Vaccine compositions and uses thereof

Abstract

The present invention relates generally to vaccine compositions and their use in methods of treating cancer, such as melanoma, in a subject. The present invention relates to a vaccine composition comprising one or more nucleic acids encoding a plurality of tumor antigen T cell epitopes, where the T cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3), and administered to a subject in combination with: (a) a Programmed Death-1 (PD-1) inhibitor, such as Nasumab; and (b) a cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor, such as ipirazumab. One or more nucleic acids may encode an antibody comprising a heavy chain and a light chain into which a T cell epitope is inserted or substituted.

Description

Technical Field

[0001] This invention generally relates to vaccine compositions and their use in methods of treating cancer in subjects. Background Technology

[0002] Immunotherapy using checkpoint inhibitors (CPIs), including monotherapy with programmed death-1 (PD-1) inhibitors and combination therapy with cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitors, has altered first-line treatment outcomes in 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 PlusIpilimumab 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, surrogate survival endpoints such as overall response rate (ORR) and 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 real-world settings, the combined nivolumab and ipilimumab achieved an ORR close to 50% (P. Serra-Bellver et al. Real-world outcomes with ipilimumab and nivolumab in advanced melanoma: a multicentreretrospective study European Journal of Cancer 176 (2022) 121e132), and some practitioners have reported 28% in real-world settings (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). Recently, a nucleic acid therapeutic vaccine, in combination with pembrolizumab, has shown efficacy in delaying recurrence in the unresectable stage of melanoma (LJ Albrecht et al., The Latest Option: Nivolumab and Relatlimabin Advanced Melanoma. Curr Oncol Rep 25, 647–657 (2023)). However, in the unresectable stage of the disease, the nucleic acid vaccine has not yet shown a meaningful effect in reducing tumor burden. Summary of the Invention

[0003] In one aspect, the present invention provides a composition for a method of treating cancer, wherein the composition:

[0004] (i) is a vaccine composition comprising one or more nucleic acids encoding multiple tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3), and wherein the method comprises administering the composition in combination with the following substances to a subject:

[0005] (a) Programmed death-1 (PD-1) inhibitors; and

[0006] (b) Cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors;

[0007] (ii) Containing a programmed death-1 (PD-1) inhibitor, wherein the method comprises administering the PD-1 inhibitor in combination with the following substances to a subject:

[0008] (a) A vaccine composition comprising one or more nucleic acids encoding multiple tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3); and

[0009] (b) Cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors; or

[0010] (iii) Containing a cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitor, wherein the method comprises administering the CTLA-4 inhibitor in combination with the following substances to the subject:

[0011] (a) A vaccine composition comprising one or more nucleic acids encoding multiple tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3); and

[0012] (b) Programmed death-1 (PD-1) inhibitors.

[0013] In another aspect, the present invention provides a composition for a method of treating cancer in a subject, the composition comprising one or more nucleic acids encoding a plurality of tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3), and wherein the method comprises administering the composition in combination with the following substances to the subject:

[0014] (a) Programmed death-1 (PD-1) inhibitors; and

[0015] (b) Cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors.

[0016] In another aspect, the present invention provides a composition comprising a programmed death-1 (PD-1) inhibitor in a method for treating cancer in a subject, wherein the method comprises administering the PD-1 inhibitor in combination with the following substances to the subject:

[0017] (a) A vaccine composition comprising one or more nucleic acids encoding multiple tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3); and

[0018] (b) Cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors.

[0019] In another aspect, the present invention provides a composition comprising a cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitor in a method for treating cancer in a subject, wherein the method comprises administering the CTLA-4 inhibitor in combination with the following substances to the subject:

[0020] (a) A vaccine composition comprising one or more nucleic acids encoding multiple tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3); and

[0021] (b) Programmed death-1 (PD-1) inhibitors.

[0022] In another aspect, the present invention provides a method for treating cancer in a subject, wherein the method includes administering a vaccine composition to the subject, wherein the vaccine comprises one or more nucleic acids encoding a plurality of tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3), and wherein the vaccine composition is administered to the subject in combination with the following substances:

[0023] (a) Programmed death-1 (PD-1) inhibitors; and

[0024] (b) Cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors.

