Combination of poxvirus encoding HPV polypeptides and IL-2 with anti-PD-L1 antibodies

Abstract

The present invention relates to a combination of a) a poxvirus vector encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and an immunostimulatory cytokine, and b) an anti-PD-L1 antibody or an antigen-binding fragment thereof, for use in the treatment of HPV-positive cancer, wherein a first administration of the poxvirus occurs 5 to 10 days before a first administration of the anti-PD-L1 antibody, and subsequent administrations of the poxvirus and anti-PD-L1 antibody are administered.

Description

[Technical Field]

[0001] The present invention relates to a combination of a) a poxvirus vector encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and an immunostimulatory cytokine, and b) an anti-PD-L1 antibody or an antigen-binding fragment thereof, for use in the treatment of HPV-positive cancer, wherein a first administration of the poxvirus occurs 5 to 10 days before a first administration of the anti-PD-L1 antibody, and subsequent administrations of the poxvirus and anti-PD-L1 antibody are administered. [Background technology]

[0002] Human papillomavirus (HPV) is the most commonly diagnosed sexually transmitted disease. HPV is associated with genital warts, anogenital (cervix, vagina, vulva, penis, and anus) squamous intraepithelial lesions and malignancies, and also with 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 HPV genome encodes the DNA sequences of six early (E) proteins and two late proteins that form the viral shell, which are involved in viral gene regulation and cell transformation, as well as a region of regulatory DNA sequence known as the long regulatory region (LCR) or upstream regulatory region (Palefsky JM and Holly EA, Cancer Epidemiol Biomarkers Prev. (1995) 4(4): 415-428).

[0004] HPV genotypes can be broadly divided into "high-risk" (16, 18, 31, 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) HPV 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 cervical cancer, HPV has been linked to anal and penile cancer. Patients infected with high-risk (carcinogenic) HPV subtypes, i.e., HPV-16 genotype, plus HPV-18, 31, or 33, also have a two- to three-fold increased risk of oral and oropharyngeal cancer. HPV-associated tumors occur primarily at the base of the tongue or in the tonsillar region, although a small percentage of tumors in other sites are also HPV-positive. It is unclear why the oropharynx is more susceptible to HPV transformation than other sites.

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

[0006] High-risk HPV infection is not only associated with various cancers but is also known to be involved in the oncogenesis of 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 should be considered along with traditional risk factors such as tobacco abuse or alcohol abuse (D'Souza G. et al., The New England Journal of Medicine. (2007) 356(19): 1944-1956).

[0007] The mechanism linking carcinogenesis and HPV infection has been shown to involve two viral proteins, E6 and E7. E6 and E7 are said to be oncoproteins because they have the ability to disrupt normal replication control in infected cells by inhibiting key regulatory factors. The E6 oncoprotein binds to the p53 tumor suppressor protein and induces its degradation through a ubiquitin-mediated process that disrupts the p53 pathway, resulting in uncontrolled cell cycle progression (Chung CH and Gillison ML, Clin Cancer Res. (2009) 15(22): 6758-6762). The HPV E7 protein binds to and inhibits the retinoblastoma protein (pRb), preventing it from inhibiting the transcription factor E2F, resulting in loss of cell cycle control. Furthermore, functional inactivation of Rb leads to upregulation of the p16 protein. P16 is encoded by the CDKN2A tumor suppressor gene and regulates the activity of the cyclin D-CDK4 / 6 complex, which phosphorylates Rb, thereby leading to the release of the transcription factor E2F, which initiates cell cycle progression.

[0008] Two preventive vaccines have been developed against HPV infection: a quadrivalent vaccine (Gardasil) and a bivalent vaccine (Cervarix). Both are approved for the prevention of HPV infection and HPV-associated disease in subjects who have not previously been exposed to HPV.

[0009] However, to date, few antitumor vaccines for HPV-positive cancers, particularly SCCHN, have been clinically evaluated. Vaccine-induced generation and long-term maintenance of tumor-specific immune cells is a goal that an antitumor vaccine must achieve to be clinically beneficial in HPV-positive cancers, particularly SCCHN.

[0010] TG4001 (corresponding to the research name MVATG8042) is a therapeutic recombinant vaccine / immunotherapy product based on the highly attenuated, non-transmissible vaccinia vector, Modified Vaccinia Virus Ankara (MVA). Its genome, a single linear double-stranded DNA molecule of approximately 178 kilobase pairs, contains inserted transgenes encoding three proteins: the HPV E6 and E7 oncoproteins, which have been modified to eliminate their oncogenic potential, and human interleukin-2 (IL-2) as an adjuvant.

[0011] TG4001 has been clinically investigated in gynecological conditions. Of the five Phase II studies conducted, four included patients with precancerous lesions, specifically cervical intraepithelial neoplasia (CIN) grade 2 / 3 and vulvar intraepithelial neoplasia (VIN) grade 3, and one study included patients with cervical cancer. Two Phase II studies, including 21 (TG4001.07) and 206 (NV25025) patients with HPV-16-associated CIN grade 2 / 3, demonstrated proof-of-concept that TG4001 had high activity and efficacy compared with placebo in terms of histologic resolution and response rates, as well as viral clearance. The dose used in these Phase II studies in patients with HPV-16-associated CIN grade 2 / 3 was 5 × 10 administered subcutaneously. 7 Regarding the safety profile, the therapeutic vaccine product was well tolerated (no major toxicities were observed), with the most common adverse events being injection site reactions (Brun JL et al., Am J Obstet Gynecol. (2011) 204(2): 169 e161-168, Harper DM et al., Gynecol Oncol. (2019) 153(3): 521-529).

[0012] The overall safety profile of TG4001, based on data from a total of 313 subjects (healthy volunteers or patients with CIN 2 / 3, cervical cancer, or VIN treated with TG4001 via the intramuscular or subcutaneous route as monotherapy or in combination with the immunomodulator imiquimod), was evaluated in patients receiving 5x10 TG4001 weekly or every 3 weeks for up to 7 weeks in a maximum of 6 injections. 7 The study showed that TG4001 was well tolerated up to the highest dose tested, i.e., 100 pfu. Consistent with its immunostimulatory properties, administration of TG4001 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 is a negative regulator of T cell activity, limiting T cell activity at various stages of the immune response when it interacts with its two ligands, PD-L1 and PD-L2. Upon ligand binding, PD-1 inhibits, via phosphatase activity, the kinase signaling pathway that normally leads to T cell activation. Several antibodies that disrupt the PD-1 axis are in clinical development. PD-L1 is also thought to negatively signal T cells by interacting with B7, and PD-L1-blocking antibodies prevent this interaction. Immune checkpoint inhibitors also enhance the function of tumor-infiltrating lymphocytes (TILs), which enhance antitumor immunity within the tumor microenvironment. The presence of TILs correlates with a relatively favorable prognosis in many cancer types. Thus, PD-L1+ TILs have been shown to be an indicator of response to immune checkpoint inhibition, and the absence of TILs may be a predictive marker of lack of response to PD-1 / L1 inhibition (Herbst RS et al., Nature. (2014) 515(7528): 563-567).

[0014] Anti-PD-L1 antibodies have been clinically investigated in the treatment of various solid tumors and have been found to provide clinical benefit in a variety of cancers (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. It has been shown in preclinical models to mediate both adaptive and innate immune functions. Avelumab has been shown to reverse suppression of T cell-mediated antitumor immune responses in preclinical models by blocking the interaction between the PD-1 receptor and PD-L1. Avelumab has also been shown to induce NK cell-mediated direct tumor cell lysis in vitro via antibody-dependent cellular cytotoxicity (ADCC). When combined with axitinib, avelumab is indicated in the United States for the first-line treatment of patients with advanced renal cell carcinoma (RCC). The U.S. Food and Drug Administration (FDA) has also granted accelerated approval to avelumab for (i) the treatment of adult and pediatric patients 12 years of age and older with metastatic Merkel cell carcinoma (mMCC) and (ii) patients with locally advanced or metastatic urothelial carcinoma (mUC) who have disease progression during or after platinum-containing chemotherapy or who have disease progression within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

[0016] Avelumab has demonstrated an acceptable safety profile in cancer patients. Warnings and precautions for avelumab include immune-mediated adverse reactions (pneumonitis and hepatitis, including fatalities, colitis, endocrinopathy, nephritis and renal impairment, and other adverse reactions, which may be severe and have included fatalities), infusion-related reactions, major adverse cardiac events (MACE), and embryo-fetal toxicity.

[0017] Clinical trials testing avelumab in HPV-positive cancers, including SCCHN, are currently underway, but no results have been published at this time.

[0018] Despite the results obtained with TG4001 in precancerous lesions or with anti-PD-L1 antibodies alone in cancer, there remains a significant need for novel treatments to improve anti-tumor efficacy and responder rates. Thus, there remains a need to develop novel therapeutic options for treating cancer. In particular, there exists a need for (a) poxvirus vectors encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines, or (b) methods of treating cancer that improve the efficacy of anti-PD-L1 antibodies in one or more types of cancer. The present invention provides combination products for use in the treatment of HPV-positive cancer that address the above needs.

[0019] Immune checkpoint inhibitors (including anti-PD-L1 antibodies) have been proposed for combination with many other types of anticancer therapies, including vaccine-based immunotherapies such as poxvirus vectors. In most cases, experiments have been performed in animal models of cancer not associated with HPV infection, and the toxicity of the tested combinations has not been specifically analyzed (WO 2016 / 128542, WO 2015 / 175334, WO 2015 / 069571, Remy-Ziller et al., Hum Vaccin Immunother. (2018) 14(1): 140-145). Two phase II clinical trials (NCT03353675 and NCT02823990) are ongoing combining TG4010 (an MVA vector encoding MUC1 and interleukin-2) and nivolumab (an anti-PD-1 antibody) in the treatment of non-small cell lung cancer (NSCLL) (Oliveres H. et al., J Thorac Dis. (2018) 10(Suppl 13): S1602-S1614), but the results of the trials have not been published.

[0020] In the context of HPV-positive cancer, various vaccine combinations, including viral vaccines, intended to stimulate immune responses to HPV antigens and combinations of immune checkpoint inhibitors have also been proposed (Gildener-Leapman et al., Oral Oncol. (2014) 50(9): 780-4; WO 2015 / 103602; Rice et al., Cancer Gene Ther. (2015) 22(9): 454-62; WO 2016 / 071306; U.S. Patent No. 2017 / 051019; U.S. Patent No. 2019 / 142933). To date, no experimental data have been disclosed demonstrating an acceptable toxicity profile and improved therapeutic efficacy for HPV-positive cancers of a combination of a viral vaccine encoding an HPV antigen and an anti-PD-L1 antibody. An ongoing phase Ib / II clinical trial (NCT03260023) of the combination of TG4001 and avelumab in the treatment of HPV-positive SCCHN 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 surprisingly found that the combination of a poxvirus vector, preferably TG4001, encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines with an anti-PD-L1 antibody or antigen-binding fragment thereof, preferably avelumab, using a specific administration scheme in HPV-positive cancer patients resulted in acceptable toxicity and improved immune responses to HPV E6 and E7 polypeptides. Notably, despite the immunostimulatory effects of both avelumab and TG4001 (live vector), no interactions between the two products resulting in unacceptable toxicity were observed, which was not reasonably foreseeable. In particular, given the fact that the U.S. Food and Drug Administration (FDA) issued a clinical hold on a study combining an anti-PD-L1 antibody with a Listeria monocytogenes vaccine (NCT02291055) after a patient died from complications of an adverse event, there was a risk of an interaction between the two products that could result in unacceptable toxicity, especially given the immunostimulatory effects of both products. This case suggested that the vaccine (another type of live vector) may have amplified known side effects of immune checkpoint inhibitors. Furthermore, promising beneficial immune changes were observed with this combination, including induction of immune responses to HPV E6 and E7 polypeptides, an increase in CD8 T cells and a decrease in CD4 regulatory T cells in the circulation and tumor tissue, and upregulation of genes associated with a "hot" rather than a "cold" tumor profile and a relatively favorable prognosis, which were not observed with either treatment alone.

[0022] The enhancement may be additive or synergistic. The enhancement effect of the combination therapy is at least additive. The inventors surprisingly found that the combination of (a) a poxvirus vector encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines with (b) an anti-PD-L1 antibody results in improved treatment. Initial results of clinical trials indicate that the combination therapy is effective in treating cancers, such as recurrent / metastatic HPV16-positive cancers (see Example 1), and that the combination therapy is well tolerated (see Example 1). Furthermore, effects attributable to each of the two treatments in the combination were observed (see Example 1). This indicates at least an additive enhancement effect of the combination therapy.

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

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

[0025] The present invention provides For the manufacture of a medicament for use in treating HPV-positive cancer or HPV-positive cancer intraepithelial precancerous lesions, a) a poxvirus vector encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines; b) an anti-PD-L1 antibody or antigen-binding fragment thereof The use of a combination of The present invention also relates to a use wherein the first administration of the poxvirus is administered 5 to 10 days before the first administration of the anti-PD-L1 antibody, followed by subsequent administrations of the poxvirus and anti-PD-L1 antibody.

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

[0027] In the combination, method, or use, the poxvirus is preferably a vaccinia virus, more preferably a modified vaccinia virus Ankara (MVA), preferably encoding membrane-bound HPV (preferably HPV-16) non-oncogenic E6 and E7 polypeptides and human interleukin-2 (IL-2). Most preferably, the poxvirus is an MVA virus encoding membrane-bound HPV-16 non-oncogenic E6 and E7 polypeptides and human IL-2. Each dose of poxvirus administered to a subject is preferably 3 x 10 7 From 7 x 10 7 pfu, more preferably about 5 x 10 7 pfu. Each dose of poxvirus administered to a subject is preferably administered subcutaneously.

[0028] In the combination, method, or use, the anti-PD-L1 antibody or antigen-binding fragment thereof preferably mediates antibody-dependent cellular cytotoxicity (ADCC). Structurally, the anti-PD-L1 antibody or antigen-binding fragment thereof preferably comprises a heavy chain comprising three complementarity-determining regions having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and a light chain comprising three complementarity-determining regions having the amino acid sequences of SEQ ID NOs: 4, 5, and 6. More preferably, the anti-PD-L1 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 7 or 8, and a light chain having the amino acid sequence of SEQ ID NO: 9. Most preferably, the anti-PD-L1 antibody is avelumab. Each dose of the anti-PD-L1 antibody or antigen-binding fragment thereof is preferably about 10 mg / kg or about 800 mg. Each dose of the anti-PD-L1 antibody or antigen-binding fragment thereof is preferably administered intravenously, more preferably by intravenous infusion.

[0029] The targeted therapeutic application is the treatment of HPV-positive cancer, preferably HPV-positive oropharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, vulvar cancer, penile cancer, mucosal cancer, or non-melanoma skin cancer, or HPV-positive intraepithelial precancerous lesions. The cancer or intraepithelial precancerous lesion is preferably HPV-16 positive, and the cancer may particularly be HPV-16-positive squamous cell carcinoma of the head and neck (HPV-16+ SCCHN). The targeted cancer is more preferably recurrent and / or metastatic HPV-positive cancer (preferably recurrent and / or metastatic HPV16-positive cancer, most preferably recurrent and / or metastatic HPV16-positive SCCHN).

