Vaccine composition and its use

Abstract

The present invention relates, in general terms, to a vaccine composition and its use in the treatment of cancer such as melanoma in a subject. The vaccine composition contains one or more nucleic acids encoding a plurality of tumor antigen T cell epitopes, the T cell epitopes being GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3), and is administered to the subject in combination with (a) a programmed cell death 1 (PD-1) inhibitor such as nivolumab; and (b) a cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor such as ipilimumab. One or more nucleic acids may encode antibodies having heavy and light chains into which the T cell epitopes are inserted or substituted.

Description

[Technical Field]

[0001] This invention generally relates to vaccine compositions and their use in the treatment of cancer in subjects. [Background technology]

[0002] Immunotherapy with checkpoint inhibitors (CPIs), including programmed cell death 1 (PD-1) inhibitors, administered alone or in combination with cytotoxic T lymphocyte antigen 4 (CTLA-4) inhibitors, has revolutionized first-line treatment outcomes for patients with unresectable or metastatic advanced melanoma (J. Larkin et al. Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma N Engl J Med 2019; 381:1535-1546; Jedd D. Wolchok et al. Long-Term Outcomes With Nivolumab Plus Ipilimumab or Nivolumab Alone Versus Ipilimumab in Patients With Advanced Melanoma Journal of Clinical Oncology 2022 40:2, 127-137). In the pivotal trial Checkmate-067, the overall response rate (ORR) and survival surrogate endpoints such as progression-free survival (PFS) at 3 years were 58% and 34%, respectively (Errico, A. CheckMate 067 - frontline nivolumab improves PFS alone or in combination with ipilimumab. Nat Rev Clin Oncol 12, 435 (2015)).In actual clinical practice, the objective response rate (ORR) achieved with the combination of nivolumab and ipilimumab is close to 50% (P. Serra-Bellver et al. Real-world outcomes with ipilimumab and nivolumab in advanced melanoma: a multicentre retrospective study European Journal of Cancer 176 (2022) 121e132), while some practitioners report an ORR of 28% in actual clinical practice (Parakh S, Randhawa M, Nguyen B, Warburton L, Hussain MA, Cebon J, Millward M, Yip D, Ali S. Real-world efficacy and toxicity of combined nivolumab and ipilimumab in patients with metastatic melanoma. Asia Pac J Clin Oncol. 2019 Feb;15(1):26-30). In recent years, nucleic acid vaccines in melanoma have shown efficacy in delaying the time to recurrence in combination with pembrolizumab in unresectable disease stages (LJ Albrecht et al., The Latest Option: Nivolumab and Relatlimab in Advanced Melanoma. Curr Oncol Rep 25, 647-657 (2023)). However, in unresectable disease stages, nucleic acid vaccines have not shown a significant effect in reducing tumor burden. [Overview of the project]

[0003] In one aspect, the present invention relates to a composition used for the treatment of cancer in a subject, wherein the composition is (i) A vaccine composition containing one or more nucleic acids encoding multiple tumor antigen T cell epitopes (wherein the T cell epitopes are GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3)), wherein the method comprises the composition, (a) Programmed cell death 1 (PD-1) inhibitors; and, (b) cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor; This includes administering the substance to the target in combination with other substances; (ii) A method comprising a programmed cell death 1 (PD-1) inhibitor, wherein the PD-1 inhibitor is (a) A vaccine composition containing one or more nucleic acids encoding multiple tumor antigen T cell epitopes (wherein the T cell epitopes are GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3)); and (b) cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor; This includes administering it to the target in combination with; or (iii) A cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor is included in the method, and the CTLA-4 inhibitor is used (a) A vaccine composition containing one or more nucleic acids encoding multiple tumor antigen T cell epitopes (wherein the T cell epitopes are GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3)); and (b) Programmed cell death 1 (PD-1) inhibitors; This includes administering the substance to the target in combination with other substances; A composition is provided.

[0004] In another aspect, the present invention provides a composition for use in the treatment of cancer in a subject, the composition comprising one or more nucleic acids encoding a plurality of tumor antigen T cell epitopes, wherein said T cell epitopes have the sequences GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2) and WNRQLYPEWTEAQRLD (SEQ ID NO: 3), and said method comprises administering said composition to the (a) a programmed cell death 1 (PD-1) inhibitor; and, (b) a cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor; subject in combination therewith.

[0005] In another aspect, the present invention provides a composition for use in the treatment of cancer in a subject, which composition comprises a programmed cell death 1 (PD-1) inhibitor, wherein said method comprises administering the PD-1 inhibitor to the (a) a vaccine composition comprising one or more nucleic acids encoding a plurality of tumor antigen T cell epitopes, wherein the T cell epitopes have the sequences GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2) and WNRQLYPEWTEAQRLD (SEQ ID NO: 3); and, (b) a cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor; subject in combination therewith.

[0006] In another aspect, the present invention provides a composition for use in the treatment of cancer in a subject, which composition comprises a cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor, wherein said method comprises administering the CTLA-4 inhibitor to the (a) a vaccine composition comprising one or more nucleic acids encoding a plurality of tumor antigen T cell epitopes, wherein the T cell epitopes have the sequences GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2) and WNRQLYPEWTEAQRLD (SEQ ID NO: 3); and, (b) a programmed cell death 1 (PD-1) inhibitor; subject in combination therewith.

[0007] In another aspect, the present invention relates to a method for treating cancer in a subject, comprising administering a vaccine composition to the subject, wherein the vaccine contains one or more nucleic acids encoding a plurality of tumor antigen T cell epitopes, the T cell epitopes being GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3), and the vaccine composition is (a) Programmed cell death 1 (PD-1) inhibitors; and, (b) cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor; This provides a treatment method that involves administering the treatment to the target in combination with the other treatments.

[0008] This invention is at least in part based on the inventors' astonishing discovery that melanoma patients who received a nucleic acid (referred to as "SCIB1") encoding three T-cell epitopes GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in combination with ipilimumab and nivolumab unexpectedly showed an overall response rate of 81.8%. This is significantly higher than the approximately 50% overall response rate previously reported for ipilimumab and nivolumab combinations without nucleic acids.

[0009] In another aspect, the present invention relates to a vaccine composition for use in the treatment of unresectable stage III or stage IV melanoma in a subject, comprising a DNA molecule encoding an antibody in which multiple tumor antigen T cell epitopes are inserted or substituted, The antibody has a T cell epitope of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) inserted or substituted in CDR-H1, a T cell epitope of SVYDFFVWL (SEQ ID NO: 2) inserted or substituted in CDR-H2, and a T cell epitope of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) inserted or substituted in CDR-L1. The first dose of the vaccine composition is administered to the subject in week 0, followed by doses in weeks 4, 7, 13, and 25, and then every 12 weeks thereafter. The vaccine composition is administered intramuscularly in doses of approximately 8 mg of total DNA per dose. The vaccine composition is administered in combination with nivolumab and ipilimumab. Nivolumab is administered to patients in the first phase at a dose of approximately 1 mg / kg every 3 weeks for 4 doses, followed by the second phase. (i) A dose of approximately 240 mg is administered every two weeks, with the first dose of the second stage being given approximately three weeks after the fourth dose of the first stage; or, (ii) A dose of approximately 480 mg is administered every four weeks, with the first dose of the second stage being given approximately six weeks after the fourth dose of the first stage; Ipilimumab will be administered to the subjects at a dose of approximately 3 mg / kg every 3 weeks for 4 doses, and each dose of ipilimumab will be administered on the same day as the first stage of nivolumab administration, The first doses of ipilimumab and nivolumab should be administered approximately one week after the first dose of the vaccine composition. A vaccine composition is provided.

[0010] In another aspect, the present invention relates to a method for treating unresectable stage III or stage IV melanoma in a subject, comprising administering to the subject a vaccine composition containing a DNA molecule encoding an antibody in which multiple tumor antigen T cell epitopes are inserted or substituted. The antibody has a T cell epitope of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) inserted or substituted in CDR-H1, a T cell epitope of SVYDFFVWL (SEQ ID NO: 2) inserted or substituted in CDR-H2, and a T cell epitope of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) inserted or substituted in CDR-L1. The first dose of the vaccine composition is administered to the subject in week 0, followed by doses in weeks 4, 7, 13, and 25, and then every 12 weeks thereafter. The vaccine composition is administered intramuscularly in doses of approximately 8 mg of total DNA per dose. The vaccine composition is administered in combination with nivolumab and ipilimumab. Nivolumab is administered to patients in the first phase at a dose of approximately 1 mg / kg every 3 weeks for 4 doses, followed by the second phase. (i) A dose of approximately 240 mg is administered every two weeks, with the first dose of the second stage being given approximately three weeks after the fourth dose of the first stage; or, (ii) A dose of approximately 480 mg is administered every four weeks, with the first dose of the second stage being given approximately six weeks after the fourth dose of the first stage; Ipilimumab will be administered to the subjects at a dose of approximately 3 mg / kg every 3 weeks for 4 doses, and each dose of ipilimumab will be administered on the same day as the first stage of nivolumab administration, The first doses of ipilimumab and nivolumab should be administered approximately one week after the first dose of the vaccine composition. We provide treatment methods.

[0011] These and other aspects of the present invention are described in further detail below. The features of each aspect of the present invention apply to each of the other aspects with necessary modifications, unless otherwise required by the context. [Modes for carrying out the invention]

[0012] Detailed description of the invention As described above, the present invention is partly based on positive interim data obtained from Stage 1 of a Phase 2 trial called "SCOPE," which is investigating "SCIB1" in combination with the checkpoint inhibitors (CPIs) ipilimumab and nivolumab in advanced melanoma. Initial data from 11 patients showed an objective response rate (ORR) of 82% to treatment. The Phase 2 SCOPE trial was designed to determine whether SCIB1 in combination with CPIs could improve the ORR in patients with unresectable metastatic melanoma. This concept is whether the vaccine induces a new immune response or boosts an existing immune response, which is subsequently protected by the CPI in the tumor environment. In Stage 1 of the SCOPE trial, patients received SCIB1 via a needle-free device in combination with the most effective treatment currently available, namely the CPIs nivolumab and ipilimumab. The first indicator in the SCOPE trial was that a response was achieved in more than 8 out of 15 patients, suggesting that SCIB1, in combination with dual CPI therapy, can significantly improve current treatment outcomes for these patients. Sixteen patients with stage IV metastatic disease received this combination. To date, 11 of these patients have reached 13 weeks and have been evaluated by radiographic imaging, with 9 already showing an objective response, an ORR of 82%, and no increase in toxicity. At this point, the tumor volume reduction was 31%–94%. Four patients who reached 25 weeks of imaging evaluation and two patients who reached 37 weeks of evaluation showed total tumor volume reductions of 69%–94% and 87%–94%, respectively. This is significantly better than the 50% ORR reported in patients receiving this dual CPI therapy (without SCIB1) alone in clinical practice, with a progression-free survival of 11.5 months.

[0013] Unless otherwise specifically defined, all technical and scientific terms in this specification shall be construed as having the same meaning as those commonly understood by those skilled in the art (for example, in immunology, molecular biology, cancer therapy, and / or biochemistry).

[0014] Unless otherwise indicated, the techniques used in this invention are standard procedures and are well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), FM Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley - Interscience (1988, including all revisions to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JE Coligan et al. This is described and explained in publications such as *Current Protocols in Immunology*, John Wiley & Sons (including all revisions to date).

[0015] In this specification, the term “about” includes ±10%, more preferably ±5%, of the specified value, unless otherwise indicated.

[0016] Throughout the specification, the terms “includes” or “contains” are understood to indicate the inclusion of the elements, integers, or steps or groups of elements, integers, or steps mentioned, but not to exclude other elements, integers, or steps or groups of elements, integers, or steps.

[0017] In one aspect, the present invention relates to a vaccine composition for use in the treatment of cancer in a subject. The term "vaccine composition" refers to an immune response, preferably CD8 + and / or CD4 + This refers to a composition that can promote the T cell response.

[0018] The vaccine composition used in the present invention contains one or more nucleic acids encoding the following tumor antigen T cell epitopes: GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3). The term "T cell epitope" refers to a T cell (preferably CD8) whose amino acid sequence, when presented by the major histocompatibility complex (MHC), is associated with T cells. + or CD4 + This refers to a peptide containing an amino acid sequence that can be recognized by T cells. In this specification, the term "T cell epitope" encompasses peptides that may contain multiple different short amino acid sequences recognized by T cells within their entire peptide sequence. However, the term "T cell epitope" does not include large polypeptide sequences, such as sequences with more than 40 amino acids. For example, the "T cell epitope" of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) contains three identified epitope sequences within its entire peptide sequence, as shown in the table below.

[0019] [Table A]

[0020] The references in the table above are listed below: Tsai V, Southwood S, Sidney J, Sakaguchi K, Kawakami Y, Appella E, Sette A, Celis E. Identification of subdominant CTL epitopes of the GP100 melanoma-associated tumor antigen by primary in vitro immunization with peptide-pulsed dendritic cells. J Immunol. 1997 Feb 15;158(4):1796-802. PMID: 9029118. Lapointe R, Royal RE, Reeves ME, Altomare I, Robbins PF, Hwu P. Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100. J Immunol. 2001 Oct 15;167(8):4758-64. doi: 10.4049 / jimmunol.167.8.4758. PMID: 11591807. Kobayashi H, Lu J, Celis E. Identification of helper T-cell epitopes that encompass or lie proximal to cytotoxic T-cell epitopes in the gp100 melanoma tumor antigen. Cancer Res. 2001 Oct 15;61(20):7577-84. PMID: 11606397. Parkhurst MR, Fitzgerald EB, Southwood S, Sette A, Rosenberg SA, Kawakami Y. Identification of a shared HLA-A*0201-restricted T-cell epitope from the melanoma antigen tyrosinase-related protein 2 (TRP2). Cancer Res. 1998 Nov 1;58(21):4895-901. PMID: 9809996. Touloukian CE, Leitner WW, Topalian SL, Li YF, Robbins PF, Rosenberg SA, Restifo NP. Identification of a MHC class II-restricted human gp100 epitope using DR4-IE transgenic mice. J Immunol. 2000 Apr 1;164(7):3535-42. doi: 10.4049 / jimmunol.164.7.3535. PMID: 10725708; PMCID: PMC2241739.

[0021] Nucleic acids are administered to a target as a vaccine composition that is taken up by cells, and as a result, T cell epitopes are expressed in the target and presented to T cells. T cell epitopes may be encoded by multiple different nucleic acids, for example, a total of two or three nucleic acid molecules, preferably all encoded by a single nucleic acid molecule. Therefore, the term “nucleic acid” in this specification refers to a nucleic acid molecule having a polynucleotide sequence. For example, a vaccine composition may contain a single nucleic acid molecule containing a polynucleotide sequence encoding three T cell epitopes.

[0022] The nucleic acid may be RNA such as DNA, cDNA, or mRNA, obtained, for example, by cloning or produced entirely or partially by chemosynthesis. Preferably, the nucleic acid is DNA. For therapeutic use, the nucleic acid is preferably in a form expressible in the target to be treated. The nucleic acid may be recombinant or may be provided as an isolate in an isolated and / or purified form. It may not contain, or substantially does not contain, nucleic acids adjacent to the gene encoding the T cell epitope, except possibly one or more regulatory sequences for expression. If the nucleic acid contains RNA, the sequences shown in this specification should be interpreted as RNA equivalents having U instead of T.

[0023] Suitable nucleic acids can be readily prepared by those skilled in the art, based on available nucleic acid sequences and clones, using, for example, the information and references contained herein and techniques known in the art (see, for example, Sambrook, Fritsch and Maniatis, “Molecular Cloning”, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989 and Ausubel et al, Short Protocols in Molecular Biology, John Wiley and Sons, 1992). These techniques include (i) using polymerase chain reaction (PCR) to amplify a sample of such nucleic acid, for example, from a genomic source, (ii) chemical synthesis, or (iii) preparation of a cDNA sequence. The polypeptide-encoding DNA may be generated and used in a manner preferred by those skilled in the art, including by using encoding DNA to identify preferred restriction enzyme recognition sites on both sides of the portion to be expressed and cleaving the portion from the DNA. This portion may then be operably ligated to a suitable promoter in a standard, commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA using suitable PCR primers. Modifications to the sequence can be made, for example, using site-directed mutagenesis to result in the expression of the modified peptide or to take into account codon preference in the host cell used to express the nucleic acid.

[0024] To obtain expression of a nucleic acid sequence, the sequence can be incorporated into a vector having one or more control sequences (e.g., promoters) operably linked to a sequence encoding a T cell epitope to control its expression. The vector may also contain other sequences, such as promoters or enhancers, to activate the expression of the nucleic acid sequence. The nucleic acid may encode a secretion signal (also referred to in this specification as a “leader sequence”) so that a T cell epitope produced by a host cell is secreted from the cell. A preferred leader sequence includes the amino acid sequence shown in SEQ ID NO: 16. The promoter may be nonspecific (a promoter that is active regardless of the cellular environment) and / or a constitutive promoter that does not require a specific cell type or tissue type to activate the expression of a T cell epitope or a polypeptide containing a T cell epitope. The promoter may be a CMV-IE promoter, preferably a promoter containing the nucleotide sequence shown in SEQ ID NO: 15 or a functional variant thereof.

[0025] If desired, the polypeptide encoded by the nucleic acid can be obtained by transforming the vector into a host cell in which the vector is functional, culturing the host cell to produce the polypeptide, and recovering the polypeptide from the host cell or surrounding medium. Prokaryotic and eukaryotic cells are used in the art for this purpose, and these include strains of Escherichia coli, yeast, insect cells, and animal cells, eukaryotic cells such as COS, CHO cells, Bowes melanoma, and other suitable human cells. If the present invention is a nucleic acid encoding the heavy and light chains of an antibody, each nucleic acid may be present in the same expression vector activated by the same or different promoters, or in separate expression vectors. Preferably, the heavy and light chain sequences of the antibody are encoded in the same nucleic acid (e.g., a vector) and activated by separate promoters.

[0026] Nucleic acids are used to stimulate an immune response to a T cell epitope in patients, such as mammals including humans. Helper and / or cytotoxic T cell responses may also be stimulated. The avidity of the T cell response to a particular epitope may be higher than that obtained by immunization with the same epitope as a simple peptide, or by immunization with the same epitope encoded in an antigen as a peptide or nucleic acid. Nucleic acids may be administered as combination therapy, i.e., as a light chain encoding nucleic acid and a heavy chain encoding nucleic acid. Nucleic acids may be administered intravenously, intradermally, intramuscularly, orally, or by other routes. Intradermal or intramuscular administration is particularly preferred because these tissues contain dendritic cells. Preferably, the vaccine composition containing nucleic acids is administered intramuscularly. Preferably, the vaccine composition is administered by needle-free injection.

[0027] The nucleic acid may be a vector. The vector may be used for the expression of the T cell epitope encoded by the nucleic acid. The nucleic acid may be a DNA molecule. For example, the nucleic acid may be a DNA vector. Preferably, the nucleic acid is a DNA plasmid. Preferably, a single DNA plasmid encodes all of the T cell epitopes. Alternatively, the nucleic acid may be a doggybone vector.

[0028] Nucleic acids or vectors may be used in combination with pharmaceutically acceptable carriers. Such carriers may include, but are not limited to, physiological saline, buffered saline, dextrose, liposomes, water, glycerin, ethanol, and combinations thereof.

[0029] Nucleic acids and / or vectors useful in the present invention may be formulated as pharmaceutical compositions. These compositions may contain, in addition to one or more of the above substances, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials must be non-toxic and must not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials may depend on the route of administration, e.g., intradermal, oral, intravenous, cutaneous or subcutaneous, intranasal, intramuscular, or intraperitoneal routes. The formulation is preferably a solution of DNA mixed with nucleic acids or GET peptides as a stable, dry powder deposited on the surface of microscopic gold particles, suitable for injection by gene gun. The formulation may be suitable for intradermal or intramuscular administration using electroporation. The formulation may be suitable for administration using needle-free injection.

[0030] The compositions described in this specification are preferably administered to an individual in a “therapeutic dose,” which is an amount sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate and process of administration, depends on the nature and severity of what is being treated. The determination of the treatment prescription, e.g., dosage, is within the purview of the practicing physician and other physicians, and usually takes into account the disorder to be treated, the individual patient’s condition, the site of delivery, the method of administration, and other factors known to healthcare professionals. The nucleic acids of this invention relate specifically to the treatment of existing cancers and the prevention of cancer recurrence after initial treatment or surgery. Examples of the above techniques and protocols are described in Remington’s Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980.

[0031] Preferably, the nucleic acid stimulates helper and / or cytotoxic T cells that can kill tumors when administered to humans in an effective dose. The optimal dose may be determined by a physician based on several parameters, including, for example, age, sex, weight, severity of the condition being treated, the active ingredient to be administered, and the route of administration. For example, a dose of 1 to 10,000 ug of DNA may be sufficient to stimulate the helper and cytotoxic T cell response.

[0032] The vaccine compositions described in this specification are administered in combination with programmed cell death 1 (PD-1) inhibitors and cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitors. These may be administered simultaneously or sequentially, depending on the condition to be treated. They may also be administered in combination with other treatments. Other cancer treatments include other monoclonal antibodies, other chemotherapeutic agents, other radiotherapy, or other immunotherapy known in the art.

[0033] The dosage of nucleic acid depends on the characteristics of the drug used, such as its binding activity and in vivo plasma half-life, the concentration of nucleic acid in the formulation, the route of administration, the site and rate of administration, the patient's clinical tolerance, and the pathological condition the patient is suffering from, and this is well within the scope of the physician's skill. Different dosages may be used in a series of sequential inoculations; the practitioner may administer the initial dose and then boost with a relatively lower dose of nucleic acid.

[0034] The vaccine composition may be administered in a total nucleic acid amount of 1 to 20 mg, 5 to 15 mg, or 6 to 10 mg per dose. The vaccine composition may also be administered to a subject in a total nucleic acid amount of approximately 8 mg per dose. Preferably, the vaccine composition is administered to a subject in a total nucleic acid amount of 8 mg per dose. The term "dose" refers to the total amount of the active ingredient administered to the subject, which may be administered, for example, in multiple separate injections. For example, a dose of 8 mg may be administered as four separate 2 mg injections to different injection sites.

[0035] The timing of administering the vaccine composition may be determined by those skilled in the art. Preferably, the first dose of the vaccine composition is administered to the subject in week 0, and subsequent doses are administered between weeks 4, 7, 13, and 25, and thereafter every 12 weeks. In this specification, "week 0" refers to the week in which the subject receives the first dose. Similarly, "week 4" refers to a point in time approximately 4 weeks after week 0, and so on. More preferably, the first dose of the vaccine composition is administered to the subject on day 1, and subsequent doses are administered between days 29, 50, 92, and 176, and thereafter every 84 days. In this context, "day 1" refers to the day in which the subject receives the first dose, and "day 29" refers to a point in time 28 days (i.e., 4 weeks) later, and so on.

[0036] Preferably, the vaccine composition is administered to the subjects according to the administration schedule described for "SCIB1" in the clinical trial protocol shown in Example 3 ("SCOPE" clinical trial).

[0037] Nucleic acids may be introduced into host cells. The introduction may be generally referred to as "transformation" (especially in vitro), and any available technique may be used. For eukaryotic cells, preferred techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transformation using retroviruses or other viruses, such as baculoviruses in the case of vaccinia or insect cells. For bacterial cells, preferred techniques include calcium chloride transformation, electroporation, and bacteriophage-mediated transfection. Alternatively, direct injection of nucleic acids may be used.

[0038] Marker genes, such as antibiotic resistance or susceptibility genes, may be used to identify clones containing the target nucleic acid, as is well known in the art.

[0039] The introduction may be followed by inducing or enabling expression from nucleic acids, for example, by culturing host cells (which may actually include cells being transformed, but the cells are likely to be descendants of the transformed cells) so that a polypeptide (or peptide) encoding the gene is produced under conditions of gene expression. If the polypeptide is expressed ligated to a suitable signal leader peptide, it may be secreted from the cells into the culture medium. After production by expression, the polypeptide or peptide may be isolated and / or purified from the host cells and / or culture medium as necessary and subsequently used as desired in formulations of compositions which may include one or more additional components, such as a pharmaceutical composition containing one or more pharmaceutically acceptable excipients, vehicles or carriers (see, for example, below).

[0040] The pharmaceutical composition may contain, in addition to the active ingredient, pharmaceutically acceptable excipients, diluents, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials must be non-toxic and must not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials depends on the route of administration, which may be orally or, for example, by intradermal or intramuscular injection.

[0041] Injection is assumed to be the primary route of therapeutic administration of the composition, but delivery by catheter or other surgical tubing may also be used. Some preferred routes of administration include intravenous, subcutaneous, intraperitoneal, and intramuscular administration. Liquid formulations may be used after being reconstituted from powder formulations. A preferred route of administration is, for example, intramuscular administration by needle-free injection.

[0042] The nucleic acids, vectors, peptides, and / or vaccines of the present invention may be administered to a subject using needle-free injection. As is known to those skilled in the art, needle-free injectors (also known as "jet injectors") use a narrow, high-pressure liquid stream to penetrate the outermost layer of skin (stratum corneum) and deliver the composition to the underlying epidermis or dermis (i.e., intradermal injection), fat (i.e., subcutaneous injection), or muscle (i.e., intramuscular injection) tissues.

[0043] For intravenous injection or injection at the site of the disease, the active ingredient is in the form of a parenterally acceptable aqueous solution that does not contain pyrogens and has a suitable pH, isotonicity, and stability. Those skilled in the art can easily prepare a suitable solution using an isotonic vehicle such as sodium chloride injection, Ringer's injection, or Ringer's lactate injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0044] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain saline solution, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. If the formulation is liquid, it may be, for example, saline solution containing a non-phosphate buffer with a pH of 6.8 to 7.6, or a lyophilized powder.

[0045] The vaccine composition may contain nucleic acid at a concentration of approximately 4 mg / mL. The vaccine composition may also contain approximately 8.1 mM disodium hydrogen phosphate, approximately 1.5 mM potassium dihydrogen phosphate, approximately 2.7 mM potassium chloride, and approximately 137 mM sodium chloride, and the pH of the vaccine composition is approximately 7.4. The vaccine composition may also contain nucleic acid in Dulbecco's phosphate-buffered saline that does not contain calcium or magnesium.

