficient dendritic cells Mice were completely unresponsive to the antibody-interferon beta, whereas mice with type I interferon receptor-deficient T cells showed only slightly reduced tumor suppression. Furthermore, dendritic cells from mice treated with antibody interferon beta were able to directly activate tumor-specific T cells, suggesting that treatment with antibody therapy (e.g. antibody-interferon beta) combined with interferon (e.g. type I interferon) Increased dendritic cell antigen presentation and T cell activation. Thus, it is likely that dendritic cells respond directly to antibody therapy combined with interferon (eg, antibody-interferon beta) to increase antigen presentation and T cell activation, which reactivates T cells.

In the present invention, the inventors have also shown that antibody treatment itself is capable of inducing the production of interferon (eg, type I interferon), which contributes to dendritic cell activation and subsequent anti-tumor T cell immunity. This also suggests that the main suppressor cell type in the tumor microenvironment is dendritic cells rather than T cells, as antibody therapy combined with type I interferon when type I interferon receptors are selectively deleted in dendritic cells (such as antibody-interferon beta fusion protein) the therapeutic effect disappeared. Therefore, targeting dendritic cells in addition to T cells can further increase the efficacy of antibody therapy.

To balance immune-mediated damage with an ongoing immune response, host immunity often induces inhibitory signaling following immune activation. Of these, upregulation of PD-L1 in tumor tissue is the most notable as a protective mechanism during cancer immunotherapy, and the inventors are working with antibody therapy combined with interferon (such as type I interferon) This phenomenon was observed after treatment. Indeed, blocking the PD-L1/PD-1 pathway further enhanced the antitumor effect and cleared the tumor. This is specific for PD-L1/PD-1 because when combining antibody therapy with type I interferon with blocking signaling from two other important inhibitory molecules on T cells (CTLA-4 or BTLA) No synergistic effect was observed when the antibodies were combined. This clearly demonstrates that targeting inhibitory pathways specifically induced by active immunotherapy could guide future clinical treatments.

From the above analysis, it can be understood that antibodies (Abs) that preferentially target oncogenic receptors are increasingly used in cancer therapy, but after long-term and expensive treatment, tumors often develop resistance to these antibodies. sex. The present inventors have armed antibodies with interferons (e.g. type I interferons, such as interferon beta) as a new generation of biologics that are far superior to first generation antibody therapies and independent of various resistance mechanisms. For the control of tumors resistant to antibody therapy. This novel strategy is to control antibody resistance by reactivating and rebridging suppressed innate and acquired immunity. Mechanistically, the combination of antibodies and type I interferons primarily targets intratumoral dendritic cells, which reactivate cytotoxic T cells by increasing cross-induction in the tumor-associated microenvironment. Furthermore, intratumoral blockade of PD-1/PD-L1 pathway signaling induced by the combination of antibodies with type I interferons further overcomes resistance that hinders therapy and completely clears larger established tumors . Thus, the present invention establishes a new generation of antibody-based immunotherapy capable of eradicating antibody-resistant tumors.

Therefore, the new generation of immune-based antibody therapy of the present invention is able to overcome the above-mentioned use of antibodies by reactivating innate and adaptive immune cells in tumors and generating specific T cell responses targeting multiple tumor mutant antigens. The problem of therapy. Therefore, this new strategy may be able to eliminate tumor cells that antibodies cannot directly target.

On the other hand, the PD-1/PD-L pathway not only plays an important role in regulating self-tolerance, but also plays a key role in immunity against microbial infection. Many microorganisms that cause chronic infection may mostly use the PD-1/PD-L pathway to weaken anti-infection immunity and promote persistent infection. Unlike acute infection or vaccination, which produce potent effector and memory CD8+ T cells, the function of virus-specific CD8+ T cells is usually weakened during chronic viral infection. In chronic infection models, such as mouse LCMV infection or primate SIV infection, or HIV, HBV, HCV and other viral infections, the persistence of non-functional virus-specific CD8+ T cells can usually be observed, a phenomenon known as Exhaustion (exhaustion), T cell exhaustion represents a series of immune deficiencies. With the increase of viral load, the function of virus-specific T cells is more seriously impaired, the ability to proliferate and produce IL-2 disappears in the early stage, and the function to produce effector cytokines and cytolysis disappears in the later stage.

At the same time, interferon (IFN), as a broad-spectrum antiviral agent, has also been shown to be able to inhibit the replication of viruses; at the same time, it can also enhance the viability of natural killer cells (NK cells), macrophages and T lymphocytes, thereby It plays an immune regulatory role and enhances antiviral ability. Therefore, according to the present invention, it can also be understood that the combined use of interferon (such as type I interferon, such as interferon beta), especially interferon combined with antibodies (such as fusion, especially in the form of fusion protein) and Agents that block signaling of the PD-1/PD-L pathway (eg, anti-PDL1 antibodies) can also be used to treat microbial infections, viral infections, and/or other infectious diseases.

Accordingly, one aspect of the invention relates to the use of an interferon, such as a type I interferon, eg IFNβ or IFNα5, for the manufacture of a medicament for overcoming tumor resistance to antibody therapy.

In one embodiment, the interferon (e.g., type I interferon) is linked (e.g., to form a fusion protein) to a targeting moiety (e.g., an antibody) that binds a tumor-associated antigen, wherein the targeting moiety is directly linked to the interferon. Linked or linked via a linker. In a specific embodiment, said targeting moiety is located at the N-terminus of said interferon. In another specific embodiment, said targeting moiety is located at the C-terminus of said interferon. In a specific embodiment, the interferon can be linked to two or more of the targeting moieties to form a multispecific (eg bispecific) molecule (eg multispecific fusion protein, eg bispecific specific fusion protein).

In the case where the interferon and the targeting moiety are connected by a linker to form a fusion protein, the linker may comprise amino acid sequences of any suitable length, for example, the linker may comprise 1-10, 1- 20, 1-30 or more amino acids (or consist thereof), for example the linker may 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 amino acids, or consist of said amino acids.

In specific embodiments, the targeting moiety is an antibody, such as an anti-EGFR antibody or an anti-Neu antibody.

In other specific embodiments, the tumor is 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.

In another aspect, the invention relates to the antagonism of interferon (e.g. type I interferon, such as IFN-β or IFNα5) with a compound that blocks the PD-1/PDL signaling pathway (e.g. antibody, such as anti-PD1 or PD-L1 Antibodies) for the preparation of medicines (such as pharmaceutical kits), wherein the medicines are used for the treatment of tumors (such as malignant solid tumors, especially breast cancer, lung cancer, prostate cancer, colon cancer, skin cancer, head and neck cancer , lymphoma or melanoma), for example to overcome tumor resistance to antibody therapy. Wherein, the interferon and the compound blocking the PD-1/PDL signal transduction pathway may exist independently of each other in a form not mixed with each other in the drug, or may exist in admixture in a manner that does not affect each other's efficacy.

In a specific embodiment, the above-mentioned tumor resistant to antibody therapy or the host carrying the tumor is deficient in one or more of the following: 1) interferon (such as type I interferon), such as interferon Expression and/or function of α5 or interferon β; 2) expression and/or function of interferon receptors (such as type I interferon receptors), especially the expression and/or function of interferon receptors on dendritic cells Function; 3) Cross-presentation of dendritic cells and the resulting anti-tumor cytotoxic T cells.

Among them, in a specific embodiment, the interferon (such as type I interferon) can be linked to a targeting moiety (such as an antibody) that binds a tumor-associated antigen (such as forming a fusion protein), wherein the targeting moiety and the The above-mentioned interferon is connected directly or through a linker. In a specific embodiment, said targeting moiety is located at the N-terminus of said interferon. In another specific embodiment, said targeting moiety is located at the C-terminus of said interferon. In a specific embodiment, the interferon can be linked to two or more of the targeting moieties to form a multispecific (eg bispecific) molecule (eg multispecific fusion protein, eg bispecific specific fusion protein).