[0030] The combination of (a) a poxvirus vector encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines and (b) an anti-PD-L1 antibody can be provided in a single dosage form or in separate unit dosage forms. The combination is administered according to a specific administration scheme, in which the first administration of the poxvirus is administered 5 to 10 days before the first administration of the anti-PD-L1 antibody, and subsequent administrations of the poxvirus and anti-PD-L1 antibody are administered. Preferably, the subsequent administrations of the poxvirus and anti-PD-L1 antibody are administered until disease progression. More preferably, the combination is administered according to the following administration scheme:

[0031] a) 3 x 10 of the poxvirus 7 From 7 x 10 7 A first dose of 3 x 10 pfu was administered subcutaneously, followed by 3 x 10 7 From 7 x 10 7 Subsequent poxvirus doses of pfu progress until 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 intravenously 5-10 days after the first poxvirus dose, followed by subsequent doses of about 10 mg / kg or about 800 mg of anti-PD-L1 antibody administered intravenously every two weeks until disease progression.

[0032] Particularly preferred embodiments according to the present invention are as follows. a) the poxvirus is an MVA virus encoding membrane-associated HPV-16 non-oncogenic E6 and E7 polypeptides 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) MVA virus encoding membrane-associated HPV-16 non-oncogenic E6 and E7 polypeptides and human IL-2 was administered in a volume of 5 x 10 7 pfu once weekly for 6 weeks, then every 2 weeks until 6 months, and then every 12 weeks thereafter until disease progression. ii) Avelumab is administered by intravenous infusion at a dose of about 10 mg / kg or about 800 mg every 2 weeks starting on day 8 until disease progression.

[0033] The combination, method or use according to the present invention preferably induces a positive immune response against cancer or precancerous lesions in the subject being treated. In particular, in the circulation, the combination for use according to the present invention preferably increasing the proportion of circulating HPV E6 and / or E7 polypeptide-specific CD8 and / or 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 Increased tumor tissue infiltration by HPV E6 and / or E7 polypeptide-specific CD8 and / or CD4 T cells; and / or ·Reduce tumor tissue infiltration by regulatory T cells.

Brief Description of 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 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, shaded gray) according to the dosing scheme described in Example 1. Partial response (PR) according to RECIST v1.1 is indicated by *(B). Change in individual long axis direction: % change from baseline (change from 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 the 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 of individual patients. [Figure 3] It is a figure showing the analysis of gene expression changes in tumor tissue during treatment. The expression of a panel of 770 genes related to the immune response was evaluated at baseline and after treatment (day 43). Volcano plots of changes in gene expression of T cell activation (A), cytotoxic cells (B), pathogen defense (C), and NK cell function (D) after treatment compared to before treatment. 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," unless otherwise indicated by context, are used throughout this application to mean "at least one," "at least a first," "one or more," or "plurality" of the compounds or steps to which they refer. Thus, the terms include both only one of the referenced compounds or steps, and two or more of the referenced compounds or steps.

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

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

[0039] As used herein, the term "combination" refers to any possible assortment of two or more entities (e.g., at least a poxvirus and an anti-PD-L1 antibody as described herein). In particular, a "combination" refers to (i) a product comprised of two or more coordinated components physically, chemically, or otherwise combined, or mixed and produced as a single entity; (ii) two or more separate products packaged together in a single package or as a unit and comprised of a drug and device product, a device and biological product, or a biological and drug product; or (iii) a separately packaged drug, device, or biological product intended solely for use with an approved, individually designated drug, device, or biological product by its investigational plan or proposed labeling, and intended for its intended use, application, or other similar purpose. or (iv) any separately packaged investigational product, device, or biological product that is intended for use only with another individually designated investigational product, device, or biological product by its proposed labeling, and both are required to achieve its intended use, application, or effect.

[0040] As used herein, the terms "combination therapy," "in combination with," or "in conjunction with" refer to any form of combined, parallel, simultaneous, sequential, or intermittent treatment with at least two different treatment modalities (i.e., compounds, components, targeting agents, or therapeutic agents). Thus, the term refers to the administration of one treatment modality before, during, or after the administration of another treatment modality to a subject. The combined modalities may be administered in any order. The therapeutically active modalities are administered together (e.g., simultaneously in the same or separate compositions, formulations, or unit dosage forms) or separately (e.g., according to appropriate administration protocols for separate compositions, formulations, or unit dosage forms, on the same or different days, in any order), in a manner and dosage regimen prescribed by a healthcare provider or in accordance with a regulatory agency. Generally, each treatment modality is administered at a dose and / or time schedule determined for that treatment modality. Optionally, three or more modalities may be used in the combination therapy. In addition, the combination therapies provided herein may be used in conjunction with other types of treatments, for example, other anti-cancer treatments selected from the group consisting of chemotherapy, surgery, radiotherapy (radiation), and / or hormonal therapy, and other treatments associated with the current standard of care for a subject.

[0041] As used herein, in each instance, the terms "comprise" (and any form of "comprise," such as "comprises" and "comprising"), "having" (and any form of "having," such as "had" and "having"), "including" (and any form of "comprising," such as "included" and "comprising"), or "containing" (and any form of "containing," such as "contained" and "containing"), when used to define products, compositions, and methods, are open-ended and do not exclude additional, unrecited elements or method steps. Thus, a polypeptide "comprises" an amino acid sequence if that amino acid sequence could be part of the final amino acid sequence of that polypeptide. "Consisting essentially of" means excluding any other essential, significant components or steps. Thus, a composition consisting essentially of the recited components would not exclude trace amounts of contaminants and pharmaceutically acceptable carriers, but would exclude other active ingredients. A polypeptide "consists essentially of" an amino acid sequence if that amino acid sequence is present, optionally with insignificant additional amino acid residues. "Consisting of" means excluding more than trace amounts of other ingredients or steps. For example, a polypeptide "consists of" an amino acid sequence if it contains no amino acids other than those recited. In the context of this disclosure, whenever a product or method or use is indicated as "comprising" something, embodiments in which the product or method consists essentially of or consists of the same something are also contemplated in the context of the present invention.

[0042] The terms "mutant," "analog," or "variant," as used herein, refer to a component (polypeptide or nucleic acid) that exhibits one or more modifications relative to its unmodified counterpart. Any modification can be envisioned, including substitution, insertion, and / or deletion of one or more nucleotide / amino acid residues. When several mutations are contemplated, they can involve consecutive and / or non-consecutive residues. Mutations can be introduced by several methods known to those skilled in the art, such as site-directed mutagenesis (e.g., using the Sculptor™ in vitro mutagenesis system, Amersham, Les Ullis, France), PCR mutagenesis, DNA shuffling, and chemical synthesis techniques (e.g., resulting in synthetic nucleic acid molecules). Preferred analogs retain a degree of sequence identity with their unmodified counterparts, having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. Generally, the term "identity" refers to amino acid-to-amino acid or nucleotide-to-nucleotide correspondence between two polypeptide or nucleic acid sequences. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to achieve optimal global alignment (i.e., optimal alignment of both full-length sequences). Various computer programs and mathematical algorithms are available in the art for determining the percentage identity between amino acid or nucleic acid sequences.

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

[0044] The terms "polypeptide," "peptide," and "protein" refer to a polymer of amino acid residues comprising at least nine or more amino acids linked through peptide bonds. The polymer may be linear, branched, or cyclic, may comprise naturally occurring amino acids and / or 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 preferably referred to as a polypeptide or protein, while if it is 50 amino acids or less in length, it is referred to as a "peptide."

[0045] The term "subject" generally refers to an organism that is in need of or could benefit from any of the products and methods of the present invention. Typically, the organism is a mammal. Preferably, the subject is a human who has or has been diagnosed as being at risk for a pathological condition, such as an infectious disease caused by or associated with a pathogenic organism, or a proliferative disease such as cancer. The terms "subject" and "patient" can be used interchangeably when referring to a human organism and include males and females. The subject being treated can be a neonate, infant, young adult, or adult.

[0046] The terms "treatment" and "therapy," as used herein, refer to a set of hygienic, pharmacological, surgical, and / or physical measures used with the intention of curing and / or alleviating diseases and / or symptoms, with the goal of improving a health problem. Preventive and curative methods are both included within the terms "treatment" and "therapy," as they aim to maintain and / or restore the health of an individual or animal. The administration of appropriate medicines to alleviate and / or cure a health problem, regardless of the origin of the symptoms, diseases, and disorders, should be considered within the context of this application as a form of treatment or therapy.

[0047] Poxvirus vectors The combination, method or use according to the invention contains as a first component a poxvirus vector encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and an immunostimulatory cytokine.

[0048] Thus, the term "poxvirus vector" or "poxviral vector" should be broadly understood to include nucleic acid vectors (e.g., DNA poxviral vectors) that contain at least one element of a poxvirus genome and may be packaged in poxviral particles, and the poxviral particles resulting therefrom. The terms "poxvirus," "poxvirion," "poxviral particle," and "poxviral vector particle" are used interchangeably to refer to poxviral particles formed when a nucleic acid vector is transduced into an appropriate cell or cell line with conditions suitable to allow the production of poxviral particles. The term "infectious" refers to the ability of a poxviral vector to infect and enter a host cell or subject. Poxviral vectors can be replication-competent, replication-selective (e.g., engineered to replicate better or preferentially in specific host cells), or can be replication-deficient or genetically disabled so as to be replication-impaired.

[0049] Types of poxviruses As used herein, the term "poxvirus" refers to viruses belonging to the Poxviridae family, of which the Chordopoxvirinae subfamily, which targets vertebrate hosts, is preferred. The Chordopoxvirinae subfamily includes several genera, such as Orthopoxvirus, Capripoxvirus, Avianpoxvirus, Parapoxvirus, Leporipoxvirus, and Swinepoxvirus. In the context of the present invention, Avianpoxviruses, including Orthopoxvirus, Canarypoxvirus (e.g., ALVAC), and Fowlpoxvirus (e.g., FP9 vector), are preferred.

[0050] In a preferred embodiment of the combination, method, or use according to the invention, the poxvirus belongs to the genus Orthopoxvirus, more preferably to the species Vaccinia virus (VV). Vaccinia virus is a large, complex, enveloped virus with a linear, double-stranded DNA genome approximately 200 kb in length, which encodes numerous viral enzymes and factors that enable the virus to replicate independently of the host cell machinery. Two different infectious virus particles exist: the intracellular IMV (representing the intracellular mature virion), surrounded by a single lipid envelope, which remains in the cytosol of the infected cell until lysis, and the double-enveloped EEV (representing the extracellular enveloped virion), which buds out from the infected cell.

[0051] A particularly suitable poxvirus in the context of the present invention is MVA (Modified Vaccinia Virus Ankara) due to its highly attenuated phenotype, the pronounced IFN-type 1 response generated during infection compared to non-attenuated vectors, and the availability of the sequence of its genome (see, for example, Genbank under accession number U94848).

[0052] HPV E6 and E7 polypeptides The poxvirus (preferably VV, more preferably MVA) of the combination, method or use according to the invention encodes at least human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines.

[0053] Over 100 HPV genotypes have been identified, and they are classified into "low-risk" (LR) and "high-risk" (HR) serotypes according to their oncogenic potential. LR-HPVs cause benign tumors in infected subjects, whereas HR-HPVs are at high risk for malignant progression. The gene products encoded by E6 and E7 of HR HPV genotypes are involved in the oncogenic transformation of infected cells, presumably through the binding of these viral proteins to the cellular tumor suppressor gene products p53 and retinoblastoma (Rb), respectively (Howley, 1996, Papillomaviruses and their replication, p 2045-2076. Reviewed in BN Fields, DM Knipe and PM Howley (eds), Virology, 3rd ed. Lippincott-Raven Press, New York, NY). The amino acid residues involved in binding of the unmodified HPV-16 E6 polypeptide to p53 have been clearly defined as residues 118 to 122 (+1 being the first Met residue, or, preferably, residues 111 to 115 starting from the second Met residue) (Crook et al., Cell (1991) 67, 547-556), and the amino acid residues involved in binding of the unmodified HPV-16 E7 polypeptide to Rb 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, the poxvirus (preferably VV, more preferably MVA) preferably encodes at least the E6 and E7 polypeptides of an HR-HPV, wherein the HR-HPV is preferably selected from HPV-16, HPV-18, HPV-30, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66, HPV-68, HPV-70, and HPV-85, more preferably from HPV-16 and HPV-18, and most preferably, said HR-HPV is HPV-16.

[0055] Sources of papillomaviruses include, but are not limited to, biological samples (e.g., biological samples, tissue sections, biopsy specimens, and tissue cultures collected from subjects exposed to papillomaviruses), cultured cells (e.g., CaSki cells available from ATCC), and recombinant materials available in depositories or sales catalogs or described in the literature. The nucleotide sequences and amino acid sequences of the encoded polypeptides of several papillomavirus genomes have been described in the literature and are available in specialized data banks (e.g., Genbank). For general information, the HPV-16 genome has accession numbers NC_01526 and K02718, HPV-18 has NC_001357 and X05015, HPV-31 has J04353, HPV-33 has M12732, HPV-35 has NC_001529, HPV-39 has NC_001535, HPV-45 has X74479, and HPV-46 has X74479. PV-51 is listed in Genbank as NC_001533, HPV-52 as NC_001592, HPV-56 as X74483, HPV-58 as D90400, HPV-59 as NC_001635, HPV-68 as X67160 and M73258, HPV-70 as U21941, and HPV-85 as AF131950.

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

[0057] As defined above in connection with the term "polypeptide," "papillomavirus polypeptides" include unmodified and modified papillomavirus polypeptides and peptides thereof. In particular, the present invention encompasses the use / expression of unmodified HPV E6 and E7 polypeptides and analogs thereof (e.g., fragments thereof, such as peptides, and modified versions), especially when the unmodified polypeptides exert undesirable properties (e.g., oncogenic or transforming properties, cytotoxicity, etc.). For example, to circumvent the tumorigenic properties of HPV E6 and E7 polypeptides, one can use or express non-oncogenic analogs that exhibit reduced ability to bind to p53 and Rb, respectively.

[0058] Suitable E6 polypeptides for use in the present invention include non-oncogenic mutants that are unable to bind to the cellular tumor suppressor gene product p53. Representative examples of non-oncogenic E6 polypeptides have been described in the art (see, e.g., WO 1999 / 03885). Preferred modifications in this context include deletions of one or more amino acid residues located from about position 118 to about position 122 in HPV-16 E6 (+1 represents the first methionine residue in the native HPV-16 E6 polypeptide), with deletions of residues 118-122 (CPEEK) in HPV-16 E6 (see, e.g., SEQ ID NO: 12) or residues 113-117 (NPAEK) in HPV-18 E6 being particularly preferred.

[0059] Suitable E7 polypeptides for use in the present invention include non-oncogenic mutants that are unable to bind to the cellular tumor suppressor gene product Rb. Representative examples of non-oncogenic E7 polypeptides have been described in the art (see, e.g., WO 1999 / 03885). Preferred modifications in this context include the deletion of one or more amino acid residues located from about position 21 to about position 26 in HPV-16 E7 (+1 represents the first amino acid of the unmodified HPV-16 E7 polypeptide), with the deletion of residues 21-26 (DLYCYE) in HPV-16 E7 (see, e.g., SEQ ID NO: 13) or residues 24-28 (DLLCH) in HPV-18 E7 being particularly preferred.