[0046] Nucleic acids encoding T cell epitopes can be administered to patients using one of several methods known in the art. For example, nucleic acids can be delivered directly as "naked DNA." This approach is described, for example, in Wolff et al., Science 247: 1465-1468 (1990), and in U.S. Patents 5,580,859 and 5,589,466. Nucleic acids can also be administered using gene gun delivery, for example, as described in U.S. Patent 5,204,253. Particles containing only DNA may be administered. Alternatively, the DNA may be attached to particles such as gold particles. Generally, a plasmid for a vaccine or immunological composition may include DNA encoding an antigen (e.g., one or more neoantigens) operably linked to a regulatory sequence that controls the expression or expression and secretion of the antigen from a host cell, e.g., a mammalian cell; for example, from upstream to downstream, DNA of a promoter such as a mammalian virus promoter (e.g., a CMV promoter such as the hCMV or mCMV promoter, e.g., the earliest promoter or SV40 promoter—see the documentation cited or incorporated in the specification for useful promoters), DNA of a eukaryotic leader peptide for secretion (e.g., tissue plasminogen activator), DNA of a neoantigen, and DNA encoding a termination factor (e.g., 3'UTR transcription termination factor derived from a gene encoding bovine growth hormone or bGH polyA). The composition may contain two or more plasmids or vectors, each vector containing and expressing a different T cell epitope, preferably all epitopes being encoded by a single nucleic acid (e.g., a vector). See also U.S. Patent No. 5,849,303 (Wasmoen) and No. 5,811,104 (Dale). DNA or DNA plasmid formulations may be formulated with or within cationic lipids; see also U.S. Patent Application Publication 2003 / 0104008 (Loosmore) with respect to cationic lipids and adjuvants.Furthermore, the instructions in U.S. Patent No. 6,228,846 and No. 6,159,477 (Audonnet) can be relied upon as instructions for DNA plasmids that contain sequences encoding T cell epitopes and can be used to construct and use DNA plasmids that express them in vivo.

[0047] Nucleic acids can also be delivered by forming complexes with cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for example, in International Publication No. 96 / 18372; International Publication No. 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Patent No. 5,279,833; International Publication No. 91 / 06309; and Feigner et al, Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987). RNA encoding the target peptide (e.g., mRNA) can also be used for delivery (see, e.g., Kiken et al, 2011; Su et al, 2011; also see U.S. Patent No. 8278036; Halabi et al. J Clin Oncol (2003) 21: 1232-1237; Petsch et al, Nature Biotechnology 2012 Dec 7;30(12): 1210-6).

[0048] Nucleic acids may be administered as part of a viral vector, such as an adenovirus, lentivirus, or AAV vector, or other viral vectors known in the art.

[0049] The composition may be administered by microspheres, liposomes, other microparticle delivery systems, or sustained-release formulations, which are placed in specific tissues, including blood. Preferred examples of sustained-release carriers include shared articles, e.g., semipermeable polymer matrices in the form of suppositories or microcapsules. Embodiable or microencapsulated sustained-release matrices include polylactide (U.S. Patent No. 3,773,919; European Patent Application Publication No. 58481), a copolymer of L-glutamic acid and gamma-ethyl L-glutamate, and poly(2-hydroxyethyl methacrylate). Liposomes containing polypeptides are prepared by well-known methods: European Patent Application Publications No. 52522; No. 36676; No. 88046; No. 143949; No. 142541; JP A 83 11808; U.S. Patents No. 4,485,045 and No. 4,544,545. Typically, liposomes are small (approximately 200-800 Å) and single-lamellar, with a lipid content exceeding approximately 30 mol% cholesterol, the ratio of which is adjusted to achieve an optimal polypeptide leakage rate. The composition may be administered topically to the tumor site or other desired site, or it may be delivered in a manner that targets the tumor or other cells.

[0050] The present invention relates to the use of constructs in the form of plasmids, vectors, transcriptions, or expression cassettes containing the above-mentioned nucleic acids. The present invention provides recombinant host cells comprising one or more of the above-mentioned constructs. Systems for cloning and preparing suitable constructs in various different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Suitable vectors can be selected or constructed, containing appropriate regulatory sequences, including, as necessary, promoter sequences, termination factor sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences. The vector may, as necessary, be a plasmid, virus, e.g., a phage or phagemide. For example, numerous known techniques and protocols for manipulating nucleic acids in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins are detailed in Ausubel et al., 1992.

[0051] Nucleic acids may be introduced into host cells. The introduction may be carried out using any available technique. Preferred techniques for eukaryotic cells include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transformation using retroviruses or other viruses, such as vaccinia or, in the case of insect cells, baculoviruses. Preferred techniques for bacterial cells include calcium chloride transformation, electroporation, and bacteriophage-mediated transfection. Following introduction, expression from the nucleic acids may be induced or enabled, for example, by culturing the host cells under conditions that allow gene expression. In one embodiment, the nucleic acids of the present invention are integrated into the genome (e.g., chromosomes) of the host cell. Integration may be facilitated by the inclusion of sequences that promote genomic recombination, according to standard techniques. The present invention also provides a method comprising using the construct in an expression system to express the T cell epitope.

[0052] One or more nucleic acids in the vaccine composition may encode an antibody having heavy and light chains into which a T cell epitope is inserted or substituted. In this regard, the T cell epitope is heterologous to the antibody. The term “insertion or substitution” does not require a physical cloning step to replace the sequence. These terms are simply used to identify that the T cell epitope sequence is present in the antibody sequence in a heterologous manner. Such an antibody modified to contain a heterologous T cell epitope used to stimulate an immune response may be referred to in the art as an “immunobody.” Preferred antibodies are described in International Publication No. 2008 / 116937 (incorporated by reference in this specification). A preferred antibody that may be encoded by nucleic acids in the vaccine composition is the antibody referred to as “DCIB68” in International Publication No. 2008 / 116937.

[0053] The antibody has a heavy chain and a light chain, which preferably include a variable region and a constant region. Suitable antibody sequences and the structure and position of the immunoglobulin domain therein are well known in the art and may be determined by referring to http: / / www.imgt.org / . Throughout this specification, residue numbering refers to the standardized IMGT system for antibody sequence numbering, as disclosed in Lefranc et al., 2009. Other suitable numbering systems are known to those skilled in the art.

[0054] Antibodies may be monoclonal or polyclonal, and may be IgA, IgD, IgE, IgG, or IgM, but IgG is preferred. IgG antibodies may be human IgG1, IgG2, IgG3, or IgG4, or IgG subclasses such as mouse IgG1, IgG2a, IgG2b, or IgG3. Preferably, the IgG antibody is a human IgG1 antibody. Antibodies may have a constant region of a human antibody and a variable or hypervariable region of a mouse or human monoclonal antibody into which a T cell epitope is inserted. Variable regions other than the hypervariable region may be derived from the variable region of a human antibody. Such antibodies are called humanized antibodies. Methods for producing humanized antibodies are known in the art. Such methods are described, for example, in U.S. Patent No. 5,225,539 (Winter). Variable regions of antibodies outside the mouse hypervariable region may be derived from a mouse monoclonal antibody. In such cases, the entire variable region is derived from the mouse monoclonal antibody, and the antibody is called chimeric. Methods for producing chimeric antibodies are known in the art. Such methods include, for example, those described in the U.S. patents of Boss (Celltech) and Cabilly (Genentech). See also U.S. Patent No. 4,816,397 and No. 4,816,567, respectively.

[0055] Nucleic acids in a particular aspect of the present invention are such that the heavy chain, light chain, or antibody expressed therefrom has at least one heterologous T cell epitope, so that the heavy chain, light chain, or immunoglobulin molecule does not have to take its native configuration. The T cell epitope disrupts the expressed protein, which may result in, for example, the heavy chain or immunoglobulin molecule being unable to bind to an antigen, the heavy chain and light chain (if present) being unable to associate, or the heavy chain or immunoglobulin molecule being unable to be properly secreted. The disruption may also be a disruption of the tertiary structure of the immunoglobulin molecule, thereby preventing the formation of disulfide bonds.

[0056] T cell epitopes may be inserted into or substituted into the CDR region of the antibody. CDR1 and CDR2 form part of the antibody's beta-sheet structure and are partially embedded within the folded molecule. By altering their length, amino acid composition, or charge, this structure may be disrupted, preventing the folding and association of the heavy and light chains. CDRH3 is exposed on the surface of the immunoglobulin molecule and is therefore more susceptible to modification. In this invention, it is preferable that at least CDR1 and / or CDR2 are substituted with T cell epitopes. In fact, in certain embodiments, the loss of framework regions at CDRH junctions completely disrupts antibody folding, while the insertion of epitopes in these regions induces a good T cell response. Incorporation of epitopes into CDRH1 (amino acid length 5) or CDRH2 (amino acid length 17) causes sufficient disruption, allowing heavy chain secretion but resulting in a very small amount of intact antibody, even if the light chain sequence is the native sequence. This indicates that secondary structure is important for heavy and light chain pairing. The integration of the light chain epitope into CDRL1 results in low levels of light chain secretion, even if only a single epitope integration into the heavy chain CDRH3 is present.

[0057] The term "heterogeneous T cell epitope" is intended to mean a T cell epitope that is heterogeneous to the antibody. For example, a heterogeneous T cell epitope may not have been previously present in the antibody. A heterogeneous T cell epitope may be inserted in its entirety, or it may consist of the inserted amino acid sequence and the amino acids of an adjacent second portion. This is to ensure that the inserted epitope has a similar processing profile in the heterogeneous nucleic acid as the original antigen. One or more CTL / helper epitopes may be inserted within the same variable region.

[0058] T cell epitopes can be inserted at any location in the heavy chain or light chain. While each epitope is preferably inserted into the variable region of the heavy chain and / or light chain, the present invention also includes nucleic acids encoding heavy chains or antibodies in which the T cell epitope is inserted only into the constant region of the heavy chain and / or light chain, or into both the constant and variable regions. In the nucleic acids of the present invention, the sequence encoding the T cell epitope may be inserted (i.e., added) to the sequence encoding the heavy chain and / or light chain, or it may be substituted for the sequence encoding the heavy chain and / or light chain.

[0059] In the variable region, T cell epitopes may be inserted into or substituted for one or more CDRs of the heavy and / or light chain, i.e., L1, L2, L3, H1, H2, or H3. Of these, L1, H1, and H2 are currently preferred. In certain embodiments, T cell epitopes are inserted into or substituted for L1, H1, and / or H2 of the CDR. Preferably, the incorporated T cell epitopes are not of similar size and charge to the amino acids of the antibody's original CDR, so that the antibody does not take its native configuration, for example, not fold, and not secrete correctly. Alternatively, they may be inserted into or substituted for framework regions surrounding the CDR. Epitopes may also be inserted using complementary oligonucleotides encoding antigenic epitopes, which are annealed and cloned into specific sites of the antibody framework where the CDR (or other region) is replaced with a specific restriction enzyme site. The ability of recombinant antibodies to stimulate helper and cytotoxic T cell responses can be screened as illustrated in the specification.

[0060] In this invention, various combinations are possible. In a particular embodiment, one or more CD8 epitopes are inserted and / or substituted into the CDR H1 and / or H2 or non-CDR variable regions of the heavy chain or antibody. In addition, one or more CD4 epitopes may be inserted and / or substituted into the CDR L1 or non-CDR variable regions of the light chain or antibody. If multiple T cell epitopes are present, the T cell epitopes may be the same or different. Those skilled in the art will know, • CD8 epitope in CDR H1 and CD4 epitope in CDR L1; • CD8 epitope in CDR H2 and CD4 epitope in CDR L1; • CD8 epitopes in CDR H1 and CDR H2, and CD4 epitopes in CDR L1; • Two CD8 epitopes in CDR H1 and a CD4 epitope in CDR L1; • Two CD8 epitopes in CDR H2 and a CD4 epitope in CDR L1; etc. Understand that many combinations are possible, including [this].

[0061] The nucleic acids of the present invention can incorporate multiple T cell epitopes derived from a single target antigen capable of binding to the majority of MHC molecules of both class I and class II. In this way, a vaccine suitable for widespread mass vaccination may be prepared. Alternatively, the nucleic acids useful in the present invention can incorporate multiple T cell epitopes derived from multiple target antigens that bind to the most common class I and class II phenotypes. In this way, a vaccine capable of preventing the selection of antigen-loss variants may be prepared. The target antigens may be derived from a single pathogen or tumor type, or they may be selected to induce immune responses to various pathogens or cancers. Nucleic acids useful in the present invention targeting specific common HLA phenotypes may incorporate numerous T cell epitopes derived from a wide range of cancers and / or pathogens, providing a single vaccine that prevents disease.

[0062] The antibodies encoded by nucleic acids in the vaccine composition are (i) CDR-H1 has a T cell epitope of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1); (ii) CDR-H2 has the SVYDFFVWL (SEQ ID NO: 2) T cell epitope; and, (iii) T cell epitope of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-L1; It may include.

[0063] Preferably, the antibody is (i) T cell epitopes of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) in CDR-H1 and CDR-L3, respectively; (ii) CDR-H2 has the SVYDFFVWL (SEQ ID NO: 2) T cell epitope; and, (iii) T cell epitopes of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-H3 and CDR-L1 cells, respectively; Includes.

[0064] A preferred alternative antibody is, (i) CDR-H1 has a T cell epitope of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1); (ii) T cell epitope of SVYDFFVWL (SEQ ID NO: 2) on CDR-H2; (iii) T cell epitope of VPLDCVLYRYGSFSVTLDIVQG (SEQ ID NO: 4) on CDR-H3; (iii) T cell epitope of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-L1; (iv) T cell epitope ANCSVYDFFVWLHYYSVRDTLLGPGRPYR (SEQ ID NO: 5) in CDR-L2; and, (v) T cell epitope QCTEVRADTRPWSGPYILRNQDDRELWPRKFF (SEQ ID NO: 6) in CDR-L3; Includes.

[0065] The antibody may also include VH, which contains the amino acid sequence shown in SEQ ID NO: 7 or 9, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence shown in SEQ ID NO: 7 or 9, and VL, which contains the amino acid sequence shown in SEQ ID NO: 8 or 10, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence shown in SEQ ID NO: 8 or 10.

[0066] The antibody may also include a heavy chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence shown in SEQ ID NO: 11 or 13, and a light chain containing an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence shown in SEQ ID NO: 12 or 14.

[0067] Preferred antibodies encoded by nucleic acids in the vaccine composition include the antibody referred to as "DCIB68" in International Publication No. 2008 / 116937, and antibodies encoded by DNA vectors referred to as "SCIB1" and iSCIB1+ in International Publication No. 2022 / 043400. In this regard, all contents of International Publication No. 2008 / 116937 and International Publication No. 2022 / 043400 are incorporated into this specification by reference.

[0068] The antibody sequence described above may include a leader sequence (i.e., a "signal peptide" or "section signal") that promotes antibody secretion when one or more nucleic acids in the vaccine composition are expressed. The leader sequence may be located at the N-terminus of the antibody's heavy chain and / or the N-terminus of the light chain. Preferably, the leader sequence includes the amino acid sequence shown in SEQ ID NO: 16 or a functional variant thereof.

[0069] Preferably, the vaccine composition contains a single nucleic acid encoding multiple tumor antigen T cell epitopes. Preferably, the nucleic acid is a DNA vector. In this specification, the DNA vector may include a first nonspecific promoter sequence operably linked to a sequence encoding the heavy chain of the antibody, and a second nonspecific promoter operably linked to a sequence encoding the light chain of the antibody, and the T cell epitopes are inserted into or substituted in one or more CDRs of the antibody. The first and second promoter sequences may be the same or different. The heavy chain and light chain sequences may both include a leader sequence (secretion signal) and their respective N-terminuses. The arrangement of the sequences is shown in the feature maps of a representative vector "SCIB1" in Figure 1 and another representative vector "iSCIB1+" in Figure 3.

[0070] Preferred DNA sequences are those of "SCIB1" and "iSCIB1+" (also referred to as "SCIB1plus iV1" in this specification), as shown in Sequence ID No. 17 and 18, respectively. Therefore, the nucleic acid may be SCIB1 or iSCIB1+ as described in International Publication No. 2022 / 043400, or other preferred nucleic acids as described in International Publication No. 2022 / 043400.

[0071] The vaccine composition described in this specification is administered in combination with a programmed cell death 1 (PD-1) inhibitor and a cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor. The PD-1 inhibitor and the CTLA-4 inhibitor act as immune checkpoint inhibitors.

[0072] Immune checkpoints are crucial signaling pathways in the immune system that maintain self-tolerance and regulate the duration and magnitude of physiological immune responses. Under normal conditions, these pathways prevent excessive effector activity by T cells. Two important examples of these pathways are the cell surface receptors CTLA-4 and PD-1 (Teft, 2006; Keir, 2008). In some cases, tumors express or overexpress inhibitory immune checkpoint pathways as a primary immune evasion mechanism. Since many immune checkpoints are initiated by ligand-receptor interactions, these signals can be readily blocked by antibodies or modulated by recombinant ligand or receptor forms (Pardoll, 2012).

[0073] Immune checkpoint proteins are important targets for pharmacological blockade (Teft, 2006; Keir, 2008), and dramatic clinical responses have been observed after treatment with antibodies that block PD-1 and CTLA-4 (see, e.g., Brahmer, 2010; Robert, 2011; Topalian, 2012; Powles, 2014; Topalian, 2014; Brahmer, 2015; Le, 2015; Robert, 2015; Reck, 2016; Langer, 2017). Therefore, in representative embodiments, the present invention features a nucleic acid-based vaccine and a novel combination of one or more CTLA-4 and PD-1 inhibitors.

[0074] CTLA-4 inhibitors may also be anti-CTLA-4 antibodies. Anti-CTLA-4 antibodies may be, for example, ipilimumab or tremelimumab. Anti-CTLA-4 antibodies bind to CTLA-4 and block the interaction between CTLA-4 and its ligand, CD80 / CD86. Blocking CTLA-4 has been shown to increase T cell activation and proliferation, including the activation and proliferation of tumor-infiltrating T effector cells. Inhibition of CTLA-4 signaling may also reduce regulatory T cell function, which may contribute to an overall increase in T cell responsiveness, including anti-tumor immune responses. Ipilimumab is an IgG1κ immunoglobulin with a molecular weight of approximately 148 kDa. Ipilimumab (Yervoy®, Bristol-Meyers Squibb, New York, NY) is a recombinant human mAb produced from mammalian (Chinese hamster ovary) cell cultures.

[0075] CTLA-4 functions to regulate T cell activation in peripheral tissues by modulating early T cell activation and partially modulating programmed cell death 1 (PD-1) signaling function. The PD-1 receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed in several cell types, including Tregs, activated B cells, and natural killer (NK) cells, and in vivo is primarily expressed in pre-activated T cells, binding to two ligands, PD-L1 and PD-L2. PD-L1 and PD-L2, endogenous ligands of PD1, are expressed in non-hematopoietic cells, including activated immune cells and tumor cells. In this specification, PD-1 means human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence is shown in GENBANK accession number U64863. Programmed cell death ligand 1 (PD-L1) is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), and when bound to PD-1, it downregulates T cell activation and cytokine secretion. PD-L3, as described in this specification, includes human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-LI sequence is shown in GENBANK accession number Q9NZQ7. Tumors have been shown to evade immune surveillance by expressing PD-L1 / L2 and to suppress tumor-infiltrating lymphocytes via PD-1 / PD-L1,2 interactions (Dong et al. Nat. Med. 8:793-800. 2002).

[0076] PD-1 inhibitors may also be anti-PD1 antibodies. Anti-PD-1 antibodies may include, for example, nivolumab, pembrolizumab, or pidilizumab. Nivolumab (Opdivo®, Bristol-Myers Squibb Company, NY) is a human immunoglobulin G4 (IgG4) mAb that binds to the programmed cell death 1 (PD-1) receptor, blocking interactions with PD-L1 and programmed cell death ligand 2 (PD-L2), and reversing the inhibition of PD-1 pathway-mediated immune responses, including anti-tumor immune responses. Binding of the PD-1 receptor expressed on T cells to PD-L1 and PD-L2 inhibits T cell proliferation and cytokine secretion. Upregulation of PD-1 ligands occurs in some tumors, and signaling through this pathway may contribute to the inhibition of tumor-mediated active T cell immune surveillance. The antibodies of the present invention include, but are not limited to, the anti-PD-1 and anti-PD-Ll mAbs disclosed in U.S. Patent No. 8,008,449 and No. 7,943,743, respectively. Other anti-PD-1 mAbs are described, for example, in U.S. Patent No. 7,488,802 and No. 8,168,757, and anti-PD-Ll mAbs are described, for example, in U.S. Patent No. 7,635,757 and No. 8,217,149 and U.S. Patent Application Publication No. 2009 / 0317368. U.S. Patent No. 8,008,449 describes seven anti-PD-1 HuMAb: 1708.2D3, 4M, 5C4 (also referred to in this specification as nivolumab or BMS-936558), 4A11, 7D3, and 5F4.

[0077] Nivolumab (Opdivo®) is approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) in combination with ipilimumab (Yervoy®) for the treatment of melanoma, and vice versa. The Opdivo® EMA product information (incorporated by reference in this specification), provided on the EMA website: https: / / www.ema.europa.eu / documents / product-information / opdivo-epar-product-information_en.pdf, indicates that the recommended dosage is as follows: For adults and adolescents aged 12 years and older weighing at least 50 kg, the recommended dosage and administration is the first four doses, administered intravenously at a dose of 1 mg / kg nivolumab in combination with 3 mg / kg ipilimumab every three weeks, followed by a second stage of nivolumab monotherapy, administered intravenously at a dose of 240 mg every two weeks or 480 mg every four weeks, as shown in Table 2 (see Sections 5.1 and 5.2). Regarding the monotherapy stage, the initial dose of nivolumab is: - If using 240 mg every two weeks, 3 weeks after the last dose of the nivolumab and ipilimumab combination; or, - When using 480 mg every 4 weeks, 6 weeks after the last dose of the nivolumab and ipilimumab combination, It must be administered to them.

[0078] For adolescents aged 12 years and older weighing less than 50 kg, the recommended dosage and administration are as follows: the first four doses consist of intravenous administration of 1 mg / kg nivolumab in combination with 3 mg / kg ipilimumab every three weeks, followed by a second stage of nivolumab monotherapy, which is either intravenous administration of 3 mg / kg every two weeks or 6 mg / kg every four weeks, as shown in Table 2 (see Sections 5.1 and 5.2). Regarding the monotherapy stage, the initial dose of nivolumab is: - When using 3 mg / kg every two weeks, three weeks after the last dose of the nivolumab and ipilimumab combination; or, - When using 6 mg / kg every 4 weeks, 6 weeks after the last dose of the nivolumab and ipilimumab combination, It must be administered to them. The following table, regenerated from the Opdivo® EMA product information referenced above, provides recommended doses and infusion times for intravenous administration of nivolumab in combination with ipilimumab for the treatment of melanoma.

[0079] [Table B]

[0080] The recommended dosing schedule for the above combination of nivolumab and ipilimumab is also shown in the Yervoy® EMA product information (incorporated herein by reference) provided on the EMA website: https: / / www.ema.europa.eu / en / documents / product-information / yervoy-epar-product-information_en.pdf.

[0081] In the present invention, when the PD-1 inhibitor is nivolumab and the CTLA-4 inhibitor is ipilimumab, these antibodies may be administered to subjects according to the recommended administration schedule (for combination therapy of melanoma) in the Yervoy® EMA product information (also known as the "product label") or the Opdivo® EMA product information. Preferably, ipilimumab and nivolumab are administered to subjects according to the administration schedule described in the clinical trial protocol ("SCOPE" clinical trial) shown in Example 3.

[0082] Preferably, the CTLA-4 inhibitor (e.g., ipilimumab) is administered on the same day as the PD-1 inhibitor (e.g., nivolumab). Therefore, the CTLA-4 inhibitor (e.g., ipilimumab) and the PD-1 inhibitor (e.g., nivolumab) may be administered in separate compositions or in the same composition. The first dose of the CTLA-4 inhibitor and the PD-1 inhibitor is preferably administered to the target about one week after the first dose of the vaccine composition.

[0083] The vaccine composition described in this specification, in combination with a CTLA-4 inhibitor and a PD-1 inhibitor, is particularly suitable for the treatment of melanoma (especially advanced melanoma), as shown in the results of the intermediate clinical trials described in Example 4. Specifically, the melanoma may be stage III or stage IV melanoma. The melanoma may be metastatic melanoma. In this regard, "metastatic" melanoma refers to melanoma in which primary melanoma cells have spread from the original tumor site to organs such as lymph nodes, lungs, liver, brain, and bone. The melanoma may be unresectable (i.e., cannot be removed surgically). For example, the melanoma may be unresectable stage III or stage IV melanoma. Preferably, the subject has not received systemic treatment for advanced melanoma.

[0084] Preferably, the melanoma is an unresectable stage III or stage IV melanoma, and the subjects are: (a) Being 18 years of age or older; (b) Never having received systemic treatment for an advanced disease; (c) No brain metastases, ocular melanoma, mucosal melanoma, or autoimmune disease.

[0085] Preferably, the subject is also, (d) HLA serotype class I is HLA-A * It is 02 (i.e., HLA-A2); (e) HLA serotype class II is selected from HLA-DR4, HLA-DR7, HLA-DR53, and HLA-DQ6.

[0086] Prior art references herein are incorporated to the maximum extent permitted by law. Other features or advantages of the present invention are also evident from the following drawings, examples, numbered embodiments and claims.

[0087] Array description [Table C]

[0088] Typical antibody sequences [ka]

[0089] [ka]

[0090] Typical whole DNA vector sequences [ka]

[0091] [ka]

[0092] [ka]

[0093] [ka]

[0094] [ka]

[0095] The present invention will be further described below with reference to the following embodiments and drawings. [Brief explanation of the drawing]

[0096] [Figure 1] SCIB1 plasmid map [Figure 2] Schematic diagram of modified IgG encoded by SCIB1 [Figure 3] iSCSIB1+ plasmid map [Figure 4] Schematic diagram of modified gG coded by SCIB1 and iSCSIB1+ [Figure 5] Simon Phase 1 and Simon Phase 2 study schemes for SCIB1 in patients with unresectable advanced melanoma treated with either nivolumab and ipilimumab combination or pembrolizumab. [Figure 6] Waterfall plot showing the maximum change from baseline in the total reference diameter of the target lesion in patients treated with SCIB1 in addition to nivolumab and ipilimumab. Data are shown for all patients who assessed the target lesion at baseline and underwent initial imaging by week 13. Bars in the negative quadrant indicate tumor regression, and bars in the positive quadrant indicate tumor size increase. Horizontal lines indicate a 30% reduction or 20% increase in tumor volume at the target lesion, which are response or progression thresholds. Change in tumor volume was defined as the percentage decrease (%) in the total reference diameter of the target lesion from baseline to the minimum value, as measured by September 2023. [Figure 7] Spider plots illustrating the persistence of tumor response in patients over time. Data are shown for all patients who underwent target lesion assessment at baseline and initial imaging by week 13. Change in tumor volume was defined as the percentage reduction in the total reference diameter of the target lesion from baseline to minimum, as measured by September 2023. [Figure 8] C57Bl / 6 mice were transplanted with B16F1 tumor cells, followed by administration of checkpoint inhibitor antibodies, and tumor growth and survival were monitored (n≧5). [Figure 9] (A) HLA-DR4 transgenic mice were transplanted with B16 DR4 tumor cells, or (B) transgenic HLA-DP4 mice were transplanted with B16 F1 HHDII / DP4 tumor cells, followed by treatment with checkpoint inhibitor antibodies, and tumor growth and survival were monitored (n≧5). [Figure 10]Ex vivo immune response (indicating epitope CDR location) measured by IFNγELISpot assay in C57Bl / 6 mice (A), HLA-DR4 transgenic mice (B), or HHDII / DR1 transgenic mice (C) after immunization with iSCIB1+. Data represent the mean of at least two independent experiments with n=3. Error bars indicate the standard deviation. [Figure 11] (A) C57Bl / 6 mice were transplanted with B16F1 tumor cells, or (B) transgenic HHDII / DP4 mice were transplanted with B16 F1 HHDII / DP4 tumor cells, followed by immunization with iSCIB1+, and tumor growth and survival were monitored (n=10). [Figure 12] Splenocytes from C57Bl / 6 mice immunized with iSCIb1+ alone or iSCIb1+ plus dual checkpoint therapy (anti-PD1 and anti-CTLA4 antibodies) were grown in vitro, and peptide-specific responses to sequences inserted into (A) CDR L3 (TRP-2 aa60-91) and (B) CDR H3 (gp100 aa471-192) were analyzed. Data are the mean of n=3, and error bars indicate the standard deviation. [Figure 13] C57Bl / 6 mice were transplanted with b16F1 tumor cells, and then immunized with either iSCIB1+ alone, iSCIB1+ in combination with dual checkpoint therapy (CPI therapy (anti-PD1 and anti-CTLA4 antibodies)), or dual checkpoint therapy alone. Tumor growth and survival were then monitored (n=10). [Examples]

[0097] Methods and materials (preclinical study) peptide Peptides were selected based on publicly available sequences, binding predictions from the IEDB database (http: / / www.iedb.org / ), and binding predictions from SYFPEITHI (http: / / www.syfpeithi.de). The peptides (see table below) were synthesized to a purity of over 90% (Genscript), dispensed into single-use vials, lyophilized at -80°C for storage, and reconstituted in PBS on the day of use.