[0025] This invention is based, at least in part, on the inventors' surprising discovery that administration of a nucleic acid encoding three T-cell epitopes—GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3) (referred to as "SCIB1")—in combination with ipilimumab and nivolumab unexpectedly resulted in an overall response rate of 81.8%. This is significantly higher than the approximately 50% overall response rate previously reported for the ipilimumab and nivolumab combination in the absence of the nucleic acid.

[0026] In another aspect, the present invention provides a vaccine composition for a method of treating unresectable stage III or IV melanoma, the vaccine composition comprising a DNA molecule encoding an antibody, wherein a plurality of tumor antigen T-cell epitopes are inserted into or substituted into the antibody.

[0027] The antibody contains the T-cell epitope GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) inserted or substituted into CDR-H1, the T-cell epitope SVYDFFVWL (SEQ ID NO: 2) inserted or substituted into CDR-H2, and the T-cell epitope WNRQLYPEWTEAQRLD (SEQ ID NO: 3) inserted or substituted into CDR-L1.

[0028] The first dose of the vaccine composition was administered to the subject at week 0, followed by subsequent doses at weeks 4, 7, 13, 25, and every 12 weeks thereafter. The vaccine composition was administered intramuscularly at a total DNA content of approximately 8 mg per dose.

[0029] The vaccine composition is administered in combination with nivolumab and ipilimumab.

[0030] In the first phase, nivolumab was administered to subjects at four doses of approximately 1 mg / kg every three weeks, followed by administration in the second phase:

[0031] (i) A dose of approximately 240 mg every two weeks, with the first dose of the second phase administered approximately three weeks after the fourth dose of the first phase; or

[0032] (ii) A dose of approximately 480 mg every four weeks, with the first dose of Phase II administered approximately six weeks after the fourth dose of Phase I.

[0033] Ipilimumab was administered to subjects in four doses of approximately 3 mg / kg every three weeks, with each dose of ipilimumab being administered on the same day as each of the four doses of nivolumab in the first phase.

[0034] The first dose of ipilimumab and the first dose of nivolumab were administered approximately one week after the first dose of the vaccine composition.

[0035] In another aspect, the present invention provides a method for treating unresectable stage III or IV melanoma in a subject, wherein the method comprises administering to the subject a vaccine composition comprising a DNA molecule encoding an antibody, wherein a plurality of tumor antigen T-cell epitopes are inserted into or substituted into the antibody.

[0036] The antibody contains the T-cell epitope GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) inserted or substituted into CDR-H1, the T-cell epitope SVYDFFVWL (SEQ ID NO: 2) inserted or substituted into CDR-H2, and the T-cell epitope WNRQLYPEWTEAQRLD (SEQ ID NO: 3) inserted or substituted into CDR-L1.

[0037] The first dose of the vaccine composition was administered to the subject at week 0, followed by subsequent doses at weeks 4, 7, 13, 25, and every 12 weeks thereafter. The vaccine composition was administered intramuscularly at a total DNA content of approximately 8 mg per dose.

[0038] The vaccine composition is administered in combination with nivolumab and ipilimumab.

[0039] In the first phase, nivolumab was administered to subjects at four doses of approximately 1 mg / kg every three weeks, followed by administration in the second phase:

[0040] (i) A dose of approximately 240 mg every two weeks, with the first dose of the second phase administered approximately three weeks after the fourth dose of the first phase; or

[0041] (ii) A dose of approximately 480 mg every four weeks, with the first dose of Phase II administered approximately six weeks after the fourth dose of Phase I.

[0042] Ipilimumab was administered to subjects in four doses of approximately 3 mg / kg every three weeks, with each dose of ipilimumab being administered on the same day as each of the four doses of nivolumab in the first phase.

[0043] The first dose of ipilimumab and the first dose of nivolumab were administered approximately one week after the first dose of the vaccine composition.

