Combination of poxviruses encoding HPV polypeptides and il-2 with Anti-PD l1 antibodies

Abstract

To provide a combination for use in the treatment of HPV-positive cancer.SOLUTION: The present invention relates to a combination of a) a poxviral vector encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and an immune-stimulatory cytokine, and b) anti-PD L1 antibodies or antigen binding fragments thereof, for use in the treatment of HPV-positive cancers, wherein a first administration of said poxviral vector is performed 5 to 10 days before a first administration of said anti-PD L1 antibodies, and wherein subsequent administrations of said poxviral and anti-PD L1 antibodies are performed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides a method for treating HPV-positive cancers, comprising: a) administering to a subject at least one human papillomavirus (HPV) virus; Encoding human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines and b) a poxvirus vector that binds to PD-L1 and a combination of an anti-PD-L1 antibody or an antigen-binding fragment thereof. and a first administration of the poxvirus is administered before the first administration of the anti-PD-L1 antibody. 5 to 10 days before the administration of the poxvirus and anti-PD-L1 antibody. The present invention relates to a combination of [Background technology]

[0002] Human papillomavirus (HPV) is the most commonly diagnosed sexually transmitted disease. HPV is a type of virus that causes genital warts, anogenital warts (cervix, vagina, vulva, penis, anus) and squamous intraepithelial cancer. It is associated with lesions and malignancies, including squamous cell carcinoma of the head and neck (SCCHN).

[0003] HPV is a small deoxyribonucleic acid (DNA) virus of approximately 7,900 base pairs. The PV genome contains six early (E) sequences that are associated with viral gene regulation and cell transformation. The DNA sequences of two late proteins that form the protein and shell of the virus A region of regulatory DNA sequence known as the long regulatory region (LCR) or upstream regulatory region Coding sequences (Palefsky JM and Holly EA, Cancer Epidemiol Biomarkers Prev. (1995) 4(4): 415-428).

[0004] HPV genotypes are broadly classified as “high risk” ( 16 , 18 , 31 ) based on their malignant potential. , 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and “low risk” ” (6, 11, 40, 42, 43, 44, 53, 54, 61, 72, 73, and 81) Types 16 and 18 are the most commonly found HPV types in cancer. For example, type 16 is found in approximately 50% of patients with cervical cancer. In addition to causing cancer, HPV has been linked to cancer of the anus and penis. High-risk (oncogenic) HPV subtypes, i.e., HPV-16 genotype, and in addition In patients infected with HPV-18, 31, or 33, the incidence of oral cavity and oropharyngeal cancers is The risk is also increased by about two to three times. A small percentage of tumors in other areas are also HPV-positive. However, HPV-related tumors occur primarily at the base of the tongue or tonsillar region. It is unclear whether this site is more susceptible to HPV transformation than other sites.

[0005] A review of published clinical trials, both single-arm and randomized, showed that the sex of oropharyngeal cancer ranges from 20% to 60% (Ihloff AS et al., Oral O (2010) 46(10): 705-711).

[0006] High-risk HPV infection is not only associated with various cancers, but also increases the risk of developing HPV-positive cancers. In particular, several studies have shown that infection with high-risk HPV is an independent risk factor for the development of SCCHN, and traditional This indicates that these factors should be considered together with other risk factors (D'Souza G. et al., The N ew England journal of Medicine. (2007) 356(19): 1944-1956).

[0007] The mechanism linking cancer and HPV infection is the role of two viruses, E6 and E7. E6 and E7 have been shown to be associated with proteins that play important roles in They have the ability to disrupt normal replication control in infected cells by inhibiting regulatory factors. The E6 oncoprotein inhibits the p53 tumor suppressor It binds to factor proteins and disrupts the p53 pathway through a ubiquitin-mediated process. Induce the degradation of β-glucan, which leads to uncontrolled cell cycle progression (Chung CH a nd Gillison ML, Clin Cancer Res. (2009) 15(22): 6758-6762). HPV E7 Tan Protein binds to and inhibits the retinoblastoma protein (pRb), which in turn activates the transcription factor This prevents the inhibition of E2F, resulting in loss of cell cycle control. Partial inactivation of CDK leads to upregulation of p16 protein. Cyclin DC, encoded by the N2A tumor suppressor gene, phosphorylates Rb Transcription factor E2F regulates the activity of the DK4 / 6 complex, thereby initiating cell cycle progression This results in the release of

[0008] Two types of vaccines have been developed to prevent HPV infection. The first is a vaccine called ARDASILO (Ardasil) and the second is a bivalent vaccine (Cervarix). HPV infection and HPV-associated disease in subjects without previous HPV exposure Approved for prophylaxis.

[0009] However, to date, no antitumor vaccines for HPV-positive cancers, especially SCCHN, have been clinically Few have been evaluated for their efficacy. and long-term maintenance is clinically beneficial in HPV-positive cancers, especially SCCHN. , an anti-tumor vaccine is a goal that must be realized.

[0010] TG4001 (corresponding to the research name MVATG8042) is a highly attenuated non- Based on the transmissible vaccinia vector, Modified Vaccinia Virus Ankara (MVA), It is a therapeutic recombinant vaccine / immunotherapy product based on the approximately 178 kilobase genome. The spare single linear double-stranded DNA molecule binds to three proteins, namely, HPV E6 and E7 oncoproteins modified to eliminate E6 and E7 oncoproteins and adjuvants containing an inserted transgene encoding human interleukin-2 (IL-2) as a .

[0011] TG4001 has been clinically investigated in gynecological conditions. Five Phase II studies have been conducted Of these, four were precancerous lesions, specifically cervical intraepithelial neoplasia (CIN) grade 2 / 3 and exocrine One study included patients with VIN grade 3 and one with cervical cancer. The study included 21 patients with HPV-16-associated CIN grade 2 / 3 (TG400 Two phase 2 trials involving 1.07 (TG4) and 206 (NV25025) patients were 001 compared favorably with placebo for histologic resolution and response rates, as well as viral clearance. The study demonstrated proof of concept that the HPV-16-associated C The dose used in these phase II studies in patients with IN grade 2 / 3 was 5 × 10 7 The safety profile of the therapeutic vaccine was The product was well tolerated (no major toxicities were observed), with the most common adverse events occurring after injection. It was demonstrated that this was a site reaction (Brun JL et al., Am J Obstet Gynecol. (2011) 20 4(2): 169 e161-168, Harper DM et al., Gynecol Oncol. (2019) 153(3): 521-529) .

[0012] A total of 313 subjects (healthy volunteers or those administered intramuscularly or subcutaneously) were treated with TG4001 either as monotherapy or in combination with the immunomodulator imiquimod. Based on data from patients with CIN 2 / 3, cervical cancer, or VIN The overall safety profile of TG4001 was evaluated in patients receiving either weekly or every 3 weeks. 5 × 10 administered in up to six injections for up to seven weeks. 7 The highest tested pfu Consistent with its immunostimulatory properties, TG 4001 administration was associated with the onset of injection site reactions in most treated patients. Most of these events were mild to moderate in intensity.

[0013] PD-1 interacts with its two ligands, PD-L1 and PD-L2. Negative regulator of T cell activity, limiting T cell activity at various stages of the immune response When the ligand binds, PD-1 normally activates the phosphatase Inhibits kinase signaling pathways that lead to T cell activation. PD-L1 interacts with B7, and one antibody is currently in clinical development. It is also thought to provide a negative signal to T cells, and PD-L1 blocking antibodies can block this interaction. Immune checkpoint inhibitors also enhance the function of tumor-infiltrating lymphocytes (TILs). Therefore, TILs enhance anti-tumor immunity within the tumor microenvironment. The presence of TILs is essential for many cancers. PD-L1+ TILs are associated with a relatively good prognosis in various types of cancer. have been shown to be an indicator of response to checkpoint inhibition, and the absence of TILs is associated with PD -1 / L1 inhibition (Herbst RS et al. , Nature. (2014) 515(7528): 563-567).

[0014] Anti-PD-L1 antibodies are being clinically investigated in the treatment of various solid tumors and have shown clinical efficacy in a variety of cancers. It has been found to provide benefits (Brahmer JR et al., N Engl J Med. (2012) 366(26): 2455-2465).

[0015] Avelumab is a human anti-programmed death-ligand-1 (PD-L1) antibody. Preclinical models have shown that HIV is involved in both adaptive and innate immune functions. By blocking the interaction between the PD-1 receptor and PD-L1, lumab has shown promise in preclinical models. Avelumab has been shown to reverse the suppression of T cell-mediated antitumor immune responses in tumors. In vivo analysis demonstrated that NK cell-mediated direct tumor cell lysis via antibody-dependent cellular cytotoxicity (ADCC) was induced. It has also been shown to induce progression in vitro. When combined with axitinib, avelumab It is approved in the United States for the first-line treatment of patients with renal cell carcinoma (RCC) after chemotherapy. The Food and Drug Administration (FDA) has approved the use of methadone in (i) adults aged 12 years and older with metastatic Merkel cell carcinoma (mMCC); and treatment of pediatric patients and (ii) with disease progression during or after platinum-containing chemotherapy. or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy For patients with locally advanced or metastatic urothelial carcinoma (mUC) with disease progression The company also granted accelerated approval to avelumab.

[0016] Avelumab has demonstrated an acceptable safety profile in cancer patients. Warnings and precautions regarding immune-mediated adverse reactions (pneumonitis and hepatitis, including fatal cases) , colitis, endocrine disorders, nephritis and renal impairment, and other adverse reactions [which may be severe and may result in death]. Examples include], infusion-related reactions, major adverse cardiac events (MACE), and embryo-fetal toxicity Gender is included.

[0017] Clinical trials are currently underway testing avelumab in HPV-positive cancers, including SCCHN. However, the results have not yet been made public.

[0018] obtained in precancerous lesions using TG4001 or using anti-PD-L1 antibody alone Improve antitumor efficacy and responder rates despite results obtained in cancer Therefore, there remains a great need for novel treatments for cancer. There is still a need to develop therapeutic options for (a) at least human papillomavirus (HPV) Human papilloma virus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines or (b) a poxvirus vector encoding a There is a need for methods of treating cancer that improve the effectiveness of anti-PD-L1 antibodies. Ming provides a combination product for use in the treatment of HPV-positive cancer that addresses the above needs. do.

[0019] Immune checkpoint inhibitors (including anti-PD-L1 antibodies) are administered via poxvirus vectors. It is being combined with many other types of anti-cancer therapy, including vaccine-based immunotherapies such as In most cases, the behavior of cancers not related to HPV infection has been suggested. Experiments have been conducted in animal models, and analysis of the toxicity of the tested combinations has been specifically (International Publication No. 2016 / 128542, International Publication No. 2015 / 1 Pamphlet No. 75334, Pamphlet No. WO 2015 / 069571, Remy-Z iller et al., Hum Vaccin Immunother. (2018) 14(1): 140-145). Non-small cell lung cancer (N TG4010 (encoding MUC1 and interleukin-2) in the treatment of SCLL Two Phase II clinical trials are underway to combine nivolumab (anti-PD-1 antibody) with MVA vector (a vector that uses nivolumab) Trials (NCT03353675 and NCT02823990) are ongoing (Oliver es H. et al., J Thorac Dis. (2018) 10(Suppl 13): S1602-S1614), but the results of the study have not been made public. Not represented.

[0020] In the context of HPV-positive cancer, immunization against HPV antigens, including viral vaccines, Various vaccine combinations and immune checkpoint blockades intended to stimulate an immune response Combinations of anti-inflammatory drugs have also been proposed (Gildener-Leapman et al., Oral Oncol. (2014) 50(9): 780-4, International Publication No. 2015 / 103602, Rice et al., Cancer Gene Ther. (2015) 22(9): 454-62, International Publication No. 2016 / 071306, US Japanese Patent No. 2017 / 051019, U.S. Patent No. 2019 / 142933 To date, no studies have been conducted on the combination of HPV antigen-encoding viral vaccines and anti-PD-L1 antibodies. Demonstration of an acceptable toxicity profile and improved therapeutic efficacy for HPV-positive cancers No clinical trial data are available. TG4001 and avelumab in the treatment of HPV-positive SCCHN An ongoing Phase Ib / II clinical trial (NCT03260023) of the combination has been reported. (Lin et al., Front Oncol. (2018) 8: 532), but the results of the trial have not been published. Summary of the Invention [Means for solving the problem]

[0021] In the context of the present invention, the inventors have surprisingly found that at least Human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines a poxvirus vector encoding a PD-L1 antibody, preferably TG4001, or an antigen-binding fragment thereof, preferably avelumab, administered in a specific dose in patients with HPV-positive cancer. When combined using this scheme, toxicity was tolerable and HPV E6 and E7 polioviruses were We found that the immune response to the peptides was improved, particularly with avelumab and TG. Despite the immunostimulatory effects of both 4001 (live vector) and 4002 (live vector), they resulted in unacceptable toxicity. No interactions between the two products were observed, which could not reasonably be expected. In particular, the combination of anti-PD-L1 antibodies and Listerin was not effective after a patient died from a complication of an adverse event. The U.S. Food and Drug Administration (FDA) has approved a study (NCT02291055) combining the vaccine with HIV. In light of the fact that the FDA has issued a clinical hold, particularly given the immunostimulatory effects of both products, There was a risk of an interaction between the two products that could result in unacceptable toxicity. is considering whether vaccines (another type of live vector) can prevent the known side effects of immune checkpoint inhibitors. Furthermore, promising beneficial immune changes (HPV E6 and induction of immune responses against E7 polypeptides, CD8 T cells in the circulation and tumor tissues Increase in CD4 T cells and decrease in CD4 regulatory T cells, as well as "cold" tumor proliferator-activated receptor agonists. It is associated with a "hot" tumor profile rather than a low-grade tumor profile and is associated with a relatively favorable prognosis. The combination of these two drugs resulted in significant improvements in the expression of steroid hormones, including upregulation of genes associated with steroid hormones. Treatment alone was not observed.

[0022] The enhancement may be additive or synergistic. The enhancement effect of the combination therapy is at least additive. The present inventors have surprisingly found that (a) at least human papillomavirus (H PV) POC encoding E6 and E7 polypeptides and immunostimulatory cytokines We have found that the combination of (a) a viral vector and (b) an anti-PD-L1 antibody results in improved treatment. Early results from clinical trials show that the combination therapy is effective in treating recurrent / metastatic HPV16-positive cancers. It has been shown that the combination therapy is effective in treating cancer (see Example 1) and that the combination therapy is well tolerated. (See Example 1.) Furthermore, the results show that each of the two treatments in this combination A similar effect was observed (see Example 1). This suggests at least an additive enhancing effect of the combination therapy. The results are showing.

[0023] Therefore, the present invention provides For use in the treatment of HPV-positive cancer or HPV-positive intraepithelial precancerous lesions, a) at least human papillomavirus (HPV) E6 and E7 polypeptides, and a poxvirus vector encoding an immunostimulatory cytokine in b) an anti-PD-L1 antibody or antigen-binding fragment thereof A combination of The first administration of the poxvirus occurs 5 to 10 days after the first administration of the anti-PD-L1 antibody. and a subsequent administration of the poxvirus and anti-PD-L1 antibody. Regarding combinations.

[0024] The present invention relates to a method for treating HPV-positive cancer or HPV-positive intraepithelial precancerous disease in a subject in need thereof. 1. A method for treating a disorder comprising: a) at least human papillomavirus (HPV) E6 and E7 polypeptides, and a poxvirus vector encoding an immunostimulatory cytokine in b) an anti-PD-L1 antibody or antigen-binding fragment thereof administering to said subject a combination of The first administration of the poxvirus occurs 5 to 10 days after the first administration of the anti-PD-L1 antibody. and a subsequent administration of the poxvirus and anti-PD-L1 antibody. It also concerns the law.

[0025] The present invention provides Medications for use in the treatment of HPV-positive cancer or HPV-positive cancer intraepithelial precancerous lesions For manufacturing, a) at least human papillomavirus (HPV) E6 and E7 polypeptides, and and poxvirus vectors encoding immunostimulatory cytokines. b) an anti-PD-L1 antibody or antigen-binding fragment thereof The use of a combination of The first administration of the poxvirus occurs 5 to 10 days after the first administration of the anti-PD-L1 antibody. and subsequent administration of the poxvirus and anti-PD-L1 antibody. It also relates to use.

[0026] The present invention provides For the treatment of HPV-positive cancer or HPV-positive intraepithelial precancerous lesions, a) at least human papillomavirus (HPV) E6 and E7 polypeptides, and a poxvirus vector encoding an immunostimulatory cytokine in b) an anti-PD-L1 antibody or antigen-binding fragment thereof The use of a combination of The first administration of the poxvirus occurs 5 to 10 days after the first administration of the anti-PD-L1 antibody. and subsequent administration of the poxvirus and anti-PD-L1 antibody. It also relates to use.

[0027] In said combination, method or use, said poxvirus is preferably a vaccine Vaccinia virus, more preferably modified vaccinia virus Ankara (MVA), Preferably, the non-oncogenic E6 and E7 polypeptides of membrane-associated HPV (preferably HPV-16) are Most preferably, the peptide encodes human interleukin 2 (IL-2). The poxvirus contains membrane-associated HPV-16 non-oncogenic E6 and E7 polypeptides. The poxvirus administered to the subject is an MVA virus that encodes human IL-2. Each dose of rux is preferably 3 x 10 7 From 7 x 10 7 pfu, and more preferably about 5×10 7 Each dose of poxvirus administered to a subject is preferably It is administered below.

[0028] In the combination, method or use, the anti-PD-L1 antibody or its antigen-binding The antibody fragment preferably mediates antibody-dependent cellular cytotoxicity (ADCC). The D-L1 antibody or antigen-binding fragment thereof preferably comprises the antigens of SEQ ID NOs: 1, 2, and 3. A heavy chain comprising three complementarity determining regions having the amino acid sequences of SEQ ID NOs: 4, 5, and 6. and more preferably, a light chain comprising three complementarity determining regions having the amino acid sequences of the anti-PD-L1 antibody comprises a heavy chain having the amino acid sequence SEQ ID NO: 7 or 8 and a Most preferably, the anti-PD-L1 antibody comprises a light chain having an amino acid sequence of Each dose of the anti-PD-L1 antibody or antigen-binding fragment thereof is preferably about The anti-PD-L1 antibody or its antigen-binding activity is 10 mg / kg or about 800 mg. Each dose of the fragment is preferably administered intravenously, more preferably by intravenous infusion. will be done.

[0029] Targeted therapeutic applications include HPV-positive cancers, preferably HPV-positive oropharyngeal cancers; Cervical cancer, vaginal cancer, anal cancer, vulvar cancer, penile cancer, mucosal cancer, or non-melanoma skin cancer Treatment of skin cancer or HPV-positive intraepithelial precancerous lesions. is preferably HPV-16 positive, and the cancer is particularly HPV-16 positive head and neck squamous cell carcinoma. The targeted cancers may also be epithelial carcinomas (HPV-16+ SCCHN). Preferably, recurrent and / or metastatic HPV-positive cancer (preferably recurrent and / or metastatic is metastatic HPV16-positive cancer, most preferably recurrent and / or metastatic HPV16-positive SCCHN).