In the case where the interferon and the targeting moiety are connected by a linker to form a fusion protein, the linker may comprise amino acid sequences of any suitable length, for example, the linker may comprise 1-10, 1- 20, 1-30 or more amino acids (or consist thereof), for example the linker may 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 amino acids, or consist of said amino acids.

In another aspect, the present invention relates to a kit comprising:

a) an interferon, such as a type I interferon, such as IFNβ or IFNα5; and

b) A compound that blocks the PD-1/PDL signaling pathway, such as an antibody, such as an anti-PDL1 antibody.

When using the kit, the interferon (such as type I interferon, such as IFNβ or IFNα5) and the compound that blocks the PD-1/PDL signaling pathway can be simultaneously and sequentially (in any order) ) or administered to subjects in need respectively. And in the kit, the interferon (such as type I interferon, such as IFNβ or IFNα5) and the compound that blocks the PD-1/PDL signaling pathway can exist in a form that does not mix with each other (such as each exist in a separate space); or exist in a mixture in a form that does not affect the effectiveness of each other.

Wherein, in a specific embodiment, the interferon (such as type I interferon) in the kit can be linked to a targeting moiety (such as an antibody) that binds to a tumor-associated antigen (such as to form a fusion protein), wherein the The targeting moiety is directly connected to the interferon or connected through a linker. In a specific embodiment, said targeting moiety is located at the N-terminus of said interferon. In another specific embodiment, said targeting moiety is located at the C-terminus of said interferon. In a specific embodiment, the interferon can be linked to two or more of the targeting moieties to form a multispecific (eg bispecific) molecule (eg multispecific fusion protein, eg bispecific specific fusion protein).

In the case where the interferon and the targeting moiety are connected by a linker to form a fusion protein, the linker may comprise amino acid sequences of any suitable length, for example, the linker may comprise 1-10, 1- 20, 1-30 or more amino acids (or consist thereof), for example the linker may 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 amino acids, or consist of said amino acids.

In another specific embodiment, said targeting moiety is an antibody, such as an anti-EGFR antibody or an anti-Neu antibody.

In another specific embodiment, the tumor is 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. In particular, the tumor may be a breast tumor or a melanoma.

In a specific embodiment, the tumor or the host bearing the tumor is deficient in one or more of the following: 1) the expression of interferon (such as type I interferon), such as interferon alpha 5 or interferon beta and/or function; 2) expression and/or function of interferon receptors (such as type I interferon receptors), particularly on dendritic cells; 3) tree Cross-presentation of dendritic cells and the resulting antitumor cytotoxic T cells.

In another aspect, the present invention also relates to a kit comprising:

a) an interferon as defined above; and

b) Instructions for use, which describe the use of the kit for overcoming tumor resistance to antibody therapy.

In other aspects, the invention relates to a method of treating tumors (for example, for overcoming tumor resistance to antibody therapy), said method comprising administering to a patient a therapeutically effective amount of an interferon as defined hereinabove or an interferon as hereinbefore defined The defined kit of the present invention.

In specific embodiments, said patient is resistant to antibody therapy. In other embodiments, the patient is deficient in one or more of the following: expression and/or function of interferon, e.g., type I interferon, such as interferon alpha 5 or interferon beta; 2) interferon-affected The expression and/or function of the body, especially the expression and/or function of interferon receptors on dendritic cells; 3) the cross-presentation of dendritic cells and the resulting anti-tumor cytotoxic T cells.

In further embodiments, said method of prevention and/or treatment further comprises administering to said patient simultaneously, sequentially (in any order) or separately: 1) an interferon as defined above; and 2) blocking PD -1/PDL signaling pathway compounds, such as antibodies, such as anti-PDL1 antibodies.

In yet another aspect, the invention relates to an interferon as defined above, or a kit of the invention, for use in overcoming tumor resistance to antibody therapy.

In other aspects, the type I interferons, kits or methods of the invention may be used in combination (e.g. simultaneously or in any order) with anti-tumor antibody therapy or other anti-tumor therapy (e.g. chemotherapy, radiotherapy, etc.) applied sequentially).

In addition, optionally, the reagents and/or kits of the present invention may further comprise a pharmaceutically acceptable carrier.

In summary, the present invention has several important implications for the field of cancer immunotherapy. First, it establishes a pathway to generate a new generation of antibody-based therapies (such as antibody therapy combined with type I interferon, such as antibody-interferon beta fusion protein), which enables the acquired immune response to more effectively deal with antibody resistance. sex and relapse. Enhanced cytotoxic T cell responses are then able to kill more tumor cells to create the following positive feedback loop: Cytotoxic T cell-mediated tumor cell death triggers endogenous danger/innate signaling, which further generates tumor-targeting innate and acquired immunity, and ultimately lead to more complete tumor regression. Second, the present invention provides evidence that interferons, such as type I interferons, which link innate and acquired antitumor immunity, are critical factors for antibody-mediated tumor regression, and thus provide important targets for cancer immunotherapy. Third, the present invention reveals that dendritic cells are the main resistant cell type in tumors, suggesting that they play a major role in defining the immunosuppressive tumor microenvironment. Therefore, targeting dendritic cells will be another important strategy for improving the efficacy of cancer immunotherapy. Fourth, the present invention proposes that antagonizing acquired resistance induced by immunotherapy will maximize the therapeutic effect of immunotherapy and ultimately cure the host's tumor. In summary, the strategies employed in the present invention point to several new directions for optimizing targeted immunotherapy, which can greatly impact antineoplastic drug discovery and clinical cancer treatment.

Description of drawings

Figure 1. Interferon production is induced and essential for antibody-mediated tumor regression. A) WT BALB/c mice (n=4/group) were subcutaneously injected with 5×10 ⁵ TUBO cells and administered 100 μg of anti-Neu (left column) or control IgG on day 14. WT B6 mice (n=4/group) were injected subcutaneously with 7×10 ⁵ B16-EGFR cells and administered 100 μg of anti-EGFR (right column) or control IgG on day 14. Four days later, the tumors were digested and the CD45 ⁺ and ^CD45- populations in the tumor tissue were isolated. Real-time PCR was performed to detect the expression of type I interferons at the mRNA level. B) WT BALB/c mice (n=5/group) were subcutaneously injected with 5×10 ⁵ TUBO-EGFR cells, and 100 μg of anti-Neu was administered on days 14 and 21. 200 μg of anti-type I interferon receptor or control Ig were administered intratumorally on the same day. Tumor growth was measured twice a week and compared. C) WT B6 mice (n=5/group) were subcutaneously injected with 5×10 ⁵ B16-EGFR cells, and 2×10 ⁹ adenovirus-IFNβ or control virus were administered intratumorally on days 14 and 21 . Tumor growth was measured twice a week and compared. *, P<0.05 compared with the control group. A representative set of experimental results from three sets of experiments is shown.

Figure 2. Ab-IFNβ greatly improves the anti-tumor effect of the antibody. A) WT BALB/c mice (n=5/group) were subcutaneously injected with 5×10 ⁵ TUBO-EGFR and treated with 25 μg of anti-EGFR-IFNβ or control antibody on day 14, 18 and 22. Tumor growth was measured twice a week and compared. B) WT B6 mice (n=5/group) were injected subcutaneously with 1×10 ⁶ B16-EGFR and treated with 25 μg of anti-EGFR-IFNβ or control antibody on day 14, 18 and 22. Tumor growth was measured and compared twice a week. C) Rag1KO mice (n=5/group) were subcutaneously injected with 3×10 ⁶ H460 cells, and 2×10 ⁶ OTI LN cells were adoptively transferred on day 13. On days 14, 18 and 22, 25 μg of anti-EGFR-interferon beta or control antibody was administered. Tumor growth was measured twice weekly and compared. D) Neu ^OTI/OTII -Tg female mice (n=5/group) were subcutaneously injected with 1×10 ⁶ NOP23 and treated with 25 μg of anti-Neu-IFNβ or control antibody on day 14, 18 and 22 deal with. Tumor growth was measured twice weekly and compared. *, P<0.05 compared with the control group. A representative set of three experiments is shown.