[0060] The HPV (preferably HPV-16) E6 and / or E7 polypeptides for use in the present invention may be further modified to be membrane-anchored and enhance efficient membrane display of the polypeptide on the surface of the expressing host cell. This may be achieved by fusing the HPV (preferably HPV-16) E6 and / or E7 polypeptide to a signal peptide and a membrane-associated peptide. Such peptides are known in the art. Briefly, signal peptides are generally present at the N-terminus of membrane-presented or secreted polypeptides and initiate their passage into the endoplasmic reticulum (ER). Signal peptides contain 15 to 35 essentially hydrophobic amino acids, which are then removed by specific ER-localized endopeptidases to yield the mature polypeptide. The membrane-associated peptide is usually highly hydrophobic in nature and serves to anchor the polypeptide to the cell membrane (see, e.g., Branden and Tooze, 1991, in Introduction to Protein Structure, pp. 202-214, Garland, NY). The choice of signal peptides and membrane-bound peptides that can be used in the context of the present invention is enormous. They can be obtained independently from any secreted or membrane-bound polypeptide (e.g., cellular or viral polypeptide), such as rabies glycoprotein, HIV virus envelope glycoprotein, or measles virus F protein, or can be synthetic. The preferred site for signal peptide insertion is the N-terminus downstream of the translation initiation codon, and for membrane-bound peptides, it is the C-terminus, e.g., immediately upstream of the stop codon. If necessary, a linker peptide can be used to connect the signal peptide and / or membrane-bound peptide to the encoded polypeptide.

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

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

[0063] Suitable promoters for driving expression of at least the human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines encoded by the poxvirus vectors included in the combinations, methods, or uses of the present invention are preferably poxvirus promoters, such as vaccinia virus promoters 7.5K, H5R, Tk, p.28, p.11, or K1L. Synthetic promoters are also suitable, as are chimeric promoters of late and early promoters. Such promoters are well known in the art. Preferably, expression of both the HPV E6 and E7 polypeptides is under the control of the variola p7.5 promoter, and expression of the immunostimulatory cytokine (e.g., human IL-2) is under the control of the variola pH5R promoter.

[0064] Immunostimulatory cytokines In addition to at least the human papillomavirus (HPV) E6 and E7 polypeptides (preferably as described above), the poxvirus (preferably VV, more preferably MVA) of the combination, method or use according to the invention further encodes an immunostimulatory cytokine.

[0065] As used herein, the term "immunostimulatory cytokine" refers to a cytokine capable of specifically or non-specifically stimulating the immune system. A vast number of cytokines are known in the art for their ability to exert immunostimulatory effects. Non-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 (e.g., IFNα, IFNβ, IFNγ), tumor necrosis factor (TNF), colony-stimulating factors (e.g., GM-CSF, C-CSF, M-CSF), growth factors (transforming growth factor TGF, fibroblast growth factor FGF, vascular endothelial growth factor VEGF, and the like). Preferably, the immunostimulatory cytokine is an interleukin or colony-stimulating factor (e.g., GM-CSF). More preferably, the immunostimulatory cytokine is interleukin 2 (IL-2), most preferably human IL-2.

[0066] Preferred poxviruses A preferred poxvirus of the combination, method or use according to the invention is the MVA virus encoding the membrane-bound non-oncogenic HPV-16 E6 and E7 polypeptides and human IL-2, more preferably represented by TG4001 described in WO 1999 / 03885 under its scientific name MVATG8042.

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

[0068] Antibodies or antigen-binding fragments thereof The term "antibody" refers to an immunoglobulin molecule capable of specifically binding to an antigen, e.g., a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the context of the present invention, "antibody" (or "Ab") is used in the broadest sense and encompasses naturally occurring antibodies and human-engineered antibodies, including full-length antibodies or functional fragments or analogs thereof that are capable of binding to an antigen, such as PD-L1 (and thus retain the antigen-binding portion). Antibodies used in the present invention may be of any origin, e.g., human, humanized, animal (e.g., rodent or camelid antibodies), or chimeric. Antibodies may be of any isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgM, etc.). In addition, antibodies may be glycosylated or non-glycosylated. The term antibody also includes bispecific or multispecific antibodies, so long as they exhibit binding specificity for an antigen, such as PD-L1. As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen-binding fragment thereof or antibody fragment that competes for specific binding with the intact antibody, fusion proteins containing antigen-binding portions (e.g., antibody-drug conjugates), any other modified configuration of immunoglobulin molecules containing antigen recognition sites, antibody compositions with polyepitopic specificity, and multispecific antibodies (e.g., bispecific antibodies). However, intact, i.e., unfragmented, monoclonal antibodies are preferred.

[0069] For illustrative purposes, a full-length antibody is a glycoprotein comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH) made up of three CH1, CH2, and CH3 domains (optionally with a hinge between CH1 and CH2). Each light chain is composed of a light chain variable region (VL) and a light chain constant region comprising one CL domain. Each VH and VL region contains three hypervariable regions termed complementarity-determining regions (CDRs), interspersed with more conserved regions termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDR regions of the heavy and light chains are generally responsible for determining binding specificity.

[0070] The CDR of an antibody is defined by its heavy and light chain amino acid sequence, which is compared with the criteria known to those skilled in the art.Various methods have been proposed for determining CDR, and the part of the amino acid sequence of the heavy or light chain variable region of an antibody that is defined as CDR varies depending on the method selected.In this description, all CDRs are defined according to the AbM definition used by Oxford Molecular's AbM antibody modeling software (see, for example, the CDR sequence of avelumab in WO2013 / 079174).

[0071] The antibody may be monoclonal, human, chimeric, humanized, and / or human, and may comprise a human constant region that can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function).

[0072] In the context of the present invention, it is preferable to use monoclonal antibodies. As used herein, "monoclonal antibody" refers to a composition containing antibody molecules with identical and unique antigen specificity. The antibody molecules present in this composition are likely to vary in terms of their post-translational modifications, and particularly in terms of their glycosylation structure or their isoelectric point, but they are all encoded by the same heavy and light chain sequences and therefore have the same protein sequence before any post-translational modifications. Some differences in protein sequence related to post-translational modifications (e.g., cleavage of the heavy chain C-terminal lysine, deamidation of asparagine residues, and / or isomerization of aspartic acid residues) may still exist among the various antibody molecules present in the composition. Monoclonal antibodies can be produced using hybridoma technology or methods that do not use hybridoma technology (e.g., recombinant methods). Human monoclonal antibodies can be produced using transgenic mice carrying human immunoglobulin genes rather than the mouse system. Splenocytes obtained from these transgenic mice immunized with an antigen of interest are used to produce hybridomas secreting human mAbs with specific affinity for epitopes derived from human proteins.

[0073] For the treatment of human subjects, which is a preferred embodiment of the invention, the anti-PD-L1 antibodies are preferably chimeric, humanized, or fully human, thereby limiting or preventing immune responses to the non-human portions of the anti-PD-L1 antibody. The antibodies may be those in which the variable regions or portions thereof, e.g., CDRs, have been generated in non-human organisms, e.g., rats or mice. Chimeric, CDR-grafted, and humanized antibodies are within the scope of the present invention. Antibodies that have been generated in non-human organisms, e.g., rats or mice, and then modified, e.g., within the variable framework or constant regions, to reduce antigenicity in humans, are within the scope of the present invention.

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

[0075] As used herein, "humanized antibody" refers to a non-human (e.g., murine, camel, rat, etc.) antibody whose protein sequence has been modified to increase its similarity to human antibodies (i.e., those naturally produced in humans). Antibodies can be humanized by methods known in the art. For example, monoclonal antibodies developed for human use can be humanized by leaving the majority of residues in the variable regions (especially the CDRs) unaltered and corresponding to those of non-human immunoglobulins, but substituting one or more residues in the FR regions to resemble human immunoglobulin sequences. As a general guide, the number of these amino acid substitutions in the FR regions is typically 20 or less per VH or VL of each variable region. In another example, a humanized or CDR-grafted antibody has at least one or two, and generally all three, recipient CDRs (of the immunoglobulin heavy and / or light chains) replaced with donor CDRs. An antibody may have at least a portion of a non-human CDR replaced, or only some of the CDRs replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to anti-PD-L1. Preferably, the donor is a rodent antibody, e.g., a rat or mouse antibody, and the recipient is a human framework or human consensus framework. Typically, the immunoglobulin providing the CDRs is referred to as the "donor," and the immunoglobulin providing the framework is referred to as 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 or consensus framework, or a sequence that is about 85% or more, preferably 90%, 95%, or 99% or more identical thereto. Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR-replacement, in which one, two, or all CDRs of an immunoglobulin chain may be replaced. See, for example, U.S. Patent No. 5,225,539, which describes CDR-grafting methods that can be used to prepare the humanized antibodies of the invention.

[0076] As used herein, a "human antibody" refers to an antibody in which the entire amino acid sequence of the heavy and light chains is derived from human germline immunoglobulin sequences, rather than just the constant regions (as in chimeric antibodies) and FRs (as in humanized antibodies) being of human origin. Such human antibodies can be obtained, for example, from transgenic animals or human antibody libraries into which human germline immunoglobulin sequences have been inserted.

[0077] The term "antigen-binding fragment" of any antibody refers to the portion of the intact antibody that binds to the antigen. Antigen-binding fragments may contain the variable regions that determine the antigenicity of the intact antibody. Antigen-binding fragments can be engineered for use in the combinations of the invention. Representative examples include, but are not limited to, Fab, Fab', F(ab')2, dAb, Fd, Fv, scFv, di-scFv, diabodies, and any other artificial antibody. For example, a "PD-L1-binding fragment" of any anti-PD-L1 antibody refers to the portion of the intact antibody that binds to the antigen PD-L1. More specifically, the following antigen-binding fragments of full-length anti-PD-L1 antibodies may be used in the combinations, methods, or uses according to the invention: (i) The Fab fragment is represented by a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. (ii) The F(ab')2 fragment is represented by a bivalent fragment comprising two Fab fragments linked by at least one disulfide bridge in the hinge region. (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) comprises two domains of an Fv fragment, VL and VH, which are fused together, optionally with a linker, to form a single protein chain. (vii) Any other artificial antibody.

[0078] Methods for preparing antibodies, fragments, and analogs thereof are known in the art (see, e.g., Harlow and Lane, 1988, Antibodies - A laboratory manual: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). In one embodiment, such antibodies can be generated in a host animal using a PD-L1 antigen (preferably a human PD-L1 antigen for human use). Alternatively, such antibodies can be generated from hybridomas (see, e.g., Kohler and Milstein, Nature (1975) 256: 495-7), recombinant techniques (e.g., using phage display), peptide synthesis, and enzymatic cleavage. Antibody fragments can be produced by recombinant techniques described herein. Antibody fragments may also be produced by proteolytic cleavage using enzymes such as papain to produce Fab fragments or pepsin to produce F(ab')2 fragments. Analogs (or fragments thereof) can be generated by conventional molecular biology methods (PCR, mutagenesis techniques). If desired, such fragments and analogs can be screened for functionality in the same manner as intact antibodies (eg, by standard ELISA assays).

[0079] PD-1 and PD-L1 Programmed death 1 (PD-1) belongs to the immunoglobulin (Ig) gene superfamily and is a member of the CD28 family. PD-1 is a 55-kDa type 1 transmembrane protein expressed on cells that have been exposed to antigens (e.g., activated B cells, T cells, and myeloid cells). In normal contexts, PD-1 acts by limiting T cell activity during inflammatory responses, thereby protecting normal tissues from destruction. Two ligands for PD-1, PD-L1 (programmed death ligand 1) and PD-L2 (programmed death ligand 2), have been identified. PD-L1 has been identified in 20–50% of human cancers. The interaction between PD-1 and PD-L1 results in a reduction of tumor-infiltrating 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 Q15116.

[0080] The anti-PD-L1 antibodies of a combination, method or use according to the invention preferably recognise human PD-L1, and further information about human PD-L1 (including the known amino acid sequence) is also available in the UniProtKB database under accession number Q9NZQ7.

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

[0082] The term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them with cytotoxins. Antibodies are required to arm the cytotoxic cells and kill the target cells by this mechanism. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch & Kinet, Annu Rev Immunol (1991) 9: 457-92. Thus, anti-PD-L1 antibodies may contain an ADCC-competent Fc region and improve the efficacy of current therapies by promoting ADCC lysis of cancer cells. Therefore, the anti-PD-L1 antibodies of the combinations, methods, or uses according to the present invention preferably mediate ADCC. It is particularly preferred to use full-length antibodies comprising a functional Fc region. The Fc region can be modified (at the amino acid or glycosylation level) to further improve ADCC capability (such modifications include, inter alia, one or more substitutions in the Fc and / or reduced fucosylation, as are well known in the art). Nevertheless, such ADCC-mediating anti-PD-L1 antibodies are not toxic or exhibit no increased toxicity.

[0083] Structural features of anti-PD-L1 antibodies Examples of monoclonal antibodies that bind to human PD-L1 and are useful in the combinations for use in the present invention are described in WO2007 / 005874, WO2010 / 036959, WO2010 / 077634, WO2010 / 089411, WO2013 / 019906, WO2013 / 079174, WO2014 / 100079, WO2015 / 061668, and U.S. Patent Nos. 8,552,154, 8,779,108, and 8,383,796. Specific anti-human PD-L1 monoclonal antibodies useful as PD-L1 antibodies in the combinations for use in the present invention include, but are not limited to, for example, avelumab (MSB0010718C), durvalumab (MEDI4736, an engineered IgG1 kappa monoclonal antibody with a triple mutation in the Fc domain to ablate ADCC), atezolizumab (MPLDL3280A), MPDL3280A (an IgG1 engineered anti-PD-L1 antibody), and BMS-936559 (an anti-PD-L1 fully human IgG4 monoclonal antibody).

[0084] Avelumab and atezolizumab are unique among currently available anti-PD-L1 antibodies in that they are fully human IgGs with unmutated Fc regions. Thus, avelumab contains an antibody-dependent cellular cytotoxicity (ADCC)-competent Fc region that has been shown to mediate ADCC (Boyerinas et al., Cancer Immunol Res. (2015) 3(10):1148-1157). Antibodies containing ADCC-competent Fc regions may improve the efficacy of current therapies by promoting ADCC lysis of cancer cells.

[0085] In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain comprising three complementarity-determining regions having the amino acid sequences of SEQ ID NO: 1 (avelumab H-CDR1: SYIMM), SEQ ID NO: 2 (avelumab H-CDR2: SIYPSGGITFYADTVKG), and SEQ ID NO: 3 (avelumab H-CDR3: IKLGTVTTVDY), and a light chain comprising three complementarity-determining regions having the amino acid sequences of SEQ ID NO: 4 (avelumab L-CDR1: TGTSSDVGGYNYVS), SEQ ID NO: 5 (avelumab L-CDR2: DVSNRPS), and SEQ ID NO: 6 (avelumab L-CDR3: SSYTSSSTRV). Because the CDR regions are known to be particularly involved in antigen recognition, such anti-PD-L1 antibodies or antigen-binding fragments thereof are predicted to have binding to PD-L1 similar to that of avelumab.