[0044] These and other aspects of the invention are described in further detail below. Unless the context otherwise requires, features of each aspect of the invention are applicable to the other aspects with necessary modifications. Detailed Implementation

[0045] As described above, this invention is partly based on positive interim data from the first phase of a Phase II trial called "SCOPE," which investigated the combination of "SCIB1" with the checkpoint inhibitors (CPIs) ipilimumab and nivolumab for advanced melanoma. Preliminary data from 11 patients showed an objective response rate (ORR) of 82%. The SCOPE Phase II trial aimed to determine whether the combination of SCIB1 and CPIs could improve the ORR in patients with unresectable metastatic melanoma. The concept is that the vaccine induces new immune responses or enhances existing immune responses, which are then protected by CPIs in the tumor setting. During the first phase of the SCOPE trial, patients received SCIB1 via a needle-free device in combination with the most effective currently available treatments, namely the CPIs nivolumab and ipilimumab. The first milestone of the SCOPE trial was a response in 8 out of more than 15 patients, demonstrating that the SCIB1 combination with dual CPI therapy could significantly improve the current outcomes for these patients. Sixteen patients with stage IV metastases received this combination. To date, 11 patients in these studies have reached week 13 and undergone radiographic evaluation, with 9 showing an objective response (ORR) of 82% and no increase in toxicity. At this time point, tumor volume decreased by 31%–94%. Four patients reaching week 25 and two reaching week 37 showed reductions in total tumor burden of 69%–94% and 87%–94%, respectively. This is significantly superior to the 50% ORR reported in patients receiving this dual CPI therapy (without SCIB1) in a real-world setting, with a progression-free survival of 11.5 months.

[0046] Unless otherwise specifically defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., immunology, molecular biology, cancer therapy and / or biochemistry).

[0047] Unless otherwise stated, the techniques used in this invention are standard procedures well known to those skilled in the art. These techniques are described and explained in the literature, such as 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); and FM Ausubelet et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley - Interscience (1988, including all updates to date); Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory. (1988); and JE Coligan et al., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0048] As used herein, unless otherwise stated, the term “about” includes + / - 10% of the specified value, more preferably + / - 5%.

[0049] In this specification, the word “comprising” or variations thereof such as “including” or “containing” will be understood to imply inclusion of the stated elements, integers or steps or groups of elements, integers or steps, but does not exclude any other elements, integers or steps or groups of elements, integers or steps.

[0050] In one aspect, the present invention relates to vaccine compositions for use in methods of treating cancer. The term "vaccine composition" refers to a composition capable of promoting an immune response, preferably a CD8+ and / or CD4+ T cell response.

[0051] The vaccine compositions used in this invention comprise 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 peptide comprising an amino acid sequence that is recognized by T cells (preferably CD8+ or CD4+ T cells) when presented by the major histocompatibility complex (MHC). As used herein, the term "T-cell epitope" encompasses a peptide that may comprise multiple distinct, shorter amino acid sequences recognized by T cells throughout its peptide sequence. However, the term "T-cell epitope" does not include larger polypeptide sequences, such as sequences greater than 40 amino acids. For example, the "T-cell epitope" GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1) comprises at least three identified epitope sequences throughout its peptide sequence, as shown in the table below.

[0052]

[0053] The references in the table above are provided below:

[0054] Tsai V, Southwood S, Sidney J, Sakaguchi K, Kawakami Y, Appella E,Sette A, Celis E. Identification of subdominant CTL epitopes of the GP100melanoma-associated tumor antigen by primary in vitro immunization with peptide-pulsed dendritic cells. J Immunol. 1997 Feb 15;158(4):1796-802. PMID:9029118.

[0055] Lapointe R, Royal RE, Reeves ME, Altomare I, Robbins PF, Hwu P.Retrovirally transduced human dendritic cells can generate T cellsrecognizing multiple MHC class I and class II epitopes from the melanomaantigen glycoprotein 100. J Immunol. 2001 Oct 15;167(8):4758-64. doi:10.4049 / jimmunol.167.8.4758. PMID: 11591807.

[0056] Kobayashi H, Lu J, Celis E. Identification of helper T-cell epitopesthat encompass or lie proximal to cytotoxic T-cell epitopes in the gp100melanoma tumor antigen. Cancer Res. 2001 Oct 15;61(20):7577-84. PMID:11606397.

[0057] Parkhurst MR, Fitzgerald EB, Southwood S, Sette A, Rosenberg SA,Kawakami Y. Identification of a shared HLA-A*0201-restricted T-cell epitopefrom the melanoma antigen tyrosinase-related protein 2 (TRP2). Cancer Res.1998 Nov 1;58(21):4895-901. PMID: 9809996.

[0058] Touloukian CE, Leitner WW, Topalian SL, Li YF, Robbins PF, RosenbergSA, Restifo NP. Identification of a MHC class II-restricted human gp100epitope 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.