[0030] (a) at least the human papillomavirus (HPV) E6 and E7 polypeptides; and (b) poxvirus vectors encoding immunostimulatory cytokines and anti-PD-L The antibody combination may be provided in a single dosage form or in separate unit dosage forms. The first administration of the poxvirus is between 5 and 10 days after the first administration of the anti-PD-L1 antibody. 10 days prior to the administration of the poxvirus and anti-PD-L1 antibody. Preferably, the poxvirus and The subsequent administration of the anti-PD-L1 antibody is administered until disease progression. The substances are administered according to the following administration scheme:

[0031] a) 3 x 10 of the poxvirus 7 From 7 x 10 7 The first dose of pfu was administered subcutaneously. Then, 3 x 10 7 From 7 x 10 7 Subsequent doses of pfu are in, Once a week for 6 weeks Once every two weeks until the sixth month, and Subsequent poxvirus doses every 12 weeks It is administered subcutaneously, b) a first dose of about 10 mg / kg or about 800 mg of anti-PD-L1 antibody is administered to said patient; is administered intravenously 5-10 days after the poxvirus dose, followed by approximately 10 mg / kg or subsequent anti-PD-L1 antibody doses of approximately 800 mg administered intravenously every 2 weeks until disease progression. can be.

[0032] Particularly preferred embodiments according to the present invention are as follows. a) the poxvirus is a member of the HPV-16 membrane-bound non-oncogenic E6 and E7 polynucleotides MVA virus encoding a peptide and human IL-2, b) the anti-PD-L1 antibody is avelumab; c) the poxvirus and anti-PD-L1 antibody are administered according to the following administration scheme: . i) Membrane-bound HPV-16 non-oncogenic E6 and E7 polypeptides and human IL-1 MVA virus encoding -2 was detected in 5 × 10 7 pfu dose once a week for 6 weeks, It is then administered subcutaneously every 2 weeks until 6 months, then every 12 weeks until disease progression. ii) avelumab at approximately 10 mg / kg every 2 weeks starting on day 8 until disease progression or by intravenous infusion at a dose of about 800 mg.

[0033] The combination, method or use according to the present invention is preferably used in the treatment of cancer in a subject undergoing treatment. or induce a positive immune response against precancerous lesions. The combination for use according to the invention preferably comprises: Circulating HPV E6 and / or E7 polypeptide-specific CD8 and and / or increase the proportion of CD4 T cells; and / or Reduces the proportion of circulating regulatory T cells.

[0034] Similarly, in tumor tissue, the combination, method, or use according to the present invention preferably is · to increase tumor tissue infiltration by CD8 and / or CD 4 T cells specific for the HPV E6 and / or E7 polypeptide, and / or · to reduce tumor tissue infiltration by regulatory T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] [Figure 1] It is a figure showing the change in tumor size during combination therapy. (A) Best change in tumor size: % change from baseline on day 43 (change from the calculated total value) in individual patients treated with a combination of 10 mg / kg of avelumab and TG4001 of DL1 (5 × 106 pfu, solid gray) or DL2 (5 × 107 pfu, hatched gray) according to the dosing scheme described in Example 1. Partial response (PR) according to RECIST v1.1 is indicated by *(B). Change in the individual long axis direction: % change from baseline (change from the calculated total value) in individual patients treated with a combination of 10 mg / kg of avelumab and TG4001 of DL1 (5 × 106 pfu, dotted line) or DL2 (5 × 107 pfu, solid line) at various time points shown according to the dosing scheme described in Example 1. Thresholds for progression (PD: ≧ +20%), stable (-30% < SD < +20%), and partial response (PR: ≦ -30%) are shown. The cancer type of HPV-16 positive cancer in each patient is also shown. [Figure 2] It is a figure showing the stimulation of T cell immunity by combination therapy. CD8 / CD3 ratio (A) or Treg (CD4 Foxp3) / CD8 ratio (B) of tumor immune infiltrates at baseline and on day 43 based on immunohistochemical (IHC) staining of tumor FFPE specimens from individual patients. [Figure 3]

[0023] Figure 1 shows an analysis of gene expression changes in tumor tissue during treatment. Expression of a panel of 770 genes related to immune response was assessed at baseline and after treatment (day 43). Volcano plots of post-treatment vs. pre-treatment changes in T cell activation (A), cytotoxic cells (B), pathogen defense (C), and NK cell function (D) gene expression. In each volcano plot, black dots correspond to genes in the indicated category. [Figure 4]

[0023] Figure 1 shows an analysis of gene expression changes in tumor tissue during treatment. (A) Representation of gene categories included in the previously described gene signatures Immunosign® 15 and Immunosign® 21 (Galon et al., Immunity (2013) 39(1): 11-26; Marabelle et al., Society for Immunotherapy of Cancer (SITC) 32nd Annual Meeting & Pre-Conference Programs (SITC 2017) on November 8-12, 2017 at the Gaylord National Hotel & Convention Center in National Harbor, Maryland. Poster P250). (B) Volcano plots of changes in Immunosign® 15 (B) and Immunosign® 21 (C) genes. In each volcano plot, black dots correspond to genes in the indicated signature. [Figure 5] Figure 1 shows changes in immune infiltrate in patient 0101006. (A) CD3, CD8, or CD4 Foxp3 T cells / mm2 in the tumor immune infiltrate at baseline and day 43. (B) Percentage of CD8 T cells in the vicinity of PD-L1-expressing cells according to the distance between PD-L1-expressing cells and CD8 T cells in μm. [Figure 6-1]Analysis of gene expression changes in tumor tissue during treatment of patient 0101006. Expression of genes associated with antigen processing and presentation (A), genes associated with protective responses to viruses (B), genes for Toll-like receptors (C), and Immunosign® 21 genes (D). [Figure 6-2] Same as above. [Figure 7] Figure 1 shows the effect of disease / patient characteristics on objective response rate (ORR). For each characteristic, the odds ratio (OR), 95% confidence interval, and p-value are shown. Genitalia = vulva / vagina. An OR greater than 1 indicates that the presence of the characteristic is associated with a worse ORR, while an OR less than 1 indicates that the presence of the characteristic is associated with a better ORR. A p-value <0.05 indicates that the characteristic is significantly associated with either a worse or better ORR. Only one characteristic (the presence of liver metastases) was found to be significantly associated with a worse ORR (boxed). [Figure 8] Figure 1 illustrates the impact of disease / patient characteristics on progression-free survival (PFS). For each characteristic, the hazard ratio (HR), 95% confidence interval, and p-value are shown. Genitalia = vulva / vagina. An HR greater than 1 indicates that the presence of the characteristic is associated with a worse PFS, while an HR less than 1 indicates that the presence of the characteristic is associated with a better PFS. A p-value <0.05 indicates that the characteristic is significantly associated with a worse or better PFS. Two characteristics (presence of liver metastases and anal cancer) were found to be significantly associated with a worse PFS, while one characteristic (involved lymph nodes) was found to be associated with a better PFS (all three characteristics are boxed). [Figure 9]Figure 1 shows best change in tumor size for 23 pooled patients without liver metastases from Phase Ib and Phase II. % best change from baseline (change from the total calculated value) for individual patients treated with avelumab at 10 mg / kg in combination with TG4001 according to the dosing scheme described in Example 1. Progression (PD) is shown in black, stable disease (SD) in light gray, partial response (PR) in dark gray, and complete response (CR) in medium gray. [Figure 10] Figure 1 shows best change in tumor size for 9 patients with liver metastases from pooled Phase Ib and Phase II studies. % best change from baseline (change from total calculated value) for individual patients treated with avelumab at 10 mg / kg in combination with DL2 (5 x 10 pfu) of TG4001 according to the dosing scheme described in Example 1. Progression (PD) is shown in black, stable disease (SD) is shown in light gray. [Figure 11] Volcano plot showing fold change in yes vs. no (liver lesions) vs. p-value of the linear model in the baseline (visit) population. Names are shown for the most highly differentially expressed genes. DETAILED DESCRIPTION OF THE INVENTION

[0036] General definition The terms "a" and "an" are used interchangeably unless the context indicates otherwise. Throughout this application, compounds or steps to which they refer include "at least one" or "at least one" combination of these. "one," "at least a first," "one or more," or "plurality" means Therefore, the term is used to refer to the compound or step that is being referenced. and two or more of the compounds or steps referenced. Includes both.

[0037] The terms "about" or "approximately" are used interchangeably herein to refer to a given value or This means within 5%, preferably within 4%, and more preferably within 2% of the range. In the context, in each case where "about X" refers to an approximate value of X, that value is equal to X. Embodiments are also contemplated in the context of the present invention.

[0038] Whenever the term "and / or" is used herein, it means "and" "or," and "any and all other combinations of the elements connected by said terms." For example, "recurrent and / or metastatic" means recurrent or metastatic, or or recurrent and metastatic.

[0039] As used herein, the term "combination" refers to two or more entities (e.g., at least any possible combination of poxvirus and anti-PD-L1 antibodies as described herein In particular, "combination" refers to (i) anything physically, chemically, or otherwise joined together. or composed of two or more regulated components mixed and produced as one entity (ii) Products packaged together in one package or as a unit and containing pharmaceuticals and device products, device and biological products, or biological and drug products (iii) Two or more separate products, depending on their research plan or proposed tagging; Intended for use only with approved, individually specified drugs, devices, or biological products Separately packaged drugs, devices, or biological products that are or both are required to achieve the desired effect, and approval of the proposed product, e.g. For example, a change in intended use, dosage form, strength, route of administration, or a significant change in dosage. The labeling of approved products may need to be changed to reflect the or (iv) a separate, individually designated investigational product, device, or Any separately packaged investigational device or biological product intended for use only with A drug, device, or biological product that achieves its intended use, application, or action. It can refer to an investigational drug, device, or biological product that requires both to be .

[0040] As used herein, the terms "combination therapy," "in combination with," or "in conjunction with" The term refers to the combination of at least two different treatment modalities (i.e., compounds, components, targets, etc.). any form of combination, parallel, simultaneous, sequential, or intermittent use with a therapeutic agent (e.g., a therapeutic agent or therapeutic agent) Thus, the term refers to a therapeutic treatment that is administered to a subject prior to the administration of the other treatment modality. Refers to the administration of one treatment modality during or after the other. The therapeutically active modalities may be administered together (e.g., in the same or in separate compositions, formulations, or unit dosage forms) or separately (e.g., in separate compositions, formulations, or unit dosage forms). on the same day or different days according to the appropriate administration protocol for the composition, formulation, or unit dosage form. on any date, in any order), as prescribed or prescribed by a healthcare provider or in accordance with a regulatory authority Generally, each treatment modality is administered in a manner and dosage regimen appropriate for that treatment modality. Optionally, the combination therapy may be administered at a dose and / or time schedule determined for the therapy. In addition, the combination therapy presented herein may be used in combination with other types of treatment. For example, other anti-cancer treatments such as chemotherapy, surgery, radiation therapy ( radiation), and / or hormone therapy, and other treatments relevant to the current standard of care for the subject. The treatment may be selected from the group consisting of:

[0041] As used herein, "including" (as well as "including" and any form of "including," such as "including"); "having" (as well as "having" and any form of "have" such as "have"); "include" (as well as "included" and and any form of "include" such as "contain" or "contain" The term "contains" (including any form of "contains") refers to a product, When used to define compositions and methods, they are non-limiting and do not include additional recited terms. Thus, a polypeptide may be a polypeptide of any amino acid sequence. If the sequence can be part of the final amino acid sequence of the polypeptide, "Comprising" a sequence. "Consisting essentially of" means excluding any essential other component or sequence. Therefore, the ingredients listed are essentially the same as those listed in the table. The resulting composition does not exclude trace amounts of contaminants and pharmaceutically acceptable carriers, but may contain other active ingredients. Polypeptides are those in which a given amino acid sequence is optionally When present together with additional amino acid residues, such an amino acid sequence is "essentially "Consists of" means excluding more than trace amounts of other ingredients or steps. For example, a polypeptide is defined as any amino acid sequence other than that recited. A polypeptide "consists of" an amino acid sequence if the polypeptide does not contain any amino acids. In this context, when a product or process or use is said to "comprise" something, In each case, the product or process is essentially or consists of the same thing. Embodiments are also contemplated in the context of the present invention.

[0042] The terms "mutant," "analog," or "variant" are used herein. If so, the component (polypeptide) exhibits one or more modifications relative to its unmodified counterpart. It refers to the substitution, insertion, or deletion of one or more nucleotides / amino acid residues. Any alteration, including deletions and / or deletions, is contemplated. If any residues are listed, they may refer to consecutive and / or non-consecutive residues. Mutations can be made by site-directed mutagenesis (e.g., A. Brück, Les Ullis, France). mersham, using the Sculptor™ in vitro mutagenesis system) , PCR mutagenesis, DNA shuffling, and chemical synthesis techniques (e.g., synthetic nucleases can be introduced in several ways known to those skilled in the art, such as by providing an acid molecule. It can be at least 80%, preferably at least 85%, more preferably at least 85% identical to the sequence of the unmodified counterpart. Preferably, at least 90%, more preferably at least 91%, at least 92% %, at least 93%, at least 94%, at least 95%, at least 96%, at least Some degree of identity with at least 97%, at least 98%, or at least 99% Analogs that retain sequence identity are preferred. In general, the term "identity" refers to the degree to which two between the polypeptide or nucleic acid sequences of the amino acid and the amino acid or the nucleotide and the nucleotide The percentage of identity between two sequences is calculated based on the best global alignment ( That is, the gap that needs to be introduced to obtain the optimal alignment of both full-length sequences. The number of identical gaps shared by the sequences is taken into account, along with the length of each gap. Various computer programs and mathematical algorithms are used in the art to determine percent identity between amino acid or nucleic acid sequences. It is possible.

[0043] The terms "nucleic acid," "nucleic acid molecule," "polynucleotide," and "nucleotide sequence" The terms are used interchangeably and refer to polydeoxyribonucleotides (DNA) (e.g., cDNA). A, genomic DNA, plasmids, vectors, viral genomes, isolated DNA, probes primers, and any mixture thereof) or polyribonucleotides (RNA ) (e.g., mRNA, antisense RNA, siRNA) or mixed polyribo-polynucleotides They are defined as polymers of any length of hydroxyribonucleotides. They may be single-stranded or The polynucleotide may be double-stranded, linear or circular, natural or synthetic, modified or unmodified. Polynucleotides may also include non-naturally occurring nucleotides. It may be interrupted by non-nucleotide components.

[0044] The terms "polypeptide," "peptide," and "protein" refer to a group of peptide bonds. A polymer of amino acid residues containing at least nine amino acids linked via The polymer may be linear, branched, or cyclic and may be composed of naturally occurring amino acids and / or may contain amino acid analogs and may be interrupted by non-amino acids. As a general guideline, if an amino acid polymer is more than 50 amino acid residues, it is a poly It is preferred to refer to it as a peptide or protein, while it may be less than 50 amino acids in length. If it is below, it is called a "peptide."

[0045] The term "subject" generally refers to any product or method of the present invention. refers to an organism that is beneficial to or that can be beneficial to the organism. Typically, this organism is a mammal. Preferably, the subject is suffering from an infectious disease caused by or associated with a pathogenic organism. diagnosed with or at risk of having a pathological condition such as a disease or proliferative disorder such as cancer The terms "subject" and "patient" are used interchangeably when referring to a human organism. The term "human ovarian syndrome" can be used interchangeably and includes both males and females. , young adult, or adult.

[0046] The terms "treatment" and "therapy" as used herein mean improving a health problem. used with the intent to cure and / or alleviate disease and / or symptoms1 Refers to a set of hygienic, pharmacological, surgical, and / or physical measures. Both therapeutic methods aim to maintain and / or restore the health of an individual or animal. and are included in the terms "treatment" and "therapy." Regardless, the administration of suitable medications to alleviate and / or cure health problems is within the scope of the present invention. Within the scope of the text, it should be interpreted as a form of treatment or therapy.

[0047] Poxvirus vectors The combination, method or use according to the present invention comprises, as a first component, at least one human Papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines It contains a poxvirus vector encoding the gene.

[0048] Therefore, "poxvirus vector" or "poxviral vector" The term "poxvirus" refers to a vector that contains at least one element of a poxvirus genome and is Nucleic acid vectors (e.g., DNA pox) that may be packaged in viral particles. poxvirus vectors) and the poxvirus particles resulting therefrom. The terms "poxvirus," "poxvirion," and "poxvirus" should be understood broadly. The terms "poxvirus vector particle" and "poxvirus vector particle" refer to the The nucleic acid vector is transferred to suitable cells or host cells under suitable conditions to allow the production of viral particles. It is used interchangeably to refer to the poxvirus particles formed when a virus is transduced into a cell line or a cell line. The term "infectious" refers to the ability to infect and enter a host cell or subject. Refers to the ability of poxvirus vectors. Poxvirus vectors are replication-competent. or replication selective (e.g., replicate better or selectively in a particular host cell) They can be replication-deficient or genetically engineered to be replication-impaired. It can be genetically disabled.

[0049] Types of poxviruses As used herein, the term "poxvirus" refers to a virus of the Poxviridae family. It refers to viruses belonging to the family of viruses, among which chordopoxviruses targeting vertebrate hosts. The subfamily Chordopoxvirinae is preferably the Orthopoxvirus, Capsule virus, or the Chortopoxvirinae. Genus Lipoxvirus, genus Avipoxvirus, genus Parapoxvirus, genus Leporipoxvirus The virus includes several genera, such as the genus S. poxvirus, S. swinepoxvirus, and Swinepoxvirus. In this context, orthopoxviruses, as well as canarypoxviruses (e.g. avian pox viruses, including ALVAC (e.g., ALVAC) and fowlpox viruses (e.g., FP9 vectors) A virus is preferred.

[0050] In a preferred embodiment of the combination, method or use according to the invention, a poxvirus belongs to the genus Orthopoxvirus, more preferably vaccinia virus (VV) Vaccinia virus belongs to the species Vaccinia virus has a linear double-stranded DNA genome approximately 200 kb in length It is a large, complex enveloped virus, and its genome is designed to allow the virus to grow independently of the host cell machinery. It encodes many viral enzymes and factors that enable it to replicate. There are several infectious virus particles present, which remain in the cytosol of infected cells until lysis. Intracellular IMV (representing the intracellular mature virion) surrounded by a single lipid envelope and The double-enveloped EEV (extracellular enveloped virion) buds out from the infected cell. (representing the

[0051] Particularly suitable poxviruses in the context of the present invention are those expressing highly attenuated The IFN-type 1 response generated during infection was significantly higher than that of non-attenuated vectors. and availability of its genome sequence (e.g., Genomics under accession number U94848). nbank), it is MVA (Modified Vaccinia Virus Ankara).