[0098] [Table D]

[0099] Animals and cell systems We used 8-16 week old C57Bl / 6J (Charles River), HLA-DR4 (Model # 4149, Taconic), HHDII / DP4 (EM:02221, European Mouse Mutant Archive), or HHDII / DR1 (Pasteur Institute) mice. All work was carried out with ethical approval under a project license approved by the Ministry of the Interior. For all studies, mice were randomized to different groups, and this was not blinded to the principal investigator.

[0100] Cells containing B16 melanoma expressing the relevant MHCI and II alleles (as described in Pudney et al 2010; Brentville et al 2016; Brentville et al 2019) were cultured in RPMI medium 1640 containing 10% FCS and L-glutamine (2 mmol / l) with appropriate antibiotics to maintain the plasmid. Mouse splenocytes were cultured in 10% FBS (Sigma), 2 mM glutamine, 20 mM HEPES buffer, 100 units / ml penicillin, 100 mg / ml streptomycin and 10 -5 Cells were cultured in RPMI-1640 containing M-2-mercaptoethanol. The cell lines used were mycoplasma-free, certified by the supplier (STR profiling), and used after no more than 10 passages.

[0101] Immunization protocol Animal experiments were performed with ethical approval under a project license approved by the Ministry of the Interior. 8-16 week-old C57Bl / 6 (Charles River, UK), HLA-DR4 transgenic (model #4149 Taconic, USA), HHDII / DP4 (HLA-A2.1 + / + HLADP4 + / + hCD4 + / + , EM:02221, European Mouse Mutant Archive) and HLA-A2 / DR1 transgenic (HHDII / DR1, Pasteur Institute) were used. For all studies, mice were randomized into different groups, which was not blinded to the responsible physician. DNA (1 μg / mouse) was coated onto 1.0 μm gold particles (BioRad, Hemel Hempstead, UK) according to the manufacturer's instructions, and administered intradermally via a gene gun (BioRad, USA). Mice were immunized on Day 1, Day 8 and Day 15, and spleens were harvested on Day 21, and splenocytes were collected for analysis (unless otherwise indicated). For tumor challenge experiments, mice were subcutaneously inoculated with 2.5×10 in the right flank on Day 1 4 B16F1 cells, 2.5×10 4 B16 DR4 cells or 1×10 5 B16 HHDII / DP4 tumor cells, followed by intradermal immunization with DNA bullets via gene gun on Day 4, Day 11 and Day 18. Combination studies with anti-PD-1 antibody clone RMPI-14 (250 μg / administration) and anti-CTLA-4 clone 9D9 (100 μg / administration) were performed by administering DNA and antibody intraperitoneally on Day 8, Day 15 and Day 22, via gene gun or in saline respectively. Tumor growth was monitored at 3-4 day intervals by measuring tumor size with calipers. Mice were humanely euthanized when the tumor diameter reached 15 mm.

[0102] Splenocyte culture Splenocytes harvested ex vivo were adjusted to 5×10 610 pieces / well -5 The peptides were placed in 2 ml of complete medium supplemented with M 2-mercaptoethanol and 20 IU of recombinant mouse IL-2 (Miltenyi Biotech). The peptides were added to the pool at a final concentration of 10 μg / ml for each peptide. The cultures were incubated at 37°C / 5% CO2 for up to 8 days.

[0103] Elispot assay The Elispot assay was performed using mouse IFNγ capture and detection reagents according to the manufacturer's instructions (Mabtech). Briefly, IFNγ-specific antibodies were coated onto 96-well Immobilin-P plates. Synthetic peptides (Genescript) diluted to 10 μg / ml were added to RPMI medium 1640 (GIBCO / BRL) containing 10% FCS (Sigma), 2 mM L-glutamine (Sigma), and sodium bicarbonate, further buffered with 20 mM HEPES (Sigma), and containing 50 μM 2-mercaptoethanol (Thermofisher), with 5 × 10⁶ molecules per well. 5 Individual splenocytes were directly added to the wells of a plate in a quadruple-column configuration ex vivo, and the plates were incubated at 37°C / 5% CO2 for 40 hours. Cells in culture medium alone were added as a negative control, and 5 μg / ml of lipopolysaccharide (LPS, Sigma) was used as a positive control.

[0104] For assays using in vitro cultured splenocytes, count the cells and measure 3 × 10⁻⁶ 4 pieces / well ~ 1 x 10 5 Seeds were seeded at a density of 1 / well. Peptides were added as described above, and 5 μg / ml anti-mouse CD3 antibody (clone 145-2C11) was used as a positive control. Plates were incubated for 20 hours. After incubation, IFNγ captured by biotinylation-specific IFNγ antibody was detected and colored with streptavidin alkaline phosphatase and a chromogenic substrate. Spots were analyzed and counted using an automated plate reader (Cellular Technologies Ltd).

[0105] statistics Comparative analysis of ELISpot results was performed by applying paired or unpaired ANOVA or Student's t-test as appropriate, and calculating p-values ​​accordingly. Survival analysis comparisons were performed by applying the log-rank test using Graphpad Prism software.

[0106] References Pudney, VA, et al., DNA vaccination with T-cell epitopes encoded within Ab molecules induces high-avidity anti-tumor CD8+ T cells. Eur J Immunol, 2010. 40(3): p. 899-910 Brentville, VA, et al., Citrullinated Vimentin Presented on MHC-II in Tumor Cells Is a Target for CD4+ T-Cell-Mediated Antitumor Immunity. Cancer Res, 2016. 76(3): p. 548-60 Brentville, VA, et al., T cell repertoire to citrullinated self-peptides in healthy humans is not confined to the HLA-DR SE alleles; Targeting of citrullinated self-peptides presented by HLA-DP4 for tumour therapy. Oncoimmunology, 2019. 8(5): p. e1576490.

[0107] Example 1 SCIB1 The representative nucleic acid used in this invention is SCIB1. The DNA sequence of SCIB1 (SEQ ID NO: 17) is shown below (including the amino acid sequences of the light and heavy chains encoding the expression product):

[0108] [ka]

[0109] [ka]

[0110] [ka]

[0111] [ka]

[0112] Figure 1 shows the plasmid map of SCIB1, and Figure 2 shows a schematic diagram of the modified IgG encoded by SCIB1.

[0113] The amino acid sequence of the modified IgG1 light chain (SEQ ID NO: 12) encoded by pSCIB1, which has a leader peptide (SEQ ID NO: 16) at its N-terminus, is shown below: Sequence ID 30: [ka]

[0114] The melanoma T cell epitopes contained in the light chain sequence are color-coded as follows: [ka]

[0115] The amino acid sequence (SEQ ID NO: 11) of a modified IgG1 heavy chain with a leader peptide (SEQ ID NO: 16) at its N-terminus, encoded by pSCIB1, is shown below: Sequence ID 31: [ka]

[0116] The melanoma T cell epitopes contained in the heavy chain sequence are color-coded as follows: [ka]

[0117] Example 2: iSCSIB1+ Another representative nucleic acid used in this invention is iSCSIB1+. The DNA sequence of iSCSIB1+ (SEQ ID NO: 18) is shown below (including the amino acid sequences of the light and heavy chains encoding the expression product):

[0118] [ka]

[0119] [ka]

[0120] [ka]

[0121] Figure 3 shows the plasmid map of iSCSIB1+, and Figure 4 shows schematic diagrams of the modified IgG encoded by SCIB1 and iSCSIB1+.

[0122] [Table 1]

[0123] Example 3: Details of the Clinical Study table of contents List of abbreviations Definition of Terms 1. Protocol Overview 2. Introduction and Background Explanation 2.1 Introduction 2.2 Findings obtained from non-clinical and clinical studies 2.2.1 Non-clinical research 2.2.2 Clinical research 2.3 Logical basis for dose selection 2.4 Known and potential benefits and risks to patients 3. Research Objectives and Endpoints 3.1 Primary purpose and endpoint 3.1.1 Run-in subcohort 3.1.2 Main research 3.2 Secondary purposes and endpoints 3.2.1 Main research 3.3 Experimental Objectives and Endpoints 4.Research design 4.1 Overall Research Design 4.2 Number of patients and countries planned 4.3 Research Period 4.4. Patient allocation 4.5 Blinding and Randomization 4.6. Validity of Study Design and Selection of Patient Population 4.7 Periodic review of safety and efficacy 4.8 Criteria for research completion 4.9 Procedure and Evaluation Schedule 4.10 Post-treatment follow-up 5. Patient selection and withdrawal 5.1. Inclusion criteria 5.2 Exclusion criteria 5.3 Treatment discontinuation 5.4 Withdrawal Criteria 5.4.1 Replacement patients 5.4.2 Rescreening 5.5 Research completed 6. Treatment 6.1 Surveyed Pharmaceutical Products 6.1.1 Non-surveyed drug product: Combination therapy of nivolumab and ipilimumab 6.1.2 Non-surveyed medical product: Pembrolizumab 6.2 Preparation, handling, storage, management and disposal of research pharmaceutical products 6.2.1. SCIB1 6.2.2 Checkpoint Inhibitors 6.2.3 Drug Management 6.3 SCIB1 Administration Procedure 6.4. Checkpoint Inhibitors: Use and Precautions 6.5 Treatment Schedule 6.6. Dosage adjustment, modification, and delay 6.6.1 Infusion reactions 6.6.2 Dosage adjustment and modification 6.6.3. Delay and discontinuation of checkpoint inhibitor therapy 6.7 Preceding and concomitant use of medicines and treatments 6.7.1 Contraindicated Medications and Therapies 6.7.2 Permitted Medications, Prior Medications and Adjunctive Therapies 6.8. Monitoring of patient compliance 7. Evaluation of effectiveness 7.1 Tumor imaging and RECIST evaluation 7.2 Progression-free survival and overall survival 8. Pharmacodynamic evaluation and biomarker evaluation 8.1 Blood sample 8.1.1 Handling and transportation of samples 8.1.2 Evaluation and Analysis of Study-Specific Blood Samples 8.2 Tumor Samples 8.2.1 Evaluation and Analysis of Study-Specific Tumor Samples 9. Safety Assessment 9.1. Adverse Events 9.1.1 Definition of Adverse Events 9.1.2 Recording and reporting of adverse events 9.1.3 Severity of adverse events 9.1.4 Relationship between adverse events and research treatments or devices 9.1.5 Protocol deviations due to adverse events 9.2. Serious Adverse Events 9.2.1 Definition of serious adverse events 9.2.2 Predictability 9.2.3 Reporting of serious adverse events 9.3 Pregnancy 9.4 Dosage and medical errors related to research and treatment 9.5 Clinical and Laboratory Evaluation and Testing 9.5.1. Serological Chemistry, Hematology, and Urinalysis 9.5.2 Other Clinical Laboratory Tests 9.5.3 Medical History 9.5.4. Physical Examination 9.5.5 Injection site reactions 9.5.6 Vital Signs 9.5.7 Electrocardiogram (ECG) Test 9.5.8 Ophthalmic Examination 9.5.9 Performance Status Evaluation 10. Statistics 10.1 Consideration of Sample Size and Power 10.1.1 Run-in subcohort 10.1.2 Cohort 1: Combination therapy with nivolumab and ipilimumab 10.1.3 Cohort 2: Pembrolizumab 10.2 Analysis Set 10.2.1 Complete Analysis Set 10.2.2 Safety Analysis Kit 10.2.3. Sets per protocol 10.3 Data Handling Practices 10.4 Interim analysis 10.5 Research Group 10.5.1. Breakdown of patients 10.5.2 Demographic Variables and Baseline Characteristics 10.6. Effectiveness Analysis 10.6.1 Efficacy Endpoint 10.6.2 Planned Analysis Method 10.7 Safety analysis 10.7.1 Adverse Events 10.7.2 Laboratory Parameters 10.7.3 Physical Examination 10.7.4 Vital Signs 10.7.5 12-lead electrocardiogram 10.7.6 Injection site reactions 10.7.7 Concomitant medications 10.8 Pharmacodynamic analysis and biomarker analysis 10.9 Reporting deviations from the statistical analysis plan 11. Quality Control and Quality Assurance 12. Regulatory and Ethical Obligations 12.1 Compliance with Clinical Trial Implementation Standards 12.2 Informed Consent 12.3 Contact with your primary physician 12.4 Research Ethics Committee 12.5 Regulatory approvals 12.6 Periodic review of safety and efficacy 12.7 Registration of clinical trials on the clinical trial registry website 12.8 Patient confidentiality 12.9 Data Protection 12.10. Future use of stored specimens 13. Administrative and legal obligations 13.1 Duties of the Responsible Physician 13.2. Access to Source Data and Documentation 13.3 Insurance 13.4 Amendments to Protocols and Informed Consent 13.5 Documentation and data storage of the examination 13.6 Availability and storage of survey records 13.7 Data Collection 13.8 Language 13.9 Clinical monitoring, source data verification, and auditing 13.10 Protocol deviations and violations 14. References

[0124] List of abbreviations [Table E-1]

[0125] [Table E-2]

[0126] [Table E-3]

[0127] Definition of Terms [Table F]

[0128] 1. Protocol Overview [Table G-1]

[0129] [Table G-2]

[0130] [Table G-3]

[0131] [Table G-4]

[0132] [Table G-5]

[0133] [Table G-6]

[0134] [Table G-7]

[0135] [Table G-8]

[0136] [Table G-9]

[0137]

Table G-10

[0138]

Table G-11

[0139] 2. Introduction and Background Description 2.1 Introduction Malignant melanoma is an aggressive tumor of the skin. When melanoma is diagnosed at an early stage (Stage I and II), surgery alone is curative in a high proportion of patients. However, once the tumor begins to spread, the disease is difficult to treat and the mortality rate of patients is high. In the United States (US), it was estimated that more than 76,000 people were diagnosed with melanoma in 2016, and about 10,000 patients died from the disease (Siegel et al. 2016). The corresponding figures in the European Union (EU) are 82,000 new diagnoses and about 15,700 deaths (European Cancer Observatory Cancer Database [EUCAN] Database, 2012).

[0140] Until relatively recently, the treatment of patients with unresectable Stage IV melanoma generally consisted of chemotherapy, typically dacarbazine, or cytokine therapy (interferon [IFN] alpha or interleukin-2). The ORR to such treatments was low, and the 2-year OS was less than 20% (Keilholz et al. 2005). Two new classes of therapies have significantly improved the treatment outcomes of Stage IV patients. For patients with a mutated BRAF gene, combination therapy with BRAF and MEK kinase inhibitors resulted in an ORR of over 60% and a 2-year OS of 50% (Long et al. 2015, Ascierto et al. 2016).

[0141] Another new therapeutic approach for advanced melanoma is checkpoint inhibition. By blocking immune checkpoints such as PD-1 and CTLA-4, which are selected by melanoma cells to evade the immune system, these therapies have significantly improved clinical outcomes. The anti-PD-1 antibody nivolumab was compared to dacarbazine in a randomized clinical trial of patients with previously untreated BRAF V600 wild-type melanoma (Robert et al. 2015a). The results for nivolumab and dacarbazine were 40% ORR vs. 13.9% and 5.1 months vs. 2.2 months median PFS, respectively, with a significant improvement in OS (hazard ratio for death, 0.42; p<0.001). The anti-PD-1 antibody pembrolizumab was compared to the CTLA-4 checkpoint protein antibody ipilimumab in a randomized clinical trial of patients with advanced melanoma (Robert et al. 2015b). The results for pembrolizumab and ipilimumab showed significant improvements in overall survival (OS), with an ORR of 33% vs. 12% and a median PFS of 4.1 months vs. 2.8 months, respectively (hazard ratio for death, 0.69; p=0.004).

[0142] Superior efficacy was achieved by combining anti-PD-1 and anti-CTLA-4 antibodies. The results for the combination of nivolumab and ipilimumab compared to nivolumab monotherapy: ORR was 50% vs. 40%, median PFS was 11.5 months vs. 6.9 months, and 2-year OS was 64% vs. 59%, respectively (Larkin et al. 2017). While response rates and PFS were improved with the combination compared to nivolumab monotherapy, the combination was associated with a higher incidence of CTCAE grade 3 or 4 treatment-related toxicities (55% vs. 16%). Most of these toxicities resolved within 3–4 weeks; however, approximately one-third of patients permanently discontinued the combination due to adverse events (AEs) (Larkin et al. 2015). In a separate study, the combination of nivolumab 3 mg / kg and ipilimumab 1 mg / kg resulted in an ORR of 45.6%, while nivolumab 1 mg / kg and ipilimumab 3 mg / kg resulted in an ORR of 50.6%. PFS and OS were similar between the two regimens. However, nivolumab 1 mg / kg and ipilimumab 3 mg / kg had more grade 3 / 4 adverse reactions compared to nivolumab 3 mg / kg and ipilimumab 1 mg / kg (48.3% vs. 33.9%) (Lebbe 2019).

[0143] Despite the success of immune checkpoint blockade in the treatment of melanoma, a substantial proportion of patients do not respond. One possible reason for this lack of response is an inadequate initial immune response to the developing tumor. Therefore, therapies to enhance antitumor immune activity are of interest as companion therapies to checkpoint inhibitors.

[0144] SCIB1 is a plasmid DNA designed to express a modified human immunoglobulin-1 (IgG1) antibody molecule containing two helper T cell sequences derived from the melanoma gp100 protein, in addition to a CTL epitope derived from the TRP-2 melanoma antigen. One of the gp100 sequences also incorporates a nested CTL epitope. SCIB1 is designed to be administered by needle-free injection and taken up by antigen-presenting cells that synthesize an antibody construct, process the antigen, and present the epitope to either an MHC class I or MHC class II molecule. T cells that recognize these complexes then enhance the immune response (differentiation cluster 4 positive [CD4 + Helper T cells) or direct effectors of tumor cell attack (differentiation cluster 8 positive [CD8 + It acts as a CTL (Metheringham et al. 2009). In addition to this direct presentation process, SCIB1 antibodies are secreted, and the secreted product binds to the CD64 Fc-gamma receptor (FcgR) on dendritic cells via the heavy chain Fc domain, leading to epitope uptake and CD8 + and CD4 + There is evidence that cross-presentation of epitopes to cells occurs (Pudney et al. 2010; Durrant et al. 2010). As a result of these dual processes, a high-frequency and high-avidity immune response to melanoma-associated antigens occurs.

[0145] 2.2 Findings obtained from non-clinical and clinical studies 2.2.1 Non-clinical research Primary pharmacodynamics SCIB1 codes TRP-2 CD8 +The CTL epitope is recognized in specific mouse strains, thereby enabling the evaluation of the immune response induced after SCIB1 administration in animal models. In a preliminary study using a gene gun to intradermally administer the SCIB1 plasmid, high-frequency and high-avidity responses were induced in mice, and these responses correlated with successful tumor growth inhibition and regression. Recognition of the melanoma-derived gp100 DR4 helper T cell epitope has also been confirmed in transgenic HLA-DR4 mice, demonstrating that the sequence is correctly processed and presented.

[0146] Intramuscular delivery of SCIB1 using a TDS-IM electroporation device has been shown to induce a high-frequency and high-avidity T-cell immune response and a good memory response in mice. Dose-response evaluation in mice showed that the dose selected for the initial clinical study, whether based on body weight or calculated human equivalent dose, was within a one-logarithmic range of the effective dose in mice; in addition, the three-week interval selected for clinical administration of SCIB1 using the TDS-IM device resulted in a positive response in the animal study.

[0147] In mouse studies, repeated administration over 2 or 3 weeks induced a higher immune response than a single dose, as measured by the IFNγ ELISpot assay, and the avidity of the response was higher with repeated SCIB1 administration. Evaluation after three weekly doses and a 10-week interval suggested the presence of a memory response. TRP-2 specific CD8 + Fluorescence-activated cell segregation analysis of the cell phenotype revealed that the cells were double-positive for CF62L and CD127, suggesting central memory.

[0148] In a dose-response study in which SCIB1 was administered to C57BL / 6 mice three times at one-week intervals, a dose of 10 μg / mouse induced a higher immune response in terms of both frequency and avidity compared to doses of 3 and 1 μg / mouse. The 10 μg / mouse dose is equivalent to a human dose of 2.5 mg.

[0149] SCIB1 treatment of HLA-DR4 transgenic mice subcutaneously injected with B16 mouse melanoma cells resulted in a significant delay in tumor growth compared to treatment with a control plasmid (p=0.0027). Treatment was performed by intramuscular injection using electroporation with a TDS-IM device on days 0, 6, and 13, relative to the tumor cell injection.

[0150] In experiments designed to support the combination therapy used in this study, SCIB1 was administered intradermally using a gene gun device at a dose of 1 μg / mouse on days 4, 8, and 11, in combination with an anti-PD-1 blocking mAb administered intraperitoneally at a dose of 250 μg / mouse on days 4 and 11. HLA-DR4 transgenic mice were given B16F1 DR4 mouse melanoma tumor cells (2.5 × 10⁶) on day 0. 4 SCIB1 was injected. The results showed that SCIB1 alone and anti-PD-1 alone were effective in this mouse cancer model, but the efficacy of the combination was significantly higher than that of either drug alone. 40% of mice treated with SCIB1 alone showed long-term survival compared to less than 20% of control mice (p=0.022). A similar antitumor response was induced by PD-1 blockade, which resulted in long-term survival in 50% of mice (p=0.0047). This antitumor response could be significantly enhanced by combining PD-1 blockade with SCIB1, and treated animals showed an enhanced 80% survival rate compared to controls (p<0.0001) or compared to SCIB1 (p=0.0066) or PD-1 blockade alone (p=0.0234). 6-fold higher dose of B16F1 DR4 tumor cells (1.5 × 10⁶ 5In individual cells, only combination therapy showed a significant survival benefit compared to the control (p=0.0126).

[0151] Tests of infiltrating lymphocytes in these experimental tumors showed that animals treated with SCIB1 were differentiated cluster 3 positive (CD3 + )CD8 + A significant increase in the cell ratio was observed, although the increase was milder in animals treated with PD-1 blockade alone. However, CD3 + CD8 + T cell infiltration was higher in combination with either SCIB1 (p=0.0004) or PD-1 blockade (p=0.0026) than with either drug alone. T cell proliferation, as measured by Ki67, was also increased by both SCIB1 and PD-1 blockade alone, and further increased in combination compared to each drug alone (p=0.0373 and p=0.0167, respectively). In these studies, CD4 infiltration into tumor sites was higher. + The proportion of T cells also increased with either SCIB1 alone (p=0.0086) or SCIB1 in combination with PD-1 blockade (p=0.0321). The treatment involved regulatory CD4 expressing forkheadbox P3 that infiltrates tumors. + No significant difference was induced in the number of T cells. Ultimately, SCIB1 was shown to upregulate PD-L1 expression, while PD-1 blockade did not (Xue et al. 2016).

[0152] toxicology research A single-dose toxicological study of SCIB1 administered by intramuscular injection using electroporation was conducted in C57BL / 6 mice (Report LHP0002). Tissue samples collected up to day 90 were analyzed for the SCIB1 plasmid using a validated quantitative polymerase chain reaction (QPCR) assay (Report LHP0005). No apparent treatment-related adverse effects occurred with a single dose of 40 μg / mouse. QPCR analysis revealed high copy numbers of the DNA plasmid at the injection site in all mice on day 3, with male and female mice having an average of 653,000 and 18,700,000 copies per μg of genomic DNA, respectively. By day 90, the copy number at the injection site was less than 100 copies per μg of genomic DNA in male mice and an average of 207 copies per μg of genomic DNA in female mice, indicating effective clearance of the DNA plasmid from the injection site. One ovarian sample had a copy number of 21,773 on day 3. However, all other gonadal tissues were below quantitative levels on days 3, 35, and 90, except for one test sample which showed a very low but positive signal (more than 11 copies per μg of genomic DNA) on day 90.

[0153] A pilot study (Report LHP0003) and a GLP-compliant repeated-dose study (Report LHP0001) were conducted at C57BL / 6 using SCIB1. No adverse findings were observed during the pilot study, in which a dose of 40 μg / mouse was administered on days 1, 8, and 15. An increased IFNγ response was observed in 4 out of 5 female mice treated with SCIB1, but no IFNγ response was observed in control female mice. In the GLP-compliant study, mice were administered SCIB1 at a dose of 40 μg / mouse by intramuscular injection using electroporation on days 1, 22, 43, 64, and 85. A control group was included, and the control mice were administered phosphate-buffered saline. One control female mouse and one control male mouse died immediately after the 4th and 5th doses, respectively. No clinical signs related to treatment were observed, and no effects on body weight, food intake, ophthalmoscopic tests, hematology, blood chemistry, or organ weight were detected. In both control and SCIB1-treated mice, darkening, muscle fiber necrosis, hemorrhage, inflammatory responses, and muscle regeneration were observed in the tibialis muscle on both sides of the injection site. These observed local tolerance effects were higher in SCIB1-treated mice compared to control mice, and they showed almost complete recovery after a 4-week recovery period. No significant histopathological findings were observed in the testes of male mice or the uterus of female mice, suggesting that these organs were not adversely affected by SCIB1 administration. Ovaries from female mice were not histopathologically evaluated; therefore, the findings from the biodistribution study (Report LHP0002) that SCIB1 may be delivered to and incorporated into the ovaries could not be confirmed by the histopathological results obtained from this repeated-dose toxicological study.

