Triple combination drug therapy for the treatment of head and neck cancer

Abstract

The present invention relates to a triple combination drug administration therapy for the treatment of head and neck cancer, more specifically, to a treatment method for head and neck cancer by administering an anti-PD1 antibody, a human papillomavirus (HPV) vaccine, and a persistent recombinant IL-7 fusion protein. The present invention is highly effective in treating refractory head and neck cancer that has been difficult to treat with conventional standard therapeutic agents. The invention aims to provide a pharmaceutical composition for treating head and neck cancer that has an excellent therapeutic effect on refractory head and neck cancer.

Description

[Technical field]

[0001] The present invention relates to a triple combination drug administration therapy for the treatment of head and neck cancer, and more particularly to a therapeutic method for treating head and neck cancer by administering an anti-PD1 antibody, a human papillomavirus (HPV) vaccine and a persistent recombinant IL-7 fusion protein. [Background technology]

[0002] Head and neck cancer refers to cancer that occurs in any part of the head and neck except the brain and eyes, and includes oral cancer, pharyngeal cancer (tongue cancer, tonsil cancer), laryngeal cancer, paranasal sinus cancer, salivary gland cancer, nasopharyngeal cancer, etc. Head and neck cancer has a high incidence and mortality rate, with approximately 700,000 new cases occurring worldwide each year and 350,000 dying from the disease, of which more than 90% are head and neck squamous cell carcinomas.

[0003] Various treatments, including surgical resection, radiation therapy, and chemotherapy, have been attempted for locally advanced head and neck squamous cell carcinoma (LA HNSCC). However, the disease is often diagnosed at stage 4, when the cancer is quite advanced. The disease has a complex mechanism of development, making it difficult to diagnose and predict the stage of the disease, and the survival rate is only 30%, making it difficult to treat.

[0004] Recently, the incidence of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) has been increasing, and HPV-associated HNSCC mainly originates from the oropharynx (tonsil, the base of the tongue). Oropharyngeal cancer (OPC), one of the clinical subsets of HNSCC, is one of the carcinomas with a particularly high prevalence of human papillomavirus (HPV)-associated tumor development, and the incidence of HPV-positive OPC has been increasing worldwide. Compared with HPV-negative OPC, HPV-positive OPC has different immunological characteristics, is characterized by high levels of T lymphocyte infiltration, and has a lower survival rate. In particular, forest plots from the CHECKMATE-141 and KEYNOTE-040 studies showed that the response rate of HPV-positive HNSCC to anti-PD-1 / PD-L1 therapy was relatively lower than expected. Additionally, recently published results from the KEYNOTE-412 study evaluating chemoradiotherapy and maintenance anti-PD-1 pembrolizumab in 780 patients with head and neck squamous cell carcinoma did not demonstrate statistical significance compared to the control group, suggesting that HNSCC remains a challenging disease to treat.

[0005] In addition, HPV-induced tumors express various mechanisms to evade immune surveillance. Specifically, the tumor microenvironment (TME) downregulates major histocompatibility complex (MHC) class I expression while simultaneously impairing antigen processing and presentation, (2) induces resistance to immune-mediated cytotoxic T lymphocyte (CTL) cytotoxicity, (3) expresses immunosuppressive cytokines (e.g., TGF-β and IL-10) and immune checkpoint molecules (e.g., PD-L1), and (4) induces immune cells with immunosuppressive properties (e.g., regulatory T cells, myeloid-derived suppressor cells).

[0006] HPV DNA vaccine (GX-188E), which preferentially targets HPV antigens to dendritic cells, is known to induce polyfunctional T cell immunity associated with HPV elimination in stage 3 cervical intraepithelial neoplasia patients without safety concerns. Furthermore, when administered in combination with pembrolizumab, GX-188E demonstrated remarkable anticancer efficacy (best response rate (BORR) 33.3%, progression-free survival (PFS) 2.7 months) and a manageable safety profile in patients with cervical cancer refractory to standard treatments (Korean Patent Publication No. 10-2022-0109444).

[0007] GX-I7 is a long-lasting IL-7 protein composed of human IL-7 and a hybrid Fc (hyFc) region, which can extend the half-life of IL-7. IL-7 is a potent cytokine required for thymic T cell development and homeostatic T cell expansion. For reference, it has been reported that GX-I7 administration can induce the migration of T cells to tumor sites along with the massive expansion of T cells (Korean Patent Publication No. 10-2022-0079541). In addition, it was confirmed that thymectomized animals treated with a combination treatment regimen of IL-7 protein and anti-PD-1 antibody showed a significant reduction in tumor volume compared to other treatment groups (control group, IL-7 alone, and anti-PD-1 antibody alone) (Korean Patent Publication No. 10-2021-0093950).

[0008] In the present invention, as the incidence of HPV-positive HNSCC has recently increased, there is an emerging need for customized treatment for HPV-16-positive or HPV-18-positive HNSCC. Accordingly, a method for promoting tumor-specific immunity and reversing the immunosuppressive tumor microenvironment in HPV-induced tumors was explored, and the present invention was completed by confirming that a combined therapy of anti-PD1 antibody, human papillomavirus (HPV) vaccine and persistent recombinant IL-7 fusion protein administered prior to cancer resection for anti-cancer treatment significantly improves the anti-cancer effect of head and neck cancer, which is known to have a poor prognosis according to existing treatment methods. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Korean Patent Publication No. 10-2022-0109444 [Patent Document 2] Korean Patent Publication No. 10-2021-0093950 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a pharmaceutical composition for treating head and neck cancer, which exhibits excellent therapeutic effects on refractory head and neck cancer. [Means for solving the problem]

[0011] To achieve the above objective, the present invention provides a pharmaceutical composition for treating head and neck cancer, comprising an anti-PD1 antibody, a human papillomavirus (HPV) vaccine and a persistent recombinant IL-7 fusion protein.

[0012] In the present invention, the anti-PD-1 antibody can be selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, pidilizumab, and combinations thereof.

[0013] In the present invention, the HPV vaccine may comprise polynucleotides encoding HPV-16 and HPV-18 antigens.

[0014] In the present invention, the HPV vaccine may be an isolated DNA comprising a polynucleotide encoding the antigen.

[0015] In the present invention, the HPV vaccine may comprise a polynucleotide of SEQ ID NO:15 or a functional variant having 85% or more sequence identity with SEQ ID NO:15.

[0016] In the present invention, the HPV vaccine may further comprise a nucleic acid sequence encoding a heterologous polypeptide. In the present invention, the heterologous polypeptide may comprise Fms-like tyrosine kinase 3 ligand ("FLT3L") or a portion thereof.

[0017] In the present invention, the HPV vaccine may further comprise a nucleotide sequence encoding a signal peptide.

[0018] In the present invention, the signal peptide can be selected from the signal peptide of tissue plasminogen activator (tPA), the signal peptide of herpes simplex virus glycoprotein D (HSV gD), the signal peptide of growth hormone, and any combination thereof. In the present invention, the persistent recombinant IL-7 fusion protein comprises: (a) interleukin-7 (IL-7) or modified interleukin-7 (IL-7) of the following formula (I): A-IL-7 Chemical formula (I) where A is an oligopeptide consisting of 1 to 10 amino acid residues; and

[0019] (b) a domain that extends the half-life of interleukin-7 or modified interleukin-7;

[0020] In the present invention, the IL-7 may have an amino acid sequence selected from the group consisting of SEQ ID NO:16 to SEQ ID NO:21.

[0021] In the present invention, A may be an oligopeptide composed of amino acid residues selected from the group consisting of methionine, glycine, or a combination thereof.

[0022] In the present invention, the domain may comprise an Fc region of a modified immunoglobulin.

[0023] In the present invention, the long-acting recombinant IL-7 fusion protein may comprise the amino acid sequence of SEQ ID NO:39 or a functional variant having 85% or more sequence identity with SEQ ID NO:39.

[0024] In the present invention, the anti-PD-1 antibody is pembrolizumab or nivolumab, and the single administration dose can be 50 mg to 500 mg.

[0025] In the present invention, the anti-PD-1 antibody may be for intravenous injection.