[0052] HPV E6 and E7 polypeptides The combination, method or use according to the present invention is directed to a poxvirus (preferably VV, more preferably Preferably, the MVA comprises at least the human papillomavirus (HPV) E6 and E7 polypeptides. It encodes polypeptides as well as immunostimulatory cytokines.

[0053] Over 100 HPV genotypes have been identified, which are classified according to their oncogenic potential: They are classified into "low risk" (LR) and "high risk" (HR) serotypes. V causes benign tumors in infected subjects, whereas HR-HPV carries a high risk of malignant progression. The gene products encoded by E6 and E7 of the HR HPV genotypes are likely These viral proteins bind to the cellular tumor suppressor gene product p53 and retinoblastoma (Rb). It is involved in the oncogenesis of infected cells through protein binding (Howley, 1996, Papillomavir uses and their replication, p 2045-2076. In BN Fields, DM Knipe and PM How ley (ed.), Virology, 3rd ed. Lippincott-Raven Press, New York, NY. Amino acid residues involved in binding of unmodified HPV-16 E6 polypeptide to p53 residues 118 to 122 (+1 is the first Met residue, or preferably The first Met residue is clearly defined as residues 111 to 115 (starting from the second Met residue in the (Crook et al., Cell (1991) 67, 547-556), and the unmodified HPV-16 E The amino acid residues involved in binding of the 7 polypeptide are located at residues 21 to 26. (Heck et al., Proc. Natl. Acad. Sci. USA (1992) 89, 4442-4446).

[0054] In the context of the present invention, poxviruses (preferably VV, more preferably MVA) preferably encodes at least the E6 and E7 polypeptides of HR-HPV. The HR-HPV is preferably HPV-16, HPV-18, HPV-30, HPV -31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, H PV-52, HPV-56, HPV-58, HPV-59, HPV-66, HPV-68 , HPV-70, and HPV-85, more preferably HPV-16 and HPV- 18, and most preferably, the HR-HPV is HPV-16.

[0055] Sources of papillomavirus include, but are not limited to, biological samples (e.g., , biological samples, tissue sections, biopsy specimens collected from subjects exposed to papillomavirus, and tissue culture), cultured cells (e.g., CaSki cells available from ATCC), and Recombinant material available in contract laboratories or commercial catalogs, or described in the literature The nucleotide sequences of several papillomavirus genomes and the encoded The amino acid sequences of the polypeptides involved are available in the literature and in specialized data banks (e.g. For general information, the HPV-16 genome is available at The accession numbers are NC_01526 and K02718, and HPV-18 is NC_001357 and and X05015, HPV-31 is J04353, and HPV-33 is M12732. HPV-35 is NC_001529, HPV-39 is NC_001535, and HPV- 45 is X74479, HPV-51 is NC_001533, and HPV-52 is NC_0 01592, HPV-56 is X74483, HPV-58 is D90400, HPV -59 is NC_001635, HPV-68 is X67160 and M73258, H PV-70 is listed as U21941 and HPV-85 is listed as AF131950 in Genbank. It has been done.

[0056] For illustrative purposes, the amino acid sequences of the unmodified HPV-16 E6 and E7 polypeptides are shown below. They are shown in SEQ ID NOs: 10 and 11, respectively.

[0057] As defined above in relation to the term "polypeptide," "papillomavirus "Polypeptide" refers to unmodified and modified papillomavirus polypeptides and their peptides. In particular, the present invention relates to polypeptides that are modified to have undesirable properties, such as phenotypes, which may be undesirable, e.g., unmodified HPV E6 and E7 if they exert any of the following effects: oncogenic or transforming properties, cytotoxicity, etc. Polypeptides and their analogs (e.g., fragments thereof, such as peptides, and modified For example, the use / expression of HPV E6 and E7 polypeptides in tumors. Non-oncogenic, showing reduced ability to bind p53 and Rb, respectively, to avoid toxicity Analogs can be used or expressed.

[0058] Suitable E6 polypeptides for use in the present invention bind to the cellular tumor suppressor gene product p53. The term "non-oncogenic E6 polypeptide" encompasses non-oncogenic mutants that are unable to bind to the E6 polypeptide. Examples are described in the art (e.g., WO 1999 / 03885, (See FRET). Preferred modifications in this context include the From approximately position 118 to approximately position 122 (+1 is the native HPV-16 E 6) one or more amino acid residues located at the first methionine residue of the polypeptide The deletions include residues 118–122 (CPEEK) in HPV-16 E6 (e.g., For example, see SEQ ID NO: 12) or residues 113-117 in HPV-18 E6 ( Deletions of NPAEK are particularly preferred.

[0059] Suitable E7 polypeptides for use in the present invention bind to the cellular tumor suppressor gene product Rb. The non-oncogenic E7 polypeptide includes non-oncogenic mutants that cannot bind to the E7 polypeptide. Representative examples are described in the art (e.g., WO 1999 / 03885). (See pamphlet). Preferred modifications in this context include: from about position 21 to about position 26 (+1 is the unmodified HPV-16 E7 position) The deletion of one or more amino acid residues at positions (representing the first amino acid of the polypeptide) Rarely, residues 21-26 (DLYCYE) in HPV-16 E7 (e.g., SEQ ID NO: 13) or deletion of residues 24-28 (DLLCH) in HPV-18 E7 Loss is particularly preferred.

[0060] HPV (preferably HPV-16) E6 and / or E7 polypeptides for use in the present invention The polypeptide is membrane anchored and efficiently expressed on the surface of the expression host cell. This may be further modified to enhance effective membrane presentation of HPV (preferably HPV-16) E6 and / or E7 polypeptides containing signal peptides and membrane-bound forms This may be achieved by fusing the peptide to a target molecule. Such peptides are well known in the art. Briefly, signal peptides are generally used to signal proteins that are either membrane-presented or secreted. It is present at the N-terminus of polypeptides that initiate their passage into the endoplasmic reticulum (ER). The peptides contain 15 to 35 essentially hydrophobic amino acids, which are then specifically It is removed by a specific ER-localized endopeptidase to yield the mature polypeptide. Membrane-bound peptides are usually highly hydrophobic in nature and anchor the polypeptide to the cell membrane. (e.g., Branden and Tooze, 1991, in Introduction to Proteins (See, for example, NY Garland, p. 202-214, n Structure). The selection of signal peptides and membrane-bound peptides that can be used is enormous. glycoprotein, HIV viral envelope glycoprotein, or measles virus F protein Any secreted or membrane-bound polypeptide (e.g., cellular or cellular) such as a protein The signal peptide may be obtained independently from a target protein (viral polypeptide) or may be synthetic. The preferred site for peptide insertion is the N-terminus downstream of the translation initiation codon, and Optionally, the encoded polynucleotide may be C-terminal, e.g., immediately upstream of the stop codon. A linker is used to connect the signal peptide and / or membrane-binding peptide to the polypeptide. Peptides can be used.

[0061] The poxvirus of the combination therapy according to the present invention is preferably HPV (preferably HPV -16) membrane-bound non-oncogenic E6 and E7 polypeptides and human interleukin- It encodes interleukin-2 (IL-2).

[0062] In a particularly preferred embodiment, the combination for use according to the invention is Preferably, the HPV E6 polypeptide is encoded by VV, more preferably MVA. The HPV-16 E6 variant has a deletion of residues 118 to 122 (CPEEK). , a membrane-bound non-oncogenic variant of HPV-16 E6, in particular the amino acid sequence SEQ ID NO: 12 In another particularly preferred embodiment, the HPV-16 E6 variant according to the present invention is The combination for use in the poxvirus (preferably VV, more preferably MVA) The HPV E7 polypeptide encoded thereby is identical to residue 21 in HPV-16 E7. A membrane-bound, non-oncogenic variant of HPV-16 E7 with a deletion of 26 (DLYCYE) Particularly preferred is the HPV-16 E7 variant of amino acid sequence SEQ ID NO: 13. In a preferred embodiment, the combination for use according to the invention is The HPV E6 polypeptide encoded by the HPV VV, more preferably the HPV VV, is HPV with a deletion of residues 118 to 122 (CPEEK) in PV-16 E6 -16 membrane-bound non-oncogenic variant of E6, in particular HPV with amino acid sequence SEQ ID NO: 12 -16 E6 variant and poxvirus of the combination for use according to the invention (preferably VV, more preferably MVA) Tid has a deletion of residues 21 to 26 (DLYCYE) in HPV-16 E7. A membrane-bound non-oncogenic variant of HPV-16 E7, in particular the amino acid sequence SEQ ID NO: 13 It is the HPV-16 E7 variant.

[0063] The poxvirus vector contained in the combination, method or use according to the present invention At least the human papillomavirus (HPV) E6 and E7 polypeptides encoded by Suitable promoters for driving expression of the ribozyme and immunostimulatory cytokines are preferred. or poxvirus promoters, such as vaccinia virus promoter 7.5 K, H5R, Tk, p.28, p.11, or K1L. Late and early promoters Synthetic promoters are also suitable, as are chimeric promoters of the promoters. Such promoters are well known in the art. Preferably, HPV E6 and Expression of both E7 polypeptides is under the control of the varicella p7.5 promoter, resulting in immunostimulatory activity. Expression of cytokines (e.g., human IL-2) is under the control of the smallpox pH5R promoter. It will be placed.

[0064] Immunostimulatory cytokines At least human papillomavirus (HPV) E6 and E7 polypeptides (preferably In addition to those mentioned above, the poxviruses of the combinations, methods or uses according to the invention The fluoxetine (preferably VV, more preferably MVA) may further contain immunostimulatory cytokines. Load.

[0065] As used herein, the term "immunostimulatory cytokine" refers to a specific or Refers to cytokines that have the ability to nonspecifically stimulate the immune system. are known in the art for their ability to exert immunostimulatory effects. Although not intended to be limiting, examples of suitable immunostimulatory cytokines in the context of the present invention include: interleukins (e.g., IL-2, IL-6, IL-12, IL-15, IL- 24), chemokines (e.g., CXCL10, CXCL9, CXCL11), interferons IFNα, IFNβ, IFNγ), tumor necrosis factor (TNF), colony Stimulatory factors (e.g., GM-CSF, C-CSF, M-CSF), growth factors (transforming TGF, fibroblast growth factor FGF, vascular endothelial growth factor VEGF, and Preferably, the immunostimulatory cytokine is an interleukin or or colony stimulating factors (e.g., GM-CSF). More preferably, immunostimulatory factors The cytokine is interleukin 2 (IL-2), most preferably human IL-2. be.

[0066] Preferred poxviruses A preferred poxvirus of the combination, method or use according to the invention is a membrane-bound non- Encoding oncogenic HPV-16 E6 and E7 polypeptides and human IL-2 MVA virus, more preferably MVA 1, which is identified in the International Publication No. 2001 / 000240 ... It is represented by TG4001, which is described in brochure no. 999 / 03885.

[0067] Anti-PD-L1 antibody or antigen-binding fragment thereof The combination, method or use according to the invention may further comprise, as a second component, an anti-PD-L1 antibody or or an antigen-binding fragment thereof.

[0068] Antibodies or antigen-binding fragments thereof The term "antibody" refers to an antibody that contains at least one antigen located in the variable region of an immunoglobulin molecule. antigens, e.g., carbohydrates, polynucleotides, lipids, polypeptides, via the original recognition site Context of the invention In the present specification, "antibody" (or "Ab") is used in the broadest sense and includes naturally occurring antibodies, and full-length antibodies, or PD-L1 (hence, by humans, including functional fragments or analogs thereof (which retain the original binding moiety) The antibodies used in the present invention may be of any origin, e.g., human, Antibodies may be human, animal (e.g., rodent or camelid antibodies), or chimeric. , of any isotype (e.g., IgG1, IgG2, IgG3, IgG4, I In addition, the antibody may be glycosylated or non-glycosylated. As long as it shows binding specificity to an antigen such as PD-L1, it can be called an antibody. The term also includes bispecific or multispecific antibodies. The term "antibody" refers not only to intact polyclonal or monoclonal antibodies, but also to other antibodies. Unless otherwise specified, any antigen-binding fragment that competes for specific binding with the intact antibody. fragments or antibody fragments, fusion proteins (e.g., antibody-drug conjugates) containing the antigen-binding portion conjugates), any other modified immunoglobulin molecule containing an antigen recognition site, poly Antibody compositions with epitope specificity, and multispecific antibodies (e.g., bispecific antibodies) are also available. However, intact, i.e., unfragmented, monoclonal antibodies are preferred. .

[0069] For purposes of explanation, a full-length antibody consists of two heavy (H) chains interconnected by a disulfide bond. and two light (L) chains. Each heavy chain contains a heavy chain variable region (VH), and three CH1, CH2, and CH3 domains (optionally CH1 Each light chain is composed of a heavy chain constant region (CH) with a hinge between CH1 and CH2. Each VH consists of a variable region (VL) and a light chain constant region containing one CL domain. The VL region contains three hypervariable regions named complementarity-determining regions (CDRs). Between them are more conserved regions called framework regions (FR). Each VH and VL has a FR1-CDR1-FR2-CDR2-FR3- It consists of three CDRs and four FRs in the order CDR3-FR4. The CDR regions of the light chain are generally determinative of binding specificity.

[0070] The CDRs of an antibody are determined by comparing the amino acids of its heavy and light chains with those of its amino acids, as determined by criteria known to those skilled in the art. CDRs are defined by their amino acid sequence. Various methods for determining CDRs have been proposed. The portion of the amino acid sequence of the heavy or light chain variable region of an antibody, defined as a CDR, is selected from the This description uses Oxford Molecular's AbM antibody model. All CDRs are defined according to the AbM definition used by the ring software (e.g. For example, the CDR sequence of avelumab in WO 2013 / 079174 (See

[0071] The antibody may be a monoclonal antibody, a human antibody, a chimeric antibody, a humanized antibody, and / or a human antibody. The constant region of an antibody may be human or may contain a human constant region. The constant region of an antibody may be a region that modifies the properties of the antibody. (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector or complement function. It can be, for example, mutated.

[0072] In the context of the present invention, it is preferred to use monoclonal antibodies. As used herein, "monoclonal antibody" refers to an antibody molecule having identical and unique antigen specificity. The antibody molecules present in this composition are and may vary, especially with respect to their glycosylation structure or their isoelectric point. likely all be encoded by the same heavy and light chain sequences and therefore It has the same protein sequence before any post-translational modifications (e.g., heavy chain Cleavage of C-terminal lysine, deamidation of asparagine residues, and / or deamidation of aspartic acid residues Certain differences in protein sequence related to isomerization of amino acid residues, etc., may still exist in the composition. Monoclonal antibodies can be produced using hybridoma technology, Alternatively, they can be produced by methods that do not involve hybridoma technology (e.g., recombinant methods). Human monoclonal antibodies are produced using a human immunoglobulin gene system rather than a mouse system. It can be produced using transgenic mice immunized with the antigen of interest. Splenocytes obtained from these transgenic mice were used to generate human protein-derived Hybridomas secreting human mAbs with specific affinity for epitopes were produced. To live.

[0073] For the treatment of human subjects, which is a preferred embodiment of the invention, anti-PD-L1 The antibody may be chimeric, humanized, or fully human, thereby eliminating the non-human portion of the anti-PD-L1 antibody. The antibody is a variable region or a portion thereof, For example, the CDRs may be generated in a non-human organism, such as a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the scope of the present invention. It is produced in a human organism, e.g., a rat or mouse, and then reduced in antigenicity in humans. Antibodies that have been modified, for example, within the variable framework or constant region, to reduce It is within the scope of the present invention.

[0074] As used herein, a "chimeric antibody" refers to an antibody that combines one or more elements of one species. and one or more elements of another species, e.g., human immunoglobulins. A chimeric antibody refers to a non-human antibody that contains at least a portion of the constant region (Fc) of a human antibody. It can be produced by recombinant DNA techniques known in the art.

[0075] As used herein, a "humanized antibody" refers to an antibody whose protein sequence is similar to that of a human antibody (such as a humanized antibody). modified to increase its similarity to that of a polypeptide (i.e., that which is naturally produced in humans) Antibodies are non-human (e.g., mouse, camel, rat, etc.) antibodies that are produced by the methods of the art. The humanized polypeptides can be prepared by methods known in the art. For example, the variable regions (especially the CDRs) can be humanized. The majority of residues in the human immunoglobulin are unaltered and correspond to those in non-human immunoglobulins. by substituting one or more residues in the FR region to resemble the immunoglobulin sequence. Monoclonal antibodies developed for human use can be humanized. For guidance, the number of these amino acid substitutions in the FR region is typically In another example, a humanized or CDR-grafted antibody may have at least 20 or fewer VH or VL. At least one or two, and generally all three recipient CDRs (immunoglobulin heavy chains) are involved. The antibody is a non-human CDR, in which the CDRs of ... At least a portion of the DRs may be replaced, or only some of the CDRs may be replaced. Human CDRs may be substituted. The number required for humanized antibody binding to anti-PD-L1 It is only necessary to replace the CDRs of the antibody. Preferably, the donor is a rodent antibody, e.g. For example, for rat or mouse antibodies, the recipient may use a human framework or human core. Typically, the immunoglobulins that provide the CDRs are The immunoglobulin that provides the framework is called the "acceptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent) immunoglobulin. The acceptor framework is a naturally occurring (e.g., human) framework. a work or consensus framework, or about 85% or more thereto, preferably These are sequences that are 90%, 95%, or 99% or more identical. The body can generate Cs in which one, two, or all CDRs of an immunoglobulin chain may be replaced. For example, the humanized antibodies of the present invention can be produced by CDR grafting or CDR replacement. US Patent Nos. 5,245,629 and 5,326,729 describe CDR grafting methods that can be used to prepare See specification No. 25,539.

[0076] As used herein, a "human antibody" refers to an antibody that contains constant regions (as with chimeric antibodies). Not only the FR and FR (as in humanized antibodies) are of human origin, but the entire heavy and light chains are human. Human antibodies refer to antibodies whose amino acid sequences are derived from human germline immunoglobulin sequences. For example, transgenic animals or human animals into which human germline immunoglobulin sequences have been inserted. The antibodies can be obtained from a human antibody library.

[0077] The term "antigen-binding fragment" of any antibody refers to the portion of the intact antibody that binds to the antigen. The antigen-binding fragment may contain the variable region that determines the antigenicity of the intact antibody. Antigen-binding fragments can be engineered for use in the combinations of the present invention. Representative examples include, but are not limited to, Fab, Fab', F(ab')2, dAb, Fd, This includes Fv, scFv, di-scFv, diabodies, and any other artificial antibody. For example, a "PD-L1-binding fragment" of any anti-PD-L1 antibody may bind to the antigen PD-L1. More specifically, the combinations, methods and compositions of the present invention are The following antigen-binding fragments of full-length anti-PD-L1 antibodies may be used: (i) Fab fragments are monovalent fragments consisting of the VL, VH, CL, and CH1 domains. This is represented by the following. (ii) F(ab')2 fragments have at least one disulfide bridge in the hinge region. It is typified by a bivalent fragment containing two Fab fragments linked by a bridge. (iii) Fd fragments consist of the VH and CH1 domains. (iv) An Fv fragment consists of the VL and VH domains of a single arm of an antibody. (v) dAb fragments consist of a single variable domain fragment (VH or VL domain). (vi) Single-chain Fv (scFv) consists of two domains of the Fv fragment, VL and VH. These are optionally fused together with a linker to create a single protein chain. It is. (vii) Any other artificial antibody.