[0086] It has been observed that the C-terminal lysine (K) of the heavy chain is often cleaved off during antibody production. This modification does not affect antibody-antigen binding. Therefore, in some preferred embodiments, 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 an antibody or antigen-binding fragment thereof having structural similarity to avelumab. Avelumab, its sequence, and many of its properties are described in International Publication No. WO 2013 / 079174, in which avelumab is designated A09-246-2, which has heavy and light chain sequences set forth in SEQ ID NOS: 32 and 33 (corresponding to SEQ ID NOS: 7 and 9 herein). Avelumab has two main mechanisms of action for exerting its anti-tumor effects. First, PD-L1 on tumor cells can interact with PD-1 or B7-1 on activated T cells. These interactions have been shown to significantly inhibit T cell activity. Therefore, blocking PD-L1 interaction with PD-1 or B7-1 with anti-PD-L1 can relieve T cells from immune suppression, leading to the elimination of tumor cells by T cells. Second, tumor cells may express higher levels of PD-L1 on their surface compared to normal tissues. As a fully human IgG1 monoclonal antibody, avelumab has ADCC capabilities. Avelumab binds to PD-L1 on tumor cells, and when its Fc portion binds to Fc-gamma receptors on leukocytes, it can induce antitumor ADCC.

[0091] In a preferred embodiment of the invention, the anti-PD-L1 antibody comprises the six CDRs of avelumab (SEQ ID NOs: 1-6) and blocks the interaction between human PD-1 and human PD-L1. Preferably, the anti-PD-L1 antibody also mediates ADCC. More preferably, the anti-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 amino acid sequences of the heavy chain (SEQ ID NO: 7 or 8) and light chain (SEQ ID NO: 9) of avelumab and blocks the interaction between human PD-1 and human PD-L1. Preferably, the anti-PD-L1 antibody also mediates ADCC. More preferably, the anti-PD-L1 antibody is an IgG antibody, with an IgG1 antibody being 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" refers to a group of diseases that can be defined as any abnormal malignant new growth of tissue without a physiological function, resulting from uncontrolled, usually rapid, cell proliferation and having the potential to invade or spread to other parts of the body. The term "precancerous lesion" refers to a benign lesion involving abnormal cells and associated with an increased risk of developing into cancer.

[0095] In one embodiment of the present invention, the targeted therapeutic application is the treatment of HPV-positive cancer or intraepithelial precancerous lesions.

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

[0097] HR-HPV produces two oncoproteins, E6 and E7, which are required for viral replication through their growth-stimulating activity and play a key role in malignant transformation. The E6 oncoprotein binds to the p53 tumor suppressor protein and induces its degradation via a ubiquitin-mediated process, disrupting the p53 pathway and resulting in uncontrolled cell cycle progression. The HPV E7 protein binds to and degrades the retinoblastoma protein (pRb), preventing it from inhibiting the transcription factor E2F, resulting in loss of cell cycle control. Furthermore, functional inactivation of Rb leads to the upregulation of the p16 protein. p16 is encoded by the CDKN2A tumor suppressor gene and regulates the activity of the cyclin D-CDK4 / 6 complex, which phosphorylates Rb and releases the transcription factor E2F, which initiates cell cycle progression. HPV-positive tumors are characterized by high levels of expression of p16 (Nevins JR, Hum Mol Genet. (2001) 10(7): 699-703).

[0098] The presence of HPV virus can be detected indirectly by various methods based on the detection of HPV DNA, HPV RNA, or HPV oncoproteins, or by looking for altered cellular protein expression, such as overexpression of the p16 protein. The p16 protein can be detected by immunohistochemistry (IHC), and several studies have shown a high correlation (>90%) with HPV positivity in oropharyngeal tumors, suggesting it may be a clinically useful surrogate marker (Mellin Dahlstrand H. et al., Anticancer Res. (2005) 25(6C): 4375-4383). The presence of HPV (and therefore the HPV-positive nature of cancer or intraepithelial precancerous lesions) can also be determined by detecting changes in cellular protein expression, such as (1) HPV DNA, (2) post-integration transcription of viral E6 and / or E7 mRNA, (3) viral oncoproteins E6 and E7, or (4) overexpression of p16 protein (Kim et al., J Pathol Clin Res. (2018) 4(4): 213-226). HPV DNA can be detected using, among other methods, polymerase chain reaction (PCR) or in situ hybridization (ISH). HPV RNA can be detected using, among other methods, polymerase chain reaction (RT-PCR) or in situ hybridization (ISH). Various kits for determining the HPV status (positive or negative) of cancerous or precancerous lesions are commercially available and can be used in the context of the present invention (see Table 1 in Kim et al., J Pathol Clin Res. (2018) 4(4): 213-226).

[0099] In a preferred embodiment, since HPV-16 is the main HR-HPV detected in HPV-positive cancers, the HPV status of cancerous or precancerous lesions is determined by detecting HPV-16 E7 DNA by PCR using HPV-16-specific primers. In a more preferred embodiment, the DNA of the subject to be treated is extracted from a tumor sample (e.g., a fixed tumor sample, such as a formol- or formalin-fixed paraffin-embedded (FFPE) tumor sample) by conventional methods, and then HPV-16 E7 DNA is amplified by PCR using HPV-16-specific primers. If amplification is detected (e.g., by immunofluorescence means such as SYBRgreen), the sample is considered HPV-16 positive. If amplification is not detected by this method, HPV E7 DNA is amplified by PCR using consensus primers that can amplify approximately 50 HPV genotypes, and then the amplified sequence is sequenced using Sanger sequencing. The sequences obtained then make it possible to confirm negatives and identify patients whose sample quality did not allow a result to be obtained in the primary PCR, or to detect positive patients with relatively rare genotypes.

[0100] Preferred HPV-positive cancers include HPV-positive oropharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, vulvar cancer, penile cancer, mucosal cancer, or non-melanoma skin cancer. Among HPV-positive oropharyngeal 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 have shown that HPV-positive anal cancer is significantly associated with a relatively low progression-free survival (PFS, see Figure 8), and this is actually due to the relatively high prevalence of liver metastasis in anal cancer patients, rather than anal cancer itself.In fact, some patients with anal cancer but without liver metastasis (but have other metastases) respond to treatment.Therefore, because HPV-positive cancer, particularly among the above-listed ones, HPV-positive anal cancer has a relatively high prevalence of liver metastasis, the HPV-positive cancer is preferably not HPV-positive anal cancer, particularly not anal cancer with liver metastasis, and therefore is preferably selected from HPV-positive oropharyngeal cancer (particularly SCCHN), cervical cancer, vaginal cancer, vulvar cancer, penile cancer, mucosal cancer, or non-melanoma skin cancer.

[0102] In contrast, we observed a high response rate in patients with HPV-positive vulvar / vaginal cancer (see Table 11), and a non-significant trend toward an association of HPV-positive genital (meaning vulvar / vaginal) cancer with relatively good objective response rate (ORR, see Figure 7) and PFS (see Figure 8). Thus, among HPV-positive cancers, HPV-positive vulvar and vaginal cancers are preferred.

[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 intraepithelial neoplasia (CIN) is a premalignant lesion that can exist at any one of three stages: CIN1, CIN2, or CIN3. If left untreated, CIN2 or CIN3 (collectively referred to as CIN2+) can progress to cervical cancer. Similarly, vulvar intraepithelial neoplasia (VIN) is a premalignant lesion that can exist at any one of three stages: VIN1, VIN2, or VIN3. If left untreated, VIN2 or VIN3 (collectively referred to as VIN2+) can progress to vulvar cancer.

[0104] The cancer or intraepithelial precancerous lesion to be treated is preferably positive for HR-HPV, which preferably corresponds to the HR-HPV from which the HPV E6 and E7 polypeptides encoded by the poxvirus are derived. The cancer or intraepithelial precancerous lesion to be treated is preferably positive for an HR-HPV selected from HPV-16, HPV-18, HPV-30, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-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 cancer or intraepithelial precancerous lesion to be treated is positive for HPV16. If the cancer or intraepithelial precancerous lesion to be treated is positive for HPV16, the poxvirus preferably encodes HPV-16 E6 and E7 polypeptides (more preferably the non-oncogenic versions thereof disclosed above).

[0105] Therefore, in a preferred embodiment, the targeted therapeutic application is HPV-16-positive cancer (preferably HPV16-positive anal cancer with liver metastasis, or more generally, not HPV16-positive anal cancer due to its high prevalence of liver metastasis) or HPV-16-positive intraepithelial precancerous lesions, which may be selected from HPV-positive oropharyngeal cancer (particularly SCCHN), cervical cancer, vaginal cancer, vulvar cancer, penile cancer, mucosal cancer, or non-melanoma skin cancer, among others. In a preferred embodiment, the cancer may be selected from HPV-16-positive squamous cell carcinoma of the head and neck (HPV-16+ SCCHN), HPV-16-positive vulvar cancer, and HPV16-positive vaginal cancer. In this case, the poxvirus (preferably MVA) preferably encodes HPV-16 E6 and E7 polypeptides (more preferably, the non-oncogenic versions thereof disclosed above).

[0106] In addition to its HPV-positive nature, the targeted cancer is further preferably a recurrent and / or metastatic HPV-positive cancer (more preferably a recurrent and / or metastatic HPV-16-positive cancer, most preferably a recurrent and / or metastatic HPV-16-positive SCCHN). As used herein, the term "cancer" encompasses all primary or recurrent and / or metastatic cancers. A "primary cancer" refers to a cancer growing in the original anatomical site (organ or tissue) where tumor progression began and led to the production of a cancerous mass. A "recurrent cancer" refers to a cancer that has recurred (come back), usually after a period during which the cancer was undetectable. Cancer cells from a primary cancer can spread to other parts of the body and form new cancers or "metastatic cancers" (also called secondary cancers).

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

[0108] In their pooled interim analysis of phase Ib and phase II clinical trials, the inventors surprisingly showed that lymph node metastasis is significantly associated with relatively better PFS (see Figure 8). Thus, in a preferred embodiment, the HPV-positive cancer is an HPV-positive (preferably HPV16-positive) metastatic cancer with lymph node metastasis.

[0109] The inventors also surprisingly found that lung and bone metastases were not significantly associated with lower ORR or PFS (see Figure 8). In fact, the inventors showed that only one type of metastasis was significantly associated with lower ORR and PFS: liver metastasis, particularly when patients had multiple liver metastases, i.e., liver metastases occurring at multiple sites (at least two) within the liver, particularly in at least two different lung lobes. The presence of liver metastases has been suggested to be associated with lower response to anti-PD-L1 treatment, primarily in patients with lung or mixed-type cancers (Sridhar S., et al. Clin Lung 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 were made in treatments without the combination of at least human papillomavirus (HPV) E6 and E7 polypeptides and poxvirus vectors encoding immunostimulatory cytokines, and therefore could not have been predicted to apply to the specific treatments according to the present invention. Also, such observations were not made in HPV-positive cancers, and could not have been predicted to apply to such specific cancers. In view of the inventors' observation that relatively low ORR and PFS are significantly associated with liver metastasis, but relatively low ORR and PFS are not associated with lung or bone metastasis, and relatively high PFS is associated with lymph node metastasis, the HPV-positive cancer may preferably be an HPV-positive (preferably HPV-16-positive) cancer (such as oropharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, vulvar cancer, penile cancer, mucosal cancer, or non-melanoma skin cancer) that does not have multiple liver metastases (preferably does not have liver metastases), in particular a metastatic HPV-positive (preferably HPV-16-positive) cancer (such as oropharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, vulvar cancer, penile cancer, mucosal cancer, or non-melanoma skin cancer) that does not have multiple liver metastases (preferably does not have liver metastases).

[0110] More preferably, the HPV-positive cancer may be metastatic HPV-positive (preferably HPV-16 positive) cancer, preferably without liver metastasis and with lymph node metastasis.

[0111] Dosage and Route of Administration "Administering" a drug or "administering" a drug to a patient (and grammatical equivalents of this phrase) refers to direct administration, which may be administration by a healthcare professional to the patient or self-administration, and / or indirect administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer a drug or provides a patient with a prescription for a drug is administering that drug to the patient.

[0112] "Dose" and "dosage" refer to a specific amount of an active or therapeutic agent for administration. Such amount is included in a "dosage form," which refers to physically discrete units suitable as unit dosages for human subjects and other mammals, each unit containing a predetermined amount of active agent, together with one or more suitable pharmaceutical excipients, such as carriers or adjuvants, calculated to produce a desired onset, tolerability, and therapeutic effect.

[0113] A "pharmaceutically acceptable adjuvant" refers to any substance that enhances the body's immune response to an antigen. Non-limiting examples of pharmaceutically acceptable adjuvants include alum, Freund's incomplete adjuvant, MF59, synthetic analogs of dsRNA such as poly(I:C), bacterial LPS, bacterial flagellin, imidazoquinoline, oligodeoxynucleotides containing certain CpG motifs, bacterial cell wall fragments such as muramyl dipeptide, and Quil-A®.

[0114] "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" refers to any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include, without limiting the scope of the invention, additional buffering agents; preservatives; cosolvents; antioxidants, including ascorbic acid and methionine; chelating agents, such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers (e.g., polyesters); salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; lactitol, stachyose, mannose, etc. Examples of suitable pharmaceutically acceptable carriers include organic sugars or sugar alcohols such as sucrose, sorbose, xylose, ribose, ribitol, myoinisitose, myo-inositol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), and polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers such as polyvinylpyrrolidone. Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), may also be included in the pharmaceutical compositions described herein, provided they do not adversely affect the desired characteristics of the pharmaceutical composition.

[0115] A "therapeutically effective amount" refers to an amount of a poxvirus (preferably VV, more preferably MVA, encoding at least HPV E6 and E7 polypeptides and immunostimulatory cytokines, such as TG4001 described in WO 1999 / 03885 under its scientific name MVATG8042) and / or an anti-PD-L1 antibody or antigen-binding fragment thereof (such as avelumab) described herein that has a therapeutic effect and is capable of treating cancer or precancerous lesions. In the case of cancer, e.g., advanced solid malignant tumors, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size or tumor burden; inhibit (i.e., slow to some extent, and in certain embodiments, stop) cancer cell invasion into surrounding organs; inhibit (i.e., slow to some extent, and in certain embodiments, stop) tumor metastasis; inhibit tumor growth to some extent; alleviate to some extent one or more symptoms associated with cancer; and / or produce a favorable response, such as an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), a complete response (CR), a partial response (PR), or, in some cases, stable disease (SD), a reduction in progressive disease (PD), a reduction in time to tumor progression (TTP), or any combination thereof. To the extent that a drug can prevent growth and / or kill existing cancer cells, the drug may be cytostatic and / or cytotoxic. In the case of precancerous lesions, a therapeutically effective amount of a drug can inhibit (i.e., slow to some extent, and in certain embodiments, stop) evolution into cancer. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Because a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will typically, but not necessarily, be less than the therapeutically effective amount.