[0154] As a conclusion of this study, SCIB1 did not cause any signs of systemic toxicity when administered at a dose of 40 μg / mouse via intramuscular injection using electroporation for 5 administrations at 3-week intervals, based on the results from various parameters evaluated. Findings at the injection site were of higher incidence and severity in SCIB1-treated mice compared to control mice; these changes were almost completely reversible during the 4-week recovery period. In addition, SCIB1-treated mice had increased IFNγ responses as measured by ELISpot assay, demonstrating that SCIB1 elicited the desired immune system response.

[0155] 2.2.2 Clinical Study SCIB1 has been investigated in a single open-label, non-randomized Phase 1 / 2 study (SCIB1-001) conducted in melanoma patients in the United Kingdom. This study was designed to determine the safety and tolerability of four dose levels of SCIB1 administered via the intramuscular route using the TDS-IM electroporation device, and to assess immunogenicity and antitumor activity. In Part 1 of the study, patients were enrolled into administration cohorts with a minimum of 3 patients per cohort. The dose of SCIB1 was escalated from 0.4 mg to 2 mg, 4 mg, and ultimately to 8 mg via protocol amendment. The treatment schedule consisted of administrations at Week 0, Week 3, Week 6, Week 12, and Week 24. Part 2 of the study was an expansion phase with patients treated at the 4 mg dose. After the protocol amendment, additional patients were enrolled into Part 2 at the 8 mg dose. Further details of the study design and evaluations are available in the SCIB1 IB.

[0156] Patient Population For cohorts 1-3 of Part 1, patients had to have histological confirmation of AJCC stage III or IV melanoma, either with or without a tumor at the time of screening; patients with a tumor had to have measurable disease according to RECIST. Patients in cohort 4 (8 mg dose) of Part 1 and cohort 2a (8 mg dose) of Part 2 had to have measurable disease at the time of screening. Patients in cohorts 1 (4 mg dose) and 2b (8 mg dose) of Part 2 had to have resected disease. Patients had to be HLA-A2 positive and also positive for HLA-DR4, HLA-DR7, HLA-DR53, or HLA-DQ6.

[0157] A total of 35 patients (18 males and 17 females) were enrolled in the study, with a median age of 60 years (25–75 years). Fifteen patients had tumors at baseline, and 20 patients had resected disease at baseline. Of the patients with tumors at screening, five had received chemotherapy, one had received chemotherapy and ipilimumab, one had received a vaccine, and one had received local limb perfusion. Of the 20 patients with resected disease, one had received treatment (vaccine). No dose-limiting trials (DLTs) were observed in the dose escalation phase of Part 1 of the study (n=11), and the 4 mg dose was selected for expansion in Part 2 (n=14). The 8 mg dose was subsequently studied in Part 1 (n=5) and then in Part 2 (n=5).

[0158] safety Overall, 23 patients completed all planned injections for the primary portion of the study (up to week 24 and including week 24). Twelve patients received one or more doses of SCIB1 during the continuation phase of treatment, including nine patients with resected disease. The median exposure period for primary and continuation treatment combined was 170 days, with a maximum of 1107 days (16 doses).

[0159] The most common adverse events (AEs) during the primary study (>10%) were injection site hematoma (77%), injection site pain (37%), fatigue (26%), headache (20%), procedural pain (17%), arthralgia, blurred vision, constipation, limb pain (14%), depressed mood, dizziness, dyspnea, nausea, and rash (11%). Overall, 28 patients reported AEs associated with the study drug. The most common events considered to be associated with the study drug were injection site hematoma (37%), injection site pain (20%), fatigue (14%), blurred vision, headache, and procedural pain (11%). In total, 32 patients (91%) experienced AEs related to the electroporation device and procedure. SAEs were most commonly associated with the progression of malignant melanoma, and none were considered likely to be related to either the study drug or its administration process. Severe, life-threatening (grades 3 and 4) adverse events were infrequent, with 14 events in 5 patients meeting these criteria in the main study. Two grade 3 / 4 events were considered related to SCIB1: a grade 3 injection site hematoma in one patient and a grade 3 anxiety disorder in a different patient; these patients were being treated with 4 mg of SCIB1. Five grade 3 / 4 events in two patients were considered related to the electroporation device or procedure: a grade 3 injection site hematoma and a grade 3 anxiety disorder (two events), injection site pain, and anticipatory anxiety. The injection site pain and anticipatory anxiety events led to discontinuation of SCIB1 treatment in the patient. The second patient refused to continue treatment due to pain and discomfort from the electroporation procedure.

[0160] Three deaths were recorded in the database during the primary study phase, but these occurred after patients had withdrawn from the primary study and were recorded as follow-up information; two were disease-related (one patient on 0.4 mg; one patient on 4 mg), and one was due to community-acquired pneumonia (8 mg). Two more deaths were recorded after patients had discontinued continued administration, and both were disease-related: one patient treated on 4 mg and one patient treated on 8 mg. All of these patients had tumors at the time of screening.

[0161] Effectiveness In immune response assays, 88% of patients responded to the ELISpot assay and 82% responded to the proliferation assay during the follow-up phase in addition to the main study period. The number of responders to individual melanoma-specific peptides in the ELISpot assay increased with continued administration of SCIB1. Comparison of ELISpot responses between the indicated dose groups showed a stronger response at 8 mg than at 4 mg. The number of responders to individual peptides in the proliferation assay also increased with continued administration of SCIB1. Patients who did not have a tumor at screening and received an 8 mg dose showed a significantly stronger proliferation response to a single peptide compared to patients who received a 2 / 4 mg dose. Furthermore, patients who did not have a tumor at screening showed a significantly stronger proliferation response to any peptide compared to patients who had a tumor at screening.

[0162] Of the 15 patients who had tumors at the time of study enrollment, one patient treated with an 8 mg dose met the RECIST criteria for partial response (PR) and had a duration of 204 days (29 weeks). A second patient treated with a 4 mg dose experienced a reduction of more than 30% in the size of the target lesion, but there was progression of non-target lesions.

[0163] Patients who had no detectable tumors at the time of enrollment and were treated with SCIB1 in an adjuvant setting were followed for disease-free survival. The longest follow-up is available for 16 patients treated with 2 and 4 mg of SCIB1. As of the cutoff date of October 29, 2016, all 16 patients were alive, with a median observation period of 39 months (33–52 months). At 2 years, 12 of these patients (75%) remained disease-free without additional treatment other than continued SCIB1.

[0164] 2.3 Logical basis for dose selection Based on safety findings from study SCIB1-001 and observations of a stronger immune response at the 8 mg dose compared to the 4 mg dose, the 8 mg dose was selected for this study. SCIB1 is administered intramuscularly using the PharmaJet Stratis® needle-free injection device. The 8 mg dose in patients is approximately 0.114 mg / kg or 4.44 mg / m². 2 This corresponds to the dose used in toxicological studies for mice, which is 2.0 mg / kg or 6.0 mg / m². 2 This is compared to 40 μg / mouse, which is equivalent to [a certain amount].

[0165] 2.4 Known and potential benefits and risks to patients The most commonly attributed adverse events (AEs) to SCIB1 were fatigue (14%), blurred vision, and headache (11%). Injection site hematoma and injection site pain were also attributed to the electroporation procedure used in SCIB1-001 and to SCIB1 itself, with overall incidences of 77% and 37%, respectively. SCIB1 has been shown to induce an immune response in patients with melanoma (Patel et al. 2018).

[0166] 3. Research Objectives and Endpoints 3.1 Primary purpose and endpoint 3.1.1 Run-in subcohort the purpose To evaluate the safety and tolerability of intramuscular SCIB1 in patients receiving either nivolumab and ipilimumab combination therapy or pembrolizumab.

[0167] endpoint It is measured by AE (CTCAE v5.0), vital signs, physical examination, serological and hematological tests, thyroid function tests, injection site evaluation, urinalysis, 12-lead ECG, performance status, and records of concomitant medications. The target SCIB1-related DLT rate was 0 or 1 in 6 evaluable patients.

[0168] 3.1.2 Main research the purpose To determine whether adding SCIB1 to either nivolumab and ipilimumab combination therapy or pembrolizumab increases the ORR in this patient population compared to historical data for nivolumab and ipilimumab combination therapy or pembrolizumab monotherapy.

[0169] endpoint Objective response rate measured according to the RECIST 1.1 criteria.

[0170] 3.2 Secondary purposes and endpoints 3.2.1 Main research the purpose To determine whether adding SCIB1 to either nivolumab and ipilimumab combination therapy or pembrolizumab improves the duration of response in this patient population compared to historical data for nivolumab and ipilimumab combination therapy or pembrolizumab monotherapy.

[0171] endpoint The duration of the response, measured from the time of the first response (CR or PR) to the date of disease progression (according to RECIST 1.1) or death due to any cause.

[0172] the purpose Determine the ORR measured using the iRECIST criteria (Seymour et al. 2017).

[0173] endpoint ORR determined by iRECIST.

[0174] the purpose Evaluate the PFS rate for all patients at week 49 (day 344) and week 97 (day 680).

[0175] endpoint The PFS rate is defined as the proportion of patients who, at each specified time point, are not progression-free (according to RECIST 1.1 and iRECIST), have not initiated new anticancer therapies, and have not died.

[0176] the purpose Evaluate the overall survival (OS) rate for all patients at week 49 (day 344) and week 97 (day 680).

[0177] endpoint The OS rate is defined as the proportion of patients who are still alive at each specified time point.

[0178] the purpose Further evaluation of the safety and tolerability of SCIB1 in patients receiving either nivolumab and ipilimumab combination therapy or pembrolizumab.

[0179] endpoint Safety and tolerability as defined for the above run-in subcohort.

[0180] 3.3 Experimental Objectives and Endpoints the purpose To explore the relationship between immune response and clinical outcomes.

[0181] endpoint Immune response evaluation (ELISpot and other assays) performed on peripheral blood samples obtained from patients at a limited number of facilities.

[0182] 4. Research design 4.1 Overall Research Design This is a phase 2, multicenter, open-label study of SCIB1 in patients with unresectable advanced melanoma receiving either nivolumab and ipilimumab in combination or pembrolizumab. There will be two cohorts, depending on whether patients receive dual therapy (nivolumab and ipilimumab) or monotherapy (pembrolizumab) with a checkpoint inhibitor. In the run-in subcohort, six patients will receive both SCIB1 and either nivolumab and ipilimumab or pembrolizumab to ensure there are no unexpected severe toxicities that would preclude further trials. In each of these cohorts, the first two patients will be recruited sequentially at intervals of at least 14 days to minimize the risk of unpredictable severe toxicity. If, among the six evaluable patients, fewer than two experience SCIB1-related DLTs by day 35, the remaining patients are enrolled in the cohort using Simon's optimal two-stage design (Simon 1989) (Figure 5).

[0183] Cohort 1: Combination therapy with nivolumab and ipilimumab In Phase 1, 15 patients (including 6 from the run-in subcohort) will be recruited. If there are 8 or fewer clinical responses (RECIST 1.1 objective response [CR or PR], within 25 weeks of the first dose of SCIB1), the cohort will be discontinued. Otherwise, an additional 28 patients will be recruited for Phase 2, bringing the total to 43 patients. Recruitment will continue until the decision for Phase 1 is made.

[0184] Cohort 2: Pembrolizumab In Phase 1, 14 patients (including 6 from the run-in subcohort) will be recruited. If there are 5 or fewer clinical responses (RECIST 1.1 objective response [CR or PR], within 25 weeks of the first dose of SCIB1), the cohort will be discontinued. Otherwise, an additional 30 patients will be recruited for Phase 2, bringing the total to 44 patients. Recruitment will continue until the decision for Phase 1 is made.

[0185] Recruitment will be terminated if a cohort fails to complete recruitment within a reasonable period after the other cohort has fully recruited. In addition, as part of an interim analysis, the feasibility of further recruitment of cohorts that have not met their first-stage quota will be assessed once the other cohort reaches the first stage.

[0186] DLT is defined as follows: Non-hematological adverse events related to drugs with a CTCAE v5.0 grade of 3 or higher. Excluding the following : • Grade 3 or higher gastrointestinal toxicity (nausea, vomiting, diarrhea, constipation) that can be managed with antiemetics and other therapies and does not exceed 48 hours. • Asymptomatic laboratory changes (other than laboratory values ​​of the kidneys and liver, and grade 4 lipase / amylase) that allow for successful replacement (e.g., electrolyte replacement) within 72 hours. • Grade 3 fatigue lasting less than 72 hours • Patients with liver metastases who have elevated ALT or AST levels of grade 3 or higher, and whose condition resolves to grade 2 or lower within 7 days.

[0187] Drug-related hematological adverse events of CTCAE v5.0 grade 4 or higher, grade 3 thrombocytopenia with bleeding (platelet count decreased to less than 50,000 to 25,000 / mm3), grade 3 hemolysis (indicating transfusion or medical intervention, e.g., steroids), and grade 3 febrile neutropenia (absolute neutrophil count less than 1,000 / mm3, one episode of fever above 38.3°C [101°F], or a sustained fever of 38°C [100.4°F] or higher for more than one hour).

[0188] Toxicity that meets these criteria and is considered to be associated with the administration of the study drug will be considered a DLT. Grade 3 events clearly associated with tumor flare (local pain, irritation, local rash) will not be considered DLTs. DLTs in the run-in subcohort will be evaluated up to a maximum of 35 days. Patients in the run-in subcohort who leave the study before week 5 (day 36) for reasons other than DLTs will be replaced.

[0189] If two or more of the six patients in any run-in subcohort have a SCIB1-related DLT, the available safety data will be formally reviewed by the principal investigator and sponsor to determine whether to enlist additional patients to evaluate the safety of the combination. This step is included because some events resulting from the approved use of checkpoint inhibitors meet the definition of a DLT and the contribution of SCIB1 (if any) needs to be evaluated. Relevant CTCAE grade 3 or higher events occurred in 10% of 277 melanoma patients receiving pembrolizumab monotherapy at the recommended dose and schedule (Robert et al. 2015b). In cases of nivolumab and ipilimumab combination therapy, grade 3–4 toxicity occurred in 33.9% of 180 patients receiving 3 mg / kg nivolumab and 1 mg / kg ipilimumab, and in 48.3% of 178 patients receiving 1 mg / kg nivolumab and 3 mg / kg ipilimumab (Lebbe 2019). In a SCIB1-001 monotherapy study in melanoma patients (n=35), only two grade 3 / 4 events were considered to be associated with SCIB1: a grade 3 injection site hematoma event and a grade 3 anxiety disorder in a patient associated with the electroporation procedure used in the study. This study used the PharmaJet Stratis® device, which has been associated with only mild discomfort in ongoing studies. Any patients recruited under a previous version of the protocol and who received SCIB1 using the TDS-IM electroporation device will re-consent to continue administration using the PharmaJet Stratis® device. Decisions to expand the scope of participation in these situations require confirmation and approval from the principal investigator and the clinical trial sponsor. A careful assessment of excessive severe toxicity will continue for the remainder of the study.

[0190] During a maximum 28-day screening period, patients will complete eligibility procedures and assessments. Written informed consent must be obtained before any protocol-specific procedures are performed (procedures and assessments are performed for research purposes, not as part of routine medical care). Trial results and tumor imaging obtained through routine clinical management (including those performed before written informed consent) are acceptable for screening if obtained within the designated screening timeframe. A separate consent form is to be used for HLA trials; HLA consent must be obtained before screening.

[0191] During the research treatment, patients will be monitored for safety, tumor response, disease progression, survival, and initiation of any new anti-cancer therapies.

[0192] 4.2 Number of patients and countries planned We plan to recruit 87 patients from up to 20 clinical cancer centers worldwide. Additional patients will be recruited to replace those who are ineligible for evaluation of DLT in the run-in subcohort or who are ineligible for evaluation of the primary endpoint of ORR according to RECIST 1.1 (Section 5.4.1).

[0193] 4.3 Research Period Due to COVID-19, the research has been delayed and is expected to begin recruitment in 2021 and be completed in 2025.

[0194] 4.4 Patient allocation After patients sign an informed consent form from their HLA and agree to participate in the study, they are assigned a unique three-digit identification number. This patient identification number serves as the primary method of identifying each patient throughout the study, both on the CRF and in field source documentation.

[0195] 4.5 Blinding and Randomization This is an open-label, non-randomized study involving two cohorts, and therefore, neither randomization nor blinding is required.

[0196] 4.6 Validity of study design and selection of patient population Pembrolizumab therapy has been shown to be effective in patients with advanced melanoma. However, only about one-third of treated patients respond to this checkpoint protein antibody (Robert et al. 2015b). The reason for the lack of clinical response in the remaining patients is unknown, but it is likely that the immune system's inability to recognize the patient's melanoma is partly involved. Combination therapy with antibodies against both PD-1 and CTLA-4 has been shown to increase the response rate to about 50% in patients who have never been treated for melanoma, but this combination therapy is associated with a much higher incidence of severe, life-threatening toxicity and treatment discontinuation due to these toxicities (Larkin et al. 2015).

[0197] Vaccination may provide a means to increase the immune recognition of tumors in patients and may be associated with increased response rates without the same increase in severe toxicity. SCIB1 has been shown to evoke a cellular immune response in more than 85% of treated melanoma patients and shows signs of monotherapy clinical activity.

[0198] This design for an open-label, non-randomized phase 2 study in patients with previously untreated unresectable melanoma is justifiable based on the fact that these patients receive either nivolumab and ipilimumab in combination or pembrolizumab, which are current standard checkpoint inhibitor therapies. SCIB1 has been well tolerated in its phase 1 / 2 clinical trial, and the addition of SCIB1 should not interfere with the activity of either nivolumab and ipilimumab or pembrolizumab in this population. The sample size of 87 patients is sufficient to justify a larger randomized study to determine whether SCIB1 has the ability to improve the RECIST 1.1 response rate (compared to the historical rates for nivolumab and ipilimumab combination or pembrolizumab alone in this population).

[0199] 4.7 Periodic review of safety and efficacy Safety and efficacy data obtained from the study will be formally reviewed at frequent and regular intervals, including at the end of the run-in period and at the end of the first phase of Simon's two-phase design. The review committee will include the principal investigator, the sponsor's medical monitor, and the sponsor's clinical trial director or designated person. The review plan for the run-in subcohort will be established before the enrollment of the first patients in the study and will reflect the safety review conducted during the run-in period. The complete safety and efficacy review charter will be finalized at the end of the run-in period and before further patient enrollment in the study after the run-in period.

[0200] 4.8 Criteria for research completion Each patient's study treatment period consists of 10 doses of SCIB1. Patients who complete this are considered to have completed the study treatment (week 85 [day 596]) and will enter a 12-week post-treatment follow-up, including an EOT visit (to monitor safety after the last dose of SCIB1). Patients will be evaluated for tumor response, disease progression, initiation of new cancer therapies, and survival for 97 weeks from the first dose of SCIB1.

[0201] The study will be completed when all patients have completed or discontinued the post-treatment follow-up period.

[0202] 4.9 Procedure and Evaluation Schedule The research procedures and evaluations performed for all patients are summarized in Table 2 below. Detailed procedures and evaluations are provided in Sections 7-9. Pre-screening evaluation to determine the patient's HLA histological type may be performed prior to the screening period after the patient has given separate, specific consent for this evaluation. Alternatively, HLA consent may be obtained at the discretion of the principal investigator during the screening visit.

[0203] [Table 2]

[0204] Notes to Table 2: aPTT = Activated Partial Thromboplastin Time, CPI = Checkpoint Inhibitor; CT = Computed Tomography, ECG = Electrocardiogram, ECOG = East Coast Cancer Clinical Trials Group, EOT = End of Treatment, FT3 = Free Triiodothyronine, FT4 = Free Thyroxine, HLA = Human Leukocyte Antigen, INR = International Normalized Ratio, MRI = Magnetic Resonance Imaging, TSH = Thyroid-Stimulating Hormone a) Where appropriate, checkpoint inhibitor (CPI) administration may be coordinated with SCIB1 administration to minimize visits. If CPI is interrupted or discontinued, CPI visits are not required. b) The patient will receive up to five doses of SCIB1 at weeks 37, 49, 61, 73, and 85 (days 260, 344, 428, 512, and 596, respectively). If, for example, an SCIB1 dose is not administered due to an adverse event with the combination of nivolumab and ipilimumab or pembrolizumab, the missed dose may be administered after week 13, with an interval of at least 15 days, but preferably up to 6 weeks. All procedures listed in the 12 weekly visits will be performed when the missed dose is administered, with the exception of the assessment of tumors and skin lesions. c) Three weeks after each 12-week SCIB1 administration in weeks 40, 52, 64, and 76 (days 281, 365, 449, and 533, respectively). If the patient decides to discontinue SCIB1 at the administration visit, the 12+3 evaluation visit will be replaced by the EOT visit (30 days ± 10 days) after the last SCIB1 administration. If the patient decides to discontinue SCIB1 at the 12+3 evaluation visit, all trials of the EOT visit may be conducted at the 12+3 evaluation visit if possible; if this is not possible, the EOT visit will be conducted 30 days (± 10 days) after the last SCIB1 administration or as early as possible according to the schedule. d) End-of-treatment (EOT) visits should be completed for all patients 30 days (±10 days) after the last dose of SCIB1, or as soon as possible if the decision to discontinue treatment is made more than 30 days after the last dose. For patients discontinuing SCIB1 at the time of the study evaluation visit, the EOT visit may be replaced with the 12+3 week evaluation visit (see footnote c). Adverse events should be recorded up to 30 days after the last dose of SCIB1 or the next dose of the checkpoint inhibitor, whichever is longer. Concomitant medications taken up to 30 days after the last dose of SCIB1 treatment must be recorded in the CRF. e) Post-treatment follow-up will be completed for all patients who have received at least two doses of SCIB1 (on day 1 and week 4) and have not dropped out of the study. Post-treatment follow-up will be conducted every 12 weeks (±2 weeks) from the last dose of SCIB1 up to a maximum of week 97, as needed, by on-site assessment, telephone / video call, or review of the patient's medical records. Tumor and skin lesion assessments will be conducted every 12 weeks (±2 weeks) from the last dose of SCIB1 for patients who have not recorded disease progression (according to RECIST 1.1 and iRECIST) and have not initiated any new anti-cancer treatment. f) The first dose of CPI (either nivolumab and ipilimumab or pembrolizumab) shall be administered in Week 1, following the standard treatment schedule. On days when both CPI and SCIB1 are administered, SCIB1 shall be administered first. When CPI is administered alone, the standard treatment procedure shall be followed. On days when only CPI is administered, adverse events and concomitant medication monitoring shall be performed, and blood evaluations and clinical investigations shall be based on the standard treatment. The continuation of CPI (administered according to the standard treatment) is at the discretion of the principal investigator. If CPI administration is delayed or discontinued, SCIB1 administration shall be continued according to the schedule. g) Written informed consent must be obtained before performing protocol-specific procedures. Informed consent for HLA and the primary study may be obtained simultaneously, but ideally, consent for the primary study should only be obtained after HLA eligibility has been established. Trial results and tumor imaging obtained through customary clinical management (including those performed before written informed consent) are acceptable for screening if obtained within the specified time frame. h) The medical history should include the primary diagnosis of melanoma, previous surgical interventions, previous adjuvant chemotherapy and adjuvant biological therapies, previous radiation therapy, and the start and discontinuation dates of adjuvant treatment. A baseline medication history should also be collected. i) For women of reproductive capacity (including women within 12 months of their last menstrual period), a urine pregnancy test should be performed within 72 hours prior to the first dose. If the urine pregnancy test is not negative, a serum pregnancy test will be required. The pregnancy test (serum and / or urine) should be performed again at the indicated visit. j) Patients who do not have properly stored tumor tissue samples (less than 5 years old) for PD-L1 evaluation will need to undergo a new tumor biopsy before the first dose of the study drug. k) Perform an evaluation or collect a sample before administration. l) During visits for checkpoint inhibitors only, a “limited” physical examination may be performed as needed (cardiovascular system, lungs, abdomen, skin, weight, and the extent of signs and symptoms of disease). Height should be measured only at the time of screening. m) Vital signs should be measured approximately 10–15 minutes before administration, 5 minutes, 30 minutes, and 1 hour after SCIB1 administration, and the patient should be observed for at least 1 hour after receiving CPI for at least the first two doses. Vital signs should include body temperature, heart rate, respiratory rate, and blood pressure. n) Serological chemistry tests consist of albumin, ALT, alkaline phosphatase, AST, bicarbonate or carbon dioxide, urine, calcium, chloride, creatinine, total bilirubin (and direct bilirubin if clinically indicated), gamma-glutamyl transferase, LDH, non-fasting glucose, phosphorus (measured as phosphate), potassium, sodium, uric acid (urate), and total protein. o) Serological, hematological, urinalysis, and thyroid screening performed within 48 hours of the first administration of the study drug do not need to be repeated on day 1. p) Hematological tests consist of hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin level, mean corpuscular hemoglobin concentration, platelet count, red blood cell (RBC) count, white blood cell (WBC) count, and WBC differential (basophils, eosinophils, lymphocytes, monocytes, neutrophils). q) Record protein, glucose, ketones, blood, nitrite, white blood cells, bilirubin, pH, and specific gravity using a urinalysis (dipstick). Microscopic examination is performed only if clinically indicated: bacteria, RBCs, WBCs, casts, and crystals. r) On the day of SCIB1 administration, the injection site shall be evaluated one hour after SCIB1 administration. If a moderate or severe injection site reaction is observed, a digital photograph of the injection site shall be taken at the specified time or at any other time as necessary. An anonymized copy of the digital photograph shall be sent to a dedicated database via secure file transfer. s) If SCIB1 was not administered during the previous research visit, evaluation is not necessary. t) Tumor imaging (chest, abdomen, pelvis, brain, and any known area of ​​disease) shall be performed within 28 days prior to the first dose of SCIB1. CT scans are the preferred imaging technique unless the patient has a medical reason, such as a contrast agent allergy, that would make CT scans risky, or the disease is well documented on MRI. The same technique shall be used for individual patients throughout the study. A brain MRI (preferred) or CT scan at baseline is required to establish the presence or absence of brain metastases. Repeated brain imaging is required only if the patient has brain metastases at baseline or if new brain metastases are suspected. Patients with either an objective tumor response or PD should undergo repeated scans at least 4 weeks later and up to 8 weeks thereafter to confirm the response and rule out the possibility of a tumor flare response, respectively. Response status shall be assessed by the principal investigator (according to RECIST 1.1 and iRECIST). Copies of anonymized imaging data shall be collected for the study database. The imaging visit slots are within ±7 days, and the timing of imaging visits follows the calendar; treatment delays will not be adjusted. u) Tumor evaluation shall be performed at any point during or after treatment follow-up for all patients whose disease progression has not been recorded and who have not initiated new anticancer therapy, even if the patient discontinues SCIB1 treatment earlier than week 13 (day 92). v) Skin lesions will be documented with digital photographs. Anonymized copies of the digital photographs will be collected for a research database. w) Blood samples are collected from a limited number of sites for analysis of the immunological response of cells and transported to the Scancell laboratory at ambient temperature within 8 hours of blood sample collection. Immunological blood samples taken within 7 days of the first dose of SCIB1 do not need to be taken again on day 1 (it is recommended that immunological samples taken during the screening period be taken at least 2 days before day 1 so that day 1 samples can still be taken if there are any problems with these samples). Lymphocytes and plasma are prepared and tested immediately or stored in a cold freezer until analysis. Samples for the day of administration are taken before administration. x) If no assessment requiring a clinic visit is scheduled, the assessment will be conducted remotely by a healthcare professional via telephone or video call.