[0078] Methods for preparing antibodies, fragments and analogs thereof are known in the art (e.g., For example, Harlow and Lane, 1988, Antibodies - A laboratory manual: Cold Spring Harbo (See, e.g., r Laboratory, Cold Spring Harbor, NY). In one embodiment, such antibodies The present invention relates to the use of PD-L1 antigen (preferably human PD-L1 antigen for human use) to induce inflammatory bowel disease in a host animal. Alternatively, such antibodies can be produced in hybridomas (e.g., Ko see Heller and Milstein, Nature (1975) 256: 495-7), recombinant techniques (e.g., They can be generated from peptide synthesis, enzymatic cleavage, or by other methods (e.g., using a nucleotide-display method). Antibody fragments can be produced by recombinant techniques as described herein. Fragments can be produced by enzymes such as papain, which produces Fab fragments, or pepsin, which produces F(ab')2 fragments. The analogs (or their derivatives) may be produced by proteolytic cleavage using any enzyme. fragments) can be generated by conventional molecular biology methods (PCR, mutagenesis techniques). If desired, such fragments and analogs can be synthesized in the same manner as whole antibodies. may be screened for functionality (e.g., by standard ELISA assays). stomach.

[0079] PD-1 and PD-L1 Programmed death 1 (PD-1) is a member of the immunoglobulin (Ig) gene superfamily. PD-1 is a member of the CD28 family. A 55 kDa type II protein expressed on activated B cells, T cells, and myeloid cells PD-1 is a transmembrane protein. In normal contexts, PD-1 mediates T cell proliferation during inflammatory responses. It acts by limiting the activity of steroid hormones, thereby protecting normal tissue from destruction. For PD-1, PD-L1 (programmed death-ligand 1) and PD-L2 ( Two ligands, PD-L1 and PD-L2, have been identified. It has been identified in 20-50% of cancers. The interaction between PD-1 and PD-L1 is a key factor in tumor-infiltrating lymphocytes. Reduced lymphocytes, reduced T-cell receptor-mediated proliferation, and immune evasion by cancer cells The full-length amino acid sequence of PD-1 is available in UniProtKB under accession number Q1511. It is available in 6.

[0080] The anti-PD-L1 antibody of the combination, method or use according to the invention is preferably human PD-L1 UniP also recognizes human PD-L1 and provides additional information about human PD-L1, including the known amino acid sequence. It is available in the rotKB database under accession number Q9NZQ7.

[0081] Functional characteristics of anti-PD-L1 antibodies The term "anti-PD-L1 antibody" refers to an antibody that blocks the binding of PD-L1 to PD-1. with sufficient affinity to be useful as a therapeutic agent targeting PD-L1. It refers to antibodies that can specifically bind to PD-L1 (e.g., avelumab). Anti-PD-L1 antibodies block the binding of PD-L1 expressed on cancer cells to PD-1. It means an antibody that

[0082] The term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to the activity of certain cytotoxic cells ( present on natural killer (NK) cells, neutrophils, and macrophages These cytotoxic effectors are activated by secreted Ig bound to Fc receptors (FcRs) -cells can specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins Antibodies arm cytotoxic cells and activate this mechanism. NK cells are the primary cells for mediating ADCC and are required for killing target cells. Humans express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRII. Fc expression on hematopoietic cells is discussed in Ravetch & Kinet, Annu Rev Immuno (1991) 9: 457-92, Table 3 on page 464. Therefore, anti-PD-L 1 antibody contains an ADCC-competent Fc region and promotes ADCC lysis of cancer cells. Therefore, the effectiveness of the current therapy can be improved by the combination, method and The anti-PD-L1 antibody of interest preferably mediates ADCC. In particular, functional F It is preferable to use a full-length antibody containing the Fc region. The Fc region further improves ADCC capacity. It can be modified (at the amino acid or glycosylation level) to (Such modifications include, in particular, one or more modifications in the Fc, which are well known in the art. (including multiple substitutions and / or reduced fucosylation). Such ADCC-mediated anti-PD-L1 antibodies are not toxic and do not show increased toxicity.

[0083] Structural features of anti-PD-L1 antibodies Monoclonal antibodies that bind to human PD-L1 and are useful in combinations for use in the present invention Examples of the compounds are listed in WO 2007 / 005874 and WO 2010 / 03 Pamphlet No. 6959, International Publication No. 2010 / 077634, International Publication Pamphlet No. 2010 / 089411, Pamphlet No. 2013 / 019906 Lett, International Publication No. 2013 / 079174, International Publication No. 2014 / 10 No. 0079, International Publication No. 2015 / 061668, and U.S. Patent No. 8,552,154, U.S. Patent No. 8,779,108, and and U.S. Patent No. 8,383,796, including, but not limited to, , specific anti-human PD-L1 antibodies useful as PD-L1 antibodies in combinations for use in the present invention D-L1 monoclonal antibodies include, for example, avelumab (MSB0010718C), Urvalumab (MEDI4736, triple mutation to ablate ADCC) atezolizumab (MPLDL), an engineered IgG1 kappa monoclonal antibody 3280A), MPDL3280A (IgG1 engineered anti-PD-L1 antibody), and B Includes MS-936559 (anti-PD-L1 fully human IgG4 monoclonal antibody).

[0084] Avelumab and atezolizumab have been shown to be effective against HIV-1-associated leukemia (HIV-1-associated leukemia) because they have unmutated Fc regions. It is unique among currently available anti-PD-L1 antibodies in that it is a fully human IgG. Therefore, avelumab inhibits antibody-dependent cytotoxicity, which has been shown to mediate ADCC. Contains an ADCC-competent Fc region (Boyerinas et al., Cancer Immun ol Res. (2015) 3(10):1148-1157). Antibodies containing ADCC-competent Fc regions have been shown to be effective against cancer. By promoting ADCC lysis of cells, the efficacy of current therapies may be improved.

[0085] In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 1 (antibody, Velumab H-CDR1: SYIMM), SEQ ID NO: 2 (avelumab H-CDR2: SIYP SGGITFYADTVKG), and SEQ ID NO: 3 (avelumab H-CDR3: IKLG a heavy chain comprising three complementarity-determining regions having the amino acid sequence of SEQ ID NO: 4 (avelumab L-CDR1: TGTSSDVGGYNYVS), SEQ ID NO: 5 (avelumab avelumab L-CDR2: DVSNRPS), and SEQ ID NO: 6 (avelumab L-CDR3: SS and a light chain comprising three complementarity-determining regions having the amino acid sequence: YTSSSTRV. The CDR region is known to be particularly involved in antigen recognition, so such anti-PD The PD-L1 antibody or its antigen-binding fragment has similar binding to PD-L1 as avelumab. It is predicted that this will happen.

[0086] It has been frequently observed that the C-terminal lysine (K) of the heavy chain is cleaved during antibody production. This modification does not affect antibody-antigen binding. Therefore, some preferred embodiments In this case, the anti-PD-L1 antibody is SEQ ID NO: 7 (avelumab heavy chain:

[0087] [ka] or SEQ ID NO: 8, in which lysine (K) is present

[0088] [ka] and a heavy chain having the amino acid sequence of SEQ ID NO: 9 (avelumab heavy chain:

[0089] [ka] and a light chain having the amino acid sequence:

[0090] Preferred anti-PD-L1 antibodies Preferably, the anti-PD-L1 antibody of the combination for use according to the present invention is avelumab or or an antibody or antigen-binding fragment thereof that has structural similarity to avelumab. Lumab is SEQ ID NO: 32 and 33 (corresponding to SEQ ID NO: 7 and 9 herein) and WO 99 / 09494 designated A09-246-2, which has heavy and light chain sequences as set forth in In the brochure No. 2013 / 079174, avelumab, its sequence, and many of its properties are described. Avelumab has two main mechanisms of action for its antitumor effects. First, PD-L1 on tumor cells interacts with PD-1 on activated T cells. These interactions significantly enhance T cell activity. Therefore, PD-L1 interaction with PD-1 or B7-1 has been shown to inhibit Blocking this effect with anti-PD-L1 relieves T cells from immune suppression Second, tumor cells are able to penetrate into normal tissues, leading to their elimination by T cells. It is possible that these cells express higher levels of PD-L1 on their surface compared to fully human I. As a gG1 monoclonal antibody, avelumab has ADCC capabilities. It binds to PD-L1 on tumor cells, and its Fc portion binds to Fc-gamma receptors on leukocytes In this case, anti-tumor ADCC can be induced.

[0091] In a preferred embodiment of the invention, the anti-PD-L1 antibody comprises the six CDRs (sequence number 10 ... 1 to 6) and block the interaction between human PD-1 and human PD-L1. Alternatively, the anti-PD-L1 antibody further mediates ADCC. The PD-L1 antibody is an IgG antibody, with IgG1 antibodies being particularly preferred.

[0092] In a further preferred embodiment of the invention, the anti-PD-L1 antibody comprises the heavy chain of avelumab (sequence 7 or 8) and the amino acid sequence of the light chain (SEQ ID NO: 9), Preferably, the anti-PD-L1 antibody further comprises a More preferably, the anti-PD-L1 antibody is an IgG antibody, and The G1 antibody is particularly preferred.

[0093] In the most preferred embodiment of the invention, the anti-PD-L1 antibody is avelumab.

[0094] The cancer or precancerous lesion being treated The term "cancer" is defined as any abnormal malignant new growth of tissue that has no physiological function. A group of diseases that can be defined as resulting from uncontrolled, usually rapid, cell proliferation, Refers to diseases that have the potential to invade or spread to other parts of the body. The term refers to a benign lesion involving abnormal cells that is associated with an increased risk of developing into cancer. It refers to benign lesions associated with thyroid cancer.

[0095] In one aspect of the invention, the targeted therapeutic application is HPV-positive cancer or in situ pre-cancer. Treatment of the lesion.

[0096] As used herein, "HPV-positive cancer" and "HPV-positive intraepithelial precancerous lesion" " are caused by or associated with HPV infection, and " are caused by or associated with HPV infection, respectively. It refers to a cancer or precancerous lesion in situ in which the presence of a virus can be detected.

[0097] HR-HPV produces two oncoproteins, E6 and E7, which: It is required for viral replication through its growth-stimulating activity and plays a key role in malignant transformation The E6 oncoprotein binds to the p53 tumor suppressor protein and It induces its degradation through a biquitin-mediated process, disrupting the p53 pathway and thereby HPV E7 protein is a marker for retinoblastoma tumors, resulting in uncontrolled cell cycle progression. It binds to and degrades the protein (pRb), preventing it from inhibiting the transcription factor E2F. This results in loss of cell cycle control. Furthermore, functional inactivation of Rb leads to the loss of p1 P16 results in upregulation of the CDKN2A tumor suppressor gene. It is encoded by the transcription factor E2F, which phosphorylates Rb and initiates cell cycle progression. It regulates the activity of cyclin D-CDK4 / 6 complexes, leading to the release of cyclin D-CDK4 / 6. , characterized by high levels of p16 expression (Nevins JR, Hum Mol Genet. (200 1) 10(7): 699-703).

[0098] The presence of the HPV virus is determined by the expression of HPV DNA, HPV RNA, and HPV oncoproteins. by various methods based on the detection of p16 or overexpression of p16 protein. It can be detected indirectly by looking for cellular proteins whose expression is altered. Proteins can be detected by immunohistochemistry (IHC), which has been shown to As studies have shown a very high correlation (>90%) of HPV positivity in oropharyngeal tumors, It has been suggested that it is a clinically useful surrogate marker (Mellin Dahlstrand H. et al., A Cancer Res. (2005) 25(6C): 4375-4383). The presence of HPV (and therefore cancer or The HPV-positive nature of intraepithelial precancerous lesions is determined by: (1) HPV DNA; (2) virus E6 and / or postintegrative transcription of E7 mRNA, (3) viral oncoprotein E (4) overexpression of cellular proteins, such as E6 and E7, or p16 protein It can also be determined by detecting changes in the current state (Kim et al., J Pathol Clin Res. (2018) ) 4(4): 213-226). HPV DNA can be detected by, among other methods, polymerase chain reaction (PCR) or or in situ hybridization (ISH). V RNA can be detected by, among other methods, polymerase chain reaction (RT-PCR) or in situ hybridization. Cancerous or precancerous diseases can be detected using ISH (Integrated Synthetic Immunosorbent Assay). Various kits for determining the HPV status (positive or negative) of a patient are commercially available. It can be used in the context of Ming (Kim et al., J Pathol Clin Res. (2018) 4( 4): see Table 1 on pages 213-226.

[0099] In a preferred embodiment, HPV-16 is the predominant HR-HPV detected in HPV-positive cancers. Therefore, HPV-16 E7 D was detected by PCR using HPV-16 specific primers. By detecting NA, the HPV status of cancerous or precancerous lesions is determined. In a preferred embodiment, the DNA of the subject to be treated is extracted by conventional methods from a tumor sample (e.g., For example, fixed samples, such as formol- or formalin-fixed paraffin-embedded (FFPE) tumor samples, The HPV-16 gene was extracted from the tumor sample and then subjected to PCR using HPV-16 specific primers. Thus, HPV-16 E7 DNA is amplified. Amplification is detected (e.g., SYBR green) the sample is considered HPV-16 positive. If no amplification is detected by this method, approximately 50 HPV genotypes can be amplified. HPV E7 DNA was amplified by PCR using consensus primers that can , and then sequence the amplified sequences using Sanger sequencing. The resulting sequence is then used to confirm the negative results and to confirm the quality of the sample to obtain a result in the primary PCR. Identifying patients who would not have been able to identify positive genotypes or who have relatively rare genotypes This allows for the detection of patients.

[0100] Preferred HPV-positive cancers include HPV-positive oropharyngeal cancer, cervical cancer, vaginal cancer, and anal cancer. HPV-positive cancers include genital, vulvar, penile, mucosal, or non-melanoma skin cancers. Among pharyngeal cancers, squamous cell carcinoma of the head and neck (SCCHN) is preferred.

[0101] In their pooled interim analysis of Phase Ib and Phase II clinical trials, the inventors found that HPV-positive anal cancer is associated with relatively poor progression-free survival (PFS, see Figure 8) and This is actually related to liver metastases in patients with anal cancer, not anal cancer itself. In fact, there were 100 patients with anal cancer who had liver metastases. Some patients with no metastases (but with other metastases) respond to treatment. Among the above-listed cancers, HPV-positive anal cancer has a relatively high prevalence of liver metastasis. Therefore, HPV-positive cancers are preferably not HPV-positive anal cancers, especially anal cancers with liver metastases. not ocular cancer, and therefore preferably HPV-positive oropharyngeal cancer (especially SCCHN), Choose from cervical cancer, vaginal cancer, vulvar cancer, penile cancer, mucosal cancer, or non-melanoma skin cancer It is selected.

[0102] In contrast, we have demonstrated high response rates in patients with HPV-positive vulvar / vaginal cancer (see Table 11). ), as well as relatively favorable objective response rate (ORR, see Figure 7) and PFS (Figure 8 observed a non-significant trend for the association between HPV-positive genital (meaning vulvar / vaginal) cancer and Therefore, among HPV-positive cancers, HPV-positive vulvar and vaginal cancers are preferred. stomach.

[0103] Preferred HPV-positive intraepithelial precancerous lesions include cervical intraepithelial neoplasia (CIN) grade 2. or 3 or vulvar intraepithelial neoplasia (VIN) grade 2 or 3. Cervical epithelium Cancerous Intrauterine Neoplasia (CIN) can be in one of three stages: CIN1, CIN2, or CIN3. If left untreated, it can progress to CIN2 or CIN3. IN3 (collectively referred to as CIN2+) may progress to cervical cancer. Vulvar intraepithelial neoplasia (VIN) can occur in three stages: VIN1, VIN2, or VIN3. If left untreated, VIN is a premalignant lesion that can exist in any one of the following areas: 2 or VIN3 (collectively referred to as VIN2+) may progress to vulvar cancer .

[0104] The cancer or intraepithelial precancerous lesion to be treated is preferably positive for HR-HPV. and the HR-HPV is preferably encoded by the poxvirus. The resulting HPV E6 and E7 polypeptides correspond to the HR-HPV from which they arise. The cancer or intraepithelial precancerous lesion to be treated is preferably HPV-16, HPV-18, HPV-19, or HPV-20. V-30, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-6 6, HPV-68, HPV-70, and HPV-85, more preferably HPV-1 6 and HPV-18, and most preferably , the cancer or precancerous lesion to be treated is positive for HPV16. If the cancer or precancerous lesion in situ is positive for HPV16, the poxvirus , preferably HPV-16 E6 and E7 polypeptides (more preferably the The gene encodes a non-oncogenic version of the gene.

[0105] Thus, in a preferred embodiment, the targeted therapeutic application is directed to HPV-16 positive cancers ( Preferably HPV16-positive anal cancer with liver metastases, or more generally, its prevalence of liver metastases HPV-16 positive anal cancer (not HPV-16 positive anal cancer due to high rates) or HPV-16 positive intraepithelial precancer This cancer is particularly common in HPV-positive oropharyngeal cancer (especially SCCHN), cervical cancer, Choose from cervical, vaginal, vulvar, penile, mucosal, or non-melanoma skin cancer In a preferred embodiment, the cancer is HPV-16 positive head and neck squamous cell carcinoma. (HPV-16+ SCCHN), HPV-16 positive vulvar cancer, and HPV16 positive vaginal In this case, the poxvirus (preferably MVA) can be selected from the group consisting of: Preferably, HPV-16 E6 and E7 polypeptides (more preferably, those disclosed above) It encodes a non-carcinogenic version of the

[0106] In addition to its HPV-positive nature, the targeted cancer is further preferably a recurrent and and / or metastatic HPV-positive cancer (more preferably, recurrent and / or metastatic HPV-1 HPV-16 positive cancer, most preferably recurrent and / or metastatic HPV-16 positive SCCHN) As used herein, the term "cancer" refers to primary or recurrent and / or "Primary cancer" refers to the stage at which tumor progression begins and a cancerous mass forms. This means that the cancer is growing in the anatomical site (organ or tissue) where it originated. "Recurrent cancer" is when cancer recurs (comes back), usually after a period of time when it was undetectable. Cancer cells from the primary cancer can spread to other parts of the body and It can form new cancers or "metastatic cancers" (also called secondary cancers).