Figure 3. Anti-EGFR-Interferon beta is able to induce anti-tumor specific cytotoxic T cell responses that contribute to tumor regression. A) WT and Rag1KO B6 mice (n=5/group) were subcutaneously injected with 5×10 ⁵ B16-EGFR-SIY cells and treated with 25 μg of anti-EGFR-IFNβ or Control antibody was used for treatment. Tumor growth was monitored twice weekly. B) On day 7 after the last treatment, dLN cells were harvested and subjected to an interferon gamma ELISPOT assay. C) WT B6 mice (n=5/group) were subcutaneously injected with 5×10 ⁵ B16-EGFR-SIY and treated with 25 μg of anti-EGFR-IFNβ or control antibody on day 14, 18 and 22 . CD8-depleting antibody (200 μg/mouse) was administered on the same day as anti-EGFR-interferon beta. Tumor growth was measured twice weekly and compared. D) On day 7 after the last treatment, dLN cells were harvested and subjected to an interferon gamma ELISPOT assay. *, P<0.05 compared to the control group. A representative set of three experiments is shown.

Figure 4. The antitumor effect of anti-EGFR-interferon beta requires the expression of type I interferon receptors on host hematopoietic cells. A) WT and type I interferon receptor 1KO B6 mice (n=5/group) were subcutaneously injected with 5×10 ⁵ B16-EGFR-SIY cells and treated with 25 μg of anti-EGFR-SIY on days 14, 18 and 22. Interferon β or control antibody for treatment. Tumor growth was monitored twice weekly. B) On day 7 after the last treatment, dLN, spleen and tumor infiltrating cells were harvested and interferon production in the supernatant was detected by CBA assay. C) Thirty days after bone marrow chimeric reconstitution as indicated, mice were subcutaneously injected with 5×10 ⁵ B16-EGFR-SIY and treated with 25 μg of anti-EGFR-IFNβ or Treatment was performed against Ig. Tumor growth was measured twice weekly and compared. *, P<0.05 compared to the control group. One of two representative experiments is shown.

Figure 5. Anti-EGFR-IFNβ restores the cross-presenting ability of dendritic cells. A) WTB6 mice (n=5/group) were injected subcutaneously with 5×10 ⁵ B16-EGFR-SIY and treated on day 14 and 18 with 25 μg of anti-EGFR-IFNβ or control antibody. On day 7 after the last treatment, dLN cells were harvested and subjected to an interferon gamma ELISPOT assay. B) WT B6 mice (n=5/group) were injected subcutaneously with 5×10 ⁵ B16-EGFR-SIY and treated with 25 μg of anti-EGFR-IFNβ or control antibody on day 14 and 18. On day 8 after the first treatment, dendritic cells from dLNs were purified by CD11c ⁺ positive selection and incubated with purified naive 2C T cells. Interferon production was measured 2 days later. C) Dendritic cell activation markers were measured by flow cytometry. D) Thirty days after bone marrow chimeric reconstitution as indicated, mice were subcutaneously injected with 5×10 ⁵ B16-EGFR-SIY and treated with 25 μg of anti-EGFR-IFNβ on days 14, 18 and 22 Or control Ig was processed. DT or PBS was administered on the same day as the treatment. Tumor growth was measured twice weekly and compared. *, P<0.05 compared with the control group. A representative set of two sets of experiments is shown.

Figure 6. Dendritic cells are the major cell type that directly respond to anti-EGFR-interferon beta treatment. A) WT and CD11c-Cre type I interferon receptor 1 ^flox/flox mice (n=5/group) were subcutaneously injected with 5×10 ⁵ B16-EGFR-SIY and treated with 25 μg Anti-EGFR-IFN-β or control antibody were treated. Tumor growth was measured twice weekly and compared. B) WT and CD4-Cre type I interferon receptor 1 ^flox/flox mice (n=5/group) were subcutaneously injected with 5×10 ⁵ B16-EGFR-SIY and treated with 25 μg Anti-EGFR-IFN-β or control antibody were treated. Tumor growth was measured twice weekly and compared. *, P<0.05 compared with the control group. A representative set of two sets of experiments is shown.

Figure 7. Results of antagonizing PD-L1 expression induced by anti-EGFR-IFNβ achieved complete tumor regression. A) WT B6 mice were injected subcutaneously with 5×10 ⁵ B16-EGFR-SIY cells and treated with 25 μg of anti-EGFR-IFNβ or control antibody on day 14. Two days later, tumor cells were harvested and analyzed for PD-L1 expression by flow cytometry. The red line represents the control Ig-treated group, while the blue line represents the anti-EGFR-interferon beta-treated group. B) B16-EGFR cells treated with 0.02 μg/ml anti-EGFR-IFNβ during in vitro cell culture. One day later, tumor cells were collected and analyzed for PD-L1 expression by flow cytometry. The red line represents cells stimulated with control Ig, while the blue line represents cells stimulated with anti-EGFR-interferon beta. C) WT B6 mice (n=5/group) were subcutaneously injected with 5×10 ⁵ B16-EGFR-SIY cells and treated with 25 μg of anti-EGFR-IFNβ or control antibody on day 14, 18 and 22 processed. PD-L1 blocking antibody (400 μg/mouse) was administered on the same day as anti-EGFR-interferon beta. Tumor growth was measured twice weekly and compared. D) On day 14 after the last treatment, splenocytes were harvested and subjected to an interferon gamma ELISPOT assay. *, P<0.05 compared to the control group. A representative set of three experiments is shown.

Figure 8. Proposed model of how type I interferons link innate and acquired antitumor immunity. Following antibody therapy, DAMPs released from antibody-responsive but not antibody-resistant tumors will induce type I interferon production. Ab-interferon mimics type I interferon production in situ in antibody-resistant tumors. Type I interferons simultaneously increase dendritic cell cross-presentation for better cytotoxic T cell responses, which directly activate T cells and also induce PD-L1 expression to impair antitumor immunity. The combination of Ab-interferon and PD-L1 blockade maximizes the antitumor immune response.

Figure 9. Anti-EGFR-IFNβ is able to deliver IFNβ to EGFR+ cells. A) Structure of anti-EGFR-IFNβ fusion protein. B) A431 cells were stained with hIg, anti-EGFR or anti-EGFR-IFNβ and anti-human IgG Fcγ-PE. C) WT B6 mice (n=5/group) were injected subcutaneously with 5×10 ⁵ B16-EGFR cells and treated on day 14 with 25 μg of anti-EGFR-IFNβ. At different time points, the concentration of anti-EGFR-IFNβ in different tissues was measured by hIg ELISA. One set of three representative experiments is shown.

Figure 10. Side effects of anti-EGFR-IFNβ. B6 mice were injected intravenously with different doses of anti-EGFR-IFNβ. At the indicated time points, the concentrations of the indicated cytokines in serum were measured by BD CBA kit.

Figure 11. CD20 ⁺ , NK1.1 ⁺ and Ly-6G ⁺ cells are not required for anti-EGFR-IFNβ-induced tumor regression. WT B6 mice (n=5/group) were injected subcutaneously with 5×10 ⁵ B16-EGFR and treated on days 14, 18 and 22 with 25 μg of anti-EGFR-IFNβ or control Ab. The indicated Abs (200 μg/mouse) to delete specific cell populations were administered on the same day as anti-EGFR-IFNβ. Tumor growth was measured twice weekly and compared.