[0026] In the present invention, the single administration dose of the HPV vaccine may be 0.5 mg to 5 mg.

[0027] In the present invention, the HPV vaccine may be for intramuscular injection.

[0028] In the present invention, the single administration dose of the long-acting recombinant IL-7 fusion protein may be 60 ug / kg to 5,000 ug / kg.

[0029] In the present invention, the long-acting recombinant IL-7 fusion protein can be for intramuscular injection.

[0030] In the present invention, the anti-PD1 antibody, HPV vaccine, and long-acting recombinant IL-7 fusion protein can each be administered once or multiple times.

[0031] In the present invention, the anti-PD1 antibody, HPV vaccine and long-acting recombinant IL-7 fusion protein can be administered simultaneously, separately, or sequentially.

[0032] In the present invention, the anti-PD1 antibody is pembrolizumab, and the pembrolizumab may be administered twice, the HPV vaccine three times, and the long-acting recombinant IL-7 fusion protein once.

[0033] In the present invention, the anti-PD1 antibody is nivolumab, and the nivolumab can be administered at two-week intervals, the HPV vaccine can be administered eight times, and the long-acting recombinant IL-7 fusion protein can be administered three times.

[0034] In the present invention, the pharmaceutical composition can be administered before cancer resection surgery.

[0035] In the present invention, the head and neck cancer may be HPV positive.

[0036] In the present invention, the head and neck cancer may be head and neck squamous cell carcinoma.

[0037] In the present invention, the head and neck cancer may be oropharyngeal cancer.

[0038] In the present invention, the head and neck cancer may be metastatic, recurrent and / or advanced.

[0039] In the present invention, the head and neck cancer may be locally advanced and resectable.

[0040] In the present invention, the pharmaceutical composition may be administered to a subject who has not previously received other anti-cancer treatments.

[0041] The present invention also provides a kit for treating head and neck cancer, comprising an anti-PD1 antibody, a human papillomavirus (HPV) vaccine and a persistent recombinant IL-7 fusion protein.

[0042] The present invention also provides the use of a combination of an anti-PD1 antibody, a human papillomavirus (HPV) vaccine and a long-acting recombinant IL-7 fusion protein for the manufacture of a medicament for the treatment of head and neck cancer.

[0043] The present invention also provides the use of a combination of anti-PD1 antibody, human papillomavirus (HPV) vaccine and long-acting recombinant IL-7 fusion protein for the treatment of head and neck cancer.

[0044] The present invention also provides a method for treating head and neck cancer, comprising administering to a subject in need thereof an anti-PD1 antibody, a human papillomavirus (HPV) vaccine, and a persistent recombinant IL-7 fusion protein.

[0045] In the present invention, the anti-PD1 antibody, human papilloma virus (HPV) vaccine and persistent recombinant IL-7 fusion protein can be administered simultaneously, separately, or sequentially.

[0046] In the present invention, the anti-PD1 antibody, human papilloma virus (HPV) vaccine and long-acting recombinant IL-7 fusion protein can be administered before cancer resection surgery. Effect of the Invention

[0047] The present invention has the advantage of exhibiting clinically remarkably excellent therapeutic effects on refractory head and neck cancers that are difficult to treat with conventional standard therapeutic agents or surgical therapy alone, such as HPV-positive head and neck cancers. [Brief description of the drawings]

[0048] [Figure 1] FIG. 1 is a diagram showing a composite drug administration schedule in a clinical trial according to one embodiment of the present invention.

[0049] [Diagram 2] FIG. 2 shows the results of evaluating pathologic tumor response (%) in a clinical trial according to one embodiment of the present invention.

[0050] [Diagram 3]FIG. 3 shows the results of evaluating radiologic tumor response (%) in a clinical trial according to one embodiment of the present invention.

[0051] [Figure 4] FIG. 4 shows the results of photographing the effect of head and neck cancer treatment on subjects in a clinical trial according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] (Best Mode for Carrying Out the Invention) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are those well known and commonly used in the art. definition

[0053] Unless otherwise stated or implicit in the text, the following terms and phrases include the meanings provided below. Unless otherwise expressly stated or explicitly stated in the text, the following terms and phrases do not exclude the meaning that the term or phrase has acquired in the art. The definitions are provided to help describe specific embodiments and are not intended to limit the aspects provided in the present application, since the scope of the aspects provided in the present application is limited only by the claims. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the aspects provided in the present application belong.

[0054] The term "pharmaceutical composition" is defined herein to mean a mixture or solution containing at least one therapeutic agent that is administered to a mammal to prevent or treat a particular disease or disorder affecting the mammal. In one embodiment, the mammal may be a human.

[0055] The term "pharmaceutical acceptable" is defined herein to mean a compound, substance, composition and / or dosage form that is compatible with contact with the tissues of a human patient without undue toxicity, irritant allergic response and other problematic complications that are commensurate with a reasonable benefit / risk ratio within the scope of sound medical judgment.

[0056] The term "treating" or "treatment" as used herein includes treatment that relieves, decreases, reduces, or alleviates at least one symptom or delays the progression of a disease in a human patient. For example, treatment can be a reduction in one or various symptoms of a disorder or a complete eradication of a disorder. Within the meaning of this disclosure, the term "treat" also means arresting or delaying the onset (i.e., the period before clinical signs of a disease) and / or reducing the risk of development or worsening of a disease. The term "protect" is used herein to mean preventing, delaying, or treating the development or persistence or worsening of a disease in a subject, or all of these.

[0057] The term "pharmacologically effective amount" or "clinically effective amount" is an amount sufficient to provide an observable improvement in the underlying clinically observable signs and symptoms of the disorder being treated.

[0058] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject in need of diagnosis, prognosis or treatment, particularly a mammalian subject. Mammalian subjects include, but are not limited to, humans, livestock, farm animals, zoo animals, sport animals, pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows; primates such as apes, monkeys, orangutans and chimpanzees; canines such as dogs and wolves; felines such as cats, lions and tigers; equines such as horses, donkeys and zebras; food animals such as bears, cows, pigs and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and the like. In certain embodiments, the mammal is a human subject.

[0059] In the present invention, the subject may be a human patient diagnosed with or suffering from head and neck cancer.

[0060] The terms "combination treatment / therapy," "combined treatment," "combinatorial," or "in combination" refer to treatment using two or more separate therapeutic agents simultaneously and / or at different times within a defined period of time.

[0061] The term "about" is used to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used to modify the stated value above or below (higher or lower) by a variance of 10% above or below the numerical value set forth.

[0062] The terms "significant" or "significantly" indicate statistical significance, generally meaning a difference of 2 standard deviations (2 SD) or greater.

[0063] The term "decreased" or "decrease" is used herein to generally mean a decrease of about a statistically significant amount. In some embodiments, "decreased" or "decrease" means a decrease of at least 10% compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or more, including a decrease of 100% (e.g., an absent or undetectable level compared to a reference level).

[0064] The terms "increased" or "increase" are used herein to generally mean an increase of about a statistically significant amount. In some embodiments, "increased" or "increase" means an increase of at least about 10% compared to a baseline level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or more.

[0065] As used herein, the terms "comprising" or "comprises" are used in connection with compositions, methods and their respective components, and may include unspecified elements that are essential to said methods or compositions, but may not be essential.

[0066] The term "consisting of" refers to compositions, methods, and their respective components as described herein, excluding any element not mentioned in the description of the embodiment.

[0067] Unless otherwise specified, the terms "protein," "polypeptide," and "peptide" may be used interchangeably. Anti-PD-1 antibody

[0068] Programmed cell death protein 1 (PD-1, also known as CD279) encodes a cell surface membrane protein of the immunoglobulin superfamily expressed in B cells and NK cells. Anti-PD1 antibodies have been used to treat melanoma, non-small cell lung cancer, bladder cancer, prostate cancer, colorectal cancer, head and neck cancer, triple-negative breast cancer, leukemia, lymphoma and renal cell carcinoma. Exemplary anti-PD1 antibodies include pembrolizumab (KEYTRUDA®, MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), cemiplimab (LIBTAYO®) and pidilizumab (CT-011, CURETECH LTD.).