[0205] 4.10 Post-treatment follow-up Post-treatment follow-up will be completed for all patients who have received at least two doses of SCIB1 (on day 1 and week 4) and have not dropped out of the study. Patients will undergo clinic visits every 12 weeks (±2 weeks) after the last dose of SCIB1, including patients who received their last dose at week 85 (day 596), for tumor assessment (including CT / MRI scans) and skin lesion assessment, unless disease progression occurs or new anti-cancer treatment is initiated. If disease progression occurs or new anti-cancer treatment is initiated, subsequent follow-up may be conducted by a healthcare professional via telephone / video call during a routine clinic visit or review of the patient's medical records. All patients will therefore be evaluated for 97 weeks from the first dose of SCIB1. The following information will be collected: • Date of clinic visit / follow-up phone / video call • Includes the date, type, and diagnostic method of any new cancer. • Tumor and skin lesion assessments using RECIST 1.1 and iRECIST criteria for all patients with no recorded disease progression or new anticancer treatments (tumor assessments are performed during treatment or post-treatment follow-up at the points indicated in Table 2::Procedures and Assessment Schedule). • Date of disease progression and the means by which it was established • Date of commencement of new cancer treatment and generic name of the treatment agent • Date of death and primary cause of death

[0206] 5. Patient Selection and Withdrawal 5.1 Inclusion criteria 1) The patient has histologically confirmed unresectable stage III or stage IV melanoma as defined by AJCC (8th Edition; Gershenwald et al. 2017). Patients diagnosed with melanoma of unknown primary origin are eligible. 2) The patient has not received any systemic treatment for an advanced disease. Prior adjuvant treatment, as defined as treatment after resection of any detectable disease, is acceptable; the last dose must be at least 4 weeks prior to the first dose of SCIB1. 3) The patient has been clinically evaluated and it has been determined that checkpoint inhibition with either nivolumab and ipilimumab or pembrolizumab is appropriate treatment for advanced disease. 4) The patient's BRAF status must be known; patients with BRAF mutation-positive disease may be admitted without BRAF inhibitor treatment at the discretion of the attending physician, provided that the patient does not have evidence of rapid disease progression. 5) The patient has at least one measurable lesion according to RECIST 1.1 criteria, as determined by CT scan or MRI. 6) The patient is HLA-A2 positive. * 7) The patient is positive for HLA-DR4, HLA-DR7, HLA-DR53, or HLA-DQ6. * 8) The patient must be 18 years of age or older. 9) The patient has a life expectancy of more than three months. 10) The patient's ECOG performance status is 0 or 1. 11) The patient must have adequate organ function as determined by the following laboratory values:

[0207] [Table H]

[0208] 12) Patients must have the ability and willingness to provide written informed consent approved in the REC before any research-related procedure is performed. If a patient is rescreened for research participation or if ongoing patient care is changed due to protocol modifications, they must sign a new REC-approved ICF. 13) Women of childbearing potential (including women within 12 months of their last menstrual period) must have a negative serum pregnancy test during screening (within 72 hours prior to the first dose of the planned Day 1 study drug), not be breastfeeding, and have no intention of becoming pregnant during the study, and should be warned about the potential harm to the fetus from either the combination of nivolumab and ipilimumab or pembrolizumab. Women of childbearing potential must agree to use a highly effective method of contraception (Appendix E) for the entire duration of the study treatment and for 120 days after discontinuation of SCIB1, the combination of nivolumab and ipilimumab, or pembrolizumab (whichever is later). 14) Men of reproductive capacity with a fertile partner must consent to the use of a highly effective method of contraception (Appendix E) for the entire duration of the study treatment and for 120 days after discontinuation of SCIB1, nivolumab and ipilimumab combination, or pembrolizumab (whichever comes first). 15) The patient must have the willingness and ability to follow the visit schedule, treatment plan, laboratory trials and other research procedures. * HLA testing is performed in accredited laboratories using molecular (DNA) typing.

[0209] 5.2 Exclusion criteria 1) If the patient has been diagnosed with melanoma of the mucous membrane or eye.

[0210] Patients with active central nervous system metastases or carcinomatous meningitis are eligible. (Patients responding to prior treatment for brain metastases are eligible, provided they have been stable with no evidence of progression on MRI for at least four weeks prior to the first dose of the study drug, and have not received systemic steroids for at least one week prior to the first dose of the study drug. Patients who have recently received radiotherapy for brain metastases are eligible, provided they have fully recovered from postoperative complications, the screening scan must be at least four weeks after radiosurgery, and have not received systemic steroids for at least one week prior to the first dose of the study drug.)

[0211] If a patient has previously received treatment to block CTLA-4, PD-1, PD-L1, or PD-L2, with the exception of the following: Patients who have received adjuvant therapy with these treatments are eligible.

[0212] When it is anticipated that a patient will require other forms of systemic or local anti-cancer therapy while undergoing research treatment.

[0213] If a patient has received systemic steroids or other forms of immunosuppressant drugs within one week of the first dose of the study drug, for example, systemic administration of physiological doses of steroids for the management of adrenal insufficiency, as well as topical and inhaled steroids for the management of asthma, etc., is acceptable.

[0214] If the patient has received treatment with any investigational product within 28 days prior to the first dose of the research drug (or five times the half-life of the relevant treatment).

[0215] If the patient has a prior (within the last 5 years) or current malignant tumor, excluding melanoma and other locally treated tumors such as in situ carcinomas of the breast or cervix, basal cell or squamous cell carcinoma of the skin, or prostate cancer with a Gleason score of less than 6 and prostate-specific antigen within the normal range.

[0216] Patients with uncontrolled medical conditions, uncontrolled and active infections (opportunistic, life-threatening, or clinically significant), uncontrolled bleeding risk, or uncontrolled diabetes mellitus or lung disease (including a history of obstructive pulmonary disease, pulmonary fibrosis, and symptomatic bronchospasm), or alcoholic liver disease or primary biliary cirrhosis, which may interfere with the performance and evaluation of the study. Patients suspected or diagnosed with epilepsy should be given special attention.

[0217] If the patient has NYHA class III or IV cardiac disease, myocardial infarction within the past six months, a heart rate of 50 beats per minute or less, a history of significant cardiac abnormalities and / or clinically significant abnormal baseline ECG readings, other uncontrolled cardiac conditions such as active ischemia or angina, clinically significant arrhythmias requiring treatment including anticoagulants, uncontrolled hypertension (greater than 140 / 90 mmHg), significant cerebrovascular disease, or congestive heart failure.

[0218] If the patient has a history of severe hypersensitivity reactions to mAb treatment.

[0219] Patients with a history of active autoimmune disease or documented autoimmune disease or syndrome requiring systemic steroid administration or immunosuppressants are excluded (with the exception of patients with vitiligo or resolved childhood asthma / atopic dermatitis, who are not excluded). Patients who require intermittent use of bronchodilators or topical steroid injections, patients with stable hypothyroidism on hormone replacement therapy, and patients receiving physiological doses of steroids as replacement therapy, for example, for the management of adrenal insufficiency. In such cases, the recruiting physician should review the patient's eligibility with the study's medical monitor before recruitment.

[0220] If the patient has received the vaccine within 28 days of the first dose of the research drug.

[0221] If the patient has a known history of HIV, or has tested positive for HBV or HCV and is showing active acute or chronic infection.

[0222] If the patient has a known current or recent history (within the past year) of substance abuse, including illegal drugs or alcohol.

[0223] 5.3 Treatment discontinuation SCIB1 treatment will be permanently discontinued in the following cases: 1) Any DLT related to vaccines 2) Confirmation of disease progression and / or initiation of new anti-cancer treatment. 3) Pregnancy 4) Request for discontinuation of treatment by the clinical trial sponsor 5) Request for discontinuation of treatment by the patient (or their legal representative) 6) Severe or life-threatening adverse events that require permanent discontinuation of treatment in the opinion of the attending physician. 7) In the opinion of the principal investigator, the treatment, procedures and evaluation of the study were not followed; or 8) Diseases that may interfere with the continuation of SCIB1 treatment.

[0224] At the time of discontinuation of treatment, the responsible physician will record the reason for discontinuation in the patient's medical record and CRF. The patient may decide to discontinue treatment, but may continue follow-up and monitoring for disease progression, initiation of new cancer therapies, and survival.

[0225] If SCIB1 treatment is discontinued, the patient will complete the treatment completion visit and begin post-treatment follow-up.

[0226] The combination of nivolumab and ipilimumab, or pembrolizumab, should be permanently discontinued in the event of severe or life-threatening adverse reactions, as detailed in the relevant SmPC and clinical guidelines.

[0227] 5.4 Withdrawal Criteria Patients will withdraw from the study in the following cases: • If the patient (or their legal representative) withdraws their consent If the patient requests the permanent cessation of treatment and all subsequent research-related activities, including the collection of follow-up information from the patient / caregiver, relatives, medical records, or healthcare providers; or • If the clinical trial sponsor decides to discontinue the study.

[0228] Patients who permanently discontinue treatment for other reasons (as defined in Section 5.3) will be considered to have withdrawn from the study only if follow-up information cannot be obtained from the aforementioned sources.

[0229] At the time of withdrawal, the principal investigator will record the reason for discontinuation in the patient's medical record and CRF. The patient may discontinue treatment without withdrawing from the study, as outlined in Section 5.3.

[0230] 5.4.1 Replacement patients In the run-in subcohort, patients who drop out before week 5 (day 36) for reasons other than DLT, or who have not completed safety assessments to the extent that the overall safety of the vaccine cannot be assessed, will be replaced. Patients who are unassessable for the primary endpoint of ORR according to RECIST 1.1 will be replaced as needed, as there are 43 evaluable patients in cohort 1 and 44 evaluable patients in cohort 2. Patients are considered evaluable for the primary endpoint if they have received at least one dose of SCIB1, have completed at least one post-baseline tumor assessment by week 25 (day 176), have dropped out of the study due to disease progression or SCIB1 treatment-related toxicity, or have died due to melanoma-related complications or SCIB1-related toxicity.

[0231] 5.4.2 Rescreening Re-screening of patients who previously failed screening is only permitted after consultation with the sponsor's medical monitor. Screening procedures performed outside the screening time frame must be repeated.

[0232] 5.5 Research completed Grade 3 or higher adverse events (AEs), laboratory values, and SAEs (Severity-Associated Exposures) will be frequently reviewed by the principal investigator and the sponsor. The study will be temporarily suspended if a death causally related to SCIB1 occurs or if the cumulative total number of Grade 4 events causally related to SCIB1 exceeds three. The study may be resumed after evaluation of the cause of toxicity and implementation of a revised plan (if necessary), provided that the principal investigator and the sponsor deem it appropriate. The study may be terminated in whole or in part at any time if the toxicity of SCIB1 in combination with nivolumab and ipilimumab or pembrolizumab no longer supports the continuation of the investigation in terms of risk / benefit ratio and if the justification for recruiting additional patients is not demonstrated from a medical or ethical standpoint. This decision may be made by the principal investigator together with the sponsor or by the REC or the national regulatory authority. The sponsor may also decide to terminate the study if recruitment is delayed to a sufficient extent to prevent completion of the study within an acceptable timeframe or if the development of SCIB1 is discontinued. The study may also be terminated if there are five or fewer clinical responses observed within 25 weeks of the first dose of SCIB1 (RECIST 1.1 objective response [CR or PR]).

[0233] If the study is terminated, the sponsor will notify the principal investigator and the relevant national health authorities. The principal investigator must promptly notify all participating patients and clinical trial participants (RECs) of the study's termination.

[0234] 6 Treatment 6.1 Surveyed Pharmaceutical Products The IMP in this study is SCIB1, which is a plasmid DNA (laboratory designation pVaxDCIB68) designed to express a modified human IgG1 antibody molecule containing two helper T cell sequences derived from the melanoma gp100 protein, in addition to a CTL epitope derived from the TRP-2 melanoma antigen. One of the gp100 sequences also incorporates a nested CTL epitope. SCIB1 is formulated in phosphate-buffered saline at a concentration of 4 mg / mL. Each 2 mL glass vial is sealed with a FluroTec® stopper and flip-off cap, with an extractable volume of 1.0 mL.

[0235] 6.1.1 Non-surveyed drug product: Combination therapy of nivolumab and ipilimumab The combination of nivolumab and ipilimumab is indicated for the treatment of unresectable or metastatic melanoma in adult patients. This combination has since been adopted and covered internationally, including by the UK's National Health Service (NHS). The combination of nivolumab and ipilimumab is available as standard treatment in hospital pharmacies and is used according to prescription drug information and local guidelines.

[0236] 6.1.2 Non-surveyed medical product: Pembrolizumab Pembrolizumab is approved in the European Union, the United States, Australia, and the United Kingdom for the treatment of patients with unresectable or metastatic melanoma. Pembrolizumab is available as standard treatment in hospital pharmacies and is used in accordance with prescription drug information and local guidelines.

[0237] 6.2 Preparation, handling, storage, management and disposal of research pharmaceutical products 6.2.1 SCIB1 SCIB1 should be stored frozen at -20°C (±5°C) / -5°F (±10°F) and thawed before use. Further details are provided in the IMP instruction manual.

[0238] 6.2.2 Checkpoint Inhibitors Treatment with either nivolumab and ipilimumab in combination or pembrolizumab is at the discretion of the principal investigator and follows standard treatment, as detailed in the relevant SmPC and clinical guidelines.

[0239] The first dose of checkpoint inhibitors should be administered in the first week after the first dose of SCIB1 to ensure a proper primary immune response (Massarelli et al 2019). Treatment with checkpoint inhibitors beyond the 85-week study period is at the discretion of the principal investigator as part of standard care. Storage, packaging, and handling of checkpoint inhibitors should follow SmPC guidelines as appropriate.

[0240] 6.2.3 Drug Management Drug management records shall include the amount of SCIB1 received and shall be maintained for all instances in which SCIB1 is dispensed. These records shall include the patient's study identifier number; the date, time, and number of vials of drug dispensing; the date and quantity administered; and the date and number of vials of any drug returned to the pharmacy for disposal or redistribution in accordance with local standard operating procedures (SOPs). These records shall be available for periodic audits by the study monitor. The principal investigator shall be responsible for ensuring that all used and unused study supplies are managed. Unused SCIB1 vials shall not be discarded or returned to central storage until the management of the study drug is carried out.

[0241] SCIB1, an IMP (Informed Medication Program), will only be used in patients who have provided informed consent and have been enrolled in this study.

[0242] 6.3 SCIB1 Administration Procedure SCIB1 is administered to patients using the PharmaJet Stratis® needleless intramuscular injection device. The administration procedure is performed by on-site personnel who are qualified to administer intramuscular injections and have completed a training seminar for the device. Each administration consists of four injections administered at different sites. Eligible injection sites include the lateral upper arms (mid-deltoid) or the lateral thighs (vastus lateralis). Device setup and administration of SCIB1 to patients are performed as described in the PharmaJet Stratis® needleless injection instructions. To ensure patient and research personnel safety, standard techniques and precautions for intramuscular administration are used in site preparation, SCIB1 administration, and medical waste disposal.

[0243] Any patients recruited under a previous version of the protocol and who have already received SCIB1 using a TDS-IM electroporation device will re-consent to continue administration using a PharmaJet Stratis® device.

[0244] 6.4 Checkpoint Inhibitors: Use and Precautions The reconfiguration, preparation, and administration of checkpoint inhibitors must be in accordance with the SmPC's instructions. Management of adverse reactions and modification of the treatment schedule are described in Section 6.6.

[0245] 6.5 Treatment Schedule Patients will receive four intramuscular injections of 2 mg of SCIB1, administered using the PharmaJet Stratis® needleless injection device at weeks 0, 4, 7, 13, and 25 (days 1, 29, 50, 92, and 176, respectively), and thereafter every 12 weeks until week 85 (day 596). On day 1, patient eligibility must be confirmed before the first dose of SCIB1. On days when both SCIB1 and either nivolumab and ipilimumab or pembrolizumab are administered, SCIB1 must be administered before the combination of nivolumab and ipilimumab or pembrolizumab.

[0246] 6.6 Dosage adjustment, modification, and delay 6.6.1 Infusion reactions Management of infusion reactions associated with checkpoint inhibitor administration follows standard practice, as detailed in relevant SmPC and clinical guidelines. On the day of administration, monitor the patient for infusion reactions for at least one hour after the last dose of the checkpoint inhibitor.

[0247] 6.6.2 Dosage adjustment and modification Dose reduction of SCIB1 is not considered meaningful in this study. Toxicity management will be carried out by treatment delay as described in the following section.

[0248] 6.6.3 Delay in treatment and discontinuation of checkpoint inhibitors In the event of an adverse event, the responsible physician should consider interrupting, discontinuing, or resuming the checkpoint inhibitor, as detailed in the relevant SmPC and national or regional clinical treatment guidelines.

[0249] SCIB1 may be continued in patients if either nivolumab and ipilimumab combination therapy or pembrolizumab is interrupted or discontinued, provided that the physician's opinion is that the patient intends to continue. In addition, the patient must demonstrate that there is no evidence of clinical disease progression, and any adverse reactions that led to the interruption of checkpoint inhibitors must have improved by the time of the next SCIB1 administration. Patients with BRAF mutation-positive disease must be informed of the available BRAF-targeted therapy options and must re-consent to continue receiving SCIB1 as monotherapy. Administration of SCIB1 will be continued according to the schedule in Table 2.

[0250] For example, if SCIB1 is not administered due to an adverse event, the missed dose may be administered after week 13, at least 15 days apart, but preferably with a maximum interval of 6 weeks, according to the schedule in Table 2.

[0251] Specific recommendations regarding immune-related adverse events Immunotoxicities may be managed by corticosteroid immunosuppression. Specific management of immunotoxicities should follow the guidance in the SmPC for checkpoint inhibitors.

[0252] 6.7 Preceding and concomitant use of medicines and treatments Details of prior medical treatments administered to the patient during the 30 days prior to Day 1 will be collected. All concomitant medications administered during the study treatment and during the 30 days following the last dose of SCIB1 treatment must be recorded in the CRF. The record of concomitant medications must include the generic name, indication, dosage, start and end dates, and route of administration.

[0253] 6.7.1 Contraindicated Medications and Therapies Other forms of systemic treatment for melanoma are not permitted while the patient is receiving treatment under this protocol. This includes chemotherapy, biological therapies, other forms of immunotherapy, and gene therapy. Surgical and local radiotherapy for skin lesions are not permitted and should be discussed in advance with the medical monitor of the study. Systemic corticosteroids and other immunosuppressants are not permitted, except as described in Section 6.7.2.

[0254] 6.7.2 Permitted Medications, Prior Medications and Adjunctive Therapies Systemic corticosteroids are permitted only for the management of immune-related adverse events as described in Section 6.6 (excluding physiological replacement therapy). For patients whose immune-related adverse reactions cannot be controlled by corticosteroids, other systemic immunosuppressants may be considered. Systemic administration of physiological doses of steroids, such as for the management of adrenal insufficiency, as well as topical and inhaled steroids, such as for the management of chronic medical conditions like asthma, are permitted. Vaccines are permitted (including influenza and COVID-19 vaccines, unless they are live vaccines), but must be scheduled between SCIB1 administrations starting from week 7 (day 50) so as to be 28 days before the next SCIB1 administration. Live vaccines, including intranasal influenza vaccines, are not permitted.

[0255] 6.8 Monitoring of patient compliance The principal investigator and clinical staff are responsible for monitoring patient compliance. Study drug compliance checks should be conducted after the initial dispensing of the study drug and during each visit, and study drug management records should be completed. Patients who do not comply with the study's treatment, procedures, and evaluations should be withdrawn after consultation between the principal investigator and the sponsor, and the REC should be notified.

[0256] 7. Evaluation of effectiveness 7.1 Tumor imaging and RECIST evaluation The primary efficacy measure of the study is the tumor response, measured according to the RECIST 1.1 criteria (Eisenhauer et al. 2009, Appendix A). The response using iRECIST (Seymour et al. 2017, Appendix B) is also assessed as a secondary efficacy measure.

[0257] Tumor imaging (chest, abdomen, pelvis, brain, and any known area of ​​disease) should be performed within 28 days prior to the first dose of SCIB1. CT scans are the preferred imaging technique unless the patient has a medical reason, such as a contrast agent allergy, that makes CT scans risky, or the disease is well documented by MRI. The same technique must be used for individual patients throughout the study. A baseline brain MRI (preferred) or CT scan is required to establish the presence or absence of brain metastases. Repeated brain imaging is required only if the patient has brain metastases at baseline or if new brain metastases are suspected. Patients with either an objective tumor response or PD should undergo repeat scans at least 4 weeks and at a maximum of 8 weeks to confirm the response and rule out the possibility of a tumor flare response, respectively. Response status will be assessed by the principal investigator (according to RECIST 1.1 and iRECIST). Anonymized copies of imaging data will be collected for the study database. Imaging visit slots are within ±7 days, and the timing of imaging visits will follow the calendar; treatment delays will not be adjusted. Tumor evaluation will be performed during treatment or post-treatment follow-up at the times shown in Table 2: Procedure and Evaluation Schedule.

[0258] At least one measurable lesion according to RECIST 1.1 criteria must be present at baseline. A measurable lesion is defined as a lymph node with a longest diameter of at least 10 mm measured by CT scan, or a short axis of at least 15 mm (at a 5 mm CT scan slice thickness). Skin lesions should be recorded with digital photographs. Copies of the digital photographs should be collected for the research database.

[0259] The iRECIST assessment will be used to determine patient management in the study and to decide on discontinuation. If a patient is determined to have iRECIST PD, confirmation of this will be required in a second study treatment scan. The second scan will take place at least 4 weeks and at most 8 weeks later. Patients may continue to receive study treatment during this period at the discretion of the principal investigator, provided that the patient is still adequately tolerant of study treatment and has not experienced any deterioration in performance status, any clinical increase in disease-related symptoms, or any need for enhanced management of disease-related symptoms. Detailed criteria for determining and confirming disease progression using iRECIST are provided in Appendix B.

[0260] 7.2 Progression-free survival and overall survival Patients will be evaluated for disease progression and survival for 97 weeks from the first dose of SCIB1. The following will be recorded: • Disease status and date of evaluation • Dates of disease progression according to RECIST 1.1 and iRECIST • Date of commencement of any new cancer treatment and generic name of the treatment agent • Date of death due to any cause, primary cause

[0261] 8. Pharmacodynamic evaluation and biomarker evaluation 8.1 Blood sample In a limited number of facilities, blood samples (60 mL) will be collected for the analysis of cellular immune responses and liquid biomarkers.

[0262] 8.1.1 Handling and transportation of samples Blood samples are transported to the Scancell laboratory at ambient temperature within 8 hours of collection. Lymphocytes and plasma are prepared and tested immediately, or stored frozen in a cryogenic freezer until analysis.

[0263] 8.1.2 Evaluation and Analysis of Study-Specific Blood Samples Samples are evaluated for cellular immunity to TRP-2 and gp100 peptides by antigen-specific T cell activation of lymphocytes measured by the IFNγ ELISpot assay. Immunological response data, including frequency and avidity, are tabulated and plotted, where applicable, to descriptively assess the response pattern across the study.

[0264] The T cell frequency for each antigen before and after treatment is evaluated using the ELISpot assay. Patient designation as an immunologist is defined in the SAP.

[0265] Additional analyses may be performed on a subset of the sample, as follows: CFSE proliferation assay, CTL lysis assay, MHC multimer staining of memory cells, assay to assess epitope expansion, Luminex assay for cytokine secretion and T cell subset analysis, and liquid biomarker analysis. Assays should be performed in accordance with the response confirmed by the ELISpot assay and when sufficient cells and plasma have been recovered from the blood sample.

[0266] 8.2 Tumor Samples Formalin-fixed, paraffin-embedded (FFPE) storage (within 5 years) or a fresh tumor biopsy is required at baseline for PD-L1 expression analysis. PD-L1 analysis may be performed in an accredited laboratory.

[0267] 8.2.1 Evaluation and Analysis of Study-Specific Tumor Samples FFPE tumor tissue samples will be submitted for PD-L1 expression analysis using appropriate quantitative immunohistochemical assays.

[0268] 9. Safety Evaluation 9.1 Adverse Events 9.1.1 Definition of Adverse Events An adverse event (AE) is any undesirable medical experience or change in pre-existing condition that occurs during or after administration of SCIB1, nivolumab and ipilimumab in combination, or pembrolizumab, regardless of whether it is considered to be related to these products or other test procedures (in accordance with Code of Federal Regulations 21 [CFR] 312.32a and ICH E2A guidance). Abnormal laboratory findings and other abnormal investigational findings (e.g., ECG traces) shall be recorded as AEs if the competent physician considers them to be medically significant.

[0269] In this study, disease progression is predicted and will not be recorded as an adverse event (AE). However, medical events and conditions associated with disease progression (e.g., respiratory failure, weight loss) will be recorded as AEs.

[0270] 9.1.2 Recording and reporting of adverse events AEs will be collected from the time of the initial study-related procedure (including study-specific screening procedure) up to 30 days after the last dose of SCIB1 or until the next dose of the checkpoint inhibitor, whichever is longer.

[0271] For patients who have signed informed consent for the primary study, all adverse events (AEs) must be recorded in the patient's CRF, including a brief medical description of the event, the date of occurrence, the date of resolution or, if unresolved, its status, severity, relationship to SCIB1 (IMP), relationship to needle-free injection procedures, relationship to either nivolumab and ipilimumab combination therapy or pembrolizumab (non-IMP), actions taken, and details of treatment outcomes. The principal investigator and clinical staff will work with the sponsor to investigate and follow up on any AEs of concern.

[0272] All AEs are evaluated to determine whether they meet the criteria for reporting as SAEs, as defined in Section 9.2.1.

[0273] 9.1.3 Severity of adverse events The severity of AEs is graded according to CTCAE v5.0. For AEs not included in CTCAE, the following general guidelines for CTCAE grading are followed: Grade 1: Mild, asymptomatic, or mildly symptomatic; clinical or diagnostic observation only; no intervention indicated. Grade 2: Moderate, minimal local or non-invasive intervention is indicated; for example, age-appropriate instrumental activities of daily living (ADL) such as preparing meals, shopping for groceries or clothing, using the telephone, and managing money are limited. Grade 3: Severe or medically significant but not immediately life-threatening; hospitalization or extended hospitalization is indicated; functional impairment; ADLs such as bathing, dressing, eating, toilet use, and medication are limited, but the patient is not bedridden. Grade 4: Life-threatening consequences; emergency intervention is indicated. Deaths related to Grade 5 AEs

[0274] 9.1.4 Relationship between adverse events and research treatments or devices An AE is determined by the responsible physician to be related to or unrelated to each of the following: IMP, non-IMP, or PharmaJet Stratis® needle-free injection procedures. For an event to be considered related, there must be evidence of exposure to the product or procedure with a reasonable temporal relationship. An AE is considered more likely to be explained by the product or procedure than by another cause; this includes events that are considered potentially related to the product or procedure. Events that do not meet these conditions are reported as unrelated.