[0107] Indeed, recurrent and / or metastatic cancer generally carries a relatively poor prognosis and a high susceptibility to treatment. As metastases are associated with poor response rates, new combination therapies for these cancers are particularly needed. It can affect various organs, including the lymph nodes, lungs, bones, and liver.

[0108] In their pooled interim analysis of Phase Ib and Phase II clinical trials, the inventors Surprisingly, lymph node metastasis was significantly associated with a relatively favorable PFS (see Figure 8). Therefore, in a preferred embodiment, the HPV-positive cancer is The cancer is HPV-positive (preferably HPV16-positive) metastatic cancer with nodal metastasis.

[0109] The inventors also found, surprisingly, that lung and bone metastases showed relatively low ORR or We also found that the PFS (see Figure 8) was not significantly associated with PFS. Only one type of metastasis was significantly associated with a relatively poor ORR and PFS, However, liver metastasis, especially when the patient has multiple liver metastases, i.e., multiple sites (at least two) in the liver, This is especially true in cases with liver metastases occurring in at least two different lung lobes. The presence of liver metastases is a predictor of response to anti-PD-L1 treatment, primarily in patients with lung or mixed cancers. It has been suggested that this is associated with a relatively low response rate (Sridhar S., et al. Clin L ung Cancer 2019: e601 - e608, Bilen M., et al. BMC Cancer. 2019: 19: 857, Reck M ., et al. Lancet Respir Med 2019: 7: 387-401). However, such observations are rare. At least human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulation Treatment without the use of a poxvirus vector encoding a cytokine and therefore cannot be expected to apply to any particular treatment according to the present invention. Furthermore, such observations have not been made in HPV-positive cancers, and no specific The relatively low ORR and PFS and liver metastases were also unexpected. There was a significant association with ORR and PFS, but no association with lung or bone metastases However, our findings regarding the association between relatively high PFS and lymph node metastasis In light of the observations, the HPV-positive cancer is preferably free of multiple liver metastases (preferably liver metastases). HPV-positive (preferably HPV-16 positive) cancer (oropharyngeal cancer, cervical cancer, vaginal, anal, vulvar, penile, mucosal, or non-melanoma skin cancer), especially No multiple liver metastases (preferably no liver metastases) and metastatic HPV positive (preferably HPV -16 positive) Cancer (oropharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, vulvar cancer, penile cancer) , mucosal cancer, or non-melanoma skin cancer).

[0110] More preferably, the HPV-positive cancer is preferably free of liver metastases and has lymph node metastases. It may be a metastatic HPV-positive (preferably HPV-16 positive) cancer.

[0111] Dosage and route of administration "Administering" a drug or drug to a patient (and the grammatical equivalents of this phrase) "Administration of" refers to direct administration, which may be by a healthcare professional to a patient or self-administration; and This refers to indirect administration, which can be the act of prescribing a drug and / or administering a drug. For example, A physician who instructs a patient or provides a patient with a prescription for a drug is not authorized to administer that drug to the patient. There are.

[0112] "Dose" and "dosage" refer to a specific amount of an active or therapeutic agent for administration. Such an amount is included in a "dosage form," and a "dosage form" is a form in which each unit has the desired effect, tolerability, and one or more predetermined amounts of active agents calculated to produce a therapeutic effect. and administering to a human subject, together with a suitable pharmaceutical excipient, such as a carrier or adjuvant, It refers to physically discrete units suited as unitary dosages for other mammals.

[0113] A "pharmaceutically acceptable adjuvant" is any substance that enhances the body's immune response to an antigen. Non-limiting examples of pharmaceutically acceptable adjuvants include alum, fructooligosaccharides, and sorbitol. incomplete adjuvants, MF59, synthetic analogs of dsRNA such as poly(I:C), Bacterial LPS, bacterial flagellin, imidazoquinoline, oligonucleotides containing specific CpG motifs Bacterial cell wall fragments such as godeoxynucleotides, muramyl dipeptides, and Quil- A (registered trademark).

[0114] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" is a substance that is suitable for pharmaceutical administration. Any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption agents The use of such media and agents for pharmaceutical active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are well known in the art. and concentrations that are non-toxic to the recipient, and without limiting the scope of the invention, additional buffering agents may be used. preservatives; co-solvents; antioxidants including ascorbic acid and methionine; raters; metal complexes (e.g., Zn-protein complexes); biodegradable polymers (e.g., poly esters); sodium, salt-forming counterions such as polyhydric sugar alcohols; alanine, glycine , glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine amino acids such as 2-phenylalanine, glutamic acid, and threonine; lactitol cellulose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoglobin Initose (myoinisitose), myo-inositol, galactose, galactitol , glycerol, cyclitols (e.g., inositol), polyethylene glycol, etc. Organic sugars or sugar alcohols; urea, glutathione, thioctic acid, sodium thioglycolate sodium, thioglycerol, [alpha]-monothioglycerol, and sodium thiosulfate Sulfur-containing reducing agents such as sodium; human serum albumin, bovine serum albumin, gelatin or other low molecular weight proteins such as immunoglobulins; as well as polyvinylpyrrolidone Contains hydrophilic polymers. Remington's Pharmaceutical Sciences 16th edition, Osol, A. Other pharmaceutically acceptable carriers, excipients, such as those described in Ed. (1980), or Stabilizers may also be used as described herein, provided they do not adversely affect the desired characteristics of the pharmaceutical composition. The pharmaceutical composition may be comprised of:

[0115] A "therapeutically effective amount" is a therapeutically effective amount of a compound having the ability to treat cancer or precancerous lesions. The poxviruses described in the specification (WO 1999 / 03885) At least HPV E6, such as TG4001, described under the study name MVATG8042 and E7 polypeptide and an immunostimulatory cytokine, preferably VV , more preferably MVA) and / or an anti-PD-L1 antibody or antigen-binding fragment thereof ( In the case of cancer, e.g., advanced solid malignant tumors, a therapeutically effective amount of a drug The substance can reduce the number of cancer cells; reduce tumor size or tumor burden; inhibits (i.e., slows to some extent, and in certain embodiments, inhibit (i.e., slow to some extent, and in certain embodiments, stop) tumor metastasis; inhibit tumor growth to some extent; alleviate one or more symptoms associated with cancer to some extent and / or progression-free survival (PFS), disease-free survival (DFS), if or increased overall survival (OS), complete response (CR), partial response (PR), or In some cases, stable disease (SD), reduced progression (PD), and reduced time to progression (TTP) were observed. A drug can prevent growth and produce a favorable response, such as a decrease in the number of To the extent that drugs can induce and / or kill existing cancer cells, May be cytostatic and / or cytotoxic. In the case of precancerous lesions, therapeutic The effective amount of the drug inhibits (i.e., slows to some extent, and in certain embodiments, A "prophylactically effective amount" is a dose that is administered at a required dose for a required period of time. A prophylactic dose refers to an amount effective to achieve the desired prophylactic result. For use in subjects, a prophylactically effective amount will typically be less than a therapeutically effective amount, although not necessarily. That's not necessarily the case.

[0116] "Unit dosage form," as used herein, refers to the physical dosage of a therapeutic formulation appropriate for the subject being treated. However, the poxvirus vectors and The use of anti-PD-L1 compositions will be determined by the attending physician within the scope of sound medical judgment. It will be understood that a particular effective dose level for any particular subject or organism may be determined. the disorder being treated and the severity of the disorder; the activity of the particular active agent being used; the particular composition of the subject; the age, weight, health, sex, and diet of the subject; the time of administration and use the rate of excretion of the particular active agent used; the duration of treatment; the particular compound and combination used Drugs and / or additional therapies used in conjunction with or concurrently with the use of The risk of developing a serious illness depends on a variety of factors, including similar factors that may be present in the body.

[0117] poxvirus In the combination, method or use according to the present invention, at least one human papillomavirus ( HPV) E6 and E7 polypeptides and polypeptides encoding immunostimulatory cytokines coccus virus (preferably under its research name MVATG8042 and International Publication No. 1999 / 03 Membrane-bound non-expressing steroids, such as TG4001, which is described in the 885 pamphlet, M encoding cancerous HPV-16 E6 and E7 polypeptides and human IL-2 VA virus), preferably 10 6 From 10 8 Up to pfu, more preferably 5 x 10 6 From 8 x 10 7 up to pfu, most preferably 3 x 10 7 From 7 x 10 7 Until pfu, Preferably 4 x 10 7 From 6 x 10 7 Up to pfu, very particularly preferably about 5 x 10 7 pfu dose.

[0118] In the combination, method or use according to the present invention, at least one human papillomavirus ( HPV) E6 and E7 polypeptides and polypeptides encoding immunostimulatory cytokines coccus virus (preferably under its research name MVATG8042 and International Publication No. 1999 / 03 Membrane-bound non-expressing steroids, such as TG4001, which is described in the 885 pamphlet, M encoding cancerous HPV-16 E6 and E7 polypeptides and human IL-2 VA virus) is preferably administered by subcutaneous, intramuscular, intratumoral, or intravenous routes. A particularly preferred route of administration is the subcutaneous route.

[0119] Anti-PD-L1 antibody or antigen-binding fragment thereof In the combination, method or use according to the invention, an anti-PD-L1 antibody (particularly at least An antibody containing six CDRs, or the heavy and light chains of, for example, avelumab, is preferably is administered in the following amounts:

[0120] In certain embodiments, a therapeutically effective amount of an anti-PD-L1 antibody (e.g., avelumab) or its is administered in the combination, method or use of the present invention. , sufficient to treat one or more symptoms of HPV-positive cancer. In some embodiments using an anti-PD-L1 antibody, the dosing regimen is , about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, or 20 mg / kg body weight, approximately 7 days (± 2 days), approximately 14 days (±2 days), approximately 21 days (±2 days), or approximately 30 days (±2 days) intervals. In certain embodiments, the method comprises administering an anti-PD-L1 antibody (e.g., avelumab) to the subject. ) or its antigen-binding fragment is about 5 to 20 mg / kg, more preferably 5 In some embodiments, the anti-PD- The L1 antibody is avelumab, and the therapeutically effective amount of avelumab is approximately 10 mg / kg. In some embodiments, avelumab is administered once every two weeks. Pharmacokinetic studies were conducted on days 1 and 15 of a 28-day cycle. kg dose of avelumab demonstrated excellent receptor occupancy with a predictable pharmacokinetic profile (Heery et al., 2015. Proc ASCO Annual Meeting: Abstract 3 055) This dose was well tolerated and evidence of antitumor activity, including durable responses, was observed. It was.

[0121] In some embodiments, the anti-PD-L1 antibody (e.g., avelumab) is selected from the group consisting of those described above and below. Throughout the course of treatment, approximately 80, 150, 160, 200, 240, 250, 300, 32 0, 350, 400, 450, 480, 500, 550, 560, 600, 640, 65 0, 700, 720, 750, 800, 850, 880, 900, 950, 960, 10 00, 1040, 1050, 1100, 1120, 1150, 1200, 1250, 12 80, 1300, 1350, 1360, 1400, 1440, 1500, 1520, 15 50, or 1600 mg, preferably 800 mg, 1200 mg, or 1600 mg At a fixed dose of about 7 days (± 2 days), about 14 days (± 2 days), about 21 days (± 2 days), or about It is administered at intervals of 30 days (± 2 days). Thus, in a preferred embodiment, anti-PD-L One antibody (e.g., avelumab) is preferably administered once a week (QW), once every two weeks (Q2 W), or once every three weeks (Q3W), approximately 400 to 1600 mg, more preferably approximately 800 ~1600mg, most preferably about 800-1200mg, very preferably about 800mg In certain preferred embodiments, an anti-PD-L1 antibody (e.g., abeta- lumab) is administered at a dose of approximately 800 mg Q2W.

[0122] In the combination, method or use according to the invention, an anti-PD-L1 antibody (particularly at least An antibody containing six CDRs, or the heavy and light chains of, for example, avelumab, is preferably is administered intravenously (e.g., as an intravenous infusion) or subcutaneously. PD-L1 antibodies (particularly those with at least six CDRs, or the heavy chain of, for example, avelumab and Most preferably, the anti-PD-L1 antibody is administered as an intravenous infusion. and antibodies (particularly those containing at least six CDRs, or the heavy and light chains of, for example, avelumab). The antibody (antibody) is administered as an intravenous infusion over 50 to 80 minutes, most preferably about 1 hour. .

[0123] In one embodiment, avelumab is administered as a sterile, clear, colorless solution intended for IV administration. The contents of the avelumab vial are non-pyrogenic and do not contain bacteriostatic preservatives. Lumab was formulated as a 20 mg / ml solution, stoppered with a rubber septum, and sealed in an aluminum foil container. Supplied in a disposable glass vial sealed with a polypropylene flip-off seal. For administration, avelumab must be diluted with 0.9% sodium chloride (normal saline). During administration, in-line, low protein binding, polyethersulfone ( A tube with a 0.2 micron filter made of PES was used.

[0124] Number and frequency of combination administrations In one aspect of the present invention, a) at least human papillomavirus (HPV) E6 and E7 polypeptides, and poxvirus vectors, preferably membrane-bound, encoding immunostimulatory cytokines in Encoding non-oncogenic HPV-16 E6 and E7 polypeptides and human IL-2 an MVA virus, preferably TG4001, b) an anti-PD-L1 antibody or antigen-binding fragment thereof, preferably avelumab the first administration of the poxvirus is administered before the first administration of the anti-PD-L1 antibody. 5 to 10 days before the administration of the poxvirus and anti-PD-L1 antibody. It is administered according to the particular administration scheme being implemented.

[0125] That is, the administration scheme used for the combination, method or use according to the present invention is: at least, 5 to 10 days before the first administration of the anti-PD-L1 antibody a first administration of the poxvirus, and subsequent administration of the poxvirus and anti-PD-L1 antibody Includes:

[0126] In the administration scheme used for the combination, method or use according to the present invention, The first administration of the cosvirus is administered before the first administration of the anti-PD-L1 antibody. Although not intended to be a treatment for HPV, this setting is based on the initial anti-HPV immunization with the first poxvirus administration. This anti-HPV immune response then alters the initial proliferation of the poxvirus. Without the first anti-PD-L1 treatment, the PD-1 / PD-L1 in the tumor microenvironment (by reducing immunosuppression via the L1 pathway). Since there is no anti-PD-L1 administration for 5-10 days after administration, the anti-poxvirus immune response Subsequent administration of the poxvirus and anti-PD-L1 antibody prevents potential amplification. maintains anti-HPV immune responses.

[0127] Therefore, in the combination, method or use according to the invention, the first poxvirus is administered about 5 to 10 days (i.e., 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, 32, 33, 8, 9, or 10 days, preferably 1 week) before the procedure.

[0128] In some embodiments, the combination regimen is administered to a subject (a) under the direction or supervision of a physician. , and at least HPV E6 and E7 approximately 5-10 days before the first dose of the PD-L1 antibody. Injection of a poxvirus vector encoding a polypeptide and an immunostimulatory cytokine (b) administering the PD-L1 antibody to the subject under the direction or supervision of a physician. In some embodiments, the combination regimen comprises administering to the subject at least Also included are HPV E6 and E7 polypeptides and polypeptides encoding immunostimulatory cytokines. Approximately 5-10 days after receiving the first dose of the viral vector, subjects were administered an anti-PD-L1 antibody. This includes administering the drug to the body.

[0129] The number and frequency of subsequent administrations of the poxvirus and anti-PD-L1 antibody may vary. However, the number of subjects receiving treatment may be such that the combination therapy results in unacceptable toxicity. The poxvirus and anti-PD-L Preferably, subsequent administrations of one antibody are administered.

[0130] In one embodiment, the subsequent administration of the poxvirus and anti-PD-L1 antibody is RECIST v1.1 criteria (Eisenhauer EA. et al., Eur J Cancer (2009) 45(2) This can be performed until disease progression as defined by the International Journal of Clinical Oncology (ICH 2019) 228-47.

[0131] "Disease progression," "progression," or "advanced disease" are defined in accordance with the RECIST v1.1 guide. The appearance of one or more new lesions or tumors and / or refers to a definite progression of pre-existing non-target lesions. Disease progression, progression, or advanced disease refers to the progression of tumor More than 20 percent of the time treatment began, either by mass growth or diffusion It can also refer to tumor growth.

[0132] "RECIST" stands for Response Evaluation Criteria in Solid Tumors. RECIST guidelines, The criteria, or standards, are for solid tumor measurements for use in adult and pediatric cancer clinical trials. Describes standard approaches and definitions for objective assessment of changes in tumor size RECIST v1.1 is the revised version 1.1 of the RECIST guidelines. means.

[0133] In other embodiments, beneficial biological effects (see dedicated section below) are observed in patients. As far as possible, subsequent administration of the poxvirus and anti-PD-L1 antibody is recommended. can.

[0134] With regard to frequency of administration, the following schedule is preferably used:

[0135] The frequency of subsequent poxvirus administrations can vary from about 1 week to about 3 months. The frequency of X-virus administration does not have to be constant throughout the treatment period, but instead varies. Preferably, the frequency of subsequent poxvirus administrations is reduced over time. .

[0136] In particular, when the first poxvirus administration is included, the first administration is 4 to 8 times (i.e., 4, 5 , 6, 7, or 8 times, preferably 6 times) of poxvirus administration every 5 to 10 days ( (including every 5, 6, 7, 8, 9, or 10 days, preferably weekly) (Optionally, a single initial poxvirus administration may be administered, followed by weekly administrations. It is particularly preferred to administer five subsequent poxvirus doses at a frequency of (The first group to receive the virus).

[0137] This first group of poxvirus doses is then followed by subsequent doses of poxvirus. A second group may follow at a reduced frequency. Preferably, the frequency of subsequent poxvirus administrations is This second group is administered every 1 to 3 weeks (including every 1, 2, or 3 weeks, preferably every 2 weeks). (Optionally, subsequent administration of poxvirus every 2 weeks for up to 6 months is particularly preferred) Six to ten (i.e., six, seven, eight, nine, or ten, preferably eight) subsequent potions and poxvirus administration ("second group of poxvirus administrations").

[0138] This second group of poxvirus doses is then followed by subsequent doses of poxvirus. A third group may follow at a further reduced frequency. This third arm of fluticasone administration is continued until disease progression (or, optionally, until disease progression as described later in this specification). every 10–14 weeks (10, 11, 12) as long as at least one of the biological effects is present , 13, or 14 weeks, preferably every 12 weeks) (see "Third group of poxvirus administration").

[0139] In a particularly preferred embodiment, the poxvirus is Once a week for 6 weeks Every 2 weeks until 6 months, and Until disease progression (or, optionally, at least one of the biological effects described below) Every 12 weeks (as long as one exists) It is administered.

[0140] The frequency of anti-PD-L1 administration (including the first administration and subsequent administrations) is preferably 1 to 3 weekly (including every week or every 2 or 3 weeks, preferably every 2 weeks).

[0141] The anti-PD-L1 antibody is preferably administered until disease progression (or, optionally, The drug is administered as long as at least one of the biological effects described below is present.