Figure 12. Direct activation of DC and T cells in vitro by anti-EGFR-IFNβ. Splenocytes from WT B6 mice were cultured in vitro and treated with the indicated concentrations of anti-EGFR-IFNβ. One day later, cells were harvested for analysis of the expression of CD69 on T cells and CD86 on dendritic cells by flow cytometry.

Figure 13. No synergistic antitumor effect between anti-EGFR-IFN[beta] and anti-CTLA4 and anti-BTLA. WT B6 mice (n=5/group) were injected subcutaneously with 5×10 ⁵ B16-EGFR cells and treated on days 14, 18 and 22 with 25 μg of anti-EGFR-IFNβ or control antibody. CTLA4 or BTLA blocking antibody (400 μg/mouse) was administered on the same day as anti-EGFR-IFNβ. Tumor growth was measured twice weekly and compared.

Figure 14. Summary of tumor models that responded to antibody therapy in combination with interferon in the examples of the present invention.

specific implementation plan

Terms and Definitions

Unless otherwise specified, the terms and definitions used in this application have meanings commonly used in this field and are known to those skilled in the art.

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 solid tumors as well as locations close to or adjacent to tumor growth.

As used in this application, the term "administration" refers to systemic and/or local administration. The term "systemic administration" means administration that is not localized, whereby the administered substance may affect several organs or tissues throughout the body; or whereby the administered substance may travel across several organs or tissues throughout the body to reach the target site point. For example, circulatory administration to a subject can result in expression of the therapeutic product from the administered vector in more than one tissue or organ, or can result in expression of the therapeutic product from the administered vector at specific sites, such as , either due to natural tropism or due to operably linked tissue-specific promoter elements. Those skilled in the art will appreciate that the systemic administration encompasses various forms of administration including, but not limited to, parenteral, intravenous, intramuscular, subcutaneous, transdermal, oral, and the like.

The term "topical administration" means administration at or around a specific site. Those skilled in the art will appreciate that topical administration encompasses various forms of administration, such as injection directly at or around a specific site (eg, intratumoral administration).

As used herein, the term "therapeutically effective amount" refers to an interferon of the invention required to achieve the intended disease or condition (e.g. tumor/cancer, e.g. for tumor regression or reduction in tumor size), or Amounts of components in kits of the invention. The effective amount can be determined for a particular purpose by practice, in a routine manner. In particular, the therapeutically effective amount may be that amount required to: reduce the number of cancer cells; reduce tumor size; inhibit (i.e. slow or stop) infiltration of cancer cells into peripheral organs; inhibit (i.e. slow or stop) ) tumor metastasis; inhibiting tumor growth; and/or alleviating 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 this application, the term "immunoglobulin" (Ig) is used interchangeably with antibody. The antibodies can specifically target tumor antigens, such as surface tumor antigens, such as EGFR, CD4, CD8, Neu, and the like.

The term "interferon" includes intact interferon molecules (e.g. human interferon, mouse interferon, e.g. human or mouse type I interferon, such as interferon alpha or beta), functional fragments, derivatives, equivalents thereof Or any functional variant, which is able to achieve the desired biological function, such as inducing the expansion of tumor-specific T cells, especially the cross-presentation of dendritic cells and thus the generation of anti-tumor cytotoxic T cells. In the context of the present invention, said interferon may be selected from type I, type II and/or type III interferons, such as IFN-α, IFN-β, IFN-γ, IFN-λ1 (IL-29), IFN -λ2 (IL-28a), IFN-λ (IL-28b) and IFN-ω etc.

The term "functional variant" refers to a molecule that has been modified (e.g. mutation, insertion, deletion, fusion, conjugation, cross-linking, etc.) to differ from the parent molecule (e.g. interferon), but retains the desired biological activity thereof variant of .

Interferon, its fragments or functional variants thereof can be linked to the targeting moiety by various conventional methods known in the art. The linkage can be direct or indirect (eg via a linker), eg, can be achieved by formation of a fusion protein, conjugation or chemical linkage. When such linking 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 destroy the desired properties of the resulting product (eg, induces the generation of anti-tumor cytotoxic T cells). For example, the targeting moiety can be located at the N- or C-terminus of the interferon. In a specific embodiment, the interferon can be linked to two or more of the targeting moieties to form a multispecific (eg bispecific) molecule (eg multispecific fusion protein, eg bispecific specific fusion protein). In the case where the interferon and the targeting moiety are connected by a linker to form a fusion protein, the linker may comprise amino acid sequences of any suitable length, for example, the linker may comprise 1-10, 1- 20, 1-30 or more amino acids (or consist thereof), for example the linker may 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 amino acids, or consisting of said amino acids

Those skilled in the art can select the appropriate dosage form and dosage form according to the specific condition, disease type (such as tumor type, tumor development stage, etc.), severity, patient constitution, other therapies that may be administered in combination, and therapies that have been administered before. Mode of application.

The term "cancer" refers to a condition typically characterized by unregulated cell growth (eg in mammals, eg humans). The cancer includes, but is not limited to, breast cancer, lung cancer, prostate cancer, colon cancer, skin cancer, head and neck cancer, lymphoma or melanoma, for example.

The term "antibody therapy" refers to methods of treatment through the use of antibodies that specifically target a tissue or site of interest. In the context of the present invention, it refers in particular to methods of treating tumors, such as cancer, with specific antibodies targeted to tumor cells.

In the context of the present invention, "resistance to antibody therapy" means that, after treatment with a particular antibody preparation, a tumor (e.g. cancer) or a patient bearing said tumor does not respond to said treatment (e.g. tumor cells are not killed, the tumor does not regress), and/or the therapy fails to cause regression of the tumor, reduce or inhibit metastasis or further growth of the tumor.

In this application, the term "tumor-associated antigen" includes, for example, tumor surface antigens, including but not limited to, members of the epidermal growth factor receptor family (EGFR), including EGFR, HER1, HER2, HER4 and HER8, etc. (Nam, N.H., & Parang, K. (2003), Current targets for anti cancer drμg discovery. Current Drμg Targets, 4(2), 159-179), STEAP (six-transmembrane epithelial antigen of the prostate; Hubert et al., STEAP: a prostate -specificcell-surface antigen highly expressed in human prostatetumors., Proc Natl Acad Sci U S A.1999; 96(25):14523-8.), CD55 (Hsu et al., Generation and characterization of monoclonal antibodies directed against the surface antigens of Cervical cancer cells., Hybrid Hybridomics. 2004;23(2):121-5).

Other suitable antibodies that can be used in the present invention include Rituximab (Rituxan™, chimeric anti-CD20 antibody), Campath-1H (anti-CD52 antibody), and antibodies to any cancer-specific cell surface antigen. The following exemplarily lists antibodies against specific cancer types suitable for the purposes of the present invention in combination with interferon: Alemtuzumab (Campath ^™ ) for chronic leukemia; Bevacizumab (Avastin ^TM ) for colon and lung cancer; cetuximab (Erbitux ^TM ) for colon and head and neck cancer; gemtuzumab (Mylotarg ^TM ) for acute myeloid leukemia; Ibritumomab (Zevalin ^TM ) for non- Hodgkin lymphoma; panumumab (Vectibix ^TM ) for colon cancer; rituximab (Rituxan ^TM ) for non-Hodgkin lymphoma; tositumomab (Bexxar ^TM ) for non-Hodgkin lymphoma Chickkin's lymphoma; and trastuzumab (Herceptin ^™ ) for breast cancer.