[0116] "Unit dosage form," as used herein, refers to a physically discrete unit of therapeutic formulation appropriate for the subject being treated. It will be understood, however, that the use of the poxvirus vectors and anti-PD-L1 compositions described herein will be determined by the attending physician within the scope of sound medical judgment. The specific effective dosage level for any particular subject or organism will depend on a variety of factors, including the disorder being treated and the severity of that disorder; the activity of the specific active agent used; the particular composition used; the age, weight, health, sex, and diet of the subject; the time of administration and excretion rate of the specific active agent used; the duration of treatment; drugs and / or additional therapies used in combination or concomitantly with the particular compound used, and similar factors well known in the medical arts.

[0117] poxvirus In the combination, method or use according to the invention, a poxvirus encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and an immunostimulatory cytokine (preferably an MVA virus encoding membrane-bound non-oncogenic HPV-16 E6 and E7 polypeptides and human IL-2, represented by TG4001 described in WO 1999 / 03885 under its scientific name MVATG8042) is 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 Up to pfu, very 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, a poxvirus encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and an immunostimulatory cytokine (preferably an MVA virus encoding membrane-bound non-oncogenic HPV-16 E6 and E7 polypeptides and human IL-2, represented by TG4001 described in WO 1999 / 03885 under its research name MVATG8042) is preferably administered subcutaneously, intramuscularly, intratumorally or intravenously. A particularly preferred administration route is the subcutaneous route.

[0119] Anti-PD-L1 antibody or antigen-binding fragment thereof In the combinations, methods, or uses according to the invention, the anti-PD-L1 antibodies (particularly antibodies containing at least six CDRs, or the heavy and light chains of, for example, avelumab) are preferably administered in the following amounts:

[0120] In certain embodiments, a therapeutically effective amount of an anti-PD-L1 antibody (e.g., avelumab) or an antigen-binding fragment thereof is administered in the combinations, methods, or uses of the invention. A therapeutically effective amount is sufficient to treat one or more symptoms of HPV-positive cancer. In some embodiments using an anti-PD-L1 antibody in combination therapy, the dosing regimen comprises administering the anti-PD-L1 antibody at a dose of 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 at intervals of about 7 days (± 2 days), about 14 days (± 2 days), about 21 days (± 2 days), or about 30 days (± 2 days) throughout the course of treatment. In certain embodiments, the therapeutically effective amount of an anti-PD-L1 antibody (e.g., avelumab) or an antigen-binding fragment thereof is about 5-20 mg / kg, more preferably 5-15 mg / kg, and most preferably 10 mg / kg. In some embodiments, the anti-PD-L1 antibody is avelumab, and the therapeutically effective amount of avelumab is about 10 mg / kg. In some embodiments, avelumab is administered once every two weeks. In some embodiments, avelumab is administered on days 1 and 15 of a 28-day cycle. Pharmacokinetic studies have shown that a 10 mg / kg dose of avelumab achieves excellent receptor occupancy with a predictable pharmacokinetic profile (Heery et al., 2015. Proc ASCO Annual Meeting: abstract 3055). This dose was well tolerated, and signs of antitumor activity, including durable responses, were observed.

[0121] In some embodiments, the anti-PD-L1 antibody (e.g., avelumab) is administered over the course of the above and below treatments for about 80, 150, 160, 200, 240, 250, 300, 320, 350, 400, 450, 480, 500, 550, 560, 600, 640, 650, 700, 720, 750, 800, 850, 880, 900, 950, 960, 1000, 1040, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2500, 3000, 3200, 3500, 4000, 4500, 4800, 5000, 5500, 5600, 6000, 6400, 6500, 7000, 7200, 7500, 8000, 8500, 8800, 9000, 9500, 9600, 1000, 1140, 1250, 1300, 1450, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 250 A fixed dose of 0.50, 1100, 1120, 1150, 1200, 1250, 1280, 1300, 1350, 1360, 1400, 1440, 1500, 1520, 1550, or 1600 mg, preferably 800 mg, 1200 mg, or 1600 mg, is administered at intervals of about 7 days (± 2 days), about 14 days (± 2 days), about 21 days (± 2 days), or about 30 days (± 2 days). Thus, in a preferred embodiment, the anti-PD-L1 antibody (e.g., avelumab) is preferably administered once weekly (QW), once every two weeks (Q2W), or once every three weeks (Q3W) at a dose of about 400-1600 mg, more preferably about 800-1600 mg, most preferably about 800-1200 mg, and most preferably about 800 mg. In a particularly preferred embodiment, the anti-PD-L1 antibody (e.g., avelumab) is administered Q2W at a dose of about 800 mg.

[0122] In the combinations, methods, or uses according to the invention, the anti-PD-L1 antibodies (particularly antibodies containing at least six CDRs, or the heavy and light chains of, for example, avelumab) are preferably administered intravenously (e.g., as an intravenous infusion) or subcutaneously. More preferably, the anti-PD-L1 antibodies (particularly antibodies containing at least six CDRs, or the heavy and light chains of, for example, avelumab) are administered as an intravenous infusion. Most preferably, the anti-PD-L1 antibodies (particularly antibodies containing at least six CDRs, or the heavy and light chains of, for example, avelumab) are administered as an intravenous infusion over a period of 50 to 80 minutes, most preferably about 1 hour.

[0123] In one embodiment, avelumab is a sterile, clear, colorless solution intended for IV administration. The contents of the avelumab vial are non-pyrogenic and contain no bacteriostatic preservatives. Avelumab is formulated as a 20 mg / ml solution and supplied in a disposable glass vial stoppered with a rubber septum and sealed with an aluminum polypropylene flip-off seal. For administration, avelumab must be diluted with 0.9% sodium chloride (normal saline). Tubing with an in-line, low-protein-binding, 0.2-micron polyethersulfone (PES) filter is used during administration.

[0124] Number and frequency of combination administrations In one aspect of the present invention, a) a poxvirus vector encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and an immunostimulatory cytokine, preferably an MVA virus encoding membrane-bound non-oncogenic HPV-16 E6 and E7 polypeptides and human IL-2, more preferably TG4001; b) an anti-PD-L1 antibody or antigen-binding fragment thereof, preferably avelumab is administered according to a specific dosing scheme in which the first administration of the poxvirus is given 5-10 days before the first administration of the anti-PD-L1 antibody, followed by subsequent administrations of the poxvirus and anti-PD-L1 antibody.

[0125] That is, the administration scheme used for the combination, method or use according to the present invention comprises 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 poxvirus is administered before the first administration of the anti-PD-L1 antibody. Without being bound by theory, this setup first stimulates an anti-HPV immune response with the first administration of the poxvirus, which is then amplified by the first administration of anti-PD-L1 (by reducing immunosuppression via the PD-1 / PD-L1 pathway in the tumor microenvironment) without altering the initial proliferation of the poxvirus. The absence of anti-PD-L1 administration for 5 to 10 days after the first administration of the poxvirus prevents potential amplification of the anti-poxvirus immune response. Subsequent administrations of the poxvirus and anti-PD-L1 antibody maintain the anti-HPV immune response.

[0127] Thus, in a combination, method or use according to the invention, the first administration of the poxvirus occurs about 5 to 10 days (i.e. 5, 6, 7, 8, 9, or 10 days, preferably 1 week) before the first administration of the anti-PD-L1 antibody.

[0128] In some embodiments, the combination regimen comprises: (a) administering to the subject, under the direction or supervision of a physician, a poxvirus vector encoding at least HPV E6 and E7 polypeptides and immunostimulatory cytokines about 5-10 days prior to a first administration of the PD-L1 antibody; and (b) administering to the subject, under the direction or supervision of a physician, a PD-L1 antibody. In some embodiments, the combination regimen comprises administering to the subject an anti-PD-L1 antibody about 5-10 days after the subject receives the first administration of the poxvirus vector encoding at least HPV E6 and E7 polypeptides and immunostimulatory cytokines.

[0129] The number and frequency of subsequent administrations of the poxvirus and anti-PD-L1 antibody can vary, but preferably, as many subsequent administrations of the poxvirus and anti-PD-L1 antibody are administered as long as the combination therapy provides a beneficial effect in the treated subject without inducing unacceptable toxicity.

[0130] In one embodiment, subsequent administration of the poxvirus and anti-PD-L1 antibody may be administered until disease progression as defined by RECIST v1.1 criteria (Eisenhauer EA. et al., Eur J Cancer (2009) 45(2):228-47).

[0131] "Disease progression," "progression," or "advanced disease" refers to the appearance of one or more new lesions or tumors and / or definite progression of existing non-target lesions, as defined by the RECIST v1.1 guideline. Disease progression, progression, or advanced disease can also refer to tumor growth of more than 20 percent from the time treatment began, either by an increase in tumor mass or by spread.

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

[0133] In other embodiments, subsequent administrations of the poxvirus and anti-PD-L1 antibody may be administered as long as a beneficial biological effect (see dedicated section below) is observed in the patient.

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

[0135] The frequency of subsequent poxvirus administrations can vary from about one week to about three months. Also, the frequency of poxvirus administrations does not have to be constant throughout the entire treatment period, but instead can vary. Preferably, the frequency of subsequent poxvirus administrations decreases over time.

[0136] In particular, when a first poxvirus administration is included, initially, 4 to 8 (i.e., 4, 5, 6, 7, or 8, preferably 6) poxvirus administrations can be administered every 5 to 10 days (including every 5, 6, 7, 8, 9, or 10 days, preferably weekly) (optionally, a single first poxvirus administration, followed by five subsequent weekly poxvirus administrations is particularly preferred) ("first group of poxvirus administrations").

[0137] This first series of poxvirus administrations may then be followed by a second series of subsequent poxvirus administrations at a reduced frequency. Preferably, this second series of subsequent poxvirus administrations comprises 6 to 10 (i.e., 6, 7, 8, 9, or 10, preferably 8) subsequent poxvirus administrations (the "second series of poxvirus administrations") every 1 to 3 weeks (including every 1, 2, or 3 weeks, preferably every 2 weeks) (optionally, with subsequent poxvirus administrations every 2 weeks up to the 6th month being particularly preferred).

[0138] This second series of poxvirus administrations may then be followed, at a further reduced frequency, by a subsequent third series of poxvirus administrations, which may be administered every 10 to 14 weeks (including every 10, 11, 12, 13, or 14 weeks, preferably every 12 weeks) until disease progression (or, optionally, as long as at least one of the biological effects described herein below is present) (the "third series of poxvirus administrations").

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

[0140] The frequency of anti-PD-L1 administration (including the first administration and subsequent administrations) is preferably 1 to 3 weeks (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, as long as at least one of the biological effects described herein below is present).

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

[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 A first dose of 3×10 pfu of the poxvirus (preferably an MVA virus encoding membrane-bound non-oncogenic HPV-16 E6 and E7 polypeptides and human IL-2, more preferably, e.g., TG4001, described in WO 1999 / 03885 under the name MVATG8042) is administered subcutaneously, followed by 3×10 7 From 7 x 10 7 Subsequent poxvirus doses of pfu were 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 about 10 mg / kg or about 800 mg of an anti-PD-L1 antibody (preferably an antibody containing at least six CDRs, or the heavy and light chains of, for example, avelumab, more preferably avelumab) is administered intravenously 5-10 days after the first poxvirus dose, followed by subsequent anti-PD-L1 antibody doses of about 10 mg / kg or about 800 mg administered intravenously every two 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 A first dose of about 5×10 pfu of the MVA virus (preferably, e.g., TG4001, described in WO 1999 / 03885 under the scientific name MVATG8042) encoding membrane-bound non-oncogenic HPV-16 E6 and E7 polypeptides and human IL-2 is administered subcutaneously, followed by a second dose of about 5×10 7 Subsequent MVA doses of pfu were 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 about 10 mg / kg or about 800 mg of avelumab is administered intravenously one week after the first poxvirus administration, followed by subsequent avelumab doses of about 10 mg / kg or about 800 mg administered intravenously every two weeks until disease progression.

[0145] Biological effects and biomarkers of combination therapy The inventors have surprisingly found that the combination of (a) a poxvirus vector encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and an immunostimulatory cytokine, in particular TG4001 (an MVA virus encoding membrane-bound, non-oncogenic HPV-16 E6 and E7 polypeptides and human IL-2), and (b) an anti-PD-L1 antibody or antigen-binding fragment thereof, preferably avelumab, in HPV-positive cancer patients results in reduced tumor immunosuppression and improved anti-cancer responses characterized by: Induction or enhancement of an immune response to HPV16 E6 and E7 polypeptides, Intratumoral an increase in the immune cell infiltrate, mainly CD3 T cells, preferably an increase in the number of CD8 T cells and their proportion among CD3 T cells (increased CD8 / CD3 ratio), and / or a decrease in regulatory CD4 T cells, and / or a combination of an increase in CD8 T cells and a decrease in regulatory CD4 T cells (Treg), resulting in a reduced Treg / CD8 ratio, 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, Increased expression of T cell activation genes, cytotoxic cell genes, pathogen defense genes, and NK cell function genes, ○ increased expression of one or more genes selected from the group consisting of CXCL10, CXCL11, IRF1, GZMK, GZMA, CD3D, PRF1, TBX21, CXCR3, STAT1, CD69, CCL2, GZMB, CD3G, ICOS, CD8A, STAT4, GZMM, CCR2, CD3E, and IL15, and / or an increase in the expression of one or more genes selected from the group CXCL13, GNLY, GZMH, IFNG, CXCL9, CCL5, and ITGAE, and / or a decrease in the expression of one or more genes selected from the group VEGFA, IHH, IL17A, PROM1, REN, PF4, TSLP, and LAG3. Remodeling characterized by.

[0146] Unless otherwise stated, all comparisons of increases or decreases were made relative to baseline (ie, before the combination therapy was administered to the patient).

[0147] Thus, in one embodiment of the combination therapy, the combination induces or enhances an immune response against HPV-16 E6 and E7 proteins. The immune response against HPV-16 E6 and E7 proteins can be measured by any suitable method known in the art. Suitable methods can be based on detecting CD8 and / or CD4 T cell responses against HPV-16 E6 and E7 proteins, including cytokine (especially interferon gamma (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNFα)) secretion and cytotoxicity. Cytokine secretion can be measured in vitro from peripheral blood mononuclear cell (PBMC) samples using conventional assays such as ELISA or ELISPOT. Cytotoxicity can be measured in vitro using conventional assays. In a preferred embodiment, the immunity against HPV-16 E6 and E7 proteins measured is IFNγ secretion by PBMCs, measured by ELISA, flow cytometry, or immunostaining by ELISPOT, preferably the ELISPOT technique.

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

[0149] Most preferably, the combination induces a decrease in the Treg / CD8 ratio within the tumor, which indicates a decrease in immunosuppression and a stimulation of anti-cancer immune responses within the tumor.

[0150] In tumors, immune cell infiltrates, and in particular the number of CD3 T cells, CD8 T cells, and CD4 T cells (Tregs), can be characterized by any suitable method known in the art. T cells are characterized by the surface expression of CD3 and are subdivided into two subgroups depending on their simultaneous surface expression of either CD8 or CD4. Among CD4 T cells, those that also express Foxp3 are considered regulatory CD4 T cells (Tregs). The numbers of CD3 T cells (CD3+ cells), CD8 T cells (CD3+CD8+ cells), and CD4 T cells (Tregs, CD3+CD4+Foxp3+ cells) can be measured and compared before (baseline) and after treatment using any suitable method known in the art. The CD8 / CD3 ratio, Treg / CD3 ratio, and Treg / CD8 ratio can then be easily calculated.