[0275] 9.1.5 Protocol deviations due to adverse events Deviations from the protocol may be permitted on a case-by-case basis only in patients with adverse events (AEs) or medical emergencies. The principal investigator or other attending physicians must contact the sponsor's medical expert as soon as possible to discuss the deviation and whether the patient should continue participating in the study.

[0276] 9.2 Serious Adverse Events 9.2.1 Definition of serious adverse events A SAE is any undesirable medical event that meets one or more of the following criteria: • Anything that causes death or threatens life, except when death is solely due to the progression of the disease. * • Cases requiring hospitalization or requiring an extension of existing hospitalization. ** • Those that result in significant functional impairment / loss (substantial loss of the ability to perform normal daily living activities). • Congenital abnormalities / birth abnormalities; and / or A significant medical event that does not cause death, threaten life, or require hospitalization, but which may endanger the patient and may require medical intervention to prevent one of the conditions listed in these criteria. * This refers to the risk to the patient at the time of the event and does not include events that could have been life-threatening if they had been more severe. ** Hospitalizations for scheduled surgery, rehabilitation, social reasons, or other procedures that were planned before the signing of informed consent are not classified as SAEs.

[0277] 9.2.2 Predictability For IMPs, SAEs that do not include details, severity, outcome, or frequency in the current Investigational Briefing (IB), or for non-IMPs, in the SmPC's reference safety information, are considered unpredictable.

[0278] 9.2.3 Reporting of serious adverse events SAEs will be collected from the time of the initial study-related procedure (including study-specific screening procedures) up to 30 days after the last dose of SCIB1 or until the next dose of the checkpoint inhibitor, whichever is longer. In addition, any SAEs recognized by the principal investigator or clinical practice staff and assessed as related to study participation (SCIB1 treatment or procedure) must be documented, including any follow-up communications until resolution or improvement.

[0279] Any SAEs (Surface-Affected Events) that occur while a patient is participating in the study must be reported to the safety contact within 24 hours of the incident being recognized, even if the SAE is not considered to be study-related. This is done by sending a copy of the SAE report to the safety contact via email or fax. The completion and submission of the SAE report must not be delayed, even if not all information is available. Contact details for reporting SAEs will be provided before the first patient is enrolled.

[0280] Patients exhibiting a SAE should be closely monitored until the event resolves or is deemed stable by the principal investigator, and the principal investigator and clinical staff should collaborate with the sponsor's safety officer to understand the underlying cause of the event. Any additional follow-up information related to the event should be reported to the safety contact as soon as possible after it becomes available.

[0281] The sponsor shall ensure that all ICH GCP guidelines and relevant regional and national regulatory requirements are met regarding the reporting timeline for suspected unexpected serious adverse reactions (SUSARs) and the preparation of periodic summary reports. The principal investigator shall be required to receive notification from the sponsor for all SUSARs and to comply with regional reporting requirements regarding REC notifications.

[0282] 9.3 Pregnancy Following administration of the research drug, any pregnancy in any female patient or any male patient's female partner will be tracked until the pregnancy ends. Pregnancy must be reported immediately within 24 hours of the event by submitting a completed pregnancy report to the safety office by telephone, email, or fax. The principal investigator must track the patient until the pregnancy ends and evaluate the outcome as soon as possible after delivery or completion, but within 30 days. The principal investigator will notify the safety office of the pregnancy outcome by submitting a follow-up pregnancy report. The continuation of research treatment by one or both pregnant female patients will be confirmed on a case-by-case basis by the principal investigator and the sponsor.

[0283] If the pregnancy outcome meets the criteria for immediate classification of SAE (voluntary or therapeutic abortion, stillbirth, neonatal death, or birth defects), the attending physician must report the event within 24 hours of becoming aware of the event by telephone, email, or fax, submitting a completed SAE report. Any birth defects observed in the aborted fetus must also be recorded.

[0284] 9.4 Dosage and medical errors related to research and treatment Any SCIB1 administration errors (dosage and timing other than those specified in the protocol) shall be recorded, as shall any malfunctions of the PharmaJet Stratis® needleless injection device. Any overdose (overdose on a single day or administration at a shorter frequency than specified in the protocol) shall be reported immediately to the sponsor. Administration errors related to the combination of nivolumab and ipilimumab or pembrolizumab administration during the SCIB1 treatment phase (up to end-of-treatment) shall be recorded and reported to the sponsor. Any AEs or SAEs arising from research drug errors shall be reported as described in the previous section.

[0285] 9.5 Clinical and laboratory evaluation and testing Study-specific procedures shall not be performed until the patient provides written informed consent. Clinical data obtained from procedures performed during the patient's routine treatment may be used for screening purposes, provided that they are within the specified screening time frame. Laboratory values ​​and test results after adverse events may be captured.

[0286] 9.5.1 Serological Chemistry, Hematology, and Urinalysis Serological, hematological, and urine samples should be collected according to the schedule shown in Table 2. These samples should be obtained before administration and analyzed by a local laboratory.

[0287] Serological chemistry tests consist of albumin, ALT, alkaline phosphatase, AST, bicarbonate or carbon dioxide, urine, calcium, chloride, creatinine, total bilirubin (and direct bilirubin if clinically indicated), gamma-glutamyl transferase, LDH, non-fasting glucose, phosphorus (measured as phosphate), potassium, sodium, uric acid (urate), and total protein.

[0288] Hematological tests include hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin level, mean corpuscular hemoglobin concentration, platelet count, red cell count, white cell count, and white cell differential (basophils, eosinophils, lymphocytes, monocytes, neutrophils).

[0289] A urine test (dipstick) records protein, glucose, ketones, blood, nitrite, white blood cells, bilirubin, pH, and specific gravity. Microscopic examination is performed only if clinically indicated: bacteria, RBCs, WBCs, casts, and crystals.

[0290] Serological, hematological, and urinalysis performed as part of the screening procedure within 48 hours of the first dose of the research drug do not need to be repeated on day 1.

[0291] 9.5.2 Other Clinical Laboratory Tests HLA tissue typing HLA testing will be performed in an accredited laboratory using molecular (DNA) typing. A separate consent form will be used for HLA typing to limit the number of invasive screening tests required for patients who are ineligible for the study based on their HLA type. Eligible patients must be HLA-A2 positive and also positive for HLA-DR4, HLA-DR7, HLA-DR53, or HLA-DQ6.

[0292] Pregnancy testing and verification of postmenopausal status For women of reproductive capacity (including those within 12 months of their last menstrual period), a urine pregnancy test will be performed within 72 hours prior to the first dose of the study drug on day 1. If the urine pregnancy test is not negative, a serum pregnancy test will be required. Pregnancy tests (serum and / or urine) will be performed at the intervals shown in Table 2. Serum or urine pregnancy tests will also be performed for these patients during EOT visits. In practice, it may be chosen to use serum pregnancy tests from the outset.

[0293] Hepatitis test Blood samples are obtained during screening for HBV and HCV testing.

[0294] blood coagulation test The International Normalized Ratio (INR) and activated partial thromboplastin time (aPTT) are determined during screening and EOT visits.

[0295] Thyroid function test Samples for measuring free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH) should be collected according to the schedule shown in Table 2. Samples should be collected before administration. Thyroid function tests performed as part of the screening procedure within 48 hours of the first dose of the research drug do not need to be repeated on day 1. Thyroid function tests may be performed at an accredited affiliate laboratory if these tests cannot be performed at the research facility's local laboratory.

[0296] 9.5.3 Medical History The patient's medical history will be recorded at the time of screening, and this will include details of chronic diseases, serious previous illnesses, all current illnesses, and current concomitant medications.

[0297] 9.5.4 Physical Examination A complete physical examination includes evaluation of the eyes, nervous and cardiovascular system, lungs, abdomen, head, neck, ears, nose, mouth, throat, thyroid, lymph nodes, limbs, skin, and other areas showing signs and symptoms of disease, and should be performed at the times shown in Table 2. Height and weight should be recorded at the time of screening, and weight alone should be recorded at other times as well. During visits for checkpoint inhibitors only, a “limited” physical examination may be performed as needed, as specified in Table 2 (cardiovascular system, lungs, abdomen, skin, weight, and areas showing signs and symptoms of disease). The physical examination should be performed before administration.

[0298] Skin lesion evaluation will be performed according to the schedule in Table 2. Photographs will be taken to record changes in existing skin lesions and new lesions.

[0299] 9.5.5 Injection site reactions Each injection site will be evaluated approximately one hour after the completion of SCIB1 administration. Evaluation of each injection site will also be performed during some study visits when the study drug is not administered, as outlined in Table 2. Pain, tenderness, erythema, and swelling will be evaluated on a scale of 0 to 3, where 0 is absent, 1 is mild, 2 is moderate, and 3 is severe (Appendix C). If moderate or severe injection site reactions are observed, digital photographs of the injection site will be taken at the times specified in Table 2 or at other times as necessary. Anonymized copies of the digital photographs will be sent to a dedicated database via secure file transfer.

[0300] 9.5.6 Vital Signs Vital signs should be measured approximately 10–15 minutes before administration, and then again 5 minutes, 30 minutes, and 1 hour after SCIB1 administration. Patients should be observed for at least 1 hour after receiving either nivolumab and ipilimumab combination therapy or pembrolizumab for at least the first two doses. Vital signs should also be measured during EOT visits. Vital signs include body temperature, heart rate, respiratory rate, and blood pressure.

[0301] 9.5.7 Electrocardiogram (ECG) Test A 12-lead ECG will be performed at the times shown in Table 2. The ECG findings will be interpreted by a qualified physician. Clinically significant findings obtained from the screening ECG will be recorded in the patient's medical history. Clinically significant findings obtained from the study ECG will be recorded as adverse events (AEs).

[0302] 9.5.8 Ophthalmic Examination Patients should undergo fundus examinations performed by trained healthcare professionals, such as ophthalmologists, optometrists, or other healthcare professionals, during screening and if clinically required if the patient experiences any visual impairment during the study.

[0303] 9.5.9 Performance Status Evaluation ECOG performance status should be assessed at the points shown in Table 2. Performance criteria are included in Appendix D. ECOG performance status should be assessed before administration.

[0304] 10 Statistics The following is an overview of the statistical analysis planned for this study. Further details are provided in the Statistical Analysis Plan (SAP).

[0305] 10.1 Consideration of Sample Size and Power 10.1.1 Run-in subcohort Run-in subcohorts are included to establish the safety and tolerability of SCIB1 treatment in patients receiving either nivolumab and ipilimumab combination therapy or pembrolizumab to a degree sufficient to justify additional recruitment in the overall study. For each cohort, a run-in subcohort of six patients is planned to identify DLTs. Each cohort will proceed only if no more than two of the six patients in the run-in subcohort have SCIB1-related DLTs. Run-in subcohort patients who drop out of the study before week 5 (day 36) for reasons other than DLTs will be replaced. If two or more patients in the run-in subcohort have SCIB1-related DLTs, the data will be formally reviewed by the principal investigator and sponsor to determine whether it is appropriate to recruit an additional six patients for the run-in subcohort with reduced doses of SCIB1.

[0306] 10.1.2 Cohort 1: Combination therapy with nivolumab and ipilimumab Forty-three patients will be recruited using Simon's optimal two-stage design (Simon 1989). Six of these patients will form a run-in subcohort for formal evaluation of DLT for combinations.

[0307] Patients are considered evaluable for the primary endpoint if they have received at least one dose of SCIB1, have completed at least one post-baseline tumor assessment by week 25 (day 176), have withdrawn from the study due to disease progression or SCIB1 treatment-related toxicity, or have died due to melanoma-related complications or SCIB1-related toxicity. Unevaluable patients will be replaced. This sample size will be determined based on Simon's two-stage design. The sample size calculation assumes that a 50% ORR is not of interest, as it is approximately the same as the observed ORR for nivolumab and ipilimumab combination therapy in this patient group (Lebbe 2019).

[0308] In contrast, an ORR of 70% is considered a positive signal for further development of SCIB1 administered in addition to the combination of nivolumab and ipilimumab. This clinical trial is designed with the null hypothesis that the ORR will be 50%. In Phase 1, 15 patients will be recruited, and if there are 8 or fewer clinical responses (RECIST 1.1 objective response [CR or PR], within 25 weeks of the first dose of SCIB1), further recruitment for this cohort will be stopped. Recruitment will continue until a Phase 1 decision is made. The null hypothesis will be rejected if 27 or more responses are observed in 43 patients. This design yields a Type I error rate of 5% and a power of 80% when the true response rate is 70%.

[0309] 10.1.3 Cohort 2: Pembrolizumab Across the entire cohort, a total of 44 evaluable patients (including 6 from the run-in subcohort) will be enrolled using Simon's optimal two-stage design (Simon 1989). Patients will be considered evaluable for the primary endpoint if they have received at least one dose of SCIB1, have completed at least one post-baseline tumor assessment by week 25 (day 176), have withdrawn from the study due to disease progression or SCIB1 treatment-related toxicity, or have died due to melanoma-related complications or SCIB1-related toxicity. Patients who are not evaluable will be replaced.

[0310] The sample size will be determined based on Simon's two-stage design. The sample size calculation assumes that a 30% ORR is not of interest, as it is approximately the same as the observed ORR for pembrolizumab monotherapy in this patient group (Robert et al. 2015b). In contrast, a 55% ORR is considered a positive signal for further development of the combination. This clinical trial is designed with the null hypothesis that the ORR will be 35%. In the first stage, 14 patients will be recruited, and if there are 5 or fewer clinical responses (RECIST 1.1 objective response [CR or PR], within 25 weeks of the first dose of SCIB1), further recruitment for this cohort will be stopped. Recruitment will continue until the first stage decision is made. The null hypothesis will be rejected if 21 or more responses are observed in 44 patients at the end of the study. This design yields a 5% Type I error rate and 80% power when the true response rate is 55%.

[0311] If a higher-than-expected proportion of patients were receiving PD-1 inhibitors in adjuvant settings, these assumptions are adjusted to reflect the lower expected response rate in these patients with pembrolizumab monotherapy (estimated ORR of less than 10%).

[0312] 10.2 Analysis Set 10.2.1 Complete Analysis Set The FAS consists of all admitted patients who have received at least one dose of either SCIB1, nivolumab and ipilimumab combination, or pembrolizumab.

[0313] 10.2.2 Safety Analysis Kit SAF consists of all promoted patients who have received at least one dose of either SCIB1, nivolumab and ipilimumab combination, or pembrolizumab.

[0314] 10.2.3 Sets per protocol The PPS consists of all admitted patients who are evaluable for the primary endpoint of tumor response according to the RECIST 1.1 criteria. A patient is included in the PPS if: • If SCIB1 is administered at least once and at least one post-baseline tumor assessment is completed by week 25 (day 176) • Death due to complications related to melanoma or toxicity related to SCIB1; or • If the participant withdraws from the study due to disease progression or SCIB1 treatment-related toxicity. • If there are no significant protocol deviations that are considered to have a substantial impact on safety or efficacy results.

[0315] 10.3 Data Handling Practices For all variables, only data observed from patients will be used in the statistical analysis. Unless otherwise specified in SAP, there are no plans to impute missing data.

[0316] 10.4 Interim analysis Six patients from the run-in subcohort will be included, and data safety will be confirmed after evaluation up to a maximum of 35 days.

[0317] Once 15 evaluable patients (in the nivolumab and ipilimumab combination cohort) or 14 patients (in the pembrolizumab cohort) have been recruited and followed up for response, an interim analysis will be conducted at the end of Phase 1. If there are 8 or fewer responders out of 15 patients in the nivolumab and ipilimumab combination cohort, or 5 or fewer responders out of 14 patients in the pembrolizumab cohort, the trial will be terminated. Otherwise, the trial will continue to Phase 2, recruiting an additional 28 patients (in the nivolumab and ipilimumab combination cohort) or 30 patients (in the pembrolizumab cohort).

[0318] As part of the interim analysis, the feasibility of further enrollment in cohorts that have not met their initial allocation will be assessed once the other cohort reaches the initial allocation. A Clinical Study Group (CSR) will be formed once all patients have completed research treatment. A CSR addendum will be created once all patients have completed post-treatment follow-up.

[0319] 10.5 Research Group FAS is used to present baseline and efficacy parameters. In addition, PPS is used to evaluate efficacy parameters. SAF is used to present safety parameters.

[0320] 10.5.1 Breakdown of patients Summarize the number of patients screened, enrolled, and in each analysis population for each portion of the study run-in and the overall study (i.e., all patients). In addition, present the number of patients who complete / do not complete the study, along with the main reasons for discontinuation. Discontinuation will be identified as discontinuation from treatment only or discontinuation from both treatment and the study (e.g., post-treatment follow-up). Summarize the number of patients who complete the study treatment (10 doses of SCIB1).

[0321] 10.5.2 Demographic Variables and Baseline Characteristics Demographic variables and baseline characteristics at the screening point are summarized for each patient in the run inset, and a separate summary is provided for all patients included in the FAS.

[0322] Include the medical history information collected at the time of screening in the patient list.

[0323] 10.6 Effectiveness Analysis Efficacy analysis will be conducted for patients enrolled in the study, based on both FAS and PPS. Efficacy analysis will not be performed at the end of the run-in subcohort.

[0324] 10.6.1 Efficacy Endpoint Objective response rate measured by RECIST 1.1 criteria The primary efficacy endpoint is based on ORR, measured using the RECIST 1.1 criteria. This corresponds to whether the patient achieved a PR or CR at any point after the initiation of treatment. ORR is summarized for all patients in the FAS and PPS. Each patient is summarized by their best response. Patients who are evaluable with respect to the primary endpoint but have not completed at least one post-baseline tumor assessment by week 25 (day 176) are counted as non-responders.

[0325] Response duration The duration of the response is measured from the point at which the metric is met for CR or PR (ORR definition) until the first day PD (according to RECIST 1.1) is recorded or death due to any cause, whichever occurs first. Patients who have neither progressed nor died are censored on the last day of evaluation. The duration of the overall response is estimated using the Kaplan-Meier method and summarized for all patients in the FAS and PPS. The Kaplan-Meier survival function estimates are plotted.

[0326] Immune-related ORR as measured by iRECIST criteria This endpoint is reported in a similar format to the primary endpoint (see above), except that the ORR determination is based on the use of iRECIST criteria (Seymour et al. 2017).

[0327] Progression-free survival The PFS rate at week 49 (day 344) and week 97 (day 680) is defined as the proportion of patients who, at each respective time point, have not progressed (according to RECIST 1.1 and iRECIST), have not initiated new anticancer therapy, and have not died. PFS is summarized for all patients in FAS and PPS.

[0328] Patients who have neither progressed nor died are terminated on the date of their last disease assessment. Patients who initiate new anti-cancer therapy without reporting disease progression are terminated on the date of their last disease assessment before the initiation of the new therapy. Patients who do not complete any tumor assessments and have not died are terminated on the date of their first dose of SCIB1. PFS is estimated using the Kaplan-Meier method.

[0329] Anti-cancer therapy is defined as any systemic treatment for malignant tumors, including melanoma.

[0330] overall survival rate The overall survival (OS) rates at week 49 (day 344) and week 97 (day 680) are defined as the proportion of patients who are still alive at each respective time point. OS is estimated using the Kaplan-Meier method, summarizing data for all patients, including both FAS and PPS.

[0331] 10.6.2 Planned Analysis Method This study has only one treatment group and no hypothesis-test set; therefore, no statistical trials are planned. 95% confidence intervals are presented along with summary statistics where appropriate. Further details are provided within the SAP. For all endpoints with continuous data, the following summary statistics are presented in the descriptive statistics: number of non-missing values, mean, standard deviation, median, lower quartile, upper quartile, minimum, and maximum. Categorical variables are summarized using proportions (counts and percentages). For time variables to events, the following are presented: number of events, number of censored observations, and Kaplan-Meier estimates for the median (50th), 25th, and 75th percentiles, as well as the estimated event rate at a defined time point.

[0332] 10.7 Safety analysis Safety reports will be based on Safety Afflictions (SAF) for both the run-in subset of patients and the entire patient group enrolled in the study.

[0333] 10.7.1 Adverse Events AEs will be coded using the current version of MedDRA at the start of the study. An AE will be considered treatment-induced if its occurrence date is on or after the first dose of the study treatment (SCIB1). AEs occurring before the first dose of the study treatment will be classified as pre-treatment AEs. If a patient experiences the same AE more than once, it will be counted as one AE, with the most severe AE and the AE most relevant to the study treatment being assigned to this count.

[0334] The system generates summaries of the number and percentage of patients who reported treatment-induced adverse events (TEAEs), the severity of the TEAEs, TEAEs by relationship (SCIB1, nivolumab and ipilimumab combination therapy, pembrolizumab, or PharmaJet Stratis® needleless injection procedure presented separately), and treatment-induced serious adverse events (SAEs). In addition, summaries of Grade 3 or higher TEAEs are also presented. For SAFs, separate presentations are generated for (i) patients recruited during the run-in period and (ii) all patients.

[0335] This section lists SAEs and AEs that directly lead to abandonment of research.

[0336] 10.7.2 Laboratory Parameters Summarize laboratory parameters (hematological tests, chemistry, urinalysis, and other laboratory parameters [Section 9.5.2]) for each scheduled visit. Where appropriate, calculate the corresponding CTCAE grade according to the reported values. Summarize the baseline grade and the worst grade after baseline.

[0337] Laboratory values ​​outside the normal range are identified within the patient list.

[0338] 10.7.3 Physical Examination Physical examination data is included in the patient list.

[0339] 10.7.4 Vital Signs Vital sign measurements are summarized as actual values ​​and changes from baseline across scheduled visits (and time-point assessments within each visit).

[0340] 10.7.5 12-lead electrocardiogram The 12-lead ECG data is included in the patient list.

[0341] 10.7.6 Injection site reactions Summarize injection site reactions for each scheduled visit. Record injection site reactions at four injection sites, and summarize the most severe reactions observed across all four sites.

[0342] 10.7.7 Concomitant medications Combination therapies are coded by their generic name and Anatomical Therapeutic Chemistry (ATC) class using the current version of the World Health Organization (WHO) Drug Dictionary at the start of the study. Medications are assigned either as pre- or concomitant to the study treatment. If the discontinuation date of a medication is earlier than the start date of the study treatment (SCIB1), the medication is assigned as pre-treatment. In all other circumstances, medications are assigned as concomitant with the study treatment.

[0343] Concomitant medications are summarized separately by ATC class and generic (basic) name. The primary medication is identified in the patient list. If a patient takes specific medications at different times, that medication is counted only once within the specific observation period in which it was taken (i.e., primary or concomitant).

[0344] 10.8 Pharmacodynamic analysis and biomarker analysis The immune response will be evaluated by the ELISpot assay on peripheral blood samples obtained from patients at a limited number of facilities. Additional analyses may be performed on subsets of these samples, including CFSE proliferation assays, CTL lysis assays, MHC multimer staining of memory cells, assays to assess epitope expansion, Luminex assays for cytokine secretion and T cell subset analysis, and liquid biomarker analysis.

[0345] Tumor samples are evaluated for PD-L1 expression.

[0346] 10.9 Reporting deviations from the statistical analysis plan The handling of deviations from SAP will be documented in the final CSR.

[0347] 11. Quality Control and Quality Assurance The research will be conducted in accordance with SOPs that meet the ICH and national clinical trial guidelines.

[0348] 12. Regulatory and Ethical Obligations 12.1 Compliance with Clinical Trial Implementation Standards This research will be conducted in accordance with the ICH GCP guidelines.

[0349] 12.2 Informed Consent It is the responsibility of the principal investigator to obtain written informed consent from the patient after providing a proper explanation of the study's objectives, methods, expected benefits, and potential harms. The principal investigator should also explain to the patient that they have the freedom to refuse participation in the study and to withdraw from the study at any time without any adverse effects on future treatment. Signed informed consent must be obtained before the patient receives any research treatment or undergoes any research-specific procedure or evaluation (procedures and evaluations are performed for research purposes and not as part of routine medical care). The signed ICF must be current and include any relevant information due to protocol modifications. Patients in the study will need to reconsider giving consent if the revision to the ICF is significant and includes changes that alter the benefit / risk relationship resulting from the research treatment or participation in the study.

[0350] A separate ICF is signed before any other screening procedures are performed to authorize the collection of a blood sample for HLA tissue typing. The HLA ICF may be obtained by a trained healthcare professional designated by the responsible physician. The signed ICF must be current and include any relevant information due to protocol modifications.

[0351] The original ICF, signed and dated, must be kept in the files of the principal investigator's facility and must be available for audits by the research monitor, the sponsor's representative, or the relevant national regulatory authority.

[0352] 12.3 Contact with your primary physician The ICH GCP guidelines recommend that the principal investigator inform the patient's primary physician about the patient's participation in the trial, provided the patient has a primary physician and the patient has consented to sharing information with the primary physician.

[0353] 12.4 Research Ethics Committee A copy of the protocol, along with the presented patient information sheet and ICF, will be submitted to the REC. Written approval of the protocol and the ICF must be obtained before recruiting patients for the study and transporting the study treatment to a clinical setting.

[0354] The principal investigator must inform and obtain approval from the REC for all protocol modifications and all modifications to the patient information sheet and ICF. All SUSARs that may affect patient safety or the conduct of the study will be sent to the REC. Copies of the REC notification and acknowledgment of receipt must be provided to the sponsor.

[0355] 12.5 Regulatory approvals This research will not be conducted until regulatory approval is obtained from the relevant national regulatory authorities.

[0356] 12.6 Periodic review of safety and efficacy Safety and efficacy data are formally reviewed frequently and regularly by the principal investigator and the sponsor. The review committee includes the principal investigator, the sponsor's medical monitor, and the sponsor's clinical trial director or designated person.

[0357] 12.7 Registration of clinical trials on the clinical trial registry website Research information regarding the objectives and design of the study, as well as patient eligibility criteria, will be posted in the official clinical trial register before the commencement of participation.

[0358] 12.8 Patient confidentiality In compliance with the ICH GCP guidelines, it is a requirement that the principal investigator and site grant the sponsor, regulatory authorities, and authorized REC personnel direct access to review the patient's original medical records at the site for verification of trial-related procedures and data.

[0359] To protect confidentiality, patients are identified only by their study number, patient number, and date of birth in the CRF or other documents submitted to the sponsor. This information is used for patient identification in the database. The patient's name and address are not entered in the CRF or the database. Materials containing the patient's name are not stored in files by the sponsor. Patients are informed of their rights within the ICF.

[0360] 12.9 Data Protection All personnel involved in the research will comply with national data protection guidelines.