In the present invention, "pharmaceutically acceptable carrier" refers to a carrier that does not cause allergic reactions or other uncomfortable effects in the administered cells or subjects, and 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, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, that increase the shelf life or utility of nucleic acids, polypeptides, viral particles, or cells.

In this application, "deficient" in interferon and/or its receptor means that the expression of said interferon or interferon receptor cannot reach the level required to realize its biological function, or the expressed interferon Either the interferon receptor cannot perform the desired biological function (for example, exists in a mutated form), or the interferon (or interferon receptor) cannot interact with its receptor (ligand) to cause downstream signal transduction.

The following examples are only for better explaining the present invention, and are not intended to limit the present invention in any way.

Example

Materials and methods

mouse

C57BL/6J and BALB/c mice were purchased from Harlan. Rag1 ^−/− , 2C CD8 ⁺ TCR transgenic mice, CD11c-DTR transgenic mice, CD11c-Cre and CD4-Cre transgenic mice were purchased from JAX. IFN AR1 ^-/- mice were provided by Dr. Anita Chong, University of Chicago. IFNAR1 ^fl/fl mice were provided by Dr. Ulrich Kalinke, Institute for Experimental Infection Research, Hannover, Germany (Kamphuis, Junt et al. 2006). Neu ^OT-I/OT-II transgenic mice (B6 background) were provided by Dr. Brad H. Nelson, Trev & Joyce Deeley Research Centre, British Columbia, Canada (Wall, Milne et al. 2007). All mice were housed under specific pathogen-free conditions. Animal care and use were performed in accordance with institutional and NIH protocols and guidelines, and all studies were approved by the University of Chicago Animal Care and Use Committee.

Cell Lines and Reagents

H460 was purchased from ATCC. TUBO was cloned from a spontaneous mammary tumor in BALB/c Neu-transgenic mice. TUBO-EGFR was selected after transfection with pSEB-EGFR or with a plasmid containing 2 μg/mL blasticidin (InvivoGen). After transduction with lentiviruses expressing human EGFR or EGFR-SIY, single clones of B16-EGFR and B16-EGFR-SIY were picked. NOP23 was cloned from spontaneous mammary tumors in B6Neu ^OT-I/OT-II transgenic mice and was kindly provided by Dr. Brad H. Nelson, Trev & Joyce Deeley Research Centre, Canada. H460, TUBO, B16 and their derivatives were cultured under the condition of 5% CO ₂ , and they were maintained in vitro supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 2mmol/L L-glutamine , 0.1 mmol/L MEM non-essential amino acids, 100 units/mL penicillin and 100 μg/mL streptomycin in DMEM. Anti-EGFR mAb Cetuximab was purchased from Imclone. Anti-CD8 (YTS169.4.2), anti-NK1.1 (PK136), anti-Neu (7.16.4) antibodies were produced in-house. Anti-PD-L1 (10F.9G2) and anti-Ly-6G (1A8) antibodies were purchased from BioXcell. Anti-CD20 (5D2) antibody was provided by Ouyang Wenjun (Genentech, San Francisco). Anti-type I interferon receptor (MAR-5A3) antibody was provided by Dr. Robert Schreiber (Washington University, St. Louis). Adenovirus expressing murine interferon beta was prepared as previously described (Burnette et al., 2011).

Generation of Ab-IFNβ fusion protein

The anti-EGFR V region was cloned from an anti-EGFR (LA22) hybridoma (ATCC). The V regions of the heavy and light chains were cloned into Abvec-IgG1 and Abvec-kappa, respectively (Smith et al., 2009). Then, mouse interferon beta was inserted into the C-terminus of the heavy chain as a fusion protein containing the SGGGGSGGGGSGGGGSGGGG (SEQ ID NO: 1 ) linker. The entire heavy and light chains containing interferon beta were cloned into pEE6.4 and pEE12.4 (Lonza), respectively. These two vectors were then digested with NotI and BamHI. The complete hCMV-MIE-heavy chain/SV40 transcription unit from the digested pEE6.4 plasmid was ligated with the large NotI-BamHI fragment (containing the light chain expression cassette) from the digested pEE12.4 plasmid. Plasmids containing both heavy and light chains were transfected into CHO cells and stable clones were established according to the instruction manual (Lonza). The supernatant containing anti-EGFR-interferon beta was purified by protein A column according to the instruction manual (Repligen Corporation). Anti-Neu-interferon beta was produced in the same manner except that the cDNA of the V region was from an anti-Neu (7.16.4) hybridoma.

Tumor Growth and Management

Approximately 6×10 ⁵ TUBO, TUBO-EGFR, B16-EGFR, B16-EGFR-SIY or NOP23 cells were injected subcutaneously in the right flank into 6- to 8-week-old mice. Tumor volume was measured along three orthogonal axes (a, b and c) and calculated as abc/2. Mice were treated by three intratumoral injections of 25 μg of anti-EGFR-interferon beta antibody or control antibody on days 14, 18 and 22 after tumor inoculation. For CD8 depletion experiments, 200 μg of anti-CD8 antibody was injected intraperitoneally concurrently with the anti-EGFR-interferon beta treatment. Approximately 6×10 ⁶ H460 cells were injected subcutaneously in the right flank into 6- to 8-week-old Rag1 KO mice. After tumor establishment (approximately 14 days), 2 x ¹⁰⁶ LN cells from OTI TCR transgenic mice were adoptively transferred into mice by intravenous injection. One day later, mice were treated by three intratumoral injections of 25 μg of anti-EGFR-interferon beta antibody or control antibody.

Expression of type I interferon mRNA after antibody treatment

Approximately 6 × 10 ⁵ TUBO, TUBO-EGFR or B16-EGFR cells were injected subcutaneously in the right flank into 6- to 8-week-old mice. On days 14 and 16 after tumor inoculation, mice were treated by two intratumoral injections of 100 μg of anti-Neu, anti-EGFR or control antibody. Tumors were digested with 1 mg/ml collagenase VIII (Sigma) and 200 μg/ml DNase I (Sigma) at 37°C for 30 minutes. Viable CD45 ⁺ and ^CD45- cell populations were sorted by FACS AriaII (BD Bioscience). Total RNA was isolated by RNeasy mini kit (Qiagen) and reverse transcribed into cDNA by Sensiscript RT kit (Qiagen). Expression levels of type I interferon mRNA were analyzed by quantitative real-time PCR. The primers used for the assay are as follows:

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

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

Measurement of interferon gamma secreting T cells by ELISPOT assay

SIY peptide-reactive T cells were measured by ELISPOT assay. Resuspend spleen or lymph node cells in RPMI1640 supplemented with 10% FCS, 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. A total of 1-4 x ¹⁰⁵ spleen or lymph node cells were used for this assay. SIY peptide was added at a concentration of 5 μg/mL. After 48 h of incubation, interferon gamma production was determined by interferon gamma ELISPOT kit (BD Bioscience) or CBA assay (BD Bioscience) according to the instruction manual. Cytokine spots seen were counted with an ImmunoSpot analyzer (cytotoxic T cells).

Ex vivo dendritic cell cross-presentation assay

On days 14 and 17, B16-EGFR-SIY bearing mice were treated with intratumoral injections of 25 μg of anti-EGFR, or anti-EGFR-interferon beta. Four days later, draining lymph nodes were digested with 1 mg/ml collagenase VIII (Sigma) and 200 μg/ml DNase I (Sigma) for 15 minutes at 37°C. Dendritic cells were purified by CD11c positive selection kit (Stemcell). Approximately 1 x ¹⁰⁵ dendritic cells were mixed with purified 2 x ¹⁰⁵ 2C T cells (with or without 5 μg/mL SIY peptide) to restimulate T cells. After two days, supernatants were collected and interferon gamma was measured by CBA assay (BD Bioscience).