[0151] As used herein, CD3, CD8, CD4, and Foxp3 expression refers to any detectable level of CD3, CD8, CD4, and Foxp3 protein expression on the cell surface, or CD3, CD8, CD4, and Foxp3 mRNA expression in cells or tissues. Depending on the type of sample, CD3, CD8, CD4, and / or Foxp3 protein expression on the cell surface can be detected using diagnostic CD3, CD8, CD4, and / or Foxp3 antibodies in immunohistochemistry (IHC) assays of tumor tissue sections or by flow cytometry. Alternatively, CD3, CD8, CD4, and / or Foxp3 protein expression by tumor cells can also be detected by PET imaging using binding agents (e.g., antibody fragments, affibodies, etc.) that specifically bind to CD3, CD8, CD4, and / or Foxp3. Techniques for detecting and measuring CD3, CD8, CD4, and / or Foxp3 mRNA (or cDNA) expression include RT-PCR, real-time quantitative RT-PCR (qRT-PCR), and microarray hybridization. In tumors, CD3, CD8, CD4, and / or Foxp3 expression is preferably detected using diagnostic CD3, CD8, CD4, and / or Foxp3 antibodies in immunohistochemistry (IHC) assays of tumor tissue sections. Increase / decrease is detected when the number, expression, or ratio after treatment with combination therapy is higher / lower than before treatment with combination therapy (baseline).

[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 any detectable level of expression of PD-L1 protein on the cell surface or PD-L1 mRNA in cells or tissues. PD-L1 protein expression can be detected using a diagnostic PD-L1 antibody in an immunohistochemistry (IHC) assay of tumor tissue sections or by flow cytometry, depending on the sample type. Alternatively, PD-L1 protein expression by tumor cells can be detected by PET imaging using a binding agent (e.g., an antibody fragment, affibody, etc.) that specifically binds to PD-L1. Techniques for detecting and measuring PD-L1 mRNA (or cDNA) expression include RT-PCR, real-time quantitative RT-PCR (qRT-PCR), and microarray hybridization. In tumors, PD-L1 expression is preferably detected using a diagnostic PD-L1 antibody in an immunohistochemistry (IHC) assay of tumor tissue sections. An increase is detected when PD-L1 expression after treatment with the combination therapy is higher than that before treatment with the combination therapy (baseline).High levels of PD-L1 expression on tumor cells are associated with a relatively good clinical response to anti-PD-L1 antibody 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 reduction in the Treg / CD3 ratio Induce.

[0154] In the blood circulation, CD3, CD8, CD4 and Foxp3 expression can be measured before (baseline) and after treatment by any suitable method known in the art, and the expression levels can be compared.Suitable methods include the same as those disclosed above for measuring CD3, CD8, CD4 and Foxp3 expression in tumors, but preferably, the detection of CD3, CD8, CD4 and Foxp3 expression by flow cytometry using diagnostic CD3, CD8, CD4 and / or Foxp3 antibody.Then, the CD8 / CD3 ratio, Treg / CD3 ratio and Treg / CD8 ratio can be easily calculated.

[0155] Preferably, an increase in CD8 T cells is observed both in the blood circulation and in the tumor. Similarly, a decrease in regulatory CD4 T cells is preferably observed both in the blood circulation and in the tumor. Also, preferably, both an increase in CD8 T cells and a decrease in regulatory CD4 T cells are observed in the blood circulation and / or in the tumor, more preferably both in the blood circulation and in the tumor.

[0156] At the molecular level, the inventors also surprisingly found gene expression changes consistent with the priming of innate and adaptive immunity and a transition from a "cold" to a "hot" tumor profile. A "cold tumor" is defined as a tumor with no or very limited immune infiltrate, particularly T cell immune infiltrate. At the molecular level, "cold tumors" are characterized by low levels of expression of genes associated with the presence of immune cell infiltrate, particularly genes involved in T cell activation, T cell differentiation, T cell attraction, T cell adhesion, cytotoxicity, pathogen defense, and NK cell function. In contrast, a "hot tumor" is defined as a tumor with a significant immune infiltrate, particularly T cell immune infiltrate. At the molecular level, "hot tumors" are characterized by high levels of expression of genes associated with the presence of immune cell infiltrates, particularly genes involved in T cell activation, T cell differentiation, T cell attraction, T cell adhesion, cytotoxicity, pathogen defense, and NK cell function. A transition from a "cold tumor" profile to a "hot tumor" profile is considered induced by a treatment if the treatment results in a significant increase in immune infiltrates, particularly T cell immune infiltrates. At the molecular level, this is reflected by increased levels of expression of one or more genes involved in T cell activation, T cell differentiation, T cell attraction, T cell adhesion, cytotoxicity, pathogen defense, and / or NK cell function compared to before treatment. Hot tumors are more likely to respond to therapeutic intervention and are associated with a relatively high consistency and improved clinical outcomes for patients. Conversely, cold tumors are associated with minimal immune reactivity, poor response to therapeutic interventions, and a rapid, unfavorable clinical course.

[0157] More specifically, using a panel of 770 genes involved in the immune response to cancer, we were able to demonstrate changes in gene expression within tumors between baseline and 43 days after the initiation of combination therapy. Such changes include increased expression of several T cell activation genes, cytotoxic cell genes, pathogen defense genes, and NK cell function genes. These changes include increased expression of Immunosign® by HalioDX. CR 15 and Immunosign® CR These include the expansion of gene signatures known as 21. These gene signatures reflect naturally occurring immune activity within and around tumors and are therefore thought to reflect a somewhat cold (poor prognosis) or hot (relatively good prognosis) immune status of the tumor (Galon J. et al., Immunity (2013) 39(1):11-26; Marabelle A. et al., Society for Immunotherapy of Cancer (SITC) 32 nd 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).

[0158] In particular, Immunosign® CR15 is an algorithm that integrates expression data of CXCL13, GNLY, GZMH, IFNG, CXCL9, CCL5, ITGAE, VEGFA, IHH, IL17A, PROM1, REN, PF4, TSLP, and LAG3, which are genes involved in T cell cytotoxicity, T cell differentiation, T cell attraction, T cell adhesion, immune targeting, angiogenesis suppression, immune co-suppression, and cancer stem cells. In the context of the present invention, an increase in the expression level of any one of CXCL13, GNLY, GZMH, IFNG, CXCL9, CCL5, and ITGAE and / or a decrease in the expression level of any one of VEGFA, IHH, IL17A, PROM1, REN, PF4, TSLP, and LAG3 is considered a transition to a hotter tumor state, which is beneficial for cancer treatment.

[0159] Immunosign® CR 21 is an algorithm that integrates expression data for CXCL10, CXCL11, IRF1, GZMK, GZMA, CD3D, PRF1, TBX21, CXCR3, STAT1, CD69, CCL2, GZMB, CD3G, ICOS, CD8A, STAT4, GZMM, CCR2, CD3E, and IL15, genes involved in T cell cytotoxicity, T cell activation, T cell attraction, and Th1 orientation. In the context of the present invention, increased expression levels of any one of these genes is considered a transition to a hotter tumor state, which is beneficial for cancer treatment.

[0160] Thus, in one aspect of the present invention, the combination therapy induces increased expression in one or more of the following gene categories (which the inventors have surprisingly found to be upregulated by the combination therapy): (i) T cell activation-related antibodies, preferably CD47, RPS6, CD80, IL18R1, CD7, PSEN2, TNFSF14, DPP4, STAT4, CCR1, FOXP3, CTLA4, LAG3, CD86, LILRB1, IL13, CD1C, EOMES, CCR4, CD3G, FAS, IL12B, IL18RAP, CD1D, CXCR3, TIGIT, IL4, IL12A, IFNG, CD70, CD2, CD3E, CD8A, CD8B, IL12RB2, CD5, CCR5, TB increased expression of at least one gene selected from the group of X21, IL12RB1, IRF4, ADA, CD274, LCK, F2RL1, ICOSLG, CXCL11, CXCL10, IDO1, CX3CL1, IRF1, SOCS1, IL18, SLC11A1, EGR1, ITGA1, CXCR4, CXCL9, PTPRC, LCP1, TNFRSF14, PSEN1, MAF, TP53, IL4R, STAT6, IL13RA1, and IFNGR1, and optionally the IL21R gene; (ii) increased expression of at least one gene involved in the activation of cytotoxic T cell function, preferably selected from the group of GZMM, GZMH, GZMK, GNLY, GZMB, PRF1, GZMA, HLA-C, and HLA-A genes; (iii) increased expression of at least one pathogen defense gene, preferably selected from the group of CD8A, CTSG, PRG2, CCL22, IL1B, PRF1, GNLY, CXCL10, TYK2, and OAS3 genes; (iv) increased expression of at least one NK cell function gene, preferably selected from the group of KLRC1, KLRB1, KLRC2, IL12B, KIR3DL1, KLRF1, KLRG1, NCR1, KLRK1, IL12A, and KLRD1 genes; (v) Increased expression of any combination of genes in gene categories (i), (ii), (iii), and / or (iv), preferably a combination of genes that includes at least one gene from each of the above gene categories.

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

[0162] In one embodiment of the invention, instead of or in addition to the gene expression of categories (i) to (v) and / or the genes of the Immunosign® 21 signature above, the combination therapy induces an increase or decrease in the expression of one or more of the following genes (present in the Immunosign® 15 signature): Increased expression of one or more of the following genes: CXCL13, GNLY, GZMH, IFNG, CXCL9, CCL5, and ITGAE; and / or · Reduction of the expression of one or more of the following genes: VEGFA, IHH, IL17A, PROM1, REN, PF4, TSLP, and LAG3.

[0163] In the above embodiment, the expression level of the disclosed gene category or specific gene of interest can be measured before (baseline) and after treatment by any suitable method known in the art, and the expression levels can be compared.The expression level can be measured by measuring mRNA (or cDNA) or protein expression level.Preferably, the mRNA (or cDNA) expression level is measured by techniques such as RT-PCR, real-time quantitative RT-PCR (qRT-PCR), and microarray hybridization.

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

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

[0166] In one embodiment in which biomarkers are used to determine whether to continue or discontinue combination therapy in a patient, subsequent administration of the poxvirus and anti-PD-L1 antibody can be administered as long as the combination therapy induces or enhances an immune response against the HPV-16 E6 and E7 proteins.

[0167] In another embodiment, subsequent administration of the poxvirus and anti-PD-L1 antibody can be performed, so long as the combination therapy induces: Increased immune cell infiltrate within the tumor (preferably increased CD3 T cells, more preferably increased CD8 T cells), and / or Reduction of intratumoral regulatory CD4 T cells.

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

[0169] In another embodiment, subsequent administration of the poxvirus and anti-PD-L1 antibody can be performed so long as the combination therapy induces in the circulation: an increase in CD8 T cells, preferably an increase in the CD8 / CD3 ratio, and / or Reduction of regulatory CD4 T cells (Treg), more preferably a reduction in the Treg / CD3 ratio.

[0170] In another embodiment, subsequent administration of the poxvirus and anti-PD-L1 antibody can be performed so long as the combination therapy induces increased expression of one or more of the following gene categories, which are surprisingly found by the inventors to be upregulated by the combination therapy: (i) increased expression of at least one gene involved in T cell activation, preferably selected from those disclosed above; (ii) increased expression of at least one gene involved in the activation of cytotoxic T cell function, preferably selected from those disclosed above; (iii) increased expression of at least one pathogen defense gene, preferably selected from those disclosed above; (iv) increased expression of at least one NK cell function gene, preferably selected from those disclosed above; (v) Increased expression of any combination of genes in gene categories (i), (ii), (iii), and / or (iv), preferably a combination of genes that includes at least one gene from each of the above gene categories.

[0171] In another embodiment, instead of, or in addition to, expression of genes in categories (i) to (v) above, subsequent administration of the poxvirus and anti-PD-L1 antibody can be performed so long as the combination therapy induces increased expression of one or more of the following genes (present in the Immunosign® 21 signature): CXCL10, CXCL11, IRF1, GZMK, GZMA, CD3D, PRF1, TBX21, CXCR3, STAT1, CD69, CCL2, GZMB, CD3G, ICOS, CD8A, STAT4, GZMM, CCR2, CD3E, and IL15. In particular, subsequent administration of the poxvirus and anti-PD-L1 antibody can be performed so long as the combination therapy induces increased expression of one or more of the following genes (see Figure 6D): CXCL10, CXCL11, IRF1, GZMK, GZMA, CD3D, PRF1, TBX21, and CXCR3.

[0172] In another embodiment, subsequent administration of the poxvirus and anti-PD-L1 antibody can be performed instead of, or in addition to, expression of the genes in categories (i) to (v) and / or the genes in the Immunosign® 21 signature above, so long as the combination therapy induces an increase or decrease in expression of one or more of the following genes (present in the Immunosign® 15 signature): Increased expression of one or more of the following genes: CXCL13, GNLY, GZMH, IFNG, CXCL9, CCL5, and ITGAE; and / or Reduction of the expression of one or more of the following genes: VEGFA, IHH, IL17A, PROM1, REN, PF4, TSLP, and LAG3.

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

[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 evaluated the combination of TG4001 and avelumab in HPV16-positive R / M (R / M stands for recurrent and / or metastatic) cancer for safety, efficacy, and immunological response. This example presents preliminary Phase 1b data.

[0176] Materials and methods Study design and procedures Two different dose levels (DL) of TG4001 (DL1 5 × 10 6 and DL2 5×10 7 A multicenter, open-label, single-arm study using a 3+3 design for phase Ib combining 10 mg / kg avelumab with 10 mg / kg pfu. Phase II included TG4001 at DL2.

[0177] TG4001 was administered subcutaneously (SC) once weekly on days 1, 8, 15, 22, 29, and 36, then every 2 weeks (starting on day 36) until month 6 (from day 1 of study treatment), and then every 12 weeks until disease progression, unacceptable toxicity, or patient withdrawal from the study for any reason, whichever occurred first. Avelumab was administered intravenously (IV infusion) every 2 weeks starting on day 8 (1 week after the first TG4001 dose) until disease progression, unacceptable toxicity, or patient withdrawal from the study for any reason, whichever occurred first.

[0178] Study endpoints and evaluation Safety and efficacy of the combination of TG4001 and avelumab, immune parameters (T cell responses, changes in infiltrates, and gene expression of immune-related genes).

[0179] Tumor response was assessed according to RECIST v1.1 (Eisenhauer EA. et al., Eur J Cancer (2009) 45(2):228-47). PBMC samples were collected longitudinally, and tissue samples were collected at baseline and day 43.

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

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

[0182] Tumor size measurement Tumor response was assessed by computed tomography (CT) (or magnetic resonance imaging (MRI)). Preferably, CT (or MRI) of the head and neck, chest, abdomen, and all other known sites of disease was performed within 21 days before the start of study treatment. Patients were evaluated every 6 weeks from the start of treatment until disease progression or for 9 months after the start of study treatment, whichever occurred first. After 9 months of treatment, evaluations were performed every 12 weeks until documented progression. All measurements were recorded in metric notation (mm).