[0059] Nucleic acids are administered to a subject in a vaccine composition for uptake by cells, enabling the expression and presentation of T-cell epitopes to T cells in the subject. T-cell epitopes may be encoded by multiple different nucleic acids, for example, a total of two or three nucleic acid molecules, or preferably all encoded in a single nucleic acid molecule. Therefore, the term "nucleic acid" is used herein to refer to a nucleic acid molecule having a polynucleotide sequence. For example, a vaccine composition may include a single nucleic acid molecule containing a polynucleotide sequence encoding three T-cell epitopes.

[0060] Nucleic acids can be DNA, cDNA, or RNA, such as mRNA, obtained, for example, by cloning or produced wholly or partially by chemical synthesis. Preferably, the nucleic acid is DNA. For therapeutic purposes, the nucleic acid is preferably in a form capable of being expressed in the subject of treatment. Nucleic acids can be recombinant or provided as isolates in isolated and / or purified forms. In addition to one or more regulatory sequences that may be present for expression, genes encoding T-cell epitopes may be flanked by no nucleic acids, or substantially no nucleic acids. When nucleic acids include RNA, references to the sequences shown herein should be interpreted as references to RNA equivalents in which T is replaced by U.

[0061] Given a nucleic acid sequence and available clones, suitable nucleic acids can be readily prepared by a skilled technician, for example, using the information and references contained herein and techniques known in the art (e.g., see 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) amplifying samples of such nucleic acids using polymerase chain reaction (PCR), for example, amplification from genomic sources, (ii) chemical synthesis, or (iii) preparation of cDNA sequences. DNA encoding polypeptides can be generated and used in any suitable manner known to those skilled in the art, including by acquiring the encoding DNA, identifying suitable restriction endonuclease recognition sites on both sides of the portion to be expressed, and cleaving said portion from the DNA. The cap portion can then be operatively ligated to a suitable promoter in a standard commercially available expression system. Another recombinant method is to amplify the relevant portion of the DNA using suitable PCR primers. Sequences can be modified, for example, by using site-directed mutagenesis, to result in the expression of modified peptides or by taking into account codon bias in the host cell used to express nucleic acids.

[0062] To achieve the expression of a nucleic acid sequence, the sequence can be integrated into a vector having one or more control sequences (e.g., promoters) operatively linked to a sequence encoding a T-cell epitope to control its expression. The vector may include other sequences, such as promoters or enhancers, to drive the expression of the nucleic acid sequence. The nucleic acid may encode a secretion signal (also referred to herein as a "lead sequence") that causes the T-cell epitope produced in the host cell to be secreted from the cell. A preferred leader sequence comprises the amino acid sequence provided in SEQ ID NO: 16. The promoter can be a nonspecific (active regardless of the cellular environment) and / or constitutive promoter that does not require a specific cell type or tissue type to drive the expression of the T-cell epitope or a polypeptide containing the T-cell epitope. The promoter can be a CMV-IE promoter, preferably a promoter comprising the nucleotide sequence provided in SEQ ID NO: 15 or a functional variant thereof.

[0063] If desired, a polypeptide encoded by nucleic acid can be obtained by transforming a 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 culture medium. In the art, prokaryotic and eukaryotic cells are used for this purpose, including *Escherichia coli* strains, yeast, and eukaryotic cells such as insect cells, and animal cells such as COS, CHO cells, Bowes Melanoma, and other suitable human cells. When the present invention relates to nucleic acids encoding the heavy and light chains of an antibody, the respective nucleic acids may be present in the same expression vector, driven by the same or different promoters, or in separate expression vectors. Preferably, the heavy and light chain sequences of the antibody are encoded by the same nucleic acid (e.g., the vector) and driven by separate promoters.

[0064] Nucleic acids are used to stimulate an immune response to T-cell epitopes in patients (e.g., mammals, including humans). They can stimulate helper and / or cytotoxic T-cell responses. T-cell responses targeting specific epitopes may have higher affinity than immunization with the same epitope as a simple peptide or with the same epitope encoded within an antigen (as a peptide or nucleic acid). Nucleic acids can be administered as a combination therapy, i.e., nucleic acids encoding a light chain and nucleic acids encoding a heavy chain. Nucleic acids can be administered intravenously, intradermally, intramuscularly, orally, or via other routes. Intradermal or intramuscular administration is particularly suitable because these tissues contain dendritic cells. Preferably, a vaccine composition containing nucleic acids is administered intramuscularly. Preferably, the vaccine composition is administered via needle-free injection.