[0069] In one embodiment, the anti-PD1 antibody is pembrolizumab.

[0070] Pembrolizumab (Keytruda®, formerly Lambrolizumab), also known as Merck 3745, MK-3475 or SCH-900475, is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab has been disclosed in Hamid, O. et al., New England Journal of Medicine, 2013, 369(2):134-44; WO2009 / 114335; and US8,354,509.

[0071] In another embodiment, the anti-PD1 antibody is nivolumab.

[0072] Nivolumab (Opdivo®), also known as iMDX-1106, MDX-1106-04, ONO-4538, or BMS-936558, has a CAS Registry Number of 946414-94-4. Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in US 8,008,449 and WO 2006 / 121168. Human papillomavirus (HPV) vaccine

[0073] A human papilloma virus (HPV) vaccine that may be used in one embodiment of the present application may preferably comprise a fusion protein of HPV16 and HPV18 or a polynucleotide encoding said fusion protein.

[0074] In one embodiment, the fusion protein comprises three or more amino acid sequences selected from the following examples: (1) an N-terminal portion of an HPV16 E6 protein, (2) a C-terminal portion of an HPV16 E6 protein, (3) an N-terminal portion of an HPV16 E7 protein, (4) a C-terminal portion of an HPV16 E7 protein, (5) an N-terminal portion of an HPV18 E6 protein, (6) a C-terminal portion of an HPV18 E6 protein, (7) an N-terminal portion of an HPV18 E7 protein, and (8) a C-terminal portion of an HPV18 E7 protein, wherein the fusion protein does not bind to p53 or dimerize with HPV16 or HPV18 E6 protein, and wherein the fusion protein does not bind to pRb or dimerize with HPV16 or HPV18 E7 protein. This is described in US11135262B2, the entire contents of which are incorporated herein by reference.

[0075] A fusion polypeptide that modifies the 3D structure of E6 and E7 derived from HPV types 16 and 18 and a fusion protein containing an immune enhancing peptide, and a polynucleotide encoding the same, are disclosed in US9000139B2. The fusion protein according to an exemplary embodiment of US9000139B2 may contain a recombinant fusion polypeptide to modify the 3D structure of E6 and E7 derived from HPV types 16 and 18. More specifically, the fusion polypeptide is a fusion polypeptide in which the 1st to 85th amino acids of the E6 protein derived from HPV type 16, the 1st to 65th amino acids of the E7 protein, the 71st to 158th amino acids of the E6 protein, and the 51st to 98th amino acids of the E7 protein derived from HPV type 18 are sequentially linked. US9000139B2 relates to nucleic acid molecules encoding fusion polypeptides and immune enhancing peptides. Exemplary immune enhancing peptides include CD40 ligand, Flt3 ligand, flagellin and / or OX40. Specific examples of polynucleotides encoding fusion proteins, optimized signal sequences (e.g., tPa), and immune enhancing peptides (e.g., Flt3 ligand) can be prepared with reference to Example 1 of US9000139B2. The entire contents of US9000139B2 are incorporated herein by reference.

[0076] As used herein, the term "GX-188E" refers to a nucleic acid construct having the nucleotide sequence of SEQ ID NO:15, or a variant thereof having at least about 80% or more, about 81% or more, about 82% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 100% sequence identity to SEQ ID NO:15.

[0077] In the present invention, the term "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of a second polypeptide. As discussed herein, a particular polypeptide is at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to another polypeptide, which can be determined using methods and computer programs / software known in the art, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, parameters are set such that the percentage of identity is calculated over the entire length of the reference polypeptide sequence, allowing gaps in homology of up to 5% of the total number of amino acids in the reference sequence.

[0078] The human papillomavirus (HPV) vaccine may be a substance described in International Publication Nos. 2016 / 024255 and 2021 / 215896, the entire contents of which are incorporated herein by reference. Long-acting recombinant IL-7 fusion protein

[0079] In the long-acting recombinant IL-7 fusion protein that may be used in one embodiment of the present application, interleukin-7 (IL-7) may be interleukin-7, a polypeptide capable of binding to the IL-7 receptor (also known as CD127), or a polypeptide having the activity of IL-7 or an activity similar thereto.

[0080] As used herein, the term "a polypeptide having the activity of IL-7 or a similar activity thereof" refers to a polypeptide or protein having the same or similar sequence and activity as IL-7.

[0081] In the present application, IL-7 includes polypeptides consisting of the amino acid sequences shown in SEQ ID NOs: 16 to 21. IL-7 may have about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to the sequences of SEQ ID NOs: 16 to 21. The sequence identity of the peptide sequence may be determined using known sequence alignment or comparison software. For example, the sequence identity may be determined using the BLASTP program (blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins) as the default setting.

[0082] IL-7 may include IL-7 protein or a fragment thereof, and the fragment may bind to IL-7 receptor. The term "IL-7 protein" used in this application may be used as a concept including "IL-7 protein and a fragment thereof capable of binding to IL-7 receptor". IL-7 may be obtained from humans, rats, mice, monkeys, cows or sheep.

[0083] Specifically, human IL-7 can have the amino acid sequence shown in SEQ ID NO: 16 (Genbank Accession No. P13232); rat IL-7 can have the amino acid sequence shown in SEQ ID NO: 17 (Genbank Accession No. P56478); mouse IL-7 can have the amino acid sequence shown in SEQ ID NO: 18 (Genbank Accession No. P10168); monkey IL-7 can have the amino acid sequence shown in SEQ ID NO: 19 (Genbank Accession No. NP_001279008); bovine IL-7 can have the amino acid sequence shown in SEQ ID NO: 20 (Genbank Accession No. P26895), and ovine IL-7 can have the amino acid sequence shown in SEQ ID NO: 21 (Genbank Accession No. Q28540).

[0084] The IL-7 protein or fragments thereof may also include variously modified proteins or peptides, i.e., variants. The modifications may be made in a manner that substitutes, deletes, or inserts one or more proteins into the wild-type IL-7 without modifying the function of IL-7. These variant proteins or peptides may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the wild-type protein.

[0085] Usually, the wild type amino acid residue is replaced with alanine, however substitutions can be made with conservative amino acid substitutions that have no or only a weak effect on the overall protein charge, ie, polarity or hydrophobicity.

[0086] Conservative amino acid substitutions can be seen in Table 1 below. [Table 1]

[0087] For each amino acid, further conservative substitutions include "homologs" of the amino acid. In particular, "homologs" have a methylene group (CH 2 ) is inserted. Examples of "homologs" may include, but are not limited to, homophenylalanine, homoarginine, homoserine, and the like.

[0088] In a long-acting recombinant IL-7 fusion protein that may be used in one embodiment of the present application, the modified interleukin-7 (IL-7) may have the following structure: A-IL-7 Chemical formula (I) A is an oligopeptide consisting of 1 to 10 amino acid residues, and IL-7 is interleukin 7, a polypeptide capable of binding to the IL-7 receptor (also known as CD127), or a polypeptide having IL-7 activity or an activity similar thereto.

[0089] In the modified IL-7 structure of A-IL-7, the A residue can be linked directly to the N-terminus of IL-7 or linked via a linker.

[0090] Specifically, the result may be in the form of A-IL-7 or A-linker-IL-7.

[0091] When the linker is a peptide linker, the linkage can occur at any linkage region. They can be coupled using cross-linking agents known in the art. Examples of cross-linking agents can include, but are not limited to, imidoesters, including 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as 4-azidosalicylic acid; disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate) and bifunctional maleimides such as bis-N-maleimide-1,8-octane.

[0092] The linker may be an albumin linker or a peptide linker. The peptide linker may be a peptide of 10 to 20 amino acid residues composed of Gly and Ser residues.

[0093] When the linker is formed of one selected from the group consisting of a chemical bond, the chemical bond can be a disulfide bond, a diamine bond, a sulfide-amine bond, a carboxy-amine bond, an ester bond and a covalent bond.

[0094] In one embodiment, A may be linked to the N-terminus of IL-7. A may be characterized by comprising 1-10 amino acids, which may be selected from the group consisting of methionine, glycine, and combinations thereof. In one embodiment, when A is a single amino acid residue, it is glycine.