Ex vivo T cell activation assay

On days 14 and 17, B16-EGFR-SIY bearing mice were treated by intratumoral injection with 25 μg of anti-EGFR or anti-EGFR-interferon beta. Four days later, dLN T cells were purified by a T cell negative selection kit (Stemcell). Approximately 1 x ¹⁰⁵ purified dendritic cells from naive mice were mixed with 2 x ¹⁰⁵ T cells (with or without 5 μg/mL of SIY peptide) to restimulate T cells. After two days, supernatants were collected and interferon gamma was measured by CBA assay (BD Bioscience).

Generation of bone marrow chimeras

WT mice were lethally irradiated with a single dose of 1000 rads. The next day, ^2-3x106 WT, IFN AR1 ^−/− or CD11c-DTR Tg donor bone marrow cells were adoptively transferred to the irradiated mice intravenously. After reconstitution, mice were maintained on cotrimoxazole (Bactrim) antibiotic (diluted in drinking water) for 4 weeks. 5-6 weeks after reconstitution, mice were injected with tumor cells.

Detection of endotoxin in mAb and fusion protein preparations

Endotoxin was measured by Limulus amebocyte lysate assay (Cambrex inc. MD). For all mAb preparations, the amount of endotoxin was determined to be <0.2 E.U./mg mAb.

Flow Cytometry Analysis

Single cell suspensions were incubated with anti-CD16/32 (anti-FcγIII/II receptor, clone 2.4G2) for 10 minutes and then stained with conjugated antibodies. All fluorescently labeled mAbs were purchased from Biolegend or eBioscience. Samples were analyzed on a FACSC automated flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (TreeStar, Inc.).

Statistical analysis

Means were compared using unpaired Student's two-tailed t-test. Error bars represent SD. Statistically significant differences p<0.05 and p<0.01 are marked with * and **, respectively.

Example 1

Type I interferons are required for effective tumor responses to antibody therapy in vivo

Type I interferons have been shown to serve as potentially critical danger signals in eliciting antitumor T cell responses in spontaneous tumor rejection and in various antitumor therapies. The current inventors hypothesized that anti-oncogenic receptor antibodies induce in tumor tissue Type I interferon production, which bridges innate and acquired immunity. To test whether tumor sensitivity to anti-oncogenic receptor antibodies correlates with post-treatment levels of type I interferon in the model described here, the inventors first generated two different tumor cell lines, antibody-responsive cells lines and antibody-resistant cell lines. TUBO mammary tumor cells were derived from Her2/neu Tg mice, where Neu is the main signal for cell growth, and TUBO cells were used as an anti-neu antibody-responsive tumor cell line. The EGFR-transfected B16 melanoma cell line, in which EGFR is unable to deliver growth signals, was used as a tumor cell line fully resistant to anti-EGFR treatment. The inventors treated mice bearing these tumors with the corresponding antibodies and assessed the production of type I interferons in the tumors following treatment. The inventors found that the production of interferon α5 and interferon β was increased in antibody-responsive tumor models, but not in antibody-resistant tumor models (Fig. 1A and data not shown), suggesting increased type I interference Production of the hormone is caused by antibody-induced blockade of oncogenic receptors and stress. To test whether type I interferon is required for antibody-mediated antitumor effects in vivo, the inventors used anti-type I interferon receptor blocking antibody during anti-Neu treatment in an antibody-responsive TUBO tumor mouse model Mice were treated. The inventors found that blocking type I interferon signaling impaired the therapeutic efficacy of anti-Neu antibodies (Fig. IB), suggesting that type I interferon is indeed a critical cytokine for antibody-mediated tumor regression. Thus, this raises the possibility that impaired type I interferons may limit the immune response in hosts with antibody-resistant tumors. To further test whether direct delivery of other type I interferons into tumors was sufficient to control tumor growth (even in antibody-resistant tumors), two groups were treated with an adenovirus encoding interferon beta (adenovirus-interferon beta). tumor-bearing mice. As shown in Figure 1C, Ad-interferon beta treatment alone was sufficient to control the growth of antibody-resistant and antibody-responsive tumors (data not shown). Taken together, these data suggest that targeting type I interferons to tumors may be sufficient to overcome tumor immune avoidance.

Example 2

Targeted delivery of interferon beta enhances antibody-mediated therapeutic efficacy

The data in Example 1 suggest that targeting type I interferons to tumors has potential immunotherapeutic effects, particularly for overcoming tumor resistance to antibody therapy. In order to further verify this conclusion and further improve the therapeutic effect, the current inventors prepared an anti-EGFR-interferon β (Ab-IFNβ) fusion protein to deliver interferon β directly to tumor tissues expressing EGFR in a targeted manner (Fig. 9A). The present inventors first checked whether the anti-tumor function of anti-EGFR and interferon beta in this fusion protein remained intact. The inventors found that the fusion protein can bind EGFR ⁺ cells (Fig. 9B) and activate the type I interferon receptor signaling pathway (data not shown), so it can be seen that the anti-tumor effect of anti-EGFR and interferon β in the fusion protein Functionality is well maintained. The inventors then tested whether anti-EGFR-interferon beta could specifically deliver interferon beta to EGFR ⁺ tumor sites in vivo. The results confirmed that after the initial injection, the concentration of anti-EGFR-interferon beta remained high in the tumor tissue for almost a full week (Fig. 9C), whereas it decreased in less than a week after the initial injection. Significantly lower in other tissues. Furthermore, the inventors examined the side effects of anti-EGFR-interferon beta by measuring serum cytokines, ALT and AST levels. Among the cytokines detected by the inventors were TNF, IL-12, interferon gamma, MCP-1, IL-6 and IL-10, the expression levels of interferon gamma and MCP-1 at 6 hours and one day after injection slightly increased (Figure 10). The inventors also observed no increase in the levels of ALT and AST following the treatments described above (data not shown).

In order to compare the anti-tumor therapeutic effect between the new generation anti-EGFR-IFNβ fusion protein and the first generation anti-EGFR antibody (cetuximab), the inventors treated the patients with established EFGR with each agent. ⁺ the host of the tumor. Surprisingly, the inventors observed that in a partially resistant tumor model, anti-EGFR-interferon beta was effective at a lower dose and for a shorter duration than anti-EGFR antibody alone Much more therapeutic effect (Fig. 2A). An enhanced antitumor effect was similarly observed in the intramammary fat fraction tumor injection model (data not shown). To further test whether Ab-IFNβ is able to control tumor growth in a fully resistant tumor model, the inventors tested the efficacy of anti-EGFR-IFNβ in the B16-EGFR model. As predicted, anti-EGFR treatment alone was unable to inhibit B16-EGFR tumor growth in vivo, whereas anti-EGFR-IFNβ treatment was again able to potently control tumor growth (Fig. 2B). KRAS mutation has been reported to be a key factor contributing to anti-EGFR resistance in the clinic KRAS-mutated H460 human tumors were treated with anti-EGFR-interferon-β in immune-reconstituted Rag-1 KO mice. Even in this model, anti-EGFR-IFNβ showed a superior therapeutic effect compared to anti-EGFR alone (Fig. 2C). To test whether anti-EGFR-IFNβ could control tumor metastasis, the current inventors injected B16-EGFR intravenously into WT mice to mimic tumor metastasis. The inventors found that anti-EGFR-interferon beta was able to better control metastatic tumors and also prolong the survival of mice compared to anti-EGFR alone (data not shown).