[0183] At baseline, tumor lesions and lymph nodes were classified as measurable (minimum size ≥10 mm or lymph node >15 mm) or nonmeasurable (small lesions <10 mm, nonmeasurable lesions (e.g., pleural effusion), or lymph node <15 mm). Patients admitted to the study had at least one lesion measurable by CT / MRI scan. All target lesions (all measurable lesions (nodular or nonnodular) for a total of up to five lesions) and nontarget lesions (all other lesions, measurable or nonmeasurable) were recorded. To assess tumor response, the sum of the longest diameter (SLD) of all target lesions (and the short axis of nodular lesions) was calculated at baseline and throughout the study. At each assessment, response was first assessed separately for target and nontarget lesions identified at baseline. These assessments were then used to calculate a combined lesion response that considered target and nontarget lesions and the presence or absence of new lesions. Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (target or non-target) must have a reduction in their short axis to <10 mm. Partial response (PR): At least a 30% reduction in the sum of the diameters of the target lesions, using the baseline sum of diameters as reference. - Progression (PD): An increase of at least 20% in the sum of the diameters of the target lesions and new lesions, taking as reference the smallest total in the study (including the baseline assessment). In addition to the 20% relative increase, the sum must also show an absolute increase of at least 5 mm. The appearance of one or more new lesions is also considered progression. - Stable (SD): Insufficient shrinkage to qualify as PR and insufficient growth to qualify as PD. - Not Evaluated (NE): No progression has been documented and one or more target lesions have not been assessed or have been assessed using a method different from that used at baseline. The only exception is if the SLD of an evaluable target lesion already meets the criteria for PD. In this case, the target response is PD.

[0184] All patients evaluable for response were those with at least one baseline-evaluable CT scan and one post-baseline-evaluable CT scan at week 6 after initiation of study treatment, and a best overall response assessment other than "unknown" per RECIST 1.1. Patients were to receive both IMPs (investigational drugs: TG4001 + avelumab) at the minimum compatible exposure unless they had progressed or died from underlying disease before or at the time of their first assessment.

[0185] Immunity Data Samples were collected from consenting patients after IRB approval, in accordance with all ethical guidelines related to human subject research. Peripheral blood mononuclear cells (PBMCs) were isolated using a density gradient on a Ficoll® layer. Briefly, heparinized blood was mixed with phosphate-buffered saline and Ficoll® medium. It was then centrifuged at 2300 g for 20 minutes. The PBMC layer was collected, diluted with buffered saline, and centrifuged at 1300 g for 10 minutes to remove residual Ficoll® solution. The cells were resuspended in buffered saline and recentrifuged. The cell pellet was then resuspended in storage medium (IMDM containing 10% DMSO and 20% human serum), aliquoted into cryovials, and frozen in the container using isopropyl alcohol.

[0186] Tissue samples were obtained using standard core needle biopsy with an 18G or larger needle. Sample sections 4 μm (immunohistochemistry) or 10 μm (gene expression analysis) thick were formalin-fixed and paraffin-embedded before processing.

[0187] ELISPOT T cell responses to HPV In patients in a phase I study, IFN-γ-producing cells were quantified by ELISpot after a 5-day in vitro expansion phase. Briefly, after thawing, cells were counted using an NC200 automated cell counter and plated at 2E+06 cells per well in 500 μL of X-VIVO-15 medium containing 2% CTS serum replacement solution in 24-well culture plates in the presence or absence of stimulating antigen (2 μg / mL E6 or E7 peptide pools or 1 μg / mL control peptide mixture pool per peptide). On day 5, cells were harvested, counted, and plated in quadruplicate at 2E+05 cells per well in ELISpot IFN-γ plates. After 24 h of incubation, ELISpot plates were revealed and then dried before spots were counted using an automated ELISpot reader, according to the manufacturer's instructions.

[0188] For patients in the Phase II study, the method was modified to increase specificity. IFN-γ-producing cells were quantified by ELISpot. Briefly, on days 0, 1 (pre-vaccination), and D43 (post-vaccination), PBMCs were collected in PCT tubes by venipuncture in patients and sent to a central laboratory (PPD) for extraction by centrifugation on a Ficoll gradient. Cells were washed, counted, and 10 × 10 6 The tube containing the cells was sent and the cells were frozen and stored in LN before analysis.

[0189] Cells were thawed and stimulated overnight with medium (negative control), E6 peptide pool (PepTivator, Miltenyi Biotech), E7 peptide pool (PepTivator, Miltenyi Biotech), or CEF (PepTivator, Miltenyi Biotech; positive controls: EBV, CMV, and influenza). Cells were then plated onto anti-IFN-γ-coated plates and incubated (1 × 10) before development. 5 Cells / well). PBMCs collected from patients before and after peptide vaccination and known to have developed T cell responses were used in each analytical run as assay controls. Three replicates per condition were performed. After overnight incubation, ELISpot plates were revealed according to the manufacturer's instructions and then allowed to dry before spots were counted in an automated ELISPOT reader. Patients were considered positive for a given antigen if the positive and negative controls were as expected and if the antigen spot count was higher than the negative control by +2 times the assay's coefficient of variation.

[0190] Immunohistochemistry for immunoscore assessment To characterize the tumor immune composition, 4-μm-thick formalin-fixed, paraffin-embedded tissue sections were used. Consecutive slices from the same biopsy core were used for all analyses for a given patient at baseline and day 43. The first slide of each series was used for confirmation of tumor nature by pathologist review of the tissue after hematoxylin and eosin staining. Staining for CD3 and CD8 was performed on consecutive slides according to the following protocol: antigen retrieval with Tris-base buffer (pH = 8) for 60 minutes, quenching of endogenous peroxidase activity, incubation with antibodies against CD8 for 32 minutes at 37°C and CD3 for 20 minutes at 37°C, development with the Ultraview Universal DAB IHC detection kit, and counterstaining with Mayer's hematoxylin. The primary antibodies used to stain immune cells were as follows: Rabbit monoclonal anti-human CD3 VM (clone 2GV6 Ventana), mouse monoclonal anti-human CD8 (clone C8 / 144, Dako®). Digital images of stained tissue sections were acquired at 20x magnification and a resolution of 0.45 μm / pixel. Quantification of CD8- and CD3-positive cells within the tumor and invasive margin was performed from whole-slide digital scans using the Immunoscore® module developed by HalioDx (Marseille, France).

[0191] Multispectral histological assessment of CD3 / CD8 / PD-L1 and CD3 / CD4 / FOXP3 Four-mm-thick FFPE tissue slides were deparaffinized, rehydrated through an ethanol gradient ending with a distilled water wash, and fixed in 10% neutral-buffered formalin for 20 minutes. Antigen retrieval was performed via microwave treatment in antigen retrieval solution. For each staining cycle, protein blocking was performed for 15 minutes using serum-free protein block solution, followed by incubation with the primary Abs anti-CD3 (obtained from Ventana, as above), anti-CD4 (mouse monoclonal anti-human CD4, clone UMAB64, Clinisciences), and anti-FoxP3 (mouse monoclonal anti-human FOXP3, clone 236A / E7, AbCam), or anti-CD3, anti-CD8, and anti-PDL-1 for 30 minutes at room temperature.

[0192] Next, incubation with HRP-conjugated polymer mouse or rabbit antibodies was performed for 15 minutes at room temperature, followed by a 10-minute fluorophore incubation. Finally, all slides were counterstained with DAPI for 5 minutes, followed by whole-slide scanning and quantification using proprietary digital pathology software developed by HalioDx. Results were tabulated and plotted using the Graphpad Prism software package.

[0193] Analysis of gene expression changes in tumor tissue during treatment: Multiplex immunogene expression Relative expression levels of immune genes within the tumor microenvironment were measured on formalin-fixed tumor tissue (10 μm thick) using Nanostring nCounter technology. After extraction, total RNA (300 ng) was assayed on an nCounter digital analyzer according to the manufacturer's instructions and hybridized to a pan-cancer immune profiling panel. This panel contains 770 genes, including key checkpoints, chemokines, cytokines, and related regulatory genes. Data quality control and normalization were performed using the nSolver software package. Measured expression values ​​were normalized to the geometric mean of housekeeping gene expression levels with the lowest coefficient of variation (%CV). Statistical analysis was performed using the nSolver Advanced Analysis module and the R software package. Immunosign® was defined using a commercially available, proprietary algorithm developed by HalioDx.

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

[0195] [Table 1]

[0196] Patient baseline characteristics were as follows: median age 57.8 years (range 39-78 years) with HPV-16-positive cancer of various cancer types (anal, cervical, oropharyngeal, and vaginal) primarily of squamous cell carcinoma origin. At baseline, all patients presented with distant metastases.

[0197] Overview of treatment-related adverse events Safety was assessed through reporting of adverse events (AEs) and by clinical laboratory tests, physical examinations, electrocardiograms (ECGs), and vital signs at various time points. Adverse events and laboratory abnormalities were assessed by the National Cancer Institute's 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. Specifically, a total of 68 adverse events were observed in 9 patients, 23 in DL1 and 45 in DL2. TG4001 and avelumab combination therapy was well tolerated. In fact, no treatment-related serious adverse events (SAEs) were observed, and only two grade 3 events occurred in one DL1-treated patient (5 × 10 6 PFU).

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

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

[0203] [Table 3]

[0204] The results in Table 3 and Figures 1A and 1B show that the combination therapy is able to stabilize the disease or induce a partial response in two-thirds of all patients. The efficacy was observed with the high dose DL2 of TG4001 (5 × 10 7 pfu) seems to be better.

[0205] Immunity Data Specific T cell responses to HPV Four patients were evaluable for 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 three of four patients evaluable for ELISPOT had E6 or E7 reactive T cells at day 43.

[0208] Changes in TILs under treatment The CD8 / CD3 and Treg (CD4 FoxP3) / CD8 ratios at baseline and day 43 are shown in Figures 2A and 2B, which show that the treatment period was associated with an overall increase in the CD8 / CD3 ratio infiltrate and a decrease in the Treg (CD4 FoxP3) / CD8 ratio, suggesting a more favorable immune profile (enriched stimulatory CD8 T cells and reduced immunosuppressive Tregs).

[0209] PD-L1 expression on TILs and tumor cells under treatment PD-L1 expression was assessed on TILs and tumor cells at baseline and day 43 (and also at day 85 for one patient) in seven patients, five of whom were evaluable at baseline and 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 who were evaluable at baseline and day 43 had a significant increase in PD-L1 expression on tumor cells at day 43. This is predicted to correlate with a trend toward an increase in response to immunotherapy treatment.

[0212] Analysis of gene expression changes in tumor tissue during treatment To explore therapy-induced gene expression changes in tumor tissue, the expression of a panel of 770 genes involved in immune response was assessed at baseline and after treatment (day 43). In particular, the expression of the previously described gene signatures Immunosign® 15 and Immunosign® 21 was studied (Galon et al., Immunity (2013) 9(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).

[0213] Volcano plots of gene expression and pathway changes identified as overexpressed post-treatment versus pre-treatment are shown in Figures 3 and 4. Black dots represent relevant genes involved in the indicated mechanism (e.g., T cell activation in Figure 3A), and the level of expression correlates with its position on the log scale (overexpression to the right of 0, underexpression to the left of 0).

[0214] Figure 3 shows that pathways involved in viral vaccine response (pathogen defense, see Figure 3C) and priming of antitumor immunity (T cell activation, cytotoxic cell, and NK cell function, see Figures 3A, 3B, and 3D) were overexpressed during treatment.

[0215] Additionally, Figure 4A shows a description of the gene categories included in the gene signatures previously described as Immunosign® 15 and Immunosign® 21, and Figures 4B and 4C show that many genes in the Immunosign® 15 and Immunosign® 21 signatures were overexpressed during treatment.

[0216] The observed gene expression changes are consistent with priming of innate and adaptive immunity and a shift toward a "hotter" tumor profile, which is more consistently 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 showed a tumor with low levels of infiltration, moderate PD-L1 expression on tumor cells and infiltrating immune cells, and low spatial colocalization of CD8 cells with PD-L1-expressing tumor cells, all characteristics consistent with a cold tumor at baseline.

[0219] The changes in immune infiltrate are shown in Figure 5, and PD-L1 expression on the surface of TILs and tumor cells at baseline and day 43 is shown in Table 6 below.

[0220] [Table 6]

[0221] Figure 5 and Table 6 show that tumors were significantly more infiltrated, with a greater than four-fold increase in CD3 infiltration, a three-fold increase in CD8 infiltration, and a doubling of tumor and PD-L1 immunoexpression, on day 43. No significant changes were observed in the level of infiltration by immunosuppressive cells during treatment.

[0222] Furthermore, digital pathology analysis of the samples showed that infiltrating CD8 cells clustered around PD-L1-positive tumor cells (Figure 5B), suggesting a profile more favorable for successful response to checkpoint blockade.

[0223] Changes in gene expression: Gene expression profiles in patient tumor tissues also revealed significant changes over the treatment period, as shown by a color map of 770 immune-related genes (data not shown).

[0224] Analysis of gene expression changes further demonstrated a strong increase in the expression of genes involved in antigen processing and presentation (Figure 6A), response to viruses (Figure 6B), and Toll-like receptor expression (Figure 6C). Activation of these pathways is consistent with the development of an adaptive response to a viral vaccine.

[0225] Additionally, 9 genes in the Immunosign® 21 signature were strongly overexpressed (Figure 6E), suggesting a shift from an immune-excluded phenotype to "hot" tumors likely to benefit from immunotherapeutic intervention.

[0226] conclusion Preliminary results from NCT03260023 Phase 1b indicate that: The combination of TG4001 and avelumab was safe and well tolerated in patients with HPV-positive cancer who had received multiple lines of prior therapy at both dose levels of TG4001 studied. This combination showed promising efficacy signals in DL2 and is being evaluated in an ongoing Phase II trial. This treatment is associated with tumor microenvironment changes that likely alter the course of disease by shifting tumors from a cold to a hotter immune state, even in heavily pretreated patients. This may be particularly useful in patients with an "immune-excluded" tumor phenotype, as demonstrated in individual study cases.

[0227] These results suggest that the combination of (a) a poxvirus vector encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines and (b) an anti-PD-L1 antibody is safe, well tolerated, and effective (with at least additive efficacy) in treating patients with HPV-positive cancer.

[0228] Example 1B Phase II studies and pooled interim analyses Patient / disease characteristics In phase II, TG4001 was administered at DL2. DL2 was the recommended phase II dose after the completion of phase IB, supported by results showing that the combination of TG4001 and avelumab was safe and no notable differences were observed between DL1 and DL2 in terms of the nature and severity of reported AEs. Twenty-five patients were enrolled in this study for the planned interim analysis per protocol. Of these, three were not evaluable for tumor response. Of the 22 evaluable patients, 11 patients (50%) had anal cancer, four patients (18.2%) had cervical cancer, four patients (18.2%) had vaginal / vulvar cancer, and three patients (13.6%) had oropharyngeal cancer. Seven additional patients were enrolled prior to the interim analysis, one of whom was not evaluable for tumor response. Of the six evaluable patients, four patients (66.6%) presented with anal cancer, one patient each with oropharyngeal cancer (16.6%) and cervical cancer (16.6).