[0095] Methionine and glycine do not induce immune responses in the human body. Protein therapeutics produced in E. coli always contain methionine at the N-terminus, but no abnormal reactions have been reported. Glycine is also widely used as a GS linker, and it has been reported that commercial products such as Dulaglutide do not induce immune responses (Cell Biophys. 1993 Jan-Jun; 22(103):189-224).

[0096] In an exemplary embodiment, A may be an oligopeptide containing 1 to 10 amino acids selected from the group consisting of methionine (Met, M), glycine (Gly, G), and combinations thereof. In an embodiment, A may be an oligopeptide consisting of 1 to 5 amino acids. For example, A may be methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, or glycine-glycine-glycine.

[0097] The modified IL-7 can have the structure of A-IL-7, which includes a polypeptide having the activity of IL-7 or an activity similar thereto, and an oligopeptide consisting of 1 to 10 amino acids.

[0098] In one embodiment, the modified IL-7 may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-35. In addition, the modified IL-7 may have a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NOs: 30, 31, 32, 33, 34, or 35.

[0099] A long-acting recombinant interleukin-7 (IL-7) fusion protein that can be used in one embodiment of the present invention is (a) interleukin-7 (IL-7) or modified interleukin-7 (IL-7) of the following formula (I): A-IL-7 Chemical formula (I) where A is an oligopeptide consisting of 1 to 10 amino acid residues; and (b) a domain that extends the half-life of interleukin-7 or modified interleukin-7;

[0100] When the domain that extends the half-life of said interleukin-7 or modified interleukin-7 is represented by B, B may be linked directly to IL-7 or modified IL-7 or linked via a linker, specifically, the resulting form may be A-IL-7-B, BA-IL-7, A-IL-7-linker-B, B-linker-A-IL-7, A-linker-IL-7-linker-B, or B-linker-A-IL-7.

[0101] When the linker is a peptide linker, the linkage can occur at any linkage region. They can be coupled using cross-linking agents known in the art. Examples of cross-linking agents can include, but are not limited to, imidoesters, including 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as 4-azidosalicylic acid; disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate) and bifunctional maleimides such as bis-N-maleimide-1,8-octane.

[0102] The linker may be an albumin linker or a peptide linker. The peptide linker may be a peptide of 10 to 20 amino acid residues composed of Gly and Ser residues.

[0103] When the linker is formed of one selected from the group consisting of a chemical bond, the chemical bond can be a disulfide bond, a diamine bond, a sulfide-amine bond, a carboxy-amine bond, an ester bond and a covalent bond.

[0104] The interleukin-7 or modified interleukin-7 half-life extending domain may be a fusion partner for increasing the in vivo half-life of interleukin-7 or modified interleukin-7, and preferably includes an Fc region or a portion thereof of an immunoglobulin, albumin, an albumin binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), a long unstructured hydrophilic sequence of amino acids (XTEN), hydroxyethyl starch (HES), an albumin binding small molecule, and combinations thereof.

[0105] When the domain is an immunoglobulin Fc region, it may be an Fc region of a modified immunoglobulin. In particular, the Fc region of the modified immunoglobulin may have attenuated antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) due to modification of the binding affinity with Fc receptors and / or complement. The modified immunoglobulin Fc region may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof. The modified immunoglobulin Fc region may be selected from the group consisting of IgG1, IgG2, IgG3, IgD, IgG4, and combinations thereof. In particular, the modified immunoglobulin Fc region may comprise, from the N-terminus to the C-terminus, a hinge region, a CH2 domain, and a CH3 domain. In particular, the hinge region may comprise a human IgD hinge region; the CH2 domain may comprise some of the amino acid residues of human IgD and some of the amino acid residues of a human IgG4 CH2 domain; and the CH3 domain may comprise some of the amino acid residues of a human IgG4 CH3 domain.

[0106] Furthermore, two long-acting recombinant IL-7 fusion proteins can form a dimer. For example, when the domain that extends the half-life of the IL-7 or modified IL-7 is an Fc region, the Fc regions can bind to each other to form a dimer.

[0107] As used herein, the term "Fc region," "Fc fragment," or "Fc" refers to a protein that includes the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, but does not include the variable regions of the heavy and light chains and the light chain constant region (CL1), and may further include the hinge region of the heavy chain constant region. In one embodiment, a hybrid Fc or a hybrid Fc fragment thereof may be designated "hFc" or "hyFc."

[0108] The term "Fc region variant" as used herein refers to a variant produced by substituting a portion of the amino acids in the Fc region or by combining a heterologous Fc region. The Fc region variant can be prevented from being cleaved at the hinge region. Specifically, the 144th and / or 145th amino acids of SEQ ID NO: 24 may be modified. Preferably, the variant may have the amino acid K at the 144th position substituted with G or S and the amino acid E at the 145th position substituted with G or S.

[0109] The Fc region of the modified immunoglobulin or Fc region variant may also be represented by the following chemical formula (II): Chemical formula (II) N'-(Z1)pY-Z2-Z3-Z4-C' N' is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide; p is an integer of 0 or 1; Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues from the amino acid residue at position 98 toward the N-terminus among amino acid residues at positions 90 to 98 of SEQ ID NO:22; Y is an amino acid sequence having 5 to 64 consecutive amino acid residues from the amino acid residue at position 162 toward the N-terminus among amino acid residues at positions 99 to 162 of SEQ ID NO:22; Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues from the amino acid residue at position 163 toward the C-terminus among amino acid residues at positions 163 to 199 of SEQ ID NO: 22; Z3 is an amino acid sequence having 71 to 106 consecutive amino acid residues from the amino acid residue at position 220 toward the N-terminus among the amino acid residues at positions 115 to 220 of SEQ ID NO: 23; and Z4 is an amino acid sequence having 80 to 107 consecutive amino acid residues from the amino acid residue at position 221 among amino acid residues at positions 221 to 327 of SEQ ID NO:23 toward the C-terminus.

[0110] In the present invention, the domain may have an amino acid sequence selected from the group consisting of SEQ ID NOs:24-29.

[0111] In addition, the Fc fragment may have native sugar chains, increased sugar chains, or decreased sugar chains compared to the native form, or may be deglycosylated. Immunoglobulin Fc sugar chains can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. When the sugar chains are removed from the Fc fragment, the binding affinity to the C1q portion of the first complement component C1 is rapidly reduced, and ADCC or CDC is reduced or lost, so that unnecessary immune responses are not induced in vivo. In this regard, a deglycosylated or non-glycosylated form of immunoglobulin Fc region may be more suitable for the purpose of one embodiment as a drug carrier. The term "deglycosylation" used in the present application means an Fc region from which sugars have been enzymatically removed from the Fc fragment. In addition, the term "non-glycosylated" means that the Fc fragment is produced in a non-glycosylated form by a prokaryote, preferably E. coli.

[0112] The Fc region of the modified immunoglobulin may also comprise the amino acid sequence of SEQ ID NO: 24 (hyFc), SEQ ID NO: 25 (hyFcM1), SEQ ID NO: 26 (hyFcM2), SEQ ID NO: 27 (hyFcM3), or SEQ ID NO: 28 (hyFcM4). The Fc region of the modified immunoglobulin may also comprise the amino acid sequence of SEQ ID NO: 29 (non-lytic mouse Fc).

[0113] The Fc region of the modified immunoglobulin may be as described in U.S. Pat. No. 7,867,491, and the production of the Fc region of the modified immunoglobulin may be carried out with reference to the disclosure of U.S. Pat. No. 7,867,491, the entire contents of which are incorporated herein by reference.

[0114] The long-acting recombinant IL-7 fusion protein can have an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 to 40. The IL-7 fusion protein can also have a sequence having at least about 70%, at least about 75%, at least about 80%, 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NOs: 36, 37, 38, 39, or 40.

[0115] The long-acting recombinant IL-7 fusion proteins of SEQ ID NOs: 36-40 may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 50-54.

[0116] The nucleic acid molecule may further comprise a signal or leader sequence.