[0142] In a particularly preferred embodiment, the anti-PD-L1 antibody is administered every 2 weeks until disease progression (or , optionally as long as there is at least one of the biological effects described herein below) will be done.

[0143] Preferred Administration Schemes In a preferred embodiment, the combination is administered according to the following administration scheme: a) 3 × 10 7 From 7 x 10 7 pfu of the first dose of said poxvirus (preferably The present study identified membrane-bound non-oncogenic HPV-16 E6 and E7 polypeptides and human IL- 2, more preferably an MVA virus encoding the TG4001) listed in the 85th issue pamphlet under the research name MVATG8042 subcutaneously, followed by 3 × 10 7 From 7 x 10 7 For subsequent poxviruses of pfu The amount increases until the disease progresses. Once a week for 6 weeks Every 2 weeks until 6 months, and Subsequent poxvirus doses every 12 weeks It is administered subcutaneously, b) a first dose of about 10 mg / kg or about 800 mg of an anti-PD-L1 antibody (preferably is an antibody containing at least six CDRs, or the heavy and light chains of, for example, avelumab and more preferably avelumab) administered intravenously 5 to 10 days after the first poxvirus dose. intravenously, followed by a subsequent anti-PD-L1 antibody at about 10 mg / kg or about 800 mg. The total dose is administered intravenously every 2 weeks until disease progression.

[0144] In a further preferred embodiment, the combination is administered according to the following administration scheme: a) Approximately 5 × 10 7 pfu of the first dose of membrane-bound non-oncogenic HPV-16 E6 and The MVA virus (preferably For example, the research name MVATG80 is described in the pamphlet of International Publication No. 1999 / 03885. TG4001, as described in 42), was administered subcutaneously, followed by approximately 5 × 10 7 pfu Subsequent MVA doses are administered until disease progression. Once a week for 6 weeks Every 2 weeks until 6 months, and Subsequent poxvirus doses every 12 weeks It is administered subcutaneously, b) A first dose of avelumab of about 10 mg / kg or about 800 mg is administered as a first postoperative dose. 1 week after the administration of the virus, followed by about 10 mg / kg or about 80 Subsequent avelumab doses of 0 mg will be administered intravenously every 2 weeks until disease progression.

[0145] Biological effects and biomarkers of combination therapy The present inventors have surprisingly found that (a) at least human papillomavirus (HPV) Poxviruses encoding E6 and E7 polypeptides and immunostimulatory cytokines vectors, especially TG4001 (membrane-bound non-oncogenic HPV-16 E6 and E7 polypeptides) (b) MVA virus encoding anti-PD-L1 peptide and human IL-2 Antibodies or antigen-binding fragments thereof, preferably avelumab, in combination in patients with HPV-positive cancer and reduced tumor immunosuppression and improved anti-cancer responses characterized by: We found that this can be done. Induction or enhancement of immune responses to HPV16 E6 and E7 polypeptides , Intratumoral Increased immune cell infiltrates, primarily CD3 T cells, preferably CD8 T cell numbers and and / or an increase in their proportion among CD8 and CD3 T cells (increased CD8 / CD3 ratio) leads to a decrease in regulatory CD4 T cells and / or a reduced Treg / CD8 ratio a combination of increased CD8 T cells and decreased regulatory CD4 T cells (Tregs), and / or Increased expression of PD-L1 on tumor cells In the blood circulation, Increased CD8 T cell counts, and / or Decrease in regulatory CD4 T cells, and / or Significant remodeling of gene expression in tumor cells, T cell activation genes, cytotoxic cell genes, pathogen defense genes, and NK cells Increased expression of functional genes, ○CXCL10, CXCL11, IRF1, GZMK, GZMA, CD3D, PRF 1, TBX21, CXCR3, STAT1, CD69, CCL2, GZMB, CD3G, ICOS, CD8A, STAT4, GZMM, CCR2, CD3E, and IL15 groups and / or ○CXCL13, GNLY, GZMH, IFNG, CXCL9, CCL5, and I Increased expression of one or more genes selected from the group of TGAE, and / or VEGFA, IHH, IL17A, PROM1, REN, PF4, TSLP, and L Reduction of expression of one or more genes selected from the group AG3 Remodeling characterized by.

[0146] Unless otherwise stated, all comparisons of increases or decreases are based on baseline (all i.e., before the combination therapy was administered to the patient).

[0147] Thus, in one embodiment of the combination therapy, the combination comprises HPV-16 E6 and Induce or enhance the immune response to the E7 protein. The immune response to the E7 protein can be measured by any suitable method known in the art. Suitable methods include the administration of cytokines (particularly interferon gamma ( IFNγ), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF α)) against HPV-16 E6 and E7 proteins, including secretion and cytotoxicity The method may be based on the detection of CD8 and / or CD4 T cell responses. Tokine secretion can be measured peripherally using conventional assays such as ELISA or ELISPOT. Cytotoxicity can be measured in vitro from blood mononuclear cell (PBMC) samples. In a preferred embodiment, the assay can be performed in vitro. Immunity to HPV-16 E6 and E7 proteins was assessed by IFN-γ using PBMCs. Nγ secretion and immunostaining by ELISA, flow cytometry or ELISPOT Color is measured, preferably using the ELISPOT technique.

[0148] In another embodiment of the combination therapy, the combination is administered intratumorally: Increased immune cell infiltrate, preferably CD3 T cells, more preferably CD8 T cell expansion, most preferably an increase in the CD8 / CD3 ratio, and / or Reduction of regulatory CD4 T cells (Treg), preferably a low Treg / CD3 ratio decrease Induce.

[0149] Most preferably, the combination induces a reduction in the Treg / CD8 ratio in the tumor. Such a reduction in the Treg / CD8 ratio may lead to reduced immunosuppression within the tumor and increased resistance. These studies have shown stimulation of the immune response.

[0150] Within the tumor, immune cell infiltrates, particularly CD3 T cells, CD8 T cells and the number of CD4 T cells (Tregs) can be determined by any suitable method known in the art. T cells are characterized by the surface expression of CD3 and Subdivided into two subgroups depending on their simultaneous surface expression of either CD8 or CD4 Among CD4 T cells, those that additionally express Foxp3 are classified as regulatory CD4 T cells. CD3 T cells (CD3+ cells), CD8 T cells (C D3+CD8+ cells), and CD4 T cells (Treg, CD3+CD4+Foxp3 + cells) before treatment (baseline) using any suitable method known in the art. The CD8 / CD3 ratio, T The reg / CD3 ratio and the Treg / CD8 ratio can be easily calculated.

[0151] As used herein, CD3, CD8, CD4, and Foxp3 expression are expressed in cells. CD3, CD8, CD4, and Foxp3 proteins on the surface of cells or tissues Any detectable levels of CD3, CD8, CD4, and Foxp3 mRNA in tissue It refers to the expression of CD3, CD8, CD4, and / or Foxp3 proteins on the cell surface. Protein expression is assessed by immunohistochemistry (IHC) assays of tumor tissue sections, depending on the specimen type. Diagnostic CD3, CD8, CD4, and Alternatively, the expression of IFN-γ by tumor cells can be detected using antibodies against Foxp3 and / or Foxp4. CD3, CD8, CD4, and / or Foxp3 protein expression was assessed by CD3, CD8 Binding agents (e.g., antibody fragments, antibody fragments) that specifically bind to CD4, CD4, and / or Foxp3. It can also be detected by PET imaging using CD3 , CD8, CD4, and / or Foxp3 mRNA (or cDNA) expression and techniques for measuring it include RT-PCR, real-time quantitative RT-PCR (qRT-PCR), These include PCR, T-PCR, and microarray hybridization. CD3, CD8, CD4, and / or Foxp3 expression is preferably measured in tumor tissue sections. In immunohistochemistry (IHC) assays, diagnostic CD3, CD8, CD4, and / or or Foxp3 antibody. The increase / decrease is measured by the number, occurrence, and frequency of the tumors after treatment with the combination therapy. Detected if the current or ratio is higher / lower than before treatment with the combination therapy (baseline) will be done.

[0152] In another embodiment of the combination therapy, the combination induces increased PD-L1 expression on tumor cells. As used herein, "PD-L1 expression" refers to the expression of PD-L1 proteins on the cell surface. Protein, or any detectable level of PD-L1 mRNA in cells or tissues PD-L1 protein expression was measured in tumor tissue sections, depending on the sample type. Diagnosis in immunohistochemistry (IHC) assays or by flow cytometry Alternatively, PD-L1 tagging by tumor cells can be detected using PD-L1 antibodies. Protein expression is measured by binding agents (e.g., antibody fragments, aphibody fragments) that specifically bind to PD-L1. PD-L1 m can also be detected by PET imaging using Techniques for detecting and measuring RNA (or cDNA) expression include RT-PCR, Real-time quantitative RT-PCR (qRT-PCR), and microarray hybridization Within tumors, PD-L1 expression is preferably measured by immunofluorescence of tumor tissue sections. The increase is detected using a diagnostic PD-L1 antibody in an immunohistochemistry (IHC) assay. , PD-L1 expression after treatment with combination therapy compared to before treatment with combination therapy (baseline) High levels of PD-L1 expression on tumor cells are detected when anti-PD-L1 antibodies are used. It is associated with a relatively good clinical response to systemic treatment.

[0153] In another embodiment of the combination therapy, the combination is administered in the blood circulation: an increase in CD8 T cells, preferably an increase in the CD8 / CD3 ratio, and / or Reduction of regulatory CD4 T cells (Treg), preferably a low Treg / CD3 ratio decrease Induce.

[0154] In the blood circulation, CD3, CD8, CD4, and Foxp3 expression can be measured using methods known in the art. Measure before (baseline) and after treatment using any suitable method known in the art. Suitable methods include the detection of CD3, CD4, and CD5 expression levels in tumors. 8, CD4, and Foxp3 expression. However, diagnostic CD3, CD8, CD4, and / or F by flow cytometry is required. Detection of CD3, CD8, CD4, and Foxp3 expression using oxp3 antibodies is preferred The CD8 / CD3 ratio, Treg / CD3 ratio, and Treg / CD8 ratio were then easily measured. It can be calculated.

[0155] Preferably, an expansion of CD8 T cells is observed both in the blood circulation and within the tumor. Similarly, the reduction of regulatory CD4 T cells is favorable both in the blood circulation and within tumors. Preferably, an increase in CD8 T cells and a decrease in regulatory CD4 T cells are observed. The reduction is preferably in the blood circulation and / or in the tumor, more preferably in the blood circulation and in the tumor. It is observed both within the tumor and in the

[0156] Also, at the molecular level, the present inventors have surprisingly found that the processes of innate and adaptive immunity rimming and "cold" to "hot" tumor profile They also found gene expression changes consistent with the transition to cold tumors. Defined as a tumor with no or very limited infiltrate, especially T-cell immune infiltrate. So, "cold tumors" are characterized by genes associated with the presence of immune cell infiltrates, especially T cells. activation, T cell differentiation, T cell attraction, T cell adhesion, cytotoxicity, pathogen defense, and NK cells. They are characterized by low levels of expression of genes involved in cellular function. A "hot tumor" is a tumor with a significant immune infiltrate, particularly a T cell immune infiltrate. At the molecular level, "hot tumors" are associated with the presence of immune cell infiltrates. genes, especially T cell activation, T cell differentiation, T cell attraction, T cell adhesion, cytotoxicity, High levels of expression of genes involved in pathogen defense and NK cell function Characterized from a "cold tumor" profile to a "hot tumor" profile Transition to a vasoconstrictor is considered when treatment results in a significant increase in immune infiltrate, especially T cell immune infiltrate. At the molecular level, this is thought to be induced by treatment. compared T cell activation, T cell differentiation, T cell attraction, T cell adhesion, cytotoxicity, and pathogen protection. and / or increased levels of expression of one or more genes involved in NK cell function. Hot tumors are more likely to respond to therapeutic intervention. , with relatively high consistency, is associated with improved clinical outcomes for patients. Cold tumors are associated with minimal immune reactivity, poor response to therapeutic interventions, and rapid progression It is likely to have an unfavorable clinical course.

[0157] More specifically, a panel of 770 genes involved in the immune response to cancer was used to We investigated the effect of genomic DNA expression in tumors between baseline and 43 days after the start of combination therapy. These changes included several T cell activation genes, These include increased expression of cytotoxic genes, pathogen defense genes, and NK cell function genes. These variations include Immunosign® by HalioDX CR 15 and Immunosign® CR Genetic signature known as 21 These gene signatures are related to the natural progression of tumors and tumor periphery. reflects the immune activity present in the tumor and therefore is somewhat cold (poor prognosis) or is thought to reflect a hot (relatively good prognosis) immune state (Galon J. et al., Immunity (2013) 39(1):11-26: Marabelle A. et al., Society for Immuno Therapy of Cancer (SITC) 32 nd Annual Meeting & Pre-Conference Programs (SITC 201 7) on November 8-12, 2017 at the Gaylord National Hotel & Convention Center in N ational Harbor, Maryland. Poster P250).

[0158] In particular, Immunosign® CR 15, T cell cytotoxicity, T cell differentiation, T cell attraction, T cell adhesion, immune targeting, angiogenesis suppression, immune co-suppression, and cancer Stem cell-related genes CXCL13, GNLY, GZMH, IFNG, and CXCL 9, CCL5, ITGAE, VEGFA, IHH, IL17A, PROM1, REN, P This is an algorithm that integrates the expression data of F4, TSLP, and LAG3. In the case of cytokinin, CXCL13, GNLY, GZMH, IFNG, CXCL9, CCL5, and and ITGAE, and / or VEGFA, IH H, IL17A, PROM1, REN, PF4, TSLP, and any one of LAG3 Reduction of expression levels of one gene leads to a hotter tumor state, which is beneficial in cancer treatment. It is considered to be.

[0159] Immunosign® CR 21 is T cell cytotoxicity, T cell activation, T CXCL10, CXCL11, which are genes involved in cell attraction and Th1 orientation IRF1, GZMK, GZMA, CD3D, PRF1, TBX21, CXCR3, STA T1, CD69, CCL2, GZMB, CD3G, ICOS, CD8A, STAT4, G An algorithm that integrates ZMM, CCR2, CD3E, and IL15 expression data. In the context of the present invention, an increase in the expression level of any one of these genes is This is seen as a shift to a hotter tumor state, which is beneficial in cancer treatment.

[0160] Thus, in one aspect of the invention, the combination therapy targets the following gene categories (surprisingly: (which the inventors have found to be upregulated by combination therapy) or induces increased expression in multiple genes. (i) T cell activation-related proteins, preferably CD47, RPS6, CD80, and IL18 R1, CD7, PSEN2, TNFSF14, DPP4, STAT4, CCR1, FOX P3, CTLA4, LAG3, CD86, LILRB1, IL13, CD1C, EOME S, CCR4, CD3G, FAS, IL12B, IL18RAP, CD1D, CXCR3 , TIGIT, IL4, IL12A, IFNG, CD70, CD2, CD3E, CD8A , CD8B, IL12RB2, CD5, CCR5, TBX21, IL12RB1, IRF 4, ADA, CD274, LCK, F2RL1, ICOSLG, CXCL11, CXCL 10, IDO1, CX3CL1, IRF1, SOCS1, IL18, SLC11A1, E GR1, ITGA1, CXCR4, CXCL9, PTPRC, LCP1, TNFRSF1 4, PSEN1, MAF, TP53, IL4R, STAT6, IL13RA1, and I FNGR1, and optionally at least one gene selected from the group consisting of IL21R genes. Increased gene expression, (ii) involved in the activation of cytotoxic T cell function, preferably GZMM, GZMH, GZMK, GNLY, GZMB, PRF1, GZMA, HLA-C, and HLA-A an increase in the expression of at least one gene selected from the group of genes; (iii) preferably CD8A, CTSG, PRG2, CCL22, IL1B, PR F1, GNLY, CXCL10, TYK2, and OAS3 genes; increased expression of at least one pathogen defense gene; (iv) Preferably, KLRC1, KLRB1, KLRC2, IL12B, KIR3D L1, KLRF1, KLRG1, NCR1, KLRK1, IL12A, and KLRD1 increased expression of at least one NK cell function gene selected from the group of genes (v) Any of the gene categories (i), (ii), (iii), and / or (iv) Any combination of genes, preferably at least one of each of the above gene categories. Increased expression of a combination of genes including the gene.

[0161] In another embodiment of the present invention, instead of gene expression in categories (i) to (v) above, or in addition, the combination therapy includes the following genes (Immunosign® 21 present in the signature), namely CXCL10, CXCL11, IRF1, GZM K, GZMA, CD3D, PRF1, TBX21, CXCR3, STAT1, CD69, CCL2, GZMB, CD3G, ICOS, CD8A, STAT4, GZMM, CCR2 In particular, combination therapy induces increased expression of one or more of CD3E, CD4E, and IL15. , the following genes (see Figure 6D): CXCL10, CXCL11, IRF1 , GZMK, GZMA, CD3D, PRF1, TBX21, and CXCR3 can induce an increase in the expression of multiple genes.

[0162] In one aspect of the present invention, the gene expression of categories (i) to (v) and / or the above I Instead of or in addition to the genes in the mmunosign® 21 signature In addition, the combination therapy was shown to be associated with the following genes (Immunosign® 15 signature): Induce an increase or decrease in the expression of one or more of the following: The following genes: CXCL13, GNLY, GZMH, IFNG, CXCL 9, CCL5, and ITGAE, and / or The following genes: VEGFA, IHH, IL17A, PROM1, REN, Decreased expression of one or more of PF4, TSLP, and LAG3.

[0163] In the above embodiments, the expression of the disclosed gene categories or specific genes of interest is Current levels can be measured before treatment (baseline) using any suitable method known in the art. Expression levels can be measured at (in) and (after) mRNA expression levels can be compared. A (or cDNA) or protein expression levels Preferably, mRNA (or cDNA) expression levels can be measured by RT-PCR, real-time PCR, or the like. Time-resolved quantitative RT-PCR (qRT-PCR), and microarray hybridization It is measured by techniques such as ion.

[0164] The above-mentioned biological effects of the combination therapy can be monitored as biomarkers during or before the combination therapy. It can be used.

[0165] In one embodiment, they are used to, among other things, determine whether combination therapy should be continued or discontinued in a patient. It can be used as a biomarker during combination therapy to determine

[0166] Biomarkers are used to decide whether to continue or discontinue combination therapy in patients. In one embodiment, the combination therapy is directed against HPV-16 E6 and E7 proteins. Inducing or enhancing an immune response after the poxvirus and anti-PD-L1 antibody Subsequent administrations can be performed.

[0167] In another embodiment, the combination of poxvirus and anti-P is effective in treating a patient with poxvirus, as long as the combination therapy induces: Subsequent administration of the D-L1 antibody can be performed, i.e. Increased immune cell infiltration within the tumor (preferably increased CD3 T cells, more preferably or CD8 T cell expansion), and / or Reduction of intratumoral regulatory CD4 T cells.