[0065] Nucleic acid can be a vector. The vector can be used to express T-cell epitopes encoded by the nucleic acid. Nucleic acid can be a DNA molecule. For example, nucleic acid can be a DNA vector. Preferably, the nucleic acid is a DNA plasmid. Preferably, a single DNA plasmid encodes all T-cell epitopes. Alternatively, the nucleic acid can be a doggybone vector.

[0066] Nucleic acids or vectors can be used in combination with pharmaceutically acceptable vectors. Such vectors may include, but are not limited to, saline, buffered saline, glucose, liposomes, water, glycerol, ethanol, and combinations thereof.

[0067] The nucleic acids and / or carriers used in this invention can be formulated into pharmaceutical compositions. In addition to one or more of the substances described above, these compositions may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material may depend on the route of administration, such as intradermal, oral, intravenous, skin or subcutaneous, intranasal, intramuscular, or intraperitoneal routes. The formulation is preferably a stable nucleic acid dry powder precipitated on the surface of microscopic gold particles and suitable for injection via a gene gun, or a DNA solution mixed with GET peptides. The formulation is suitable for intradermal or intramuscular administration using electroporation. The formulation is suitable for needle-free injection administration.

[0068] The compositions described herein are preferably administered to an individual in a "therapeuticly effective amount," sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate and duration of administration, will depend on the nature and severity of the disease being treated. Treatment protocols (e.g., the determination of dosage, etc.) are the responsibility of general practitioners and other physicians, and generally take into account the disease to be treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the practitioner. The nucleic acids of the present invention particularly relate to the treatment of existing cancers and the prevention of recurrence of cancer after initial treatment or surgery. Examples of the above-described techniques and solutions can be found in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980.

[0069] Preferably, when administered to humans in an effective amount, nucleic acid stimulation can kill tumor helper and / or cytotoxic T cells. The optimal dose can be determined by a physician based on a number of parameters, including, for example, age, sex, weight, severity of the disease being treated, the active ingredient being administered, and the route of administration. For example, a dose of 1–10,000 μg of DNA may be sufficient to stimulate both helper and cytotoxic T cell responses.

[0070] The vaccine compositions described herein are administered in combination with programmed death-1 (PD-1) inhibitors and cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors. These may be administered concurrently 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 chemotherapy agents, other radiation therapy techniques, or other immunotherapies known in the art.

[0071] The dosage of nucleic acid will depend on the properties of the reagent used, such as its binding activity and plasma half-life, the concentration of nucleic acid in the formulation, the route of administration, the site and rate of administration, the clinical tolerability of the patients involved, and its impact on the patient's pathological condition, all of which are within the scope of the physician's skill. Different dosages can be used in a series of consecutive vaccinations; practitioners can administer an initial vaccination followed by a booster dose of relatively small amounts of nucleic acid.

[0072] The vaccine composition can be administered at a total nucleic acid concentration ranging from 1-20 mg, 5-15 mg, or 6-10 mg per dose. The vaccine composition can also be administered to the subject at a total nucleic acid concentration of approximately 8 mg per dose. Preferably, the vaccine composition is administered to the subject at a total nucleic acid concentration of 8 mg per dose. The term "dose" refers to the total amount of active ingredient administered to the subject, which can be administered, for example, as multiple separate injections. For example, an 8 mg dose can be administered as four separate 2 mg injections at a single injection site.

[0073] The timing of administration of the vaccine composition can be determined by those skilled in the art. Preferably, the first dose of the vaccine composition is administered to the subject at week 0, followed by subsequent doses at weeks 4, 7, 13, 25, and every 12 weeks thereafter. Hereinafter, “week 0” refers to the week in which the subject receives the first dose. Similarly, “week 4” refers to approximately four weeks later than week 0, and so on. Even more preferably, the first dose of the vaccine composition is administered to the subject on day 1, followed by subsequent doses on days 29, 50, 92, 176, and every 84 days thereafter. In this context, “day 1” refers to the day in which the subject receives the first dose, “day 29” refers to 28 days (i.e., four weeks) later, and so on.