[0117] The term "signal sequence" as used herein refers to a fragment that directs the secretion of biologically active molecules, drugs, and fusion proteins, which is cleaved after translation in the host cell. In one embodiment, the signal sequence is a polynucleotide that encodes an amino acid sequence that initiates the translocation of a protein through the endoplasmic reticulum (ER) membrane. In one embodiment, useful signal sequences include antibody light chain signal sequences, such as antibody 14.18 (Gillies et al., J. Immunol. Meth 1989.125:191-202), antibody heavy chain signal sequences, such as MOPC141 antibody heavy chain signal sequence (Sakano et al., Nature, 1980.286:676-683), and other signal sequences known in the art (see, for example, Watson et al., Nucleic Acid Research, 1984.12:5145-5164).

[0118] The characteristics of signal peptides are well known in the art and signal peptides usually have 16-30 amino acids, but may contain more or fewer amino acid residues. A typical signal peptide is composed of three regions: a basic N-terminal region, a central hydrophobic region and a polar C-terminal region.

[0119] The central hydrophobic region contains 4 to 12 hydrophobic residues, which anchor the signal sequence through the membrane lipid bilayer during translocation of the immature polypeptide. After initiation, the signal sequence is primarily cleaved in the lumen of the ER by cellular enzymes known as signal peptidases. In particular, the signal sequence may be a secretory signal sequence for tissue plasminogen activation (tPa), a signal sequence of herpes simplex virus glycoprotein D (HSV gD), or growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells, including mammals, may be used. Also, as the secretory signal sequence, a signal sequence contained in wild-type IL-7 may be used or may be used after substitution with codons that are frequently expressed in the host cell.

[0120] An isolated nucleic acid molecule encoding a long-acting recombinant IL-7 fusion protein can be included in an expression vector.

[0121] The term "vector" as used herein is understood as a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell and recombined and inserted into the genome of the host cell or replicated autonomously as an episome. Vectors may include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogs thereof. Examples of viral vectors may include, but are not limited to, retroviruses, adenoviruses and adeno-associated viruses.

[0122] The term "gene expression" or "expression" of a target protein as used herein is understood to mean the transcription of a DNA sequence, translation of an mRNA transcript, and secretion of the fusion protein product or a fragment thereof.

[0123] As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, the terms "transduced," "transformed," and "transfected" refer to the introduction of a nucleic acid (e.g., a vector) into a cell using techniques known in the art.

[0124] The term "gene expression" or "expression" of a target protein as used herein is understood to mean the transcription of a DNA sequence, translation of an mRNA transcript, and secretion of an Fc fusion protein product or an antibody or antibody fragment thereof.

[0125] Useful expression vectors can be RcCMV (Invitrogen, Carlsbad) or variants thereof. The expression vector may include a polyadenylation signal sequence from human cytomegalovirus (CMV) to promote sustained transcription of the target gene in mammalian cells and bovine growth hormone to increase the stability state of the RNA after transcription. In an exemplary embodiment, the expression vector is pAD15, a modified form of RcCMV.

[0126] The expression vector may be contained in a suitable host cell adapted for expression and / or secretion of the target protein by transduction or transfection of the DNA sequence of an embodiment.

[0127] Examples of suitable host cells may include, but are not limited to, immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, human amniotic fluid-derived cells (CapT cells) or COS cells.

[0128] The modified IL-7 or long-acting recombinant IL-7 fusion protein may be a substance described in US Patent No. US10208099B2, the entire contents of which are incorporated herein by reference. Route of administration

[0129] The pharmaceutical composition according to the present invention can be administered to a subject by direct administration (e.g., locally by injection, implantation or topical administration to a tissue area) or by a system through suitable means (e.g., parenterally or orally). When the composition is administered parenterally by intravenous, subcutaneous, intraocular, intraperitoneal, intramuscular, oral, rectal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternally, intracapsular, intranasal, or aerosol administration, the composition preferably contains an aqueous or physiologically compatible suspension of body fluids or a part of these solutions. Thus, a physiologically acceptable carrier or transporter can be added to the composition and delivered to the patient, which does not negatively affect the electrolyte and / or volume balance of the patient. Thus, the physiologically acceptable carrier or transporter can be saline. The carrier or transporter can be a pharmaceutically acceptable formulation. Compositions, uses and methods of treatment

[0130] In the present invention, the pharmaceutical composition for treating head and neck cancer may be a pharmaceutical composition comprising an anti-PD1 antibody composition, a human papillomavirus (HPV) vaccine composition, and a persistent recombinant IL-7 fusion protein composition in a separated form.

[0131] The anti-PD1 antibody composition may be in the form of commercially available pembrolizumab or nivolumab. The anti-PD1 antibody may be administered at a dose of about 1 mg to 1000 mg. In one embodiment, the dose of the anti-PD1 antibody is about 10 mg to 500 mg, about 50 mg to 500 mg, about 100 mg to 500 mg, about 100 mg to 300 mg, about 150 mg to 300 mg, about 180 mg to 250 mg, about 190 mg to 250 mg, about 185 mg to 225 mg, about 185 mg to 220 mg, about 195 mg to 250 mg, about 195 mg to 225 ... The anti-PD1 antibody may be administered at intervals such as once a week, twice a week, three times a week, four times a week, every week, every 10 days, once every other week, every 20 days, every 3 weeks, every 4 weeks, once a month, once every other month, or once every 3 months. Alternatively, the anti-PD1 antibody may be administered at intervals of 5, 10, 15, 20, 30 or 40 days. In one embodiment, the anti-PD1 antibody is pembrolizumab and may be administered at weeks 1 and 4.

[0132] The anti-PD1 antibody compositions may be administered parenterally, preferably intravenously, by injection.

[0133] The HPV vaccine composition may be administered in a separate dosage form from the anti-PD1 antibody composition. The HPV vaccine composition may be GX-188E.

[0134] The composition of GX-188E can be administered parenterally, for example, subcutaneously, intracutaneously, intradermally, subdermally or intramuscularly, by injection. The composition of GX-188E refers to a composition or formulation comprising a polynucleotide comprising SEQ ID NO:9 or SEQ ID NO:15, or a variant thereof having about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 89% or more, about 90% or more, about 91% or about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with SEQ ID NO:9 or SEQ ID NO:15. It can be a GX-188E formulation.

[0135] The GX-188-containing dosage form may be administered in a manner compatible with the dosage formulation and in a therapeutically effective and immunogenic amount. The dosage depends on the subject to be treated, including, for example, the ability of the individual's immune system to mount an immune response and the degree of immunity required. A suitable dosage range is approximately several hundred micrograms of active ingredient per vaccination, with a preferred range being about 1 μg to 20 mg, e.g., about 5 μg to 10 mg. In one embodiment, the dose may be in the range of about 0.1 mg to 20 mg, 0.1 mg to 10 mg, or 0.5 mg to 10 mg, particularly about 0.5 mg to 5 mg. In yet another aspect, the dosage may be in the range of 0.5 mg to 10 mg. In yet another aspect, the dosage may be in the range of 1 mg to 10 mg. In yet another aspect, the dosage may be in the range of 1 mg to 5 mg. In still other aspects, the dosage can be in the range of 0.5 mg to 20 mg, 1 mg to 20 mg, 0.5 mg to 5 mg, 1.5 mg to 10 mg, 2 mg to 5 mg, 2.5 mg to 5 mg, 3 mg to 5 mg, or 2 mg to 10 mg. In yet another embodiment, the dosage of GX-188 is about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, about 1.9 mg, about 2 mg, about 2.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, about 1.9 mg, about 2 mg, about 2.1 mg, about 2.2 mg, About 2.3 mg, about 2.4 mg, about 2.5 mg, about 2.6 mg, about 2.7 mg, about 2.8 mg, about 2.9 mg, about 3 mg, about 3.1 mg, about 3.2 mg, about 3.3 mg, about 3.4 mg, about 3.5 mg, about 3.6 mg, about 3.7 mg, about 3.8 mg, about 3.9 mg, about 4 mg, about 4.1 mg, about 4.2 mg, about 4.3 mg, about 4.4 mg, about 4.5 mg, about 4.6 mg, about 4.7 mg, about 4.8 mg, about 4.9 mg, or about 5 mg may be administered about weekly, every other week, once every three weeks, or once every four weeks.