[0168] In another embodiment, the combination therapy induces increased PD-L1 expression on tumor cells, Subsequent administration of the poxvirus and anti-PD-L1 antibody can be performed.

[0169] In another embodiment, the combination therapy is effective to induce in the circulation: Subsequent administration of a steroid hormone and an anti-PD-L1 antibody can be administered. an increase in CD8 T cells, preferably an increase in the CD8 / CD3 ratio, and / or Reduction of regulatory CD4 T cells (Treg), preferably a low Treg / CD3 ratio Decreased.

[0170] In another embodiment, the combination therapy is directed to the following gene categories: (which have been found by the present inventors to be upregulated by Subsequent administration of the poxvirus and anti-PD-L1 antibody induces increased expression. That is, (i) at least one antibody involved in T cell activation, preferably selected from those disclosed above; Increased expression of one gene, (ii) Involved in the activation of cytotoxic T cell function, preferably one of those disclosed above. Increased expression of at least one gene selected from the group consisting of: (iii) preferably at least one pathogen defense selected from those disclosed above; Increased gene expression, (iv) preferably at least one NK cell function selected from those disclosed above. Increased gene expression, (v) Any of the gene categories (i), (ii), (iii), and / or (iv) Any combination of genes, preferably at least one of each of the above gene categories. Increased expression of a combination of genes including the gene.

[0171] In another embodiment, instead of the gene expression in categories (i) to (v) above, or In addition, the combination therapy is present in the nature), namely CXCL10, CXCL11, IRF1, GZMK , GZMA, CD3D, PRF1, TBX21, CXCR3, STAT1, CD69, C CL2, GZMB, CD3G, ICOS, CD8A, STAT4, GZMM, CCR2, and IL-15. Subsequent administration of the virus and anti-PD-L1 antibody may be performed. In particular, the combination therapy may be The following genes (see Figure 6D): CXCL10, CXCL11, IRF1, G One or more of ZMK, GZMA, CD3D, PRF1, TBX21, and CXCR3 Subsequent administration of the poxvirus and anti-PD-L1 antibody induces an increase in expression of PD-L1. Administration can be performed.

[0172] In another embodiment, the gene expression of categories (i) to (v) and / or Im above Instead of or in addition to the munosign® 21 signature genes The combination therapy was shown to be associated with the following genes (Immunosign® 15 signature): The poxvirus may be used in combination with other poxviruses, as long as it induces an increase or decrease in the expression of one or more of the poxviruses present in the poxvirus. Subsequent administration of a steroid hormone and an anti-PD-L1 antibody can be administered. The following genes: CXCL13, GNLY, GZMH, IFNG, CXCL 9, CCL5, and ITGAE, and / or The following genes: VEGFA, IHH, IL17A, PROM1, REN, P Reduction of expression of one or more of F4, TSLP, and LAG3.

[0173] All references cited herein are hereby incorporated by reference into the present disclosure. Be absorbed.

[0174] The following examples are intended to be merely illustrative of the present invention. [Example]

[0175] [Example 1] Results of the Phase 1b Clinical Trial NCT03260023 NCT03260023 showed that HPV16-positive R / M (R / M refers to recurrence and / or metastasis) The combination of TG4001 and avelumab was evaluated in patients with metastatic cancers to assess safety, efficacy, and safety. The clinical and immunological responses were evaluated. In this example, preliminary Phase Ib data are presented.

[0176] Materials and methods Study design and procedures Two different dose levels (DL) of TG4001 (DL1 5 × 10 6 and DL2 5×10 7 pfu) and 10 mg / kg avelumab in a Phase 1b 3+3 design. Phase II included a multicenter, open-label, single-arm study of TG40 in DL2. 01 was administered.

[0177] TG4001 was administered weekly on days 1, 8, 15, 22, 29, and 36, followed by Once every 2 weeks (starting on day 36) until month 6 (from day 1 of study treatment), then Progression, unacceptable toxicity, or patient withdrawal from the study for any reason, whichever occurs first Avelumab was administered subcutaneously (SC) once every 12 weeks until the onset of rheumatoid arthritis. Starting on day 1 (1 week after the first TG4001 dose), patients were randomized to receive TG4001 if they had: disease progression, unacceptable toxicity, or patient withdrawal from the study for any reason, whichever occurs first. was administered (IV infusion).

[0178] Study endpoints and evaluation The safety and efficacy of the combination of TG4001 and avelumab, as well as immune parameters (T cell response) responses, changes in the infiltrate, and gene expression of immune-related genes).

[0179] Tumor response was assessed using RECIST v1.1 (Eisenhauer EA. et al., Eur J Cancer (2 009) 45(2):228-47). PBMC samples were collected longitudinally, and tissue samples were were collected at baseline and on day 43.

[0180] Study population Important selection criteria: Oropharyngeal SCCHN, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, and anal cancer metastatic or refractory / recurrent (M / R) HPV16+ cancer, including HPV-16 E7 DNA was detected by PCR using HPV-16 specific primers. Centrally determined HPV16 positivity by detecting Up to two prior systemic therapies for the management of metastatic or recurrent disease ECOG performance status 0 or 1

[0181] Important exclusion criteria: T-cell therapy, such as anti-cancer vaccines, anti-PD-L1, anti-PD-1, or anti-CTLA-4 antibodies Prior exposure to cancer immunotherapy, including any antibody targeting a cellular coregulatory protein CNS metastases Chronic treatment with systemic corticosteroids

[0182] Tumor size measurement Tumor shrinkage can be monitored by computed tomography (CT) or magnetic resonance imaging (MRI). ) preferably in the head and neck, chest, abdomen, and all other known sites of disease. CT (or MRI) was performed within 21 days before the start of study treatment. Patients were randomly assigned to receive CT scans at the time of disease progression or or for 9 months after initiation of study treatment, whichever occurs first. After 9 months of treatment, patients were assessed every 12 weeks until documented progression. All measurements were recorded in metric notation (mm).

[0183] At baseline, tumor lesions and lymph nodes were measurable (minimum size ≥ 10 mm). or lymph nodes >15 mm) or non-measurable (small lesions <10 mm, non-measurable lesions (e.g. Patients were classified as having a disease with no or no lymph nodes (e.g., pleural effusion, lymph nodes <15 mm). Patients had at least one lesion measurable by CT / MRI scan. Target lesions (all measurable lesions (nodular or non-nodular) for a total of up to 5 lesions) and non- Target lesions (all other lesions, measurable or not) were recorded. To achieve this, the sum of the longest diameters (SLD) of all target lesions (and the short axes of nodular lesions) was used as the basis. At each assessment, responses were calculated first at baseline and throughout the study. Target and non-target lesions identified at the time of the study were assessed separately. Then, calculate the overall lesion effect taking into account the presence or absence of target and non-target lesions and new lesions. It was used to Complete response (CR): Disappearance of all target lesions. Must have a reduction in short axis <10 mm (even if non-targeted). Partial Response (PR): A minor or complete reduction in the sum of the diameters of the target lesions, using the baseline sum of the diameters as reference. At least a 30% reduction. Progression (PD): Targeted progression to disease, based on the minimum total score in the study (including baseline assessment). At least a 20% increase in the sum of the diameters of the lesion and new lesions. In addition to a 20% relative increase The sum must also show an absolute increase of at least 5 mm. The appearance of new lesions is also considered progression. - Stable (SD): Insufficient shrinkage to qualify as PR and insufficient growth to qualify as PD is. - Not Evaluated (NE): No documented progression and one or more target lesions not evaluated The only exception is that the evaluation The SLD of the target lesion that can be evaluated already meets the criteria for PD. The target response is PD.

[0184] All patients evaluable for response had at least one baseline evaluable CT scan and one post-baseline evaluable C at week 6 after initiation of study treatment Participating patients who had T-scan and best overall response assessment was RECIST 1.1 The participants were different from the "unknown" group according to the evaluation criteria. Patients should be monitored for the minimum appropriate exposure unless they have progressed or died from their underlying disease prior to the procedure. Both IMPs (investigational drugs: TG4001 + avelumab) were to be administered in Russia.

[0185] Immunity Data Samples were collected after IRB approval in accordance with all ethical guidelines related to human subjects research. Peripheral blood mononuclear cells (PBMCs) were collected from consenting patients using Ficoll (registered trademark) Briefly, heparinized blood was diluted with phosphate-buffered saline (PBS) and then separated using a density gradient in the PBS layer. and Ficoll (registered trademark) medium. It was then centrifuged at 2300 g for 20 minutes. After cardiac processing, the PBMC layer was collected, diluted with buffered saline, and centrifuged at 1300 g for 10 minutes. The cells were then treated to remove any residual Ficoll solution. The cell pellet was then resuspended and centrifuged again. The cell pellet was then resuspended in storage medium (10% DMSO and 2 Resuspend in 100% IMDM containing 0% human serum, dispense into cryovials, and The mixture was frozen in a container using alcohol.

[0186] Tissue samples were obtained using standard core needle biopsy with an 18G or larger needle. Immunohistochemistry) or 10 μm (gene expression analysis) sample sections were formalin-fixed prior to processing. The tissue was then paraffin-embedded.

[0187] ELISPOT T cell responses to HPV In patients in a phase I study, IFN-γ-producing cells were isolated from ELI after a 5-day in vitro expansion phase. Briefly, after thawing, cells were counted on an NC200 automated cell counter and quantified by Spot. The cells were counted in X-VIVO-15 medium containing 2% CTS serum replacement solution, and the stimulating antigen was 2 per 500 µL per well of a 24-well culture plate in the presence or absence of E6 or E7 peptide primers were seeded on E6 cells (2 μg / mL per peptide). On day 5, cells were harvested and counted. IFN-γ was plated in quadruplicate at 2E+05 cells per well on an ELISpot IFN-γ plate. After 24 hours of incubation, ELISA was performed according to the manufacturer's instructions. The pot plates are revealed and the spots are then counted in an automated ELISPOT reader. It was dried before.

[0188] For patients in the Phase II study, the method was modified to increase specificity. Viable cells were quantified by ELISpot. Briefly, on days 0, 1 (vaccination), PBMCs were collected by venipuncture in patients on D43 (pre-vaccination) and D44 (post-vaccination). The samples were collected in CT tubes and sent to a central laboratory (P) for extraction by centrifugation on a Ficoll gradient. The cells were washed, counted, and then transferred to a 10 x 10 6 Sent in tube containing cells Prior to analysis, cells were frozen and stored in LN.

[0189] Cells were thawed and incubated with medium (negative control), E6 peptide pool (PepTivator, Mi ltenyi Biotech), E7 peptide pool (PepTivator, Mil tenyi Biotech), or CEF (PepTivator, Milteny i Biotech, positive controls: EBV, CMV, influenza) overnight. Then, cells were plated on anti-IFN-γ coated plates and incubated for 1 hour before development. Incubated (1 × 10 5 cells / well). Before and after peptide vaccination PBMCs obtained from patients known to have developed T cell responses were used as assay controls. Three replicates were performed for each condition. After overnight incubation, Reveal the ELISpot plate according to the manufacturer's instructions, then perform automated ELISA The spots were allowed to dry before being counted in a POT reader. The positive and negative controls were as expected. and the number of antigen spots was higher than the negative control by +2 times the coefficient of variation of the assay. A patient was considered positive for a given antigen if:

[0190] Immunohistochemistry for immunoscore assessment To characterize the tumor immune composition, 4-μm-thick formalin-fixed paraffin-embedded tissue sections were prepared. All analyses for a given patient included the same data set at baseline and day 43. Serial slices from the same biopsy core were used. The first slide of each series was After staining with lecithin and eosin, the tumor nature was confirmed by pathologist examination of the tissue. CD8 staining was performed on serial slides according to the following protocol: Tris base buffer. Antigen retrieval in buffer (pH = 8) for 60 minutes, quenching endogenous peroxidase activity , antibodies against CD8 at 37°C for 32 minutes, and antibodies against CD3 at 37°C for 20 minutes. Incubation, Ultraview Universal DAB IHC Detection Kit The cells were developed with acetaminophen and counterstained with Mayer's hematoxylin. The primary antibodies used were: rabbit monoclonal anti-human CD3 VM (clone 2 GV6 Ventana), mouse monoclonal anti-human CD8 (clone C8 / 144 , Dako®). Digital images of stained tissue sections were taken at 20× magnification and The images were acquired at a resolution of 0.45 μm / pixel. Whole slide digital scanning using the Immunoscore® module developed by From this, quantification of CD8- and CD3-positive cells within the tumor and invasive margin was performed.

[0191] CD3 / CD8 / PD-L1 and CD3 / CD4 / FOXP3 multispectral tissue academic evaluation Deparaffinize 4 mm thick FFPE tissue slides with ethanol ending with distilled water washes. They were rehydrated through a gradient and fixed in 10% neutral buffered formalin for 20 minutes. Activation was achieved via microwave treatment in an antigen retrieval solution. Protein blocking was performed using serum-free protein blocking solution for 15 minutes, and the primary Ab was then detected. s anti-CD3 (obtained from Ventana as above), anti-CD4 (mouse monoclonal anti-human CD4, clone UMAB64 Clinisciences) and anti-FoxP3 (Mammalian). Mouse monoclonal anti-human FOXP3, clone 236A / E7, AbCam), or Anti-CD3, anti-CD8, and anti-PDL-1 were incubated for 30 minutes at room temperature.

[0192] Next, incubation with HRP-conjugated polymer mouse or rabbit antibody was performed at room temperature. The incubation was carried out for 15 minutes, followed by 10 minutes of fluorophore incubation. All slides were counterstained with DAPI for 5 minutes, after which whole slides were scanned and Hal Established using proprietary digital pathology software developed by ioDx The results were tabulated and analyzed using the Graphpad Prism software package. was used to plot.

[0193] Analysis of gene expression changes in tumor tissue during treatment: Multiplex immunogene expression Nanostring nCounter technology was used to measure the activity of formalin-fixed tumor tissue (thickness The relative expression levels of immune genes within the tumor microenvironment were measured on a 10 μm (microscopic) microscope. After extraction, total RNA ( 300ng) were assayed and hybridized to a pan-cancer immune profiling panel This panel includes key checkpoints, chemokines, cytokines and related regulatory genes. The data contains 770 genes, including the genomic DNA fragment. Data quality control and normalization are performed by nSolve The analysis was performed using the r software package. The measured expression values ​​were calculated using the lowest coefficient of variation (% Statistical analysis was performed using nS The analysis was carried out using the Immolver advanced analysis module and the R software package. unosign® is a commercially available, proprietary software developed by HalioDx. It was defined using a simple algorithm.

[0194] Example 1A Phase Ib Study and Results Study population Nine patients (four females and five males) were enrolled in this study and received the treatment regimen described above. In accordance with the protocol, 5 × 10 6 pfu(DL1) or 5×10 7 Patients were treated with pfu(DL2) TG4001. Table 1 shows the patient demographics. Total and baseline characteristics are shown.

[0195] [Table 1]

[0196] Baseline characteristics of patients were as follows: Various cancer types, mainly squamous cell carcinoma of origin ( Have HPV-16-positive cancers (anal, cervical, oropharyngeal, and vaginal cancers) Median age was 57.8 years (range 39–78). At baseline, all patients had distant showed metastasis.

[0197] Overview of treatment-related adverse events Safety was monitored through adverse event (AE) reporting and by clinical and physical examinations at various time points. Adverse events and laboratory findings were assessed by blood pressure, electrocardiogram (ECG), and vital signs. Abnormalities were assessed by the National Cancer Institute Common Toxicity Assessment Criteria for Adverse Events (NCI-CTCAE). Toxicity was graded according to the NCI-CTCAE (version 4.03). Treatment-related adverse event data were summarized by dose level and reported in Table 2 below.

[0198] [Table 2-1]

[0199] [Table 2-2]

[0200] As shown in Table 2, all patients experienced at least one AE. A total of 68 adverse events were observed in 10 patients, 23 in DL1 and 45 in DL2. The 4001 and avelumab combination was well tolerated. No serious adverse events (SAEs) were observed, and only two grade 3 events occurred in one DL1-treated patient (5 × 1 0 6 PFU).

[0201] Changes in tumor size Tumor size changes during combination therapy are shown in Figures 1A and 1B. As shown, DL2-treated patients Most of the patients experienced a reduction or stabilization of tumor size.

[0202] Partial response (PR) during the course of the clinical study, as assessed by RECIST 1.1 criteria The number and percentage of stable disease (SD) and progressive disease (PD) are also shown in Table 3 below.

[0203] [Table 3]

[0204] The results in Table 3 and Figures 1A and 1B demonstrate that combination therapy can stabilize the disease or It has been shown to induce partial responses in two-thirds of all patients. Efficacy is stable or partial response. Approximately 83% of patients received high-dose DL2 TG4001 (5 × 10 7 pfu) is better Yes.

[0205] Immunity Data Specific T cell responses to HPV Four patients had ELISPOT responses to HPV-16 E6 and E7 on day 43. The ELISPOT responses are shown in Table 4 below.

[0206] [Table 4]

[0207] Table 4 shows that 3 of 4 patients evaluable for ELISPOT were at E6 or E7 on day 43. These results indicate that the subjects had E7-reactive T cells.

[0208] Changes in TILs under treatment CD8 / CD3 ratio and Treg (CD4 F) at baseline and day 43 The CD8 / CD3 ratios are shown in Figures 2A and 2B. These show that the CD8 / CD3 ratios increased during the treatment period. Associated with an overall increase in the 3-specific infiltrate and a decrease in the Treg (CD4 FoxP3) / CD8 ratio This indicates a more favorable immune profile (strength of immune-stimulatory CD8 T cells) This suggests that the immune system is more susceptible to inflammatory cytokines (e.g., Tregs) than immune suppressors (e.g., Tregs).

[0209] PD-L1 expression on TILs and tumor cells under treatment PD-L1 expression was measured at baseline and day 43 (and for one patient Seven patients (also at day 85) were evaluated on TIL and tumor cells, five of whom had baseline Evaluation was possible at baseline and on day 43. The results are shown in Table 5 below.

[0210] [Table 5]

[0211] The results in Table 5 show that 4 of 5 patients were evaluable at baseline and day 43. showed that patients had a significant increase in PD-L1 expression on tumor cells at day 43 This is predicted to correlate with an increased propensity to respond to immunotherapy treatment.

[0212] Analysis of gene expression changes in tumor tissue during treatment To explore the gene expression changes in tumor tissue induced by therapy, we are conducting immunotherapy-related studies. Expression of a panel of 770 genes related to gliomas-like syndrome (GI) was assessed at baseline and after treatment (day 43). In particular, Immunosign® 15 and Immunosign® We studied the expression of a gene signature previously described as a target gene (Galon et al. , Immunity (2013) 9(1): 11-26; Marabelle et al., Society for Immunotherapy of Ca cer (SITC) 32nd Annual Meeting & Pre-Conference Programs (SITC 2017) on Novembe r 8-12, 2017 at the Gaylord National Hotel & Convention Center in National Harbo r, Maryland. Poster P250).