Anti-tumor immunity elicited by anti-Neu antibodies was greatly reduced in neu Tg mice due to high expression of neu in all mammary glands during early life stages due to the nature of transgene expression (as self and tumor-associated antigens ) Tolerant host immune cells. To test whether Ab-IFNβ could break this tolerance and induce regression of neu ⁺ tumors in neu Tg mice, the neu ⁺ tumor line NOP23 (originally generated from neu Tg mice) was established in neu Tg hosts Surprisingly, anti-Neu-interferon β inhibited tumor growth more significantly than anti-Neu antibody alone (Fig. 2D). Taken together, these data demonstrate that Ab-IFNβ fusion protein therapy is effective in controlling tumors at lower doses and in shorter Much superior results were achieved in less time (Figure 14).

Example 3

Therapeutic efficacy of an anti-EGFR-interferon beta fusion protein depends on acquired immunity

Type I interferons have multiple potential effects on tumor growth, including inhibition of proliferation, inhibition of angiogenesis, activation of innate immune cells, bridging innate and adaptive immunity, and direct activation of adaptive immune responses. To assess the relative contribution of acquired immunity among the many other anti-tumor effects of anti-EGFR-IFN-β, B16-EGFR-bearing B6 Rag1 KO mice were treated with anti-EGFR-IFN-β. In contrast to the therapeutic effect observed in WT mice, similar doses of anti-EGFR-IFNβ failed to suppress tumors in these immunocompromised Rag1 KO mice (Fig. 3A). Thus, these data support the conclusion that anti-EGFR-interferon beta mediated therapeutic effects largely require acquired immunity.

CD8 ⁺ T lymphocytes are the major cell population involved in controlling the growth of many tumors. To determine whether they are also involved in anti-EGFR-interferon beta mediated anti-tumor effects, the inventors followed the anti-tumor T cell responses during the priming phase. The inventors first established the B16-EGFR-SIY tumor cell line in which the SIY peptide (SIYRYYGL) was ectopically expressed in the human EGFR molecule; this SIY peptide was used as a surrogate marker that could be detected by endogenous or 2C Tg CD8 ⁺ T cells specifically recognize. Following treatment with anti-EGFR-IFNβ, draining lymph node (dLN) lymphocytes were isolated from tumor-bearing mice, stimulated with SIY peptide, and interferonγ production was measured as activated T Effector-function indicators of cells. As shown in Figure 3B, anti-EGFR-interferon beta treatment increased interferon gamma production from tumor antigen-specific T cells compared to anti-EGFR treatment alone. To understand whether CD8 ⁺ cells are critical for the therapeutic effect of anti-EGFR-IFNβ, the inventors administered CD8-depleted B16-EGFR-bearing WT B6 mice during anti-EGFR-IFNβ treatment. antibodies, and tumor growth was measured. CD8 ⁺ cell depletion greatly reduced the efficacy of anti-EGFR-IFNβ therapy (Fig. 3C), suggesting that an adaptive immune response is required to control tumor growth. Consistent with this finding, the inventors found that T cell responses in vitro were greatly reduced following depletion with anti-CD8 (Fig. 3D). Depletion of other cells, including NK and B cells, did not affect the anti-tumor effect of anti-EGFR-IFNβ (Fig. 11). The inventors therefore speculate that anti-EGFR-interferon beta induces a more efficient priming of tumor-specific cytotoxic T cells, leading to inhibition of tumor growth. Taken together, these data suggest that anti-EGFR-IFNβ treatment can increase T cell priming, which contributes to the anti-tumor effect of anti-EGFR-IFNβ.

Example 4

Therapeutic efficacy of anti-EGFR-interferon beta requires expression of type I interferon receptors on host hematopoietic cells

Type I interferon receptors are ubiquitously expressed in almost all cell types, thus both normal cells and tumor cells are potential anti-EGFR-IFNβ targets. It has been reported that the efficacy of anti-CD20-interferon alpha fusion protein requires the expression of type I interferon receptors on tumor cells. Therefore, the inventors reasoned that for the therapeutic effect of anti-EGFR-interferon beta, it may also be required by tumor cells. Type I interferon receptors expressed by cells. To this end, the inventors compared the antitumor effects in tumor-bearing type I interferon receptor knockout (KO) mice that lack expression of type I interferon receptors on Cells express type I interferon receptors. Surprisingly, in type I IFN receptor KO mice, the therapeutic effect of anti-EGFR-IFNβ was completely abolished (Fig. 4A), and no increased cytotoxic T cell responses were observed (Fig. 4B ). These results suggest that type I interferon receptor-mediated activation is required for the therapeutic effect of anti-EGFR-IFNβ in host cells but not in tumor cells. Since all host tissues express type I interferon receptors, the inventors constructed type I interferon receptor bone marrow chimeric (BMC) mice to further analyze whether hematopoietic cells or hosts expressing type I interferon receptors are required stromal cells to achieve antitumor effects. The inventors discovered that expression of type I interferon receptors on hematopoietic cells is required because the anti-tumor effect of anti-EGFR-interferon beta was severely impaired in type I interferon receptor KO BM reconstituted mice (Fig. 4C). These data suggest that anti-EGFR-IFNβ mediates its antitumor effects not by directly inhibiting tumor cell growth, but by activating host hematopoietic cells, which then alter the tumor microenvironment.

Example 5

Enhanced dendritic cell cross-presentation contributes to the antitumor effect of anti-EGFR-interferon-β

Given that CD8 ⁺ cells are critical for the anti-tumor effect of anti-EGFR-IFNβ therapy, the inventors further explored the underlying mechanism of how anti-EGFR-IFNβ enhances cytotoxic T cell responses. The inventors observed a significant increase in IFN-γ-producing CD8 ⁺ cells following anti-EGFR-IFN-β treatment even without stimulation with exogenous SIY peptide, suggesting increased cross-presentation of APCs (Fig. 5A) . Because cross-presentation is the main priming mechanism for activating cytotoxic T cells for anti-tumor immunity, the inventors speculate that the increase in cross-presentation may be important for restoring dendritic cell function to reactivate cytotoxic T cells in tumors and in draining lymph nodes. critical. To begin investigating the possibility that anti-EGFR-IFNβ could increase cross-presentation of APCs, the inventors used an in vivo antigen-specific system to follow the priming and activation of tumor-antigen-specific T cells. Therefore, dendritic cells from the dLN of anti-EGFR-IFNβ-treated B16-EGFR-SIY-bearing mice were assessed by incubating them with SIY-reactive 2C T cells in an ex vivo assay. Ability to enhance specific anti-tumor cytotoxic T cell responses. Indeed, dendritic cells from anti-EGFR-IFNβ-treated mice induced more IFNγ production by 2C T cells (about 33-fold) compared with anti-EGFR treatment, even in the absence of external The same was true upon restimulation with the derived SIY peptide (Fig. 5B). Taken together, these data suggest that dendritic cells activated by anti-EGFR-IFNβ enhance CD8 ⁺ T cell activation through increased cross-priming function. Furthermore, these results suggest that it is the interferon beta component of the fusion protein that is responsible for the activation of the dendritic cell cross-presentation pathway, as anti-EGFR alone did not induce strong dendritic cell activation. To test whether anti-EGFR-interferon beta also increased the direct priming function, an exogenous antigen-producing peptide (SIY) was added to the culture medium, which further increased SIY-specific 2C T cell responses. Following restimulation with exogenous SIY-peptide, dendritic cells from anti-EGFR-IFNβ-treated mice induced approximately 2-fold more IFNγ production by 2C T cells compared to anti-EGFR treatment (Fig. 5B). These results suggest that dendritic cells from the dLN of anti-EGFR-IFNβ-treated hosts are likely to be activated to a greater extent than dendritic cells from anti-EGFR-treated mice alone . Indeed, these dendritic cells had high expression of activation markers, including CD86, as assessed by flow cytometry (Fig. 5C). To further dissect whether dendritic cell activation induced by anti-EGFR-IFNβ contributed to enhanced T cell activation, tumor-bearing CD11c-DTR BMC cells were treated with DT during anti-EGFR-IFNβ treatment. mice to deplete dendritic cells. The inventors found that the therapeutic effect mediated by anti-EGFR-interferon beta was significantly impaired when dendritic cells were absent (Fig. 5D). Taken together, these data suggest that increased dendritic cell cross-presentation leads to improved CD8 ⁺ cytotoxic T cell priming and function, and this may be the main underlying mechanism for the therapeutic effect of anti-EGFR-interferon β.