[0136] The GX-188E dosage form may be administered at intervals such as once a week, twice a week, three times a week, four times a week, every week, every 10 days, once every other week, every 20 days, every three weeks, every four weeks, once a month, once every other month, or once every three months. Alternatively, the GX-188E dosage form may be administered at intervals of 5 days, 10 days, 15 days, 20 days, 30 days, or 40 days. In one embodiment, the GX-188E dosage form may be administered at week 1, week 2, or week 4.

[0137] The composition of the long-acting recombinant IL-7 fusion protein may be administered in a dosage form separate from the anti-PD1 antibody composition and the HPV vaccine composition. The composition of the long-acting recombinant IL-7 fusion protein may be administered parenterally, for example, subcutaneously, intracutaneously, intradermally, subdermally, or intramuscularly, by injection. The long-acting recombinant IL-7 fusion protein may include the amino acid sequence of SEQ ID NO: 39, for example, may have the amino acid sequence of SEQ ID NO: 39. It means a composition or dosage form containing a variant thereof having about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 89% or more, about 90% or more, about 91% or about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity. This may be in the GX-I7 dosage form.

[0138] The formulation containing the long acting recombinant IL-7 fusion protein may be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount, preferably in the range of about 10 ug / kg to about 10,000 ug / kg, about 30 ug / kg to about 9,000 ug / kg, about 50 ug / kg to about 8,000 ug / kg, about 60 ug / kg to about 7,000 ug / kg, about 70 ug / kg to about 6,000 ug / kg, about 80 ug / kg to about 5,000 ug / kg, about 90 ug / kg to about 4,000 ug / kg, or about 100 ug / kg to about 3,000 ug / kg.

[0139] In yet another aspect, the dosage is about 100ug / kg, about 150ug / kg, about 200ug / kg, about 250ug / kg, about 300ug / kg, about 350ug / kg, about 400ug / kg, about 450ug / kg, about 500ug / kg, about 550ug / kg, about 600ug / kg, about 650ug / kg, about 700ug / kg, about 750ug / kg, about 800ug / kg, about 850ug / kg, about 900ug / kg, about 950ug / kg, about 1000ug / kg, about 1050ug / kg, about 1100ug / kg, about 1200ug / kg, about 1300ug / kg, about 1400ug / kg, about 1500ug / kg, about 1600ug / kg, about 1700ug / kg, about 1800ug / kg, about 1900ug / kg, about 2000ug / kg, about 2100ug / kg, about 2200ug / kg, about 2300ug / kg, about 2400ug / kg, about 2500ug / kg, about 2600ug / kg, about 2700ug / kg, about 2800ug / kg, about 2900ug / kg, about 3000ug / kg, about 3100ug / kg, about 3200ug / kg, about 3300ug / kg, about 3400ug / kg, about 3500ug / kg, about 3600ug / kg, about 3700ug / kg, about 3800ug / kg, about 3900ug / kg, about 4000ug / kg, about 4000ug / kg, about 4500ug / kg, about or about 2000ug / kg.

[0140] The long-acting recombinant IL-7 fusion protein dosage form may be administered approximately every week, once every two weeks, once every three weeks, or once every four weeks. The long-acting recombinant IL-7 fusion protein dosage form may be administered at intervals such as once a week, twice a week, three times a week, four times a week, every week, every 10 days, once every two weeks, every 20 days, every three weeks, every four weeks, once a month, once every two months, or once every three months. Alternatively, the long-acting recombinant IL-7 fusion protein dosage form may be administered at intervals of 5 days, 10 days, 15 days, 20 days, 30 days, or 40 days. In one embodiment, the long-acting recombinant IL-7 fusion protein dosage form may be administered in the first, second, or fourth week. In one embodiment, the long-acting recombinant IL-7 fusion protein dosage form may be administered once in the second week.

[0141] The pharmaceutical composition for treating head and neck cancer according to the present invention can be administered at an appropriate time before cancer resection, including the anti-PD1 antibody composition, the human papilloma virus (HPV) vaccine composition, and the persistent recombinant IL-7 fusion protein composition. For example, the pharmaceutical composition can be initially administered about 20 weeks, about 19 weeks, about 18 weeks, about 17 weeks, about 16 weeks, about 15 weeks, about 14 weeks, about 13 weeks, about 12 weeks, about 11 weeks, about 10 weeks, about 9 weeks, about 8 weeks, about 7 weeks, about 6 weeks, or about 5 weeks before cancer resection.

[0142] In one embodiment, the pharmaceutical composition for treating head and neck cancer may include pembrolizumab as the anti-PD1 antibody, GX-188E as the human papilloma virus (HPV) vaccine, and GX-I7 as the long-acting recombinant IL-7 fusion protein. In a preferred embodiment, pembrolizumab may be administered twice, GX-188E may be administered three times, and GX-I7 may be administered once. For example, pembrolizumab may be administered intravenously (IV) at 200 mg in the first and fourth weeks, GX-188E may be administered intramuscularly (IM) at 2 mg in the first, second, and fourth weeks, and GX-I7 may be administered intramuscularly (IM) at up to 1,200 μg / kg in the second week.

[0143] In the present invention, the pharmaceutical composition for treating head and neck cancer containing pembrolizumab is characterized in that it is administered to patients who are able to undergo cancer resection.

[0144] In the present invention, the pharmaceutical composition for treating head and neck cancer containing pembrolizumab can be administered before cancer resection surgery.

[0145] In the present invention, the pharmaceutical composition for treating head and neck cancer containing pembrolizumab is characterized in that it is administered to patients who have not previously received other anti-cancer treatments.

[0146] In another embodiment, the pharmaceutical composition for treating head and neck cancer may include nivolumab as the anti-PD1 antibody, GX-188E as the human papilloma virus (HPV) vaccine, and GX-I7 as the persistent recombinant IL-7 fusion protein. In a preferred embodiment, nivolumab may be administered at two-week intervals, GX-188E may be administered three or more times, and GX-I7 may be administered one or more times. For example, nivolumab may be administered at 240 mg every two weeks, GX-188E may be administered at 2 mg at weeks 1, 2, 4, 7, 10, 13, 19, and 25, and GX-I7 may be administered at up to 1,200 μg / kg at weeks 2, 10, and 18.

[0147] In addition, the pharmaceutical composition for treating head and neck cancer according to the present invention is an anti-PD1 antibody composition, a human papillomavirus (HPV) vaccine composition and a persistent recombinant IL-7 fusion protein composition, and can be administered to patients with recurrent and / or metastatic head and neck squamous cell carcinoma at an appropriate time point as a combination therapy.

[0148] In the present invention, the dose and administration interval of each drug can be adjusted according to the condition or drug response level of the patient. For example, the dose and administration interval of GX-I7 can be adjusted according to the condition or drug response level of the patient.

[0149] The anti-PD1 antibody composition, human papillomavirus (HPV) vaccine composition and persistent recombinant IL-7 fusion protein composition may be administered in other administration modes than those mentioned above.

[0150] Additional dosage forms adapted for other modes of administration include suppositories and possibly oral, intramuscular, nasal, buccal, sublingual, intraperitoneal, intravaginal, anal, epidural, spinal and intracranial dosage forms. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10% (w / w), preferably 1% to 2% (w / w). Oral dosage forms include commonly used excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions may be in the form of solutions, suspensions, tablets, pills, capsules, sustained release dosage forms or powders, and may contain 10% to 95% (w / w), preferably 25% to 70% (w / w), of the active ingredient.

[0151] In one embodiment, the head and neck cancer may be HPV-positive head and neck cancer. In another embodiment, the head and neck cancer may be head and neck squamous cell carcinoma. In another embodiment, the head and neck cancer may be oropharyngeal cancer. In another embodiment, the head and neck cancer may be metastatic, recurrent and / or advanced head and neck cancer. In another embodiment, the head and neck cancer may be locally advanced. For example, the head and neck cancer may be resectable locally advanced.

[0152] In another aspect, the present invention provides the use of a combination of an anti-PD1 antibody, a human papillomavirus (HPV) vaccine and a long-acting recombinant IL-7 fusion protein for the treatment of head and neck cancer.