[0213] Volcano Probe of gene expression and pathway changes identified as overexpressed post-treatment vs. pre-treatment The black dots represent the mechanism shown (e.g., T in Fig. 3A). The expression levels are expressed in log-scale. The expression level correlates with the position in the matrix (overexpression to the right of 0, underexpression to the left of 0).

[0214] Figure 3 shows the mechanisms of viral vaccine responses (pathogen protection, see Figure 3C) and antitumor immunity. Timing (T cell activation, cytotoxic cell, and NK cell function, Figures 3A, 3B, and 3D) were overexpressed during treatment.

[0215] Also, Figure 4A shows the results of Immunosign® 15 and Immunosign® Gene categories included in the gene signature previously described as ®21 4B and 4C show the results of Immunosign® 15 and Many genes in the Immunosign® 21 signature are overexpressed during treatment. It shows that it has appeared.

[0216] The observed gene expression changes are associated with priming of innate and adaptive immunity and "hotter" ( This transition is consistent with a transition to a "hotter" tumor profile, which is more consistent with and is associated with improved clinical outcomes for patients.

[0217] Case study of patient 0101006 More detailed data is shown for patient 0101006, who has cervical cancer.

[0218] Changes in immune infiltrate: Patient 0101006 had a tumor with low levels of infiltration, tumor cells, and infiltrating immune cells. Moderate PD-L1 expression and low spatial distribution of CD8 cells and PD-L1-expressing tumor cells All these characteristics are consistent with baseline cold (col d) Consistent with the tumor.

[0219] The changes in immune infiltrate are shown in Figure 5, and the TIL and PD-L1 expression on the surface of tumor cells is shown in Table 6 below.

[0220] [Table 6]

[0221] Figure 5 and Table 6 show that at day 43, tumors were significantly more infiltrated, with four times the CD3 infiltration. There was a >100% increase in CD8+ T cell proliferation, a 3-fold increase in CD8+ T cell infiltration, and a doubling of tumor and PD-L1 immunoexpression. The level of infiltration by immunosuppressive cells during treatment was not significantly No changes were observed.

[0222] Furthermore, digital pathology analysis of the specimens revealed that infiltrating CD8 T cells were present around the periphery of PD-L1-positive tumor cells. This indicates that the cells are concentrated in the nucleus (Fig. 5B), which indicates that checkpoint inhibition is effective. This suggests a more favorable profile for

[0223] Changes in gene expression: As shown in the color map of 770 immune-related genes (data not shown), Gene expression profiles in patient tumor tissue also revealed significant changes over the course of treatment. It became clear.

[0224] Analysis of gene expression changes revealed that the expression of IgG1 regulates antigen processing and presentation (Figure 6A), the response to the virus, and the immune response to the virus. (Fig. 6B), and a strong increase in the expression of genes related to Toll-like receptor expression (Fig. 6C). Activation of these pathways is crucial for the adaptive response to viral vaccines. It is consistent with development.

[0225] In addition, 9 genes in the Immunosign® 21 signature were strongly overexpressed. This suggests that the immune-excluded phenotype may be related to the ability to benefit from immunotherapeutic intervention. This suggests a shift to "hot" tumors where treatment is more likely to yield significant benefit.

[0226] conclusion Preliminary results from NCT03260023 Phase 1b indicate that: The combination of TG4001 and avelumab was safe and effective at both dose levels studied. For G4001, sufficient treatment is available for patients with HPV-positive cancer who have received multiple prior lines of therapy. is tolerated. The combination showed promising efficacy signals in DL2 and is being evaluated in an ongoing Phase II study. It is said that. This procedure keeps tumors cold and resistant to chemotherapy, even in heavily pretreated patients. It is possible to alter the course of the disease by shifting the immune system from a normal to a hotter immune state. It is associated with highly altered tumor microenvironment. This is particularly true in patients with an “immune-excluded” tumor phenotype, as demonstrated in individual case studies. It may be useful for

[0227] These results suggest that (a) at least human papillomavirus (HPV) E6 and E7 Poxvirus vectors encoding polypeptides and immunostimulatory cytokines (b) The combination of anti-PD-L1 antibodies is safe in the treatment of patients with HPV-positive cancer. These findings suggest that the drug is safe, well tolerated, and effective (at least additively).

[0228] Example 1B Phase II studies and pooled interim analyses Patient / disease characteristics In Phase II, TG4001 was administered at DL2, which is recommended after completion of Phase IB. At the Phase II doses tested, the combination of TG4001 and avelumab was safe and No significant differences were observed between DL1 and DL2 in terms of the nature and severity of AEs observed. This was supported by results showing that the interim analysis planned according to the protocol The study enrolled 25 patients, of whom 3 were not evaluable for tumor response. Of the 22 evaluable patients, 11 patients (50%) presented with anal cancer. , 4 patients (18.2%) presented with cervical cancer and 4 patients (18.2%) with vaginal / vulvar cancer. 3 patients (13.6%) had oropharyngeal cancer. Prior to this, seven additional patients were enrolled, one of whom was evaluable for tumor response. Of the six evaluable patients, four patients (66.6%) presented with anal cancer. One patient each presented with oropharyngeal cancer (16.6%) and cervical cancer (16.6%). did.

[0229] For subgroup analysis, patients treated with TG4001 (N=6) in DL2 in Phase Ib part Patients treated in the Phase II part were pooled with those treated in the Phase II part (N=28, discussed immediately above). Of the 6 patients treated in Phase Ib, 4 patients (66.6%) had oropharyngeal cancer. One patient each showed vaginal cancer (16.6%) and cervical cancer (16.6%). Of the pooled dataset (N=34), 15 patients (44.1%) had anal 8 patients (23.5%) presented with oropharyngeal cancer, and 6 patients (17.6%) presented with 5 patients (14.7%) presented with cervical cancer, and 20.6% presented with vaginal / vulvar cancer. RR was observed.

[0230] The pooled patient population was analyzed by the presence of liver metastases (absence vs. presence as shown in Table 7), disease characteristics (Table 8), and prior chemotherapy treatment (Table 9).

[0231] Table 7 shows the gender and performance of subgroups of patients with and without liver metastases. Specifically, the subgroup of patients without liver metastases was 2 The subgroup of patients with liver metastases consisted of 11 patients (non-hepatic patients). It consists of patients (liver patients).

[0232] [Table 7]

[0233] Regarding the disease characteristics of the pooled dataset (N=34), as shown in Table 8, 1 Five patients presented with anal cancer, eight patients with oropharyngeal cancer, and six patients with cervical cancer. Five patients presented with vaginal / vulvar cancer. An ORR of 20.6% was observed.

[0234] [Table 8]

[0235] [Table 9]

[0236] Correlation of ORR and PFS with patient / disease characteristics Based on the above stratification, objective response rate (ORR) or progression-free survival (PFS) and , correlations of each patient / disease characteristic were assessed.

[0237] The correlation of ORR and PFS with each patient / disease characteristic is shown in Figures 7 and 8, respectively.

[0238] Regarding ORR, one disease characteristic (boxed in Figure 7), i.e., liver metastases, Only the presence of α-glucan significantly correlated with worsened ORR (see Figure 7, where Figure 7 shows an OR of 100). , 95% interval of 5–100, and p-value of 0.012).

[0239] Regarding PFS, three characteristics were significantly correlated with poor or favorable PFS. It was found that there were several (these are boxed in Figure 8). The presence of liver metastases was significantly correlated with a worse PFS (see Figure 8). The HR was 90, the 95% interval was 1.728–11.152, and the p-value was 0.002. (There are) Anal cancer was significantly associated with a poorer PFS (see Figure 8, which shows a mean PFS of 3.214 HR of 1.269, 95% interval of 1.269–8.143, and p-value of 0.014) , The presence of lymph node metastasis was significantly correlated with a relatively favorable PFS (see Figure 8). Figure 8 shows a HR of 0.388, a 95% interval of 0.151–0.998, and a p-values ​​are shown).

[0240] Regarding the association between relatively poor PFS and anal cancer, this association is unclear in patients with anal cancer. It should be noted that this may be indirectly explained by the relatively high prevalence of liver metastases in In fact, there are cases of anal cancer that have not shown metastases in the liver but have shown metastases in other organs. Patients have documented responses.

[0241] In addition, PFS was not significant for the number of patients analyzed in the pooled interim analysis. There was no correlation between genital (vulvar / vaginal) cancer and relatively good PFS (p=0.073). A tendency is observed.

[0242] Therefore, the only characteristic significantly correlated with both ORR and PFS was the presence of liver metastases. , which was significantly correlated with poorer ORR and PFS.

[0243] Therefore, this stratification was studied more thoroughly.

[0244] Table 10 below shows the efficacy parameters (RECIST 1.1, response, stable disease at 12 weeks, liver metastases according to progression before or at 12 weeks, and median PFS Further showing the distribution of patients with and without liver metastases when treated with combination therapy However, patients with liver metastases had higher response and stable disease at 12 weeks and lower response rates before or after 12 weeks. It has been shown to have a median progression-free survival (PFS) of 2 weeks and a high median PFS.

[0245] [Table 10]

[0246] Figure 9 shows the results of 23 patients from the pooled Phase Ib and Phase II interim results who were free of liver metastases. Figure 10 shows a graphical representation of the maximum change in tumor size in patients with liver metastases, pooled The same graph is shown for nine patients from the Phase Ib and Phase II interim results. There are.

[0247] Table 11 below shows the incidence of liver metastases according to primary tumor type (anal, oropharyngeal, cervical, vulvar / vaginal). The distribution of patients with and without liver metastases is shown. , and patients with liver metastases (regardless of primary tumor, no responses were observed in these patients) ) have a higher response rate than patients with vulvar / vaginal cancer without liver metastases. It also showed a high response rate (66.7%), although the number of patients analyzed was small (3 patients). Therefore, this high response rate should be interpreted with caution.

[0248] [Table 11]

[0249] Immunity Data Specific T cell responses to HPV Eleven patients were screened by ELISPOT for HPV-16 E6 and E7 on day 43. Table 12 shows that 7 of 11 patients were evaluable for response after vaccination. , showing that the mice had a detectable response in ex vivo ELISPOT. None of the patients had a pre-existing response in this experimental setting. ELISPOT responses is shown in Table 12 below.

[0250] [Table 12]

[0251] immune permeate The observations made during Phase 1 of the study were confirmed in Phase 2, with Immunosign® ) signature and an increase in the infiltrate of CD3-positive cells.

[0252] Transcriptome analysis Transcriptome analysis of tumors collected from patients with and without liver metastases was performed as described in Materials and Methods.

[0253] The present inventors were able to demonstrate gene expression variations in liver metastases. In particular, as shown in Table 13 and Figure 11, ST6GAL1 (p<0.001) and HAMP (p<0.0001) gene was overexpressed (2.74 and 7 C8A (Log2 change of 6.71; p<0.001), C8B (Log2 change of 7.25; p<0.001), C3 (Log2 change of 3.41; p<0.001) .001), C6 (Log2 change of 5.87; p<0.001), and C2 (2.06 Genes associated with the complement pathway were over-represented, including genes related to the complement pathway (Log2 change in β-glucan; p<0.001). was.

[0254] ST6GAL1 is associated with aggressiveness in many cancers, and HAMP is involved in iron metabolism. It is known to regulate immune cell activity through modification.

[0255] The effect of complement on cancer immunity is controversial, and high expression of the complement pathway is associated with F It has been shown to exert cytotoxic effects on immune cells, including CD4 and CD8. This is significant, given that the liver is the primary site of synthesis and regulation of complement factors. Interesting.

[0256] In addition, cytokines related to inflammation were also expressed, contributing to the creation of an immunosuppressive tumor environment. This immunosuppression may be detrimental to effector immune cells and may contribute to tumor These specific transactivities may promote immune escape and disease progression. Cryptome signatures are consistent with resistance to immune intervention in patients with liver metastases.

[0257] [Table 13-1]

[0258] [Table 13-2]

[0259] In conclusion, clinical observation of patients is a determinant of treatment success and clinical outcome. Genomic data suggest that tumor liver metastasis may be due to downregulation of the immune system or tumor revealed that the expression of genes and pathways associated with aggressiveness in .

[0260] conclusion In conclusion, the pooled interim analysis of phase Ib and phase II studies showed that the presence of liver metastases Indeed, liver metastases have been shown to be associated with lower ORR and shorter PFS. By stratifying patients according to the presence or absence of liver metastases, a subgroup of patients with liver metastases (N=11) was identified. In the subgroup of patients without liver metastases (N=23), the ORR was 3%, compared with 0% in the control group. An ORR of 0.4% was observed. Similarly, the median PFS in patients without liver metastases (N=23) was The median PFS for patients with liver metastases (N=11) was only 1. .4.

[0261] In addition, a pooled interim analysis of Phase Ib and II studies showed that liver Due to the relatively high prevalence of metastases, anal cancer is associated with a relatively low PFS, whereas external Vaginal / vaginal cancer has been shown to show a trend towards a relatively high PFS.

[0262] Finally, lymph node involvement is associated with a relatively favorable PFS.

Claims

1. for use in the treatment of HPV-positive cancer or precancerous lesions in situ, a) at least human papillomavirus (HPV) E6 and E7 polypeptides, and and poxvirus vectors encoding immunostimulatory cytokines. b) Anti-PD-L1 antibody or antigen-binding fragment thereof A combination of The first administration of the poxvirus is 5 to 10 days after the first administration of the anti-PD-L1 antibody. and a subsequent administration of the poxvirus and anti-PD-L1 antibody. A combination.

2. The poxvirus is a vaccinia virus, preferably a modified vaccinia virus 2. The combination of claim 1, which is Kara (MVA).

3. The poxvirus contains membrane-bound HPV-16 non-oncogenic E6 and E7 polypeptides. and human interleukin 2 (IL-2), The combination according to claim 1.

4. The anti-PD-L1 antibody of claims 1 to 3 mediates antibody-dependent cellular cytotoxicity (ADCC). A combination according to any one of claims 1 to 4.

5. The anti-PD-L1 antibody or antigen-binding fragment thereof is selected from the group consisting of amino acids 1, 2, and 3. A heavy chain containing three complementarity determining regions having the amino acid sequences of SEQ ID NOs: 4, 5 and 6. Preferably, the anti-PD-L antibody comprises a light chain having three complementarity determining regions having the amino acid sequence 1. The antibody has a heavy chain having the amino acid sequence SEQ ID NO: 7 or 8 and an amino acid sequence of SEQ ID NO: 9 The combination according to any one of claims 1 to 4, comprising a light chain having the sequence:

6. The combination of any one of claims 1 to 5, wherein the anti-PD-L1 antibody is avelumab. Fake.

7. The HPV-positive cancer is HPV-positive oropharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, vulvar cancer, cancer, penile cancer, mucosal cancer, or non-melanoma skin cancer, or the precancerous lesion in situ , cervical intraepithelial neoplasia (CIN) grade 2 or 3 or vulvar intraepithelial neoplasia (VIN) ) grade 2 or 3, and preferably the HPV-positive cancer is HPV-positive vulvar cancer or The combination according to any one of claims 1 to 6, wherein the cancer is selected from the group consisting of steroids, steroids for the treatment of rheumatoid arthritis and vaginal cancer.

8. The cancer is HPV-16 positive, preferably the cancer is HPV-16 positive head and neck cancer Squamous cell carcinoma (HPV-16+ SCCHN) or HPV-16 positive vulvar or vaginal cancer The combination according to claim 7,

9. The HPV-positive cancer is an HPV-positive cancer without multiple liver metastases, and preferably, The method according to any one of claims 1 to 8, wherein the HPV-positive cancer is an HPV-positive cancer without liver metastasis. A combination of.

10. wherein said HPV-positive cancer is a recurrent and / or metastatic HPV-positive cancer, preferably The HPV-positive metastatic cancer is an HPV-positive metastatic cancer with lymph node metastasis. The combination according to any one of claims 7 to 9.

11. The HPV-positive cancer is an HPV-positive metastatic cancer without multiple liver metastases, preferably 11. The method of claim 10, wherein the HPV-positive cancer is a metastatic HPV-positive cancer without liver metastasis. A combination of.

12. Each dose of the poxvirus is 3 x 10 ７ ~7 x 10 ７ pfu dose, The combination according to any one of claims 1 to 11.

13. a) 3 x 10 of said poxvirus ７ ~7 x 10 ７ The first dose of pfu is administered subcutaneously. Then, 3 x 10 ７ ~7 x 10 ７ Subsequent poxvirus doses of pfu were administered until disease progression. in, Once a week for six weeks Once every two weeks until the sixth month, and Subsequent poxvirus doses every 12 weeks It is administered subcutaneously, b) a first dose of about 10 mg / kg or about 800 mg of anti-PD-L1 antibody is administered to said first patient; poxvirus dose, followed by intravenous administration of up to about 10 mg / kg. or subsequent anti-PD-L1 antibody doses of approximately 800 mg are administered intravenously every 2 weeks until disease progression. can The combination according to any one of claims 1 to 12, which is administered according to the following administration scheme:

14. a) the poxvirus comprises membrane-associated HPV-16 non-oncogenic E6 and E7 polypeptides; an MVA virus encoding a peptide and human IL-2, b) the anti-PD-L1 antibody is avelumab; c) i) Membrane-bound HPV-16 non-oncogenic E6 and E7 polypeptides and human I MVA virus encoding L-2 was 5 × 10 ７ pfu dose once a week for 6 weeks , then administered subcutaneously every 2 weeks until 6 months, and then every 12 weeks until disease progression; ii) avelumab at approximately 10 mg / kg every 2 weeks starting on day 8 until disease progression or administered by intravenous infusion at a dose of about 800 mg The poxvirus and the anti-PD-L1 antibody are administered according to the following administration scheme: A combination according to any one of claims 1 to 13.

15. - increasing the immune response to HPV16 E6 and E7 polypeptides; - Within the tumor, an increase in immune cell infiltrate, preferably an increase in the CD8 / CD3 ratio, Decrease in regulatory CD4 T cells, preferably a reduction in the Treg / CD8 ratio, and / or Or Increased expression of PD-L1 on tumor cells, During blood circulation, Increased CD8 T cells, and / or Decrease in regulatory CD4 T cells, and / or - A significant remodeling of gene expression in tumor cells, T cell activation genes, cytotoxic cell genes, pathogen defense genes, and NK cells Increased expression of functional genes, ○CXCL10, CXCL11, IRF1, GZMK, GZMA, CD3D, PRF 1, TBX21, CXCR3, STAT1, CD69, CCL2, GZMB, CD3G, ICOS, CD8A, STAT4, GZMM, CCR2, CD3E, and IL15 groups and / or CXCL13, GNLY, GZMH, IFNG, CXCL9, CCL5, and I Increased expression of one or more genes selected from the group consisting of TGAE, and / or are VEGFA, IHH, IL17A, PROM1, REN, PF4, TSLP, and L Reducing the expression of one or more genes selected from the group of AG3 Remodeling characterized by The combination according to any one of claims 1 to 14, which induces

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