[0361] 12.10 Future use of stored specimens All biological specimens are collected solely for the research purposes of this clinical study. Blood samples will be discarded 12 months after the completion of the CSR. Tumor tissue blocks will be returned from the analytical laboratory to the original facility using local procedures.

[0362] If a patient withdraws their consent after a sample has been taken, those tests will be performed and the results used, as detailed in the protocol. No further samples will be taken once consent is withdrawn.

[0363] 13. Administrative and legal obligations 13.1 Duties of the Responsible Physician All principal investigators will ensure compliance with ICH GCP and provide training documentation from the start of the study.

[0364] 13.2 Access to Source Data and Documentation The principal investigator authorizes the sponsor and authorized personnel of each country's regulatory authority to have direct access to portions of the patient's medical records relevant to the study. As part of the required informed consent, the patient must be informed that their medical records will be reviewed for this purpose.

[0365] 13.3 Insurance Before the start of the trial, the sponsor must ensure that appropriate insurance is in place for conducting the trial.

[0366] 13.4 Amendments to Protocols and Informed Consent Any changes from the final approved (signed) protocol shall not be implemented without prior written approval or a favorable written opinion on the amendment by the REC and the national regulatory authorities, where applicable, except when necessary to address urgent patient safety concerns or when the changes are minor logistical or administrative modifications. The principal investigator and sponsor at each clinical site shall sign any amendments to the protocol.

[0367] 13.5 Documentation and data storage of the examination The principal investigator must store all trial-related documentation and records required for audits by the sponsor and regulatory authorities in each country within a comprehensive and centrally managed filing system.

[0368] 13.6 Availability and storage of survey records The principal investigator must retain essential documents in accordance with applicable regulations and in accordance with future developments of the research product, as follows: • At least 15 years after the completion or discontinuation of the test • For at least two years following the last approval of the right to manufacture and sell in the United States or Europe; or • At least two years after the formal discontinuation of clinical development of the product under investigation.

[0369] The clinical trial sponsor will determine the minimum retention period and notify the principal investigator when the documents can be disposed of.

[0370] Patient files and other source data (including protocols, CRFs, original reports of study results, medication dispensing logs, communication documents, informed consent records, and copies of other documents relating to the conduct of the study) must be retained for the maximum period permitted by the clinical institution.

[0371] Study documents will not be destroyed without the prior written consent of the sponsor and the principal investigator. If the principal investigator wishes to transfer study records to a third party or move them to another location, written consent must be obtained from the sponsor.

[0372] 13.7 Data Collection Data will be collected using study-specific electronic CRFs. Data collected in the CRFs will be intercepted in a clinical data management system that meets the technical requirements outlined in 21 CFR Part 11 (USA) and other anticipated requirements of eligible regulatory authorities. Data will be collected in the clinical setting by designated and trained personnel. CRFs must be completed for each patient who has provided informed consent for the primary study. Patient identity cannot be identified from the data provided on the CRFs.

[0373] Data processed at other sources (e.g., central laboratory, biological analysis laboratory) is sent to the clinical site and stored there, but is not transcribed to the CRF unless otherwise specified in the protocol. This data may be transmitted electronically to the sponsor (or the data management contracted research institution).

[0374] All patient data must have original medical records or equivalent supporting source documents before being transcribed to the CRF. Source documents are also required for post-treatment follow-up data. Data must not be recorded directly on the CRF and should not be considered source data.

[0375] 13.8 Language The CRF (Clinical Report Form) must be completed in English. The generic names of concomitant medications should be recorded in the CRF whenever possible. All documents used by the patient must be in a language that the patient can read and understand.

[0376] 13.9 Clinical monitoring, source data verification, and auditing The responsibility of a research monitor rests with a skilled clinical research monitor. The clinical research monitor is responsible for ensuring that the patient has signed the ICF and that the study is conducted in compliance with GCP guidelines, in accordance with applicable SOPs, protocols, and other written explanatory documents and regulatory guidelines.

[0377] The primary responsibility of the study monitor is to adhere to the clinical monitoring plan and visit the principal investigator and clinical staff involved in the study before, during, and after the study. This is to ensure that the protocol is followed and that all data are accurately and completely recorded and reported. The study monitor must also ensure that signed informed consent has been obtained from patients before they undergo any study-specific procedures or receive any study treatment. The monitor must also ensure that the signed ICF is up-to-date and that it complies with protocol revisions and REC approvals.

[0378] Research monitoring, source data verification, and clinical site audits shall be conducted as described in the clinical monitoring plan.

[0379] 13.10 Protocol deviations and violations Protocol deviation A protocol deviation is any failure to perform any procedure, evaluation, or schedule specified in the protocol.

[0380] Protocol violation A protocol violation is any failure to comply with the protocol's inclusion / exclusion criteria or any other material failure to comply with a procedure, evaluation, or schedule specified in the protocol that affects the primary objectives of the study or patient safety. For the purposes of this study, a protocol violation is considered a material protocol deviation.

[0381] Protocol deviations are defined in the research monitoring plan and documented in the source document by the clinical setting personnel.

[0382] If a protocol violation is reported, the sponsor will decide, with written approval from a medical professional, whether to discontinue the patient's study or allow the patient to continue the study. The decision will be based on ensuring patient safety and preserving the integrity of the study.

[0383] No requests for inclusion or exclusion of the protocol, or any deviation from the protocol, schedule, mandatory procedures, or evaluations, will be permitted by the sponsor. If a clinical practitioner becomes aware that a patient who did not meet the protocol inclusion or exclusion criteria has participated in the study, they must immediately notify the sponsor, the sponsor's representative, and the REC, as necessary, of the protocol violation.

[0384] 14. References Ascierto PA, McArthur GA, Dreno B, et al. Cobimetinib combined with vemurafenib in advanced BRAF(V600)-mutant melanoma (coBRIM): updated efficacy results from a randomised, double-blind, phase 3 trial. Lancet Oncol. 2016 Sep;17(9):1248-60. Durrant LG, Pudney V, Spendlove I, et al. Vaccines as early therapeutic interventions for cancer therapy: neutralising the immunosuppressive tumour environment and increasing T cell avidity may lead to improved responses. Expert Opin Biol Ther. 2010 May;10(5):735-48. Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45:228-47. EUCAN. Cancer factsheets. Available at: https: / / ecis.jrc.ec.europa.eu / . Gershenwald JE, Scolyer RA, Hess KR, et al. Melanoma staging: evidence-based changes in the American Joint Committee on Cancer eighth edition Cancer Staging Manual. CA Cancer J Clin. 2017;67(6):472-492. Gubin MM, Zhang X, Schuster H, et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature. 2014 Nov 27;515(7528):577-81. Keilholz U, Punt CJ, Gore M, et al. Dacarbazine, cisplatin, and interferon-alfa-2b with or without interleukin-2 in metastatic melanoma: a randomized phase III trial (18951) of the European Organisation for Research and Treatment of Cancer Melanoma Group. J Clin Oncol. 2005 Sep 20;23(27):6747-55. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med. 2015 Jul 2;373(1):23-34. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Overall survival results from a phase III trial of nivolumab combined with ipilimumab in treatment-naive patients with advanced melanoma (CheckMate 067). 2017 AACR Annual Meeting. Abstract CT075. Presented April 3, 2017. Lebbe C, Meyer N, Mortier L, et al. Evaluation of two dosing regimens for nivolumab in combination with ipilimumab in patients with advanced melanoma: results from the phase IIIb / IV CheckMate 511 trial. J Cln Oncol 2019;37:867-875. Long GV, Stroyakovskiy D, Gogas H, et al. Dabrafenib and trametinib versus dabrafenib and placebo for Val600 BRAF-mutant melanoma: a multicentre, double-blind, phase 3 randomised controlled trial. Lancet. 2015 Aug 1;386(9992):444-51. Massarelli E, William W, Johnson F, et al. Combining immune checkpoint blockade and tumor-specific vaccine for patients with incurable human papillomavirus 16-related cancer. A Phase 2 clinical trial. JAMA Oncology 2019;5:67-73. Metheringham RL, Pudney VA, Gunn B, et al. Antibodies designed as effective cancer vaccines. MAbs. 2009 Jan-Feb;1(1):71-85. Oken MM, Creech RH, Tormey DC, et al. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol. 1982;5:649-655. Patel PM, Ottensmeier CH, Mulatero C, et al. Targeting gp100 and TRP-2 with a DNA vaccine: Incorporating T cell epitopes with a human IgG1 antibody induces potent T cell responses that are associated with favourable clinical outcome in a phase I / II trial. Oncoimmunology. 2018;7(6):e1433516. Pudney VA, Metheringham RL, Gunn B, et al. DNA vaccination with T-cell epitopes encoded within Ab molecules induces high-avidity anti-tumor CD8+ T cells. Eur J Immunol. 2010 Mar;40(3):899-910. Ribas A, Hamid O, Daud A, et al. Association of pembrolizumab with tumor response and survival among patients with advanced melanoma. JAMA. 2016 Apr 19;315(15):1600-9. Robert C, Ribas A, Wolchok JD, et al. Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase 1 trial. Lancet. 2014 Sep 20;384(9948):1109-17. Robert C, Long GV, Brady B, et al. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med. 2015a Jan 22;372(4):320-30. Robert C, Schachter J, Long GV, et al. Pembrolizumab versus ipilimumab in advanced melanoma. N Engl J Med. 2015b Jun 25;372(26):2521-32. Seymour L, Bogaerts J, Perrone A, et al. iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics. Lancet Oncol. 2017 Mar;18(3):e143-e152. Simon R. Optimal two-stage designs for Phase II clinical trials. Controlled Clinical Trials. 1989; 10:1-10. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016 Jan-Feb;66(1):7-30. Snyder A, Makarov V, Merghoub T, et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med. 2014 Dec 4;371(23):2189-99. Xue W, Brentville VA, Symonds P, et al. SCIB1, a huIgG1 antibody DNA vaccination, combined with PD-1 blockade induced efficient therapy of poorly immunogenic tumors. Oncotarget. 2016 Dec 13;7(50):83088-83100.

[0385] Example 4: Results of a clinical study This embodiment presents the results of a clinical study substantially in accordance with Example 3.

[0386] Introduction Immunotherapy with checkpoint inhibitors (CPIs), including programmed cell death 1 (PD-1) inhibitors administered alone or in combination with cytotoxic T lymphocyte antigen 4 (CTLA-4) inhibitors, has revolutionized first-line treatment outcomes in patients with unresectable or metastatic advanced melanoma. 1,2 However, at the 5-year mark, the overall survival rate was 52%. 1 This means that approximately half of the patients did not receive a meaningful benefit from the treatment. Furthermore, in the pivotal Checkmate-067 trial, the surrogate survival endpoints, such as the overall response rate (ORR) and progression-free survival (PFS) at 3 years, were 58% and 34%, respectively. 3 In actual practice, the ORR achieved through combinations is only about 50%. 4 Some experts in the field report a rate of 28% in actual field operations. 5 In recent years, nucleic acid-based vaccines for melanoma have shown efficacy in delaying the time to recurrence in combination with pembrolizumab in unresectable disease stages.6 However, in unresectable disease stages, nucleic acid vaccines have not shown significant efficacy in reducing tumor burden.

[0387] method patient In the SCOPE clinical trial testing the addition of SCIB1 to standard checkpoint inhibitor regimens, eligible patients had histologically confirmed stage III (unresectable) or stage IV melanoma and had not received systemic treatment for advanced disease, although prior treatment in the adjuvant melanoma setting was acceptable. Other eligibility criteria included being 18 years of age or older; having an East Coast Clinical Oncology Group (ECOG) performance status score of 0 (asymptomatic) or 1 (mildly symptomatic); having a measurable disease assessed using computed tomography or magnetic resonance imaging according to the Response Assessment Criteria for Solid Tumors (RECIST) version 1.1; having tissue (stored or recently biopsyed) available from metastatic or unresectable tumors for assessment of PD-L1 status; and having a known BRAF V600 mutation status. Patients also had to be positive for HLA class I type HLA-A2 and at least one of the following HLA class II types: HLA-DR4, HLA-DR7, HLA-DR53, or HLA-DQ6.

[0388] The primary exclusion criteria were the presence of active brain metastases, ocular melanoma, mucosal melanoma, or autoimmune disease. Patients receiving steroids exceeding physiological doses within one week prior to the first dose of SCIB1 were excluded from the study. Patients with BRAF mutation-positive disease with evidence of rapidly progressive disease were also excluded from SCIB1 vaccination.

[0389] Design and treatment of the trial Cohort 1: Combination therapy with nivolumab and ipilimumab In Phase 1 (see Figure 5), 15 patients (including 6 from the run-in subcohort) were recruited. If there were 8 or fewer clinical responses (RECIST 1.1 objective response [CR or PR], within 25 weeks of the first dose of SCIB1), the cohort was discontinued. Otherwise, an additional 28 patients were recruited for Phase 2, bringing the total to 43 patients. Recruitment continued until the decision for Phase 1 was made. In this cohort, patients were treated as follows: The total dose of SCIB1 per administration is 8 mg, administered by four 2 mg intramuscular injections of SCIB1 using the Pharmajet Stratis needleless injection device, at weeks 0, 4, 7, 13, 25, and every 12 weeks thereafter up to week 85.

[0390] For the first four doses, 1 mg / kg of nivolumab is administered intravenously over 30 minutes every three weeks, in combination with 3 mg / kg of ipilimumab administered intravenously over 30 minutes. This is followed by a second stage, in which nivolumab monotherapy is administered intravenously at a dose of either 240 mg over 30 minutes every two weeks or 480 mg over 60 minutes every four weeks.

[0391] Regarding the monotherapy stage, the first dose of nivolumab is, • When using 240 mg every two weeks, it is administered three weeks after the last dose of the nivolumab and ipilimumab combination; or, • When using 480 mg every 4 weeks, it is administered 6 weeks after the last dose of the nivolumab and ipilimumab combination.

[0392] The first dose of the checkpoint inhibitor was administered one week after the first dose of SCIB1, and the total treatment period was 97 weeks or until disease progression or dose-limiting toxicity occurred.

[0393] Cohort 2: Pembrolizumab In Phase 1, 14 patients (including 6 from the run-in subcohort) will be recruited. If there are 5 or fewer clinical responses (RECIST 1.1 objective response [CR or PR], within 25 weeks of the first dose of SCIB1), the cohort will be discontinued. Otherwise, an additional 30 patients will be recruited for Phase 2, bringing the total to 44 patients. Recruitment will continue until the decision for Phase 1 is made. In this cohort, patients were treated as follows: The total dose of SCIB1 per administration is 8 mg, administered by four 2 mg intramuscular injections of SCIB1 using the Pharmajet Stratis needleless injection device, at weeks 0, 4, 7, 13, 25, and every 12 weeks thereafter up to week 85.

[0394] Administer either 200 mg every 3 weeks or 400 mg every 6 weeks as an intravenous infusion over 30 minutes.

[0395] The total treatment period was 97 weeks or until disease progression or dose-limiting toxicity occurred, and the first dose of checkpoint inhibitors was administered one week after the first dose of SCIB1.

[0396] The study was designed using Simon's two-step methodology, which has 80% power and an overall Type I error rate of 5% when the true response rates are 70% (Cohort 1) and 55% (Cohort 2). This clinical trial was designed with the null hypothesis that the ORR would be 50% (Cohort 1) and 30% (Cohort 2). The null hypothesis values ​​are consistent with actual field-based treatment outcomes in patients treated for unresectable melanoma.

[0397] The primary endpoint of the study is to measure the objective response rate according to the RECIST 1.1 criteria, comparing it to historical data in these separate patient populations for the combination of nivolumab and ipilimumab or pembrolizumab alone. The secondary endpoint is to determine whether adding SCIB1 to either the combination of nivolumab and ipilimumab or pembrolizumab improves the duration of response in this patient population compared to historical data for the combination of nivolumab and ipilimumab or pembrolizumab alone. Progression-free survival, defined as the proportion of patients who have not progressed (according to RECIST 1.1 and iRECIST), have not initiated new anticancer therapy, and have not died at each designated time point, is analyzed together with overall survival, defined as the patient population that is alive at each designated time point. Patient safety and tolerance are also summarized in these two cohorts. In addition, experimental endpoints evaluate the relationship between immune response and clinical outcome using ELISpot and other assays performed on peripheral blood samples obtained from patients.

[0398] result Patients and treatment From October 2019 to September 2024, a total of 17 patients were enrolled in Cohort 1 and 3 patients in Cohort 2. In Cohort 1, baseline characteristics of the patients were that 12 were male and 5 were female, and all 17 patients had metastatic disease to the lung, liver, and / or brain. Of the patients, 8 had Braf mutations, while 9 were wild-type. Eight patients had lactate dehydrogenase (LDH) levels above the upper limit of normal, and 9 patients had levels below the limit. LDH was an important prognostic factor, and elevated LDH indicated a poor prognosis. Twelve patients were under 65 years old, 2 were between 65 and 74 years old, and only 3 patients were 75 years or older. In Cohort 2, 1 patient was male and 2 were female. All 3 patients had metastatic disease to the lung or liver. Only one patient had a Braf mutation, and one patient had elevated LDH levels above the upper limit of normal. All three patients in Cohort 2 were 75 years of age or older. Across both cohorts, all patients had a total tumor volume greater than 20 cm. See Table 3.

[0399] [Table 3]

[0400] The data cut was performed in September 2023, and in Cohort 1, 11 patients had reached the time of their first computed tomography or magnetic resonance imaging 13 weeks after SCIB1 vaccination. The other 6 patients were recent participants in the study and therefore had not reached the time of their first imaging. For efficacy purposes, only patients in Cohort 1 are summarized herein. Of the 11 patients, 9 (81.8%) showed an overall response rate (ORR) as measured by RECIST 1.1 at week 13. Of these, 5 patients reached week 19, 4 reached week 25, and 2 reached week 37. The response in these patients was sustained and showed an overall reduction in total tumor volume. The greatest overall reduction in total tumor volume (-69 to -94%) was shown in patients who reached the diagnostic imaging time of week 25; see Figures 6 and 7.

[0401] The waterfall plot (Figure 6) shows the maximum change from baseline in the total reference diameter of the target lesion in patients who received SCIB1 with ipilimumab in addition to nivolumab. Data are shown for all patients who assessed the target lesion at baseline and underwent initial imaging by week 13. Bars in the negative quadrant indicate tumor regression, while bars in the positive quadrant indicate an increase in tumor size. Horizontal lines indicate a 30% reduction or 20% increase in tumor volume at the target lesion, which are the response or progression thresholds. Change in tumor volume was defined as the percentage reduction (%) in the total reference diameter of the target lesion from baseline to the minimum value observed by September 2023. As can be seen from Figure 6, only one patient experienced disease progression, while all other 10 patients experienced a reduction in total tumor volume. Of the 10 patients who showed a response, only one patient had not reached the partial response threshold at the data cut point. However, if tumor reduction continues at the time of the next scan, this patient will also be a responder and is therefore expected to contribute to the significant therapeutic effect of SCIB1 when combined with checkpoint inhibitors.

[0402] Adverse events Treatment-related adverse events of any grade that occurred in all patients treated with SCIB1 in combination with ipilimumab and nivolumab (see Table 4). The most common adverse events reported to date are injection site reactions associated with SCIB1. These reactions are mild in severity and resolve over time. Patients who experienced the greatest overall tumor volume reduction (-94%) also experienced vitiligo, characterized by pigmentation / depigmentation affecting more than 10% of the body surface area. At the last evaluation, the adverse effects persisted and were reported to be causally related to SCIB1.

[0403] Treatment-related adverse events of CTCAE grade 3 or 4 severity were reported in 16 patients. Of these, eight were causally linked to the combination of checkpoint inhibitors, and only one was thought to be related to SCIB1. One patient died during the study due to pneumonia, but this was not thought to be related to metastatic melanoma or to the treatment regimen consisting of SCIB1, ipilimumab, and nivolumab.

[0404] No serious adverse events causally linked to SCIB1 were reported (see Table 4). Serious adverse events reported to be causally linked to checkpoint inhibitors were consistent with the product characteristics summary. Adding SCIB1 to checkpoint inhibitors did not demonstrate an enhancement of checkpoint inhibitor toxicity.

[0405] [Table 4]

[0406] [Table 4-1]

[0407] Consideration In this open-label Phase II study, including patients in a first-line treatment setting for advanced / metastatic melanoma, the addition of SCIB1 to the combination of ipilimumab and nivolumab resulted in a remarkable overall response rate of 81.8%. While baseline participant characteristics were typical of patients with advanced / metastatic melanoma and reflected real-world clinical practice, patients were selected based on HLA type. The pivotal clinical trial reported an ORR of 54–58%. 1,2,3 Studies conducted in actual clinical settings report ORRs of 28% to 48%. 4,5The scale of tumor regression shown in the ORR data presented in the waterfall plot of Figure 6 and the spider plot of Figure 7 has never been demonstrated in this treatment context with any approved combination immunotherapy or any currently under development. Since reducing tumors and halting their growth is expected to extend the lifespan of these patients, those skilled in the art will use RECIST ORR as a surrogate marker. Figure 7 shows the persistence of the tumor response, which is important in the art because persistence of the response correlates with survival, as seen in long-term prognosis studies²,³. When administered with ipilimumab and nivolumab, SCIB1 has a significant impact on tumor regression and life extension in a treatment context where 50% of patients die from melanoma despite treatment with checkpoint inhibitors. Furthermore, it is hypothesized that SCIB1 can be added to any combination of checkpoint inhibitor therapies, including but not limited to the combination of reratrimab and nivolumab, to achieve similar therapeutic effects to those demonstrated with the combination of ipilimumab and nivolumab. In conclusion, SCIB1, when added to ipilimumab and nivolumab, and when discontinued from ipilimumab or discontinued from nivolumab and continued, will be as shown in long-term prognosis studies of ipilimumab and nivolumab combination therapy. 1 It has a significant impact on the overall response rate in tumors, to the extent that it can improve the overall survival rate by more than 52%.

[0408] References 1. J.Larkin et al. Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma N Engl J Med 2019; 381:1535-1546 2. Jedd D. Wolchok et al. Long-Term Outcomes With Nivolumab Plus Ipilimumab or Nivolumab Alone Versus Ipilimumab in Patients With Advanced Melanoma Journal of Clinical Oncology 2022 40:2, 127-137 3. Errico, A. CheckMate 067 - frontline nivolumab improves PFS alone or in combination with ipilimumab. Nat Rev Clin Oncol 12, 435 (2015). 4. P. Serra-Bellver et al. Real-world outcomes with ipilimumab and nivolumab in advanced melanoma: a multicentre retrospective study European Journal of Cancer 176 (2022) 121e132 5. Parakh S, Randhawa M, Nguyen B, Warburton L, Hussain MA, Cebon J, Millward M, Yip D, Ali S. Real-world efficacy and toxicity of combined nivolumab and ipilimumab in patients with metastatic melanoma. Asia Pac J Clin Oncol. 2019 Feb;15(1):26-30 6. L.J Albrecht et al., The Latest Option: Nivolumab and Relatlimab in Advanced Melanoma the Latest Option: Nivolumab and Relatlimab in ATdvanced Melanoma. Curr Oncol Rep 25, 647-657 (2023).

[0409] Example 5: Evidence for the efficacy of checkpoint inhibition in a preclinical mouse melanoma model. It is known from previous literature that mouse B16F1 melanoma does not respond to checkpoint therapy. In C57Bl / 6 mice, 2.5 × 10⁶ 4 Individual B16F1 melanoma cells were subcutaneously transplanted and then treated intraperitoneally with 100 μg of anti-mouse CTLA4 antibody (clone 9D9) or 250 μg of anti-mouse PD1 antibody (clone RMP1-14) and anti-CTLA4 antibody on days 8, 15, and 22 post-tumor transplantation. Tumor growth was monitored, and mice were euthanized when the tumor reached a 15 mm diameter endpoint. Neither checkpoint antibody therapy showed significant effect on tumor growth or survival (Figure 8).

[0410] Nevertheless, some tumor models are known to be responsive to checkpoint therapy. The B16F1 melanoma lineage was modified to express a human HLA allele (HLA-DR4 or HLA-DP4) for use in a human HLA transgenic mouse model (Brentville et al. 2016, Cancer Research 76:548-60; Brentville et al. 2019, Oncoimmunology 8:e1576490). The HLA-DR4 mouse (Taconic model 4149) has a C57Bl / 6 background, except that the mouse MHC class II allele is replaced with human HLA-DR4. For this model, the B16F1 cell lineage was modified to express human HLA-DR4 (B16 DR4). HHDII / DP4 mouse (HLA-A2.1 + / + HLADP4 + / + hCD4 + / +The European Mouse Mutant Archive (EM:02221) also has a C57Bl / 6 background, but the mouse class I allele is replaced with chimeric human HLA-A2 (HHDII), and the class II allele is replaced with HLA-DP4. For this model, B16F1 tumor cells were modified to express human HLA-A2 and HLA-DP4 (B16 HHDII / DP4) instead of mouse MHC class I or II. B16 DR4 or B16 HHDII / DP4 melanoma cells were transplanted into HLA-DR4 or HHDII / DP4 transgenic mice, respectively. 2.5 × 10⁶ cells were transplanted into HLA-DR4 transgenic mice. 4 After subcutaneous transplantation of B16 DR4 cells, patients were treated intraperitoneally with 250 μg of anti-PD1 (clone RMP1-14), 250 μg of anti-PD1 and anti-PDL1 (clone 10F.9G2), or 250 μg of anti-PD1, anti-PDL1, and 100 μg of anti-CTLA4 (clone 9D9) antibodies on days 8 and 11, or on days 4 and 11. All combinations of checkpoint antibodies significantly enhanced survival compared to the control group (Figure 9A).

[0411] HLA-DP4 transgenic mouse, 1 x 10 5 After subcutaneous transplantation of B16 HHDII / DP4 cells, the mice were treated with 250 μg of anti-PD1 (clone RMP1-14) on days 10, 14, and 17, and with 250 μg of anti-PD1 and 100 μg of anti-CTLA4 (clone 9D9) or 100 μg of anti-CTLA4 antibody intraperitoneally on days 8, 15, and 22. Anti-PD1 antibody administered at later time points did not have any effect on tumor growth and survival (Figure 9B), but anti-CTLA4 or a combination of anti-PD1 and anti-CTLA4 demonstrated evidence of tumor therapeutic activity, resulting in 100% survival at day 30 in this HLA-DP4 transgenic mouse model (Figure 9C).

[0412] These data demonstrate the effectiveness of checkpoint therapy in mouse models in which immunogenicity is enhanced by the expression of specific HLA alleles.

[0413] References 1. Brentville VA, Metheringham RL, Gunn B, et al. Citrullinated vimentin presented on MHC-II in tumor cells is a target for CD4+ T-cell-mediatedantitumor immunity. Cancer Res 2016;76:548-60. 2. Brentville VA, Symonds P, Cook KW, et al. T cell repertoire to citrullinated self-peptides in healthy humans is not confined to the HLA-DR SE alleles; targeting of citrullinated self-peptides presented by HLA-DP4 for tumour therapy. Oncoimmunology 2019;8:e1576490.