[0153] In yet another aspect, the present invention provides a use of a combination of an anti-PD1 antibody, a human papilloma virus (HPV) vaccine and a long-acting recombinant IL-7 fusion protein for the manufacture of a medicament for the treatment of head and neck cancer.

[0154] In yet another aspect, the present invention provides a method for treating head and neck cancer, comprising administering to a subject in need thereof an anti-PD1 antibody, a human papilloma virus (HPV) vaccine, and a persistent recombinant IL-7 fusion protein.

[0155] In the present invention, the anti-PD1 antibody, human papilloma virus (HPV) vaccine, and long-acting recombinant IL-7 fusion protein can be formulated separately and administered simultaneously, separately, or sequentially.

[0156] In the present invention, the anti-PD1 antibody, human papilloma virus (HPV) vaccine and long-acting recombinant IL-7 fusion protein can each be administered once or multiple times before cancer resection.

[0157] The efficacy of the combination treatment with the combination therapy of the present invention can be measured by various endpoints commonly used to evaluate cancer treatments, including, but not limited to, tumor regression, tumor weight or size shrinkage, time to disease progression, overall survival, progression-free survival, overall response rate, duration of response, inhibition of metastatic spread without tumor regression, and PET / CT imaging.

[0158] The terms "combination," "therapeutic combination," "pharmaceutical combination," and "conjugate" refer to a non-fixed-dose combination, optionally packaged with instructions for administration of the combination, where the individual therapeutic agents, anti-PD1 antibody, human papilloma virus (HPV) vaccine, and long-acting recombinant IL-7 fusion protein, may be administered simultaneously, or separately within a time interval that allows the therapeutic agents to exert a synergistic effect.

[0159] The term "concurrent" administration means that the individual therapeutic agents, the anti-PD1 antibody, the human papilloma virus (HPV) vaccine and the persistent recombinant IL-7 fusion protein, are administered to a patient in a single operation, e.g., each of the anti-PD1 antibody, the human papilloma virus (HPV) vaccine and the persistent recombinant IL-7 fusion protein is administered independently at substantially the same time or separately within a time interval that allows the anti-PD1 antibody, the human papilloma virus (HPV) vaccine and the persistent recombinant IL-7 fusion protein to exert a synergistic therapeutic effect.

[0160] The term "separate" administration means that each of the anti-PD1 antibody, human papilloma virus (HPV) vaccine and persistent recombinant IL-7 fusion protein is administered to a patient simultaneously, substantially contemporaneously, or sequentially in any order from a non-fixed dose dosage form. There may or may not be a specified time interval for administration of each of the anti-PD1 antibody, human papilloma virus (HPV) vaccine and persistent recombinant IL-7 fusion protein.

[0161] The term "sequential" administration means that each of the anti-PD1 antibody, human papilloma virus (HPV) vaccine and long-acting recombinant IL-7 fusion protein is administered to a patient in separate operations from a non-fixed (separate) dosage form.

[0162] The beneficial effect of two therapeutic agents which produces an effect greater than the simple additive effect of each therapeutic agent administered alone in a single patient can be calculated using any suitable method known in the relevant art, such as, for example, the Sigmoid-Emax equation (Holford and Scheiner, Clin. Pharmacokinet., 1981, 6:429-453), the equation of Loewe additivity (Loewe and Muischenk, Arch. Exp. Pathol. Pharmacol., 1926, 114:313-326), the mean effect equation (Chou and Talalay, Adv. Enzyme Regul., 1984, 22:27-55), and the Bliss Independence method, or equivalent methods known in the art. Applying each equation to experimental data and generating corresponding graphs can help predict the effects of drug combinations as additive, within the biologically relevant additive range, less than additive, or greater than additive. kit

[0163] The present invention can also provide a kit for treating head and neck cancer, comprising an anti-PD1 antibody, a human papillomavirus (HPV) vaccine, and a persistent recombinant IL-7 fusion protein.

[0164] The kit may contain the anti-PD1 antibody, the human papilloma virus (HPV) vaccine, and the long-acting recombinant IL-7 fusion protein, each formulated separately.

[0165] The kit may include recommended dosages of the anti-PD1 antibody, human papilloma virus (HPV) vaccine, and long-acting recombinant IL-7 fusion protein, together with instructions indicating the recommended order of administration and / or the recommended dosages. EXAMPLES

[0166] Working Example The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention. 1. Target disease and experimental design

[0167] The study aimed to explore the safety and efficacy of combination therapy with GX-188E, GX-I7 and pembrolizumab in patients with resectable locally advanced HNSCC who were HPV-16-positive and / or HPV-18-positive.

[0168] Sample size was based on an exact single-stage phase II trial design, with major pathological response (MPR) as the outcome variable. Alpha=0.05 Beta=0.20 H0=MPR≦5% H1=MPR≧35%

[0169] Based on the above statistical variables and hypotheses, a clinical trial was conducted with 11 patients as test subjects.

[0170] The study subjects were LA HNSCC patients aged 19 years or older who were confirmed by histological analysis, including HPV-positive patients (wherein HPV positivity was confirmed by p16 IHC and HPV-16+ and / or HPV-18+ confirmed by nucleic acid testing).The study subjects were those who had no history of previous anticancer treatment, had at least one measurable lesion according to RECIST v1.1 criteria, had an ECOG (Eastern Cooperative Oncology Group) activity status of 0-1, had a life expectancy of 6 months or more, had normal organ function, and agreed to provide an adequate level of fresh tumor tissue for biomarker analysis, including PD-L1 analysis staining, so that it could be available or stored. 2. Combination drug therapy

[0171] Dosing was performed according to the schedule shown in Figure 1. Pembrolizumab 200 mg IV + GX-188E 2 mg IM + GX-I7 1200 μg / kg were administered during the indicated time periods. Pembrolizumab

[0172] Pembrolizumab, used in the present invention as an example of an anti-PD1 antibody, is a potent humanized immunoglobulin G4 (IgG4) monoclonal antibody (mAb) that binds to the programmed cell death-1 (PD-1) receptor with high specificity and inhibits the interaction of PD-1 with programmed cell death ligand-1 (PD-L1) and programmed cell death ligand-2 (PD-L2). Non-clinical in vitro data shows that pembrolizumab has high affinity and potent receptor blocking activity for PD-1. Pembrolizumab has an acceptable non-clinical safety profile and is in clinical development as an intravenous (IV) immunotherapy agent for advanced malignant tumors. KEYTRUDA TM (Pembrolizumab) is approved to treat many indications. Please refer to the product information leaflet for more information on specific indications.

[0173] Pembrolizumab is provided as an infusion solution at a dose strength of 100 mg / vial. Pembrolizumab was administered intravenously (IV) at 200 mg on weeks 1 and 4. GX-188E

[0174] GX-188E, used in the present invention as an example of a human papillomavirus (HPV) vaccine, is a DNA plasmid (SEQ ID NO: 15) encoding a fusion protein containing the E6 and E7 proteins of HPV-16 and HPV-18 and human Fms-like tyrosine kinase 3 ligand (hFLT-3L).

[0175] GX-188E was administered intramuscularly (IM) at 2 mg on weeks 1, 2, and 4. GX-188E was administered intramuscularly in the deltoid or femoral muscles on each scheduled vaccination day. GX-188E was administered at 1 mg in both muscle sites for a total vaccination of 2 mg per day. GX-188E vaccination sites alternated between deltoid and femoral muscles whenever possible, but specific sites were selected between the two sites when necessary, in which case the vaccination sites were recorded separately. GX-I7

[0176] GX-I7, used in the present invention as an example of a long-acting recombinant IL-7 fusion protein, is a protein produced by inserting a gene expressing rhIL-7-hyFc into a eukaryotic expression vector. GX-I7 has a molecular weight of 104 kDa and is composed of 400 amino acids, of which 155 amino acids are derived from IL-7, 30 amino acids are derived from the IgD hinge, 8 amino acids are derived from the CH2 domain of IgD, and 207 amino acids are derived from the IgG4 region (SEQ ID NO: 39).