Example 6

Anti-EGFR-IFN-β directly targets dendritic cells to reverse a resistant tumor microenvironment for improved antitumor efficacy

The data of the present application indicate that increased cross-induction is important for the improved anti-tumor effect brought about by anti-EGFR-interferon beta treatment. However, the mechanism of how dendritic cells are activated by anti-EGFR-IFNβ treatment remains unclear. In particular, there is a need to identify cells expressing type I interferon receptors that directly contribute to the therapeutic effect of anti-EGFR-interferon beta. To address this issue, type I interferon receptor ^fl/fl mice were reared with various Cre-Tg mice. The anti-tumor effect of anti-EGFR-IFNβ was significantly reduced when the type I IFN receptor was selectively deleted in CD11c ⁺ cells in CD11c-Cre-IFNR type I ^fl/fl mice (Figure 6A ), which suggests that direct activation of dendritic cells by anti-EGFR-IFN-β may be a major contributor to its therapeutic effect. When the type I interferon receptor is selectively deleted in T cells of CD4-Cre-type I interferon receptor ^fl/fl mice, the antitumor effect of anti-EGFR-IFNβ is slightly but not significantly impaired 6B), suggesting that direct targeting of type I interferon receptors to T cells can further activate T cells that have been activated by dendritic cells, leading to improved antitumor effects. To further test this idea, the inventors assessed the effect of anti-EGFR-IFNβ stimulation on dendritic cell and T cell activation in an in vitro assay; indeed, anti-EGFR-IFNβ increased purified dendritic Activation of dendritic cells and T cells (Figure 12). Taken together, these data suggest that direct activation of type I interferon receptor-expressing dendritic cells plays a major role in anti-EGFR-IFNβ-mediated therapeutic effects, whereas expression of type I interferon on T cells The hormone receptors further enhanced this effect.

Example 7

Antagonizing anti-EGFR-IFN-β-induced PD-L1 expression achieves tumor-free outcomes

Although the anti-EGFR-interferon beta fusion achieves a more potent antitumor effect compared with anti-EGFR antibody alone, residual tumors eventually recur. The inventors therefore wondered whether anti-EGFR-interferon beta treatment might induce the expression of inhibitory molecules to prevent tissue damage, which would attenuate the antitumor effect over time. The inventors assessed PD-L1 expression after anti-EGFR-IFNβ treatment and clearly observed increased PD-L1 expression on tumor cells both in vivo and in vitro (Figures 7A and 7B). The inventors then tested whether combined anti-PD-L1 and anti-EGFR-IFNβ treatment could further enhance the long-term effect of anti-EGFR-IFNβ. Surprisingly, the long-term therapeutic effect was significantly enhanced when combined anti-PD-L1 and anti-EGFR-interferon beta treatment; mice remained tumor-free for at least 60 days after said treatment and responded to tumor stimulation Resistant (Fig. 7C, and data not shown). Furthermore, blocking the PD-1-PD-L1 interaction by anti-PD-L1 treatment further enhanced specific antitumor T cell responses (Fig. 7D). In contrast, no similar synergistic effect was observed when anti-EGFR-IFNβ was administered in combination with the antibodies anti-CTLA-4 and anti-BTLA, two other major inhibitory molecules expressed on T cells (Figure 13 ). In summary, the inventors' data demonstrate that antagonizing anti-EGFR-IFNβ-induced PD-L1 expression on tumor cells maximizes the anti-tumor effect of anti-EGFR-IFNβ with impressive tumor-free outcomes , even for antibody-resistant tumors. This combination-based strategy is likely to increase overall response and cure rates in antibody-resistant hosts, even in hosts that do not respond to anti-PD-1 antibodies that directly block T cell-expressed PD-1.

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Claims (23)

1. The use of interferon beta and an antibody blocking the PD-1/PDL signaling pathway for the preparation of a drug or a drug kit, wherein the drug or the drug kit is used to treat tumors, and the tumor is selected from: breast carcinoma, lung cancer and melanoma. 2. The use according to claim 1, wherein the antibody blocking the PD-1/PDL signaling pathway is an anti-PDL1 antibody. 3. The use of interferon beta and an antibody blocking the PD-1/PDL signal transduction pathway for the preparation of a drug or a drug kit, wherein the drug or drug kit is used to overcome the resistance of tumors to antibody therapy, so Said tumor is selected from: breast cancer, lung cancer and melanoma. 4. The use according to claim 3, wherein the antibody blocking the PD-1/PDL signaling pathway is an anti-PDL1 antibody. 5. The use according to any one of claims 1-4, wherein the interferon beta is linked to a targeting moiety that binds a tumor-associated antigen, wherein the targeting moiety is directly linked to the interferon beta or via a linker connected. 6. The use according to claim 5, wherein the targeting moiety that binds a tumor-associated antigen is an antibody that binds a tumor-associated antigen. 7. The use according to claim 6, wherein the antibody binding to a tumor-associated antigen is an anti-EGFR antibody or an anti-Neu antibody. 8. The use according to claim 5, wherein said interferon beta forms a fusion protein with a targeting moiety that binds a tumor-associated antigen. 9. The use according to any one of claims 6-7, wherein said interferon beta forms a fusion protein with said tumor-associated antigen-binding targeting moiety. 10. The use according to any one of claims 1-4, wherein the tumor or the host bearing the tumor is deficient in one or more of the following: 1) Expression and/or function of type I interferon; 2) expression and/or function of type I interferon receptors; and 3) Cross-presentation of dendritic cells and the resulting anti-tumor cytotoxic T cells. 11. The use according to claim 10, wherein said type I interferon in 1) is interferon alpha 5 and/or interferon beta. 12. The use according to claim 10, wherein said type I interferon receptor in 2) is a type I interferon receptor on dendritic cells. 13. A kit comprising: a) IFNβ; and b) Antibodies that block the PD-1/PDL signaling pathway. 14. The kit of claim 13, wherein the antibody blocking the PD-1/PDL signaling pathway is an anti-PDL1 antibody. 15. The kit of claim 13 or 14, wherein said interferon beta is linked to a targeting moiety that binds a tumor-associated antigen, wherein said targeting moiety is directly linked to said interferon beta or via a linker. 16. The kit according to claim 15, wherein said targeting moiety is an antibody. 17. The kit according to claim 15, wherein the targeting moiety is an anti-EGFR antibody or an anti-Neu antibody. 18. The kit according to claim 15, wherein said interferon beta forms a fusion protein with said targeting moiety. 19. The kit according to claim 16 or 17, wherein said interferon beta forms a fusion protein with said targeting moiety. 20. The kit according to claim 13 or 14, wherein said kit is for the treatment of a tumor which is breast cancer, lung cancer or melanoma. 21. The kit according to claim 20, wherein the tumor or the host bearing the tumor is deficient in one or more of the following: 1) Expression and/or function of type I interferon; 2) expression and/or function of type I interferon receptors; and 3) Cross-presentation of dendritic cells and the resulting generation of anti-tumor cytotoxic T cells. 22. The kit according to claim 21, wherein said type I interferon in 1) is interferon alpha 5 and/or interferon beta. 23. The kit according to claim 21, wherein said type I interferon receptor in 2) is a type I interferon receptor on dendritic cells.