[0229] For subgroup analysis, patients treated with TG4001 in DL2 (N=6) in the Phase Ib part were pooled with patients treated in the Phase II part (N=28, discussed immediately above). Of the six patients treated in Phase Ib, four patients (66.6%) presented with oropharyngeal cancer, and one patient each presented with vaginal cancer (16.6%) and cervical cancer (16.6%). Of the pooled dataset (N=34), 15 patients (44.1%) presented with anal cancer, eight patients (23.5%) presented with oropharyngeal cancer, six patients (17.6%) presented with cervical cancer, and five patients (14.7%) presented with vaginal / vulvar cancer. An ORR of 20.6% was observed.

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

[0231] Table 7 shows an overview of the gender and performance status (PS) of the subgroups of patients with and without liver metastases. Specifically, the subgroup of patients without liver metastases consisted of 23 patients (non-hepatic patients), while the subgroup of patients with liver metastases consisted of 11 patients (hepatic patients).

[0232] [Table 7]

[0233] Regarding disease characteristics of the pooled dataset (N=34), 15 patients presented with anal cancer, 8 patients presented with oropharyngeal cancer, 6 patients presented with cervical cancer, and 5 patients presented with vaginal / vulvar cancer, as shown in Table 8. 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, the correlation between objective response rate (ORR) or progression-free survival (PFS) and each patient / disease characteristic was evaluated.

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

[0238] With regard to ORR, only one disease characteristic (boxed in Figure 7 ), the presence of liver metastases, was significantly correlated with worsened ORR (see Figure 7 , which shows an OR of 100, a 95% interval of 5–100, and a p -value of 0.012).

[0239] With regard to PFS, three characteristics were found to be significantly correlated with relatively poor or good PFS (they are boxed in Figure 8). The presence of liver metastases was significantly correlated with a worse PFS (see Figure 8, which shows a HR of 4.390, a 95% interval of 1.728-11.152, and a p-value of 0.002). Anal cancer was significantly associated with a worse PFS (see Figure 8, which shows a HR of 3.214, a 95% interval of 1.269-8.143, and a p-value of 0.014). The presence of lymph node metastasis is significantly correlated with a better PFS (see Figure 8, which shows a HR of 0.388, a 95% interval of 0.151-0.998, and a p-value of 0.049).

[0240] Regarding the association of a relatively poorer PFS with anal cancer, it should be noted that this association may be indirectly explained by the relatively high prevalence of liver metastases in patients with anal cancer. Indeed, responses have been documented in patients with anal cancer that has not demonstrated liver metastases but has demonstrated metastases in other organs.

[0241] Regarding PFS, although it was not significant for the number of patients analyzed in the pooled interim analysis (p=0.073), a trend toward a relatively favorable correlation between genital (vulvar / vaginal) cancer and PFS was observed.

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

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

[0244] Table 10 below further shows the distribution of patients with or without liver metastases according to efficacy parameters (RECIST 1.1, response, stable disease at 12 weeks, progression before or at 12 weeks, and median PFS), showing that when treated with the combination therapy, patients without liver metastases have higher response and stable disease at 12 weeks, lower progression before or at 12 weeks, and higher median PFS than patients with liver metastases.

[0245] [Table 10]

[0246] Figure 9 shows a graphical representation of the maximum change in tumor size for 23 patients from the pooled Phase Ib and Phase II interim results who did not have liver metastases, and Figure 10 shows the same graphical representation for 9 patients from the pooled Phase Ib and Phase II interim results who did have liver metastases.

[0247] Table 11 below shows the distribution of patients with or without liver metastases according to the type of primary tumor (anal, oropharyngeal, cervical, vulvar / vaginal), and shows that patients without liver metastases, regardless of primary tumor, had a higher response rate than patients with liver metastases (no response was observed in these patients, regardless of primary tumor). Table 11 also shows a high response rate (66.7%) in patients with vulvar / vaginal cancer without liver metastases. However, this high response rate should be interpreted with caution due to the small number of patients analyzed (only 3 patients).

[0248] [Table 11]

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

[0250] [Table 12]

[0251] immune permeate Observations made during phase 1 of the study were confirmed in phase 2, with an increase in genes associated with the Immunosign® signature and an increase in the infiltrate of CD3-positive cells.

[0252] Transcriptome analysis Transcriptome analysis of tumors collected from patients with or without liver tumor 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) genes were overexpressed (Log2 change of 2.74 and 7.15, respectively). Genes related to the complement pathway were overrepresented, including 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), C6 (Log2 change of 5.87; p<0.001), and C2 (Log2 change of 2.06; p<0.001).

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

[0255] The effect of complement on cancer immunity is controversial, and high expression of the complement pathway has been shown to exert cytotoxic effects on immune cells, including effector CD4 and CD8, which is intriguing given that the liver is a major site of synthesis and regulation of complement factors.

[0256] Additionally, cytokines associated with inflammation were also present and may be involved in creating an immunosuppressive tumor environment. This immunosuppression may be detrimental to effector immune cells and promote tumor immune escape and disease progression. Therefore, these unique transcriptome 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 observations of patients highlighted liver metastasis as a determinant of treatment success and clinical outcome. Genomic data revealed that tumor liver metastases were characterized by the expression of genes and pathways associated with downregulation of the immune system or tumor aggressiveness.

[0260] conclusion In conclusion, a pooled interim analysis of phase Ib and phase II trials indicates that the presence of liver metastases is associated with a lower ORR and shorter PFS. Indeed, by stratifying patients for the presence or absence of liver metastases, an ORR of 30.4% was observed in the subgroup of patients without liver metastases (N=23), compared with an ORR of 0% in the subgroup of patients with liver metastases (N=11). Similarly, the median PFS for patients without liver metastases (N=23) was 5.6, while the median PFS for patients with liver metastases (N=11) was only 1.4.

[0261] Additionally, a pooled interim analysis of phase Ib and phase II trials showed that anal cancer is associated with a lower PFS due to a higher prevalence of liver metastases in patients with anal cancer, whereas vulvar / vaginal cancer shows a trend toward a higher PFS.

[0262] Finally, lymph node involvement is associated with a relatively favorable PFS. The present invention also provides the following. [1] For use in the treatment of HPV-positive cancers or precancerous lesions in situ, a) a poxvirus vector encoding at least human papillomavirus (HPV) E6 and E7 polypeptides and immunostimulatory cytokines; b) an anti-PD-L1 antibody or antigen-binding fragment thereof A combination of a combination wherein the first administration of the poxvirus occurs 5 to 10 days before the first administration of the anti-PD-L1 antibody, and subsequent administrations of the poxvirus and anti-PD-L1 antibody occur. [2] The combination according to [1], wherein the poxvirus is a vaccinia virus, preferably a modified vaccinia virus Ankara (MVA). [3] The combination according to [1] or [2], wherein the poxvirus encodes membrane-bound HPV-16 non-oncogenic E6 and E7 polypeptides and human interleukin-2 (IL-2). [4] The combination according to any one of [1] to [3], wherein the anti-PD-L1 antibody mediates antibody-dependent cellular cytotoxicity (ADCC). [5] The combination of any one of [1] to [4], wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain comprising three complementarity-determining regions having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and a light chain comprising three complementarity-determining regions having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, preferably wherein the anti-PD-L1 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 7 or 8, and a light chain having the amino acid sequence of SEQ ID NO: 9. [6] The combination product according to any one of [1] to [5], wherein the anti-PD-L1 antibody is avelumab. [7] The combination according to any one of [1] to [6], wherein the HPV-positive cancer is HPV-positive oropharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, vulvar cancer, penile cancer, mucosal cancer, or non-melanoma skin cancer, or the intraepithelial precancerous lesion is cervical intraepithelial neoplasia (CIN) grade 2 or 3 or vulvar intraepithelial neoplasia (VIN) grade 2 or 3, and preferably the HPV-positive cancer is selected from HPV-positive vulvar cancer and vaginal cancer. [8] The combination according to [7], wherein the cancer is HPV-16 positive, preferably the cancer is HPV-16 positive squamous cell carcinoma of the head and neck (HPV-16+ SCCHN) or HPV-16 positive vulvar or vaginal cancer. [9] The combination according to any one of [1] to [8], wherein the HPV-positive cancer is an HPV-positive cancer without multiple liver metastases, preferably an HPV-positive cancer without liver metastases.

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

[11] The combination according to

[10] , wherein the HPV-positive cancer is an HPV-positive metastatic cancer without multiple liver metastases, preferably an HPV-positive metastatic cancer without liver metastases.

[12] Each dose of the poxvirus is 3 x 10 7 ~7×10 7 The combination according to any one of [1] to

[11] , which is administered at a dose of pfu.

[13] a) 3 x 10 of the poxvirus 7 ~7×10 7 A first dose of 3 x 10 pfu was administered subcutaneously, followed by 3 x 10 7 ~7×10 7 Subsequent poxvirus doses of pfu were administered until disease progression. 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 intravenously 5-10 days after the first poxvirus dose, followed by subsequent doses of about 10 mg / kg or about 800 mg of anti-PD-L1 antibody administered intravenously every two weeks until disease progression; The combination according to any one of [1] to

[12] , which is administered according to the following administration scheme:

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

[13] .

[15] Increased immune response to HPV16 E6 and E7 polypeptides, Intratumoral an increase in immune cell infiltrate, preferably an increase in the CD8 / CD3 ratio, a reduction in regulatory CD4 T cells, preferably a reduction in the Treg / CD8 ratio, 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, Increased expression of T cell activation genes, cytotoxic cell genes, pathogen defense genes, and NK cell function genes, ○ increased expression of one or more genes selected from the group consisting of CXCL10, CXCL11, IRF1, GZMK, GZMA, CD3D, PRF1, TBX21, CXCR3, STAT1, CD69, CCL2, GZMB, CD3G, ICOS, CD8A, STAT4, GZMM, CCR2, CD3E, and IL15, and / or an increase in the expression of one or more genes selected from the group CXCL13, GNLY, GZMH, IFNG, CXCL9, CCL5, and ITGAE, and / or a decrease in the expression of one or more genes selected from the group VEGFA, IHH, IL17A, PROM1, REN, PF4, TSLP, and LAG3. Remodeling characterized by The combination according to any one of [1] to

[14] , which induces the above.

Claims

1. 1. A combination for use in the treatment of HPV-positive cancer or precancerous lesions in situ, comprising: a) a poxvirus vector encoding at least membrane-bound human papillomavirus (HPV) non-oncogenic E6 and E7 polypeptides and human interleukin-2 (IL-2); b) Anti-PD-L1 antibody or antigen-binding fragment thereof and the poxvirus is modified vaccinia virus Ankara (MVA); the anti-PD-L1 antibody or antigen-binding fragment thereof mediates antibody-dependent cellular cytotoxicity (ADCC); A combination wherein a first administration of said poxvirus occurs 5 to 10 days before a first administration of said anti-PD-L1 antibody, and subsequent administrations of said poxvirus and anti-PD-L1 antibody occur.

2. The combination of claim 1, wherein the poxvirus encodes membrane-bound HPV-16 non-oncogenic E6 and E7 polypeptides and human IL-2.

3. The combination according to claim 1 or 2, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain comprising three complementarity-determining regions having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and a light chain comprising three complementarity-determining regions having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, and preferably the anti-PD-L1 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 7 or 8, and a light chain having the amino acid sequence of SEQ ID NO:

9.

4. The combination according to any one of claims 1 to 3, wherein the anti-PD-L1 antibody is avelumab.

5. 5. The combination of any one of claims 1 to 4, wherein the HPV-positive cancer is HPV-positive oropharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, vulvar cancer, penile cancer, mucosal cancer, or non-melanoma skin cancer, or the intraepithelial precancerous lesion is cervical intraepithelial neoplasia (CIN) grade 2 or 3 or vulvar intraepithelial neoplasia (VIN) grade 2 or 3, preferably the HPV-positive cancer is selected from HPV-positive vulvar cancer and vaginal cancer.

6. 6. The combination of claim 5, wherein the cancer is HPV-16 positive, preferably the cancer is HPV-16 positive squamous cell carcinoma of the head and neck (HPV-16+ SCCHN) or HPV-16 positive vulvar or vaginal cancer.

7. The combination according to any one of claims 1 to 6, wherein the HPV-positive cancer is an HPV-positive cancer without multiple liver metastases, preferably an HPV-positive cancer without liver metastases.

8. The combination according to any one of claims 5 to 7, wherein the HPV-positive cancer is recurrent and / or metastatic HPV-positive cancer, preferably the HPV-positive metastatic cancer is HPV-positive metastatic cancer with lymph node metastasis.

9. The combination according to claim 8, wherein the HPV-positive cancer is an HPV-positive metastatic cancer without multiple liver metastases, preferably the HPV-positive cancer is an HPV-positive metastatic cancer without liver metastases.

10. Each dose of the poxvirus is 3 x 10 ７ ~7 x 10 ７ The combination according to any one of claims 1 to 9, which is administered in a dose of pfu.

11. a) 3 x 10 of said poxvirus ７ ~7 x 10 ７ A first dose of 3 x 10 pfu was administered subcutaneously, followed by 3 x 10 ７ ~7 x 10 ７ Subsequent poxvirus doses of pfu were administered until disease progression. 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 an anti-PD-L1 antibody is administered intravenously 5-10 days after the first poxvirus dose, followed by subsequent doses of about 10 mg / kg or about 800 mg of the anti-PD-L1 antibody administered intravenously every two weeks until disease progression. The combination according to any one of claims 1 to 10, which is administered according to the following administration scheme:

12. a) the poxvirus is an MVA virus encoding membrane-associated HPV-16 non-oncogenic E6 and E7 polypeptides and human IL-2; b) the anti-PD-L1 antibody is avelumab; c) i) MVA virus encoding membrane-bound HPV-16 non-oncogenic E6 and E7 polypeptides and human IL-2 was 5×10 ７ pfu subcutaneously once weekly for 6 weeks, then every 2 weeks until 6 months, and then every 12 weeks thereafter until disease progression; ii) avelumab is administered by intravenous infusion at a dose of about 10 mg / kg or about 800 mg every 2 weeks starting on day 8 until disease progression. 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 11.

13. - 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, a reduction in regulatory CD4 T cells, preferably a reduced Treg / CD8 ratio, and / 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, Increased expression of T cell activation genes, cytotoxic cell genes, pathogen defense genes, and NK cell function genes, increased expression of one or more genes selected from the group consisting of CXCL10, CXCL11, IRF1, GZMK, GZMA, CD3D, PRF1, TBX21, CXCR3, STAT1, CD69, CCL2, GZMB, CD3G, ICOS, CD8A, STAT4, GZMM, CCR2, CD3E, and IL15, and / or Increased expression of one or more genes selected from the group CXCL13, GNLY, GZMH, IFNG, CXCL9, CCL5 and ITGAE, and / or reduced expression of one or more genes selected from the group VEGFA, IHH, IL17A, PROM1, REN, PF4, TSLP and LAG3. Remodeling characterized by The combination according to any one of claims 1 to 12, which induces

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