[0414] Example 6: The iSCSIB1+ DNA vaccine stimulates an immune response in a mouse model. The immunogenicity of the iSCIB1+ DNA vaccine was evaluated in C57Bl / 6 mice. C57Bl / 6 mice respond to the TRP-2 aa180~188 sequence present in both SCIB1 and iSCIB1+, and the response is constrained to the mouse H-2Kb (MHC class I) allele. However, iSCIB1+ also contains three sequences not present in SCIB1: TRP-2 aa60~91 inserted in CDR L3; TRP-2 aa177~205 in CDR L2; and gp100 aa471~492 in CDR H3. C57Bl / 6 mice were immunized with 1 μg of iSCIB1+ using a gene gun device on days 1, 8, and 15, and T cell responses to specific peptides were monitored by the IFNγ ELISpot assay. High-frequency responses were generated for the TRP-2 aa180~188 peptide and the TRP-2 aa178~198 peptide (including the aa180~188 sequence), while low-frequency responses were generated for peptides covering the other two sequences (Figure 10A).

[0415] The response observed in C57Bl / 6 mice is constrained to the mouse MHC allele. To confirm that iSCIB1+ induces an immune response constrained to the human HLA allele, HLA-DR4 transgenic mice were immunized with a dose of 1 μg of iSCIB1+ using a gene gun device on days 1, 8, and 15, and the T cell response to specific peptides was monitored by an IFNγELISpot assay. HLA-DR4 mice have the C57Bl / 6 background except that the mouse MHC class II allele is replaced with that of human HLA-DR4; therefore, they still have the mouse H-2Kb (MHC class I) allele that binds to the TRP-2 aa180~188 peptide.

[0416] A strong response was generated in response to the TRP-2 aa180-188 and aa178-198 peptides. In addition, a strong response was observed in response to the gp100 aa44-59 peptides constrained to HLA-DR4 inserted into the L1 CDR of the iSCSIB1+ plasmid (Figure 10B).

[0417] The basis for a response constrained to the human HLA-A2 allele is provided by the immune response detected in HHDII / DR1 mice, in which the mouse class I allele is replaced by chimeric human HLA-A2 (HHDII) and the class II allele is replaced by HLA-DR1. Immunization of HHDII / DR1 mice and T cell responses to specific peptides monitored by the IFNγ ELISpot assay demonstrated a potent immune response to TRP-2 aa180-188 and TRP-2 aa178-198 peptides. In addition, a weak response was observed to peptides within the TRP-2 aa60-91 sequence (L3) and the gp100 aa173-190 sequence (H1) (Figure 10C).

[0418] These data demonstrate the immunogenicity of iSCIB1+ mediated by MHC alleles in both mice and humans.

[0419] Example 7: iSCIB1+ DNA vaccination mediates tumor therapy in checkpoint-nonresponsive B16F1 tumor models and checkpoint-responsive B16 HHDII / DP4 tumor models. The mouse B16F1 melanoma model is widely used in tumor research for potential melanoma therapies. In C57Bl / 6 mice, it is highly invasive and typically unresponsive to checkpoint blockade therapy (Example 5, Figure 8). In C57Bl / 6 mice, 2.5 × 10⁶ doses were administered on day 1. 4 Individual B16F1 tumor cells were subcutaneously transplanted, and then immunized with 1 μg of iSCIB1+ DNA using a gene gun device on days 4, 11, and 18. The iSCIB1+ DNA vaccine was effective and resulted in significant survival in the treated mice (Figure 11A).

[0420] A similar effect was observed in mice, with 1 x 10⁶ doses administered on day 1. 5This was also observed in HHDII / DP4 transgenic mice that were transplanted with 1 B16 HHDII / DP4 cells and then immunized with 1 μg of iSCSIB1+ DNA using a gene gun device on days 4, 11, and 18. Significantly greater survival was observed in the immunized mice compared to the control (Figure 11B).

[0421] These data demonstrate that the immune response induced by vaccination with iSCIB1+ DNA can mediate tumor therapy in checkpoint-responsive B16 HHDII / DP4 tumor models, and that the stimulation of tumor-specific responses by vaccination can also mediate treatment in checkpoint-nonresponsive B16F1 tumor models. Therefore, vaccination may be effective in tumors that are nonresponsive to checkpoint blockade therapy.

[0422] Example 8: Checkpoint blockade enhances immune response and tumor treatment induced by iSCSIB1+ DNA vaccination. The current standard of treatment for patients with metastatic melanoma is checkpoint inhibitors such as a combination of anti-PD-1 and anti-CTLA therapy. To replicate the dual regimen included in the SCIB1-002 Phase 2 clinical trial described in Example 3, the iSCIB1+ DNA product was evaluated in combination with two checkpoint inhibitors (anti-PD-1 plus CTLA-4) in C57Bl / 6 mice. The immune response was evaluated by IFNγ ELISpot assay on days 1, 8, and 15 after injection of iSCIB1+ DNA via gene gun in combination with the dual checkpoint inhibitors. Cells were collected on day 22 and further expanded in vitro for 6 days prior to analysis. Anti-PD-1 antibody and anti-CTLA-4 antibody (clone RMP1-14 and clone 9D9, respectively) were administered intraperitoneally at doses of 250 μg / mouse and 100 μg / mouse, respectively. While checkpoint blockade did not have a significant effect on the dominant immune response to the TRP-2 aa180-188 peptide, the subdominant response recognizing the TRP-2 aa69-77 and gp100 aa476-490 peptide sequences encoded by iSCIB1+ was significantly increased in the presence of checkpoint inhibitors (Figure 12A and B). Since iSCIB1+ immunization provided a significant survival benefit in the C57Bl / 6 / B16F1 tumor model (Example 7), immunization was delayed after tumor implantation to allow for confirmation of differences between treatment arms. C57Bl / 6 mice received 2.5 × 10⁶ doses on day 1. 4Nine B16F1 tumor cells were transplanted. The vaccinated group was then immunized on days 8, 15, and 22 with either iSCIB1+ alone, dual checkpoint therapy (250 μg anti-PD1 and 100 μg anti-CTLA4 antibody) alone, or a combination of iSCIB1+ and a checkpoint inhibitor. Tumor-free survival was significantly enhanced in the groups immunized with iSCIB1+ alone or iSCIB1+ in combination with dual checkpoint therapy compared to the control animals (Figure 13). Treatment with dual therapy alone showed a slight increase in survival compared to the control group, but this was not statistically significant. However, the combination of iSCIB1+ and dual therapy significantly increased survival compared to dual therapy alone (p=0.0265).

[0423] These data provide evidence for enhanced T-cell response induced by the iSCSIB1+ DNA vaccine in the presence of checkpoint inhibitor antibodies and for additional benefits to antitumor efficacy of the combination of vaccine and checkpoint therapy in a mouse melanoma model.

[0424] These data suggest that checkpoint blockade can enhance vaccine-induced responses, and that the combination of vaccines that stimulate de novo responses with checkpoint blockade, which modulates the tumor environment and protects T cells from exhaustion, may improve antitumor efficacy in clinical studies.

[0425] The present invention will be further described in the following numbered sections. 1. A vaccine composition for use in the treatment of cancer in a target, comprising one or more nucleic acids encoding multiple tumor antigen T cell epitopes, wherein the T cell epitopes include GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3), and the method comprises, (a) Programmed cell death 1 (PD-1) inhibitors; and, (b) cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor; A composition comprising administering to the subject in combination with the above. 2. A composition for use in the treatment of cancer in a subject containing a programmed cell death 1 (PD-1) inhibitor, wherein the method comprises the PD-1 inhibitor, (a) A vaccine composition containing one or more nucleic acids encoding multiple tumor antigen T cell epitopes (wherein the T cell epitopes are GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3)); and (b) cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor; A composition comprising administering to the subject in combination with the above. 3. A composition for use in the treatment of cancer in a subject containing a cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor, wherein the method comprises the CTLA-4 inhibitor, (a) A vaccine composition containing one or more nucleic acids encoding multiple tumor antigen T cell epitopes (wherein the T cell epitopes are GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3)); and (b) Programmed cell death 1 (PD-1) inhibitors; A composition comprising administering to the subject in combination with the above. 4. The composition according to any one of items 1 to 3, wherein the cancer is melanoma. 5. The composition according to item 4, wherein the melanoma is a stage III or stage IV melanoma. 6. The composition according to item 4 or 5, wherein the melanoma is metastatic melanoma. 7. The composition according to any one of items 4 to 6, wherein the melanoma is unresectable. 8. The subject has not received systemic treatment for advanced melanoma, and the composition is one of the compositions described in any one of items 1 to 7. 9. The composition according to any one of claims 4 to 8, wherein the melanoma is an unresectable stage III or stage IV melanoma, and the subject has not received systemic treatment for a progressive disease. 10. The nucleic acid is a composition according to any one of items 1 to 9, wherein the nucleic acid encodes the plurality of tumor antigen T cell epitopes. 11. The composition according to any one of items 1 to 10, wherein one or more nucleic acids encode an antibody having a heavy chain and a light chain in which the T cell epitope is inserted or substituted. 12. The composition according to item 11, wherein the T cell epitope is inserted into or replaced in the heavy chain variable region (VH) and / or light chain variable region (VL) of the antibody. 13. The composition according to claim 11 or 12, wherein the T cell epitope is inserted into or replaced in one or more CDRs of the antibody. 14. The aforementioned antibody is (i) CDR-H1 has a T cell epitope of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1); (ii) CDR-H2 has the SVYDFFVWL (SEQ ID NO: 2) T cell epitope; and, (iii) T cell epitope of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-L1; A composition according to any one of items 11 to 13, having the following characteristics: 14. The aforementioned antibody is (i) T cell epitopes of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) in CDR-H1 and CDR-L3, respectively; (ii) CDR-H2 has the SVYDFFVWL (SEQ ID NO: 2) T cell epitope; and, (iii) T cell epitopes of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-H3 and CDR-L1 cells, respectively; A composition according to any one of items 11 to 13, having the following characteristics: 15. The composition according to any one of claims 1 to 14, wherein the T cell epitope further comprises VPLDCVLYRYGSFSVTLDIVQG (SEQ ID NO: 4), ANCSVYDFFVWLHYYSVRDTLLGPGRPYR (SEQ ID NO: 5), and QCTEVRADTRPWSGPYILRNQDDRELWPRKFF (SEQ ID NO: 6). 16. The antibody is, (i) CDR-H1 has a T cell epitope of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1); (ii) T cell epitope of SVYDFFVWL (SEQ ID NO: 2) on CDR-H2; (iii) T cell epitope of VPLDCVLYRYGSFSVTLDIVQG (SEQ ID NO: 4) on CDR-H3; (iv) T cell epitope of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-L1; (v) T cell epitope of ANCSVYDFFVWLHYYSVRDTLLGPGRPYR (SEQ ID NO: 5) in CDR-L2; and, (vi) T cell epitope QCTEVRADTRPWSGPYILRNQDDRELWPRKFF (SEQ ID NO: 6) in CDR-L3; A composition having any one of items 1 to 15. 17. The composition according to any one of claims 11 to 16, wherein the antibody comprises VH having the amino acid sequence shown in SEQ ID NO: 7 and VL having the amino acid sequence shown in SEQ ID NO: 8. 18. The composition according to any one of claims 11 to 16, wherein the antibody comprises VH having the amino acid sequence shown in SEQ ID NO: 9 and VL having the amino acid sequence shown in SEQ ID NO: 10. 19. The composition according to any one of items 11 to 18, wherein the antibody is a human IgG1 antibody. 20. The composition according to any one of claims 11 to 19, wherein the antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 11 and a light chain having the amino acid sequence shown in SEQ ID NO: 12. 21. The composition according to any one of claims 11 to 19, wherein the antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 13 and a light chain having the amino acid sequence shown in SEQ ID NO: 14. 22. The composition according to any one of items 11 to 21, wherein the antibody cannot assume its original conformation when expressed. 23. The composition according to any one of claims 1 to 22, wherein the one or more nucleic acids comprises one or more nonspecific promoters that promote the expression of the T cell epitope. 24. The composition according to item 23, wherein the promoter is a CMV-IE promoter. 25. The composition according to claim 23 or claim 24, wherein the promoter has the nucleotide sequence shown in SEQ ID NO: 15. 26. The composition according to any one of claims 1 to 25, wherein the antibody comprises a leader sequence that promotes the secretion of the antibody when one or more nucleic acids are expressed. 27. The composition according to item 26, wherein the leader sequence is located at the N-terminus of the heavy chain and / or the N-terminus of the light chain of the antibody. 28. The composition according to claim 26 or 27, wherein the leader sequence comprises the amino acid sequence shown in SEQ ID NO: 16. 29. The composition according to any one of items 1 to 28, wherein one or more nucleic acids are DNA. 30. The composition according to any one of items 1 to 29, wherein one or more nucleic acids are plasmids and / or doggybone vectors. 31. The vaccine composition according to any one of claims 1 to 30, comprising a single nucleic acid encoding the plurality of tumor antigen T cell epitopes. 32. The composition according to item 31, wherein the single nucleic acid is a DNA plasmid. 33. The composition according to claim 31 or 32, wherein the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 17. 34. The composition according to claim 31 or 32, wherein the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 18. 35. The composition according to any one of items 1 to 34, wherein the PD-1 inhibitor is an anti-PD-1 antibody. 36. The composition according to any one of items 1 to 35, wherein the PD-1 inhibitor is nivolumab. 37. The composition according to any one of items 1 to 36, wherein the CTLA-4 inhibitor is an anti-CTLA-4 antibody. 38. The composition according to any one of items 1 to 37, wherein the CTLA-4 inhibitor is ipilimumab. 39. The vaccine composition according to any one of claims 1 to 38, wherein the total amount of nucleic acid is administered in a dose of 6 to 10 mg. 40. The vaccine composition according to any one of claims 1 to 39, wherein the total amount of nucleic acid is administered to the subject in a dose of approximately 8 mg. 41. The vaccine composition according to any one of claims 1 to 40, wherein each administration of the vaccine composition comprises four injections into separate injection sites. 42. The vaccine composition according to any one of claims 1 to 41, wherein the first dose of the vaccine composition is administered to the subject in week 0, and subsequent doses are administered between weeks 4, 7, 13, and 25, and thereafter every 12 weeks. 43. The vaccine composition according to any one of items 1 to 42, wherein the first dose of the vaccine composition is administered to the subject on day 1, and subsequent doses are administered on days 29, 50, 92, and 176, and thereafter every 84 days. 44. The vaccine composition according to any one of items 1 to 43, which is administered by needle-free injection. 45. The vaccine composition described in any one of items 1 to 44, which is administered intramuscularly. 46. ​​The vaccine composition is the composition described in any one of items 1 to 45, which contains nucleic acid at a concentration of approximately 4 mg / mL. 47. The vaccine composition according to any one of claims 1 to 46, wherein the vaccine composition contains about 8.1 mM disodium hydrogen phosphate, about 1.5 mM potassium dihydrogen phosphate, about 2.7 mM potassium chloride, and about 137 mM sodium chloride, and the pH of the vaccine composition is about 7.4. 48. The PD-1 inhibitor was administered to the subjects in the first stage at a dose of approximately 1 mg / kg every 3 weeks for 4 doses, followed by the second stage, (i) A dose of approximately 240 mg is administered every two weeks, where the first dose of the second stage is administered approximately three weeks after the fourth dose of the first stage; or, (ii) A dose of approximately 480 mg is administered every four weeks, with the first dose of the second stage being administered approximately six weeks after the fourth dose of the first stage; A composition as described in any one of items 1 to 47. 49. The composition according to any one of items 1 to 48, wherein the CTLA-4 inhibitor is administered to the subject at a dose of approximately 3 mg / kg every 3 weeks for 4 doses. 50. The composition according to any one of items 1 to 49, wherein the CTLA-4 inhibitor and the PD-1 inhibitor are administered on the same day. 51. The composition according to any one of claims 1 to 50, wherein the CTLA-4 inhibitor and the PD-1 inhibitor are administered in separate compositions or as a single composition. 52. The composition according to claim 49, with the addition of claim 48, wherein the CTLA-4 inhibitor is administered on the same day as the first four doses of the PD-1 inhibitor. 53. The composition according to any one of claims 1 to 52, wherein the first dose of the CTLA-4 inhibitor and the first dose of the PD-1 inhibitor are administered approximately one week after the first dose of the vaccine composition. 54. The composition according to any one of claims 1 to 53, wherein the CTLA-4 inhibitor and the PD-1 inhibitor are administered by intravenous infusion. 55. The composition according to any one of items 1 to 54, wherein the subject is a human being 18 years of age or older. 56. The HLA serotype class I of the subject is HLA-A * A composition according to any one of items 1 to 55, which is item 02. 57. The composition according to any one of claims 1 to 56, wherein the target HLA serotype class II is selected from HLA-DR4, HLA-DR7, HLA-DR53, and HLA-DQ6. 58. The composition according to any one of items 1 to 57, wherein the subject is not suffering from brain metastases, ocular melanoma, mucosal melanoma, or autoimmune disease. 59. The composition according to any one of items 1 to 58, wherein the one or more nucleic acids are vectors, preferably DNA vectors. 60. A vaccine composition for use in the treatment of unresectable stage III or stage IV melanoma in a subject, comprising a DNA molecule encoding an antibody in which multiple tumor antigen T cell epitopes are inserted or substituted, The antibody has a T cell epitope of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) inserted or substituted in CDR-H1, a T cell epitope of SVYDFFVWL (SEQ ID NO: 2) inserted or substituted in CDR-H2, and a T cell epitope of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) inserted or substituted in CDR-L1. The first dose of the vaccine composition is administered to the subject in week 0, and subsequent doses are administered between weeks 4, 7, 13, and 25, and thereafter every 12 weeks. The vaccine composition is administered intramuscularly in a total amount of DNA of approximately 8 mg per dose. The vaccine composition is administered in combination with nivolumab and ipilimumab. Nivolumab was administered to the aforementioned subjects in the first phase at a dose of approximately 1 mg / kg every 3 weeks for 4 doses, followed by the second phase. (i) A dose of approximately 240 mg is administered every two weeks, where the first dose of the second stage is administered approximately three weeks after the fourth dose of the first stage; or, (ii) A dose of approximately 480 mg is administered every four weeks, with the first dose of the second stage being administered approximately six weeks after the fourth dose of the first stage; Ipilimumab is administered to the aforementioned subjects at a dose of approximately 3 mg / kg every 3 weeks for 4 doses, and each dose of ipilimumab is administered on the same day as the first stage of 4 doses of nivolumab, A vaccine composition in which the first dose of nivolumab and the first dose of ipilimumab are administered approximately one week after the first dose of the vaccine composition. 61. The composition according to item 60, further characterized by one or more features defined in any one of items 2 to 59.

Claims

1. A composition used for the treatment of cancer in the subject, wherein the composition is (i) A vaccine composition containing one or more nucleic acids encoding multiple tumor antigen T cell epitopes (wherein the T cell epitopes are GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3)), wherein the method comprises the composition, (a) programmed cell death 1 (PD-1) inhibitors; and, (b) cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor; This includes administering it to the subject in combination with the above; (ii) A method comprising a programmed cell death 1 (PD-1) inhibitor, wherein the PD-1 inhibitor is used (a) A vaccine composition containing one or more nucleic acids encoding multiple tumor antigen T cell epitopes (wherein the T cell epitopes are GTGRAMLGHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3)); and, (b) cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor; This includes administering the above to the subject in combination with the above; or (iii) A cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor is included in the method, and the CTLA-4 inhibitor is used (a) A vaccine composition containing one or more nucleic acids encoding multiple tumor antigen T cell epitopes (wherein the T cell epitopes are GTGRAMLGHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3)); and, (b) Programmed cell death 1 (PD-1) inhibitors; A composition comprising administering to the subject in combination with the above.

2. The composition according to claim 1, wherein the cancer is melanoma, and in some cases, the melanoma is stage III or stage IV melanoma, metastatic melanoma, and / or unresectable.

3. The composition according to any one of the preceding claims, wherein the subject has never received systemic treatment for advanced melanoma, and in some cases the melanoma is an unresectable stage III or stage IV melanoma, and the subject has never received systemic treatment for a progressive disease.

4. The composition according to any one of the preceding claims, wherein the one or more nucleic acids encode an antibody having a heavy chain and a light chain in which the T cell epitope is inserted or substituted.

5. The composition according to claim 4, wherein the T cell epitope is inserted into or replaced in one or more CDRs of the antibody.

6. The aforementioned antibody is (i) A T cell epitope of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) on CDR-H1; (ii) T cell epitope of SVYDFFVWL (SEQ ID NO: 2) on CDR-H2; and, (iii) T cell epitope of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-L1; Or, (i) T cell epitopes of GTGRAMLGTHMETVTVYH (SEQ ID NO: 1) in CDR-H1 and CDR-L3, respectively; (ii) T cell epitope of SVYDFFVWL (SEQ ID NO: 2) on CDR-H2; (iii) T cell epitopes of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-H3 and CDR-L1, respectively; The composition according to claim 4 or 5, having the following characteristics.

7. The composition according to any one of the preceding claims, wherein the T cell epitope further comprises VPLDCVLYRYGFSVTLDIVQG (SEQ ID NO: 4), ANCSVYDFFVWLHYYSVRRDTLLGPGRPYR (SEQ ID NO: 5), and QCTEVRADTRPWSGPYILRNQDDRELWPRKFF (SEQ ID NO: 6).

8. The aforementioned antibody is (i) A T cell epitope of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) on CDR-H1; (ii) T cell epitope of SVYDFFVWL (SEQ ID NO: 2) on CDR-H2; (iii) A T cell epitope of VPLDCVLYRYGSFSVTLDIVQG (Sequence ID 4) on CDR-H3; (iv) T cell epitope of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-L1; (v) ANCSVYDFFVWLHYYSVRRDLLGPGRPYR (Sequence ID 5) T cell epitope in CDR-L2; and, (vi) T cell epitope QCTEVRADTRPWSGPYILRNQDDRELWPRKFF (SEQ ID NO: 6) on CDR-L3; The composition according to claim 7, which has any one of claims 4 to 6 added to it.

9. The composition according to any one of claims 4 to 8, wherein the antibody comprises (a) a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8, or (b) a VH containing the amino acid sequence shown in SEQ ID NO: 9 and a VL containing the amino acid sequence shown in SEQ ID NO:

10.

10. The composition according to any one of claims 4 to 9, wherein the antibody comprises (a) a heavy chain containing the amino acid sequence shown in SEQ ID NO: 11 and a light chain containing the amino acid sequence shown in SEQ ID NO: 12, or (b) a heavy chain containing the amino acid sequence shown in SEQ ID NO: 13 and a light chain containing the amino acid sequence shown in SEQ ID NO:

14.

11. The composition according to any one of the preceding claims, wherein the one or more nucleic acids comprises one or more nonspecific promoters that promote the expression of the T cell epitope, wherein the promoter is a CMV-IE promoter, and further optionally the promoter has the nucleotide sequence shown in SEQ ID NO:

15.

12. The composition according to any one of the preceding claims, wherein the antibody comprises a leader sequence that promotes the secretion of the antibody when one or more nucleic acids are expressed, wherein the leader sequence is located at the N-terminus of the heavy chain and / or the N-terminus of the light chain of the antibody, and further optionally the leader sequence comprises the amino acid sequence shown in SEQ ID NO:

16.

13. The vaccine composition according to any one of the preceding claims, comprising a single nucleic acid encoding the plurality of tumor antigen T cell epitopes, wherein the nucleic acid optionally comprises the nucleotide sequence shown in SEQ ID NO: 17 or SEQ ID NO:

18.

14. The composition according to any one of the preceding claims, wherein the PD-1 inhibitor is nivolumab.

15. The composition according to any one of the preceding claims, wherein the CTLA-4 inhibitor is ipilimumab.

16. The vaccine composition according to any one of the preceding claims, wherein the total amount of nucleic acid is administered in a dose of 6 to 10 mg, preferably about 8 mg, per dose.

17. The vaccine composition according to any one of the preceding claims, wherein the first dose of the vaccine composition is administered to the subject in (a) week 0, and subsequent doses are administered between weeks 4, 7, 13, and 25, and thereafter every 12 weeks, or (b) on day 1, and subsequent doses are administered on days 29, 50, 92, and 176, and thereafter every 84 days.

18. The PD-1 inhibitor was administered to the subjects in the first stage at a dose of approximately 1 mg / kg every three weeks for four doses, followed by the second stage. (i) A dose of approximately 240 mg is administered every two weeks, where the first dose of the second stage is administered approximately three weeks after the fourth dose of the first stage; or, (ii) A dose of approximately 480 mg is administered every four weeks, with the first dose of the second stage being administered approximately six weeks after the fourth dose of the first stage; The composition according to any one of the preceding claims.

19. The composition according to any one of the preceding claims, wherein the CTLA-4 inhibitor is administered to the subject at a dose of approximately 3 mg / kg every three weeks for four doses, and in some cases, each dose of the CTLA-4 inhibitor is administered on the same day as the first four doses of the PD-1 inhibitor.

20. The composition according to any one of the preceding claims, wherein the CTLA-4 inhibitor and the PD-1 inhibitor are administered on the same day, the CTLA-4 inhibitor and the PD-1 inhibitor are administered in separate compositions or as a single composition, and / or the first administration of the CTLA-4 inhibitor and the first administration of the PD-1 inhibitor are administered about one week after the first administration of the vaccine composition.

21. A vaccine composition for use in the treatment of unresectable stage III or stage IV melanoma in a subject, comprising a DNA molecule encoding an antibody in which multiple tumor antigen T cell epitopes are inserted or substituted, The antibody has a T cell epitope of GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) inserted or substituted in CDR-H1, a T cell epitope of SVYDFFVWL (SEQ ID NO: 2) inserted or substituted in CDR-H2, and a T cell epitope of WNRQLYPEWTEAQRLD (SEQ ID NO: 3) inserted or substituted in CDR-L1. The first administration of the vaccine composition is given to the subject in week 0, and subsequent administrations are given between weeks 4, 7, 13, and 25, and thereafter every 12 weeks. The vaccine composition is administered intramuscularly in a total amount of DNA of approximately 8 mg per dose. The vaccine composition is administered in combination with nivolumab and ipilimumab. Nivolumab was administered to the aforementioned subjects in the first phase at a dose of approximately 1 mg / kg every three weeks for four doses, followed by the second phase. (i) A dose of approximately 240 mg is administered every two weeks, where the first dose of the second stage is administered approximately three weeks after the fourth dose of the first stage; or, (ii) A dose of approximately 480 mg is administered every four weeks, with the first dose of the second stage being administered approximately six weeks after the fourth dose of the first stage; Ipilimumab is administered to the aforementioned subjects at a dose of approximately 3 mg / kg every three weeks for four doses, and each dose of ipilimumab is administered on the same day as the four doses of the first stage of nivolumab, and, The first dose of nivolumab and the first dose of ipilimumab shall be administered approximately one week after the first dose of the vaccine composition. composition.

22. The composition according to claim 21, further characterized by one or more of the features defined in any one of claims 1 to 20.

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