[0177] GX-I7 was administered intramuscularly (IM) at 1200 μg / kg in the second week. 3. Cancer resection and post-operative treatment

[0178] After completion of the above-mentioned combination therapy, cancer resection was performed 2 to 8 weeks later.

[0179] After surgery, patients were treated with standard cisplatin-based surgery followed by post-operative concurrent chemoradiation therapy (PO-CCRTx) based on pathological findings including high-risk factors (e.g., extracapsular extension (ECE+) or resectional margin involvement (RM+)) or at the physician's discretion. After completion of PO-CCRTx, patients were treated with postoperative adjuvant chemotherapy as deemed appropriate based on the results of surgery and the physician's discretion, and were followed up for 5 years (tumor evaluation). 4.Efficacy evaluation (1) Pathological reactions

[0180] Eleven patients achieved a pathological complete response (pCR) or major pathological response (MPR), defined as no residual disease and 10% or less residual disease, respectively.

[0181] As a result of confirming the pathological response, pathological complete response (pCR) was achieved in 4 of the 11 patients (36.3%), and major pathological response (MPR) was confirmed in the remaining 7 of the 11 patients (63.6%), indicating that the combination drug therapy of the present invention had a remarkable effect in the test group (see Figures 2 and 4). (2) Radiological reactions

[0182] In addition, radiological responses of 11 patients were analyzed using images taken before combination drug administration and before anticancer resection surgery, and follow-up images taken after surgery. Response assessment was performed according to RECIST 1.1.

[0183] Radiographic response was confirmed, and tumor regression of 30% or more was confirmed in 7 of 11 patients (see Figures 3 and 4). 5. Safety Evaluation

[0184] The study subjects were assessed for safety. Safety was assessed by recording, reporting, and analyzing the results of laboratory and physical examinations, taking into account the subjects' underlying medical conditions, abnormal reactions, and vital signs. Any abnormal reactions (SAEs) experienced by the study subjects, such as drug toxicity, were evaluated comprehensively. The researchers rated the severity of the SAEs based on the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE v4·03).

[0185] The main adverse events occurring in the 11 study subjects included fever, elevated bilirubin levels, elevated creatinine levels, hyperglycemia, and elevated alanine transferase levels; all patients recovered and were not associated with delays in surgery (data not shown).

[0186] As described above, certain parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for treating head and neck cancer, comprising an anti-programmed cell death 1 (anti-PD1 antibody), a human papillomavirus (HPV) vaccine and a long-acting recombinant interleukin-7 (IL-7) fusion protein.

2. 2. The pharmaceutical composition of claim 1, wherein the anti-PD1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, pidilizumab, and combinations thereof.

3. 2. The pharmaceutical composition of claim 1, wherein the HPV vaccine comprises polynucleotides encoding HPV-16 and HPV-18 antigens.

4. 4. The pharmaceutical composition of claim 3, wherein the HPV vaccine is an isolated DNA comprising a polynucleotide encoding the antigen.

5. 4. The pharmaceutical composition of claim 3, wherein the HPV vaccine comprises a polynucleotide of SEQ ID NO: 15 or a functional variant having 85% or greater sequence identity to SEQ ID NO:

15.

6. The pharmaceutical composition of claim 3, wherein the HPV vaccine further comprises a nucleic acid sequence encoding a heterologous polypeptide.

7. The pharmaceutical composition of claim 6 , wherein the heterologous polypeptide comprises Fms-like tyrosine kinase 3 ligand ("FLT3L") or a portion thereof.

8. The pharmaceutical composition of claim 3 , wherein the HPV vaccine further comprises a nucleotide sequence encoding a signal peptide.

9. 9. The pharmaceutical composition of claim 8, wherein the signal peptide is selected from the signal peptide of tissue plasminogen activator (tPA), the signal peptide of herpes simplex virus glycoprotein D (HSV gD), the signal peptide of growth hormone, and any combination thereof.

10. The persistent recombinant IL-7 fusion protein comprises: (a) IL-7 or a modified IL-7 having the following formula (I): A-IL-7 Chemical formula (I) where A is an oligopeptide consisting of 1 to 10 amino acid residues; and (b) a domain that extends the half-life of the IL-7 or the modified IL-7.

11. The pharmaceutical composition of claim 10, wherein the IL-7 has an amino acid sequence selected from the group consisting of SEQ ID NO:16 to SEQ ID NO:

21.

12. 11. The pharmaceutical composition of claim 10, wherein A is an oligopeptide consisting of amino acid residues selected from the group consisting of methionine, glycine, or a combination thereof.

13. The pharmaceutical composition of claim 10 , wherein the domain comprises an Fc region of a modified immunoglobulin.

14. The pharmaceutical composition of claim 13, wherein the persistent recombinant IL-7 fusion protein comprises the amino acid sequence of SEQ ID NO:39 or a functional variant having 85% or more sequence identity with SEQ ID NO:

39.

15. The pharmaceutical composition of claim 1, wherein the anti-PD-1 antibody is pembrolizumab or nivolumab, and the single administration dose is 50 mg to 500 mg.

16. The pharmaceutical composition according to claim 1, wherein the anti-PD-1 antibody is for intravenous injection.

17. 2. The pharmaceutical composition of claim 1, wherein the single administration dose of the HPV vaccine is 0.5 mg to 5 mg.

18. The pharmaceutical composition of claim 1 , wherein the HPV vaccine is for intramuscular injection.

19. The pharmaceutical composition according to claim 1, wherein the single administration dose of the long-acting recombinant IL-7 fusion protein is 60 μg / kg to 5,000 μg / kg.

20. The pharmaceutical composition according to claim 1, wherein the long-acting recombinant IL-7 fusion protein is for intramuscular injection.

21. The pharmaceutical composition according to claim 1, wherein the anti-PD1 antibody, the HPV vaccine, and the persistent recombinant IL-7 fusion protein are each administered once or multiple times.

22. The pharmaceutical composition of claim 1, wherein the anti-PD-1 antibody, the HPV vaccine, and the persistent recombinant IL-7 fusion protein are administered simultaneously, separately, or sequentially.

23. The pharmaceutical composition according to claim 1, wherein the anti-PD1 antibody is pembrolizumab, the pembrolizumab is administered twice, the HPV vaccine is administered three times, and the long-acting recombinant IL-7 fusion protein is administered once.

24. The pharmaceutical composition according to claim 1, wherein the anti-PD1 antibody is nivolumab, the nivolumab is administered at two-week intervals, the HPV vaccine is administered eight times, and the long-acting recombinant IL-7 fusion protein is administered three times.

25. The pharmaceutical composition according to claim 1 , wherein the pharmaceutical composition is administered before cancer resection.

26. The pharmaceutical composition of claim 1 , wherein the head and neck cancer is HPV positive.

27. The pharmaceutical composition of claim 1 , wherein the head and neck cancer is head and neck squamous cell carcinoma.

28. The pharmaceutical composition of claim 1 , wherein the head and neck cancer is oropharyngeal cancer.

29. 2. The pharmaceutical composition of claim 1, wherein the head and neck cancer is metastatic, recurrent and / or advanced.

30. The pharmaceutical composition of claim 1 , wherein the head and neck cancer is locally advanced and resectable.

31. A kit for treating head and neck cancer, comprising an anti-PD-1 antibody, an HPV vaccine and a long-acting recombinant IL-7 fusion protein.

32. Use of a combination of an anti-PD1 antibody, an HPV vaccine and a long-acting recombinant IL-7 fusion protein in the manufacture of a medicament for the treatment of head and neck cancer.

33. A combination of an anti-PD1 antibody, an HPV vaccine and a long-acting recombinant IL-7 fusion protein for use in the treatment of head and neck cancer.

34. A method for treating head and neck cancer, comprising administering to a subject in need thereof an anti-PD1 antibody, an HPV vaccine, and a persistent recombinant IL-7 fusion protein.

35. The method of claim 34, wherein the anti-PD1 antibody, the HPV vaccine and the long-acting recombinant IL-7 fusion protein are administered simultaneously, separately, or sequentially.

36. The method of claim 34, wherein the anti-PD1 antibody, the HPV vaccine and the persistent recombinant IL-7 fusion protein are administered prior to cancer resection.

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