[0074] Preferably, the vaccine composition is administered to the subject according to the dosing regimen described in “SCIB1” of the clinical trial protocol (“SCOPE” trial) provided in Example 3 of this document.

[0075] Nucleic acids can be introduced into host cells. Introduction (especially for in vitro introduction) can generally be referred to as "conversion" without restriction and can employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-glucan, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia virus, or, for insect cells, baculoviruses). For bacterial cells, suitable techniques may include calcium chloride conversion, electroporation, and phage transfection. As an alternative, direct injection of nucleic acids can be used.

[0076] Marker genes, such as antibiotic resistance or sensitivity genes, can be used to identify clones containing nucleic acids of interest, as is well known in the art.

[0077] The introduction may lead to or allow nucleic acid expression, for example, by culturing host cells (which may include the actual transformed cells, but more likely the cells will be progeny of the transformed cells) under conditions suitable for gene expression, resulting in the production of the encoded polypeptide (or peptide). If the polypeptide is expressed in conjugate with a suitable signal precursor 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 appropriate, and subsequently used as needed, for example, in the formulation of compositions that may contain one or more additional components, such as pharmaceutical compositions containing one or more pharmaceutically acceptable excipients, solvents, or carriers (see below, for example).

[0078] In addition to the active ingredient, the pharmaceutical composition may contain pharmaceutically acceptable excipients, diluents, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials will depend on the route of administration, which may be oral or by injection, such as intradermal or intramuscular injection.

[0079] Injection is expected to be the primary route of therapeutic administration of the composition, although delivery via catheter or other surgical conduits is also possible. Suitable routes of administration include intravenous, subcutaneous, intraperitoneal, and intramuscular administration. Liquid formulations can be used after reconstitution from powder formulations. The preferred route of administration is intramuscular, such as via needle-free injection.

[0080] The nucleic acids, vectors, peptides and / or vaccines of the present invention can be administered to the recipient using needle-free injection. As known to those skilled in the art, needle-free injectors (also known as "jet injectors") use a narrow, high-pressure liquid that penetrates the outermost layer of the skin (stratum corneum) to deliver the composition to the deep tissues of the epidermis or dermis (i.e., intradermal injection), fat (i.e., subcutaneous injection), or muscle (i.e., intramuscular injection).

[0081] For intravenous or site-specific injection, the active ingredient will be in a parenteral, pyrogen-free aqueous solution with suitable pH, isotonicity, and stability. Those skilled in the art can readily prepare suitable solutions using isotonic solvents such as sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0082] Pharmaceutical compositions intended for oral administration may be in tablet, capsule, powder, or liquid form. Tablets may contain a solid carrier, such as gelatin or an adjuvant. Liquid pharmaceutical compositions typically contain a liquid carrier, such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may include physiological saline solutions, dextran 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, a physiological saline solution containing a non-phosphate buffer solution with a pH of 6.8–7.6, or a lyophilized powder.

[0083] The vaccine composition may contain a concentration of approximately 4 mg / mL of nucleic acid. The vaccine composition may 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 contain nucleic acid in Dulbecco phosphate-buffered saline (calcium or magnesium-free).

[0084] Nucleic acids encoding T-cell epitopes can be administered to a patient using any of a variety of methods known in the art. For example, nucleic acids can be delivered directly as “naked DNA.” This method is described, for example, in Wolff et al., Science 247:1465-1468 (1990) and U.S. Patent Nos. 5,580,859 and 5,589,466. Nucleic acids can also be administered using ballistic delivery, as described, for example, in U.S. Patent No. 5,204,253. Particles containing only DNA can be administered. Alternatively, the DNA can be adhered to particles such as gold particles. Typically, plasmids used in vaccines or immunizing compositions may contain DNA encoding an antigen (e.g., one or more neoantigens) operatively linked to a regulatory sequence that controls the expression or 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 viral promoter (e.g., a CMV promoter, such as an hCMV or mCMV promoter, such as an early-intermediate promoter, or an SV40 promoter—see referenced or incorporated herein for useful promoters), DNA of a eukaryotic leader peptide for secretion (e.g., tissue plasminogen activator), DNA of the neoantigen, and DNA encoding a terminator (e.g., a 3'UTR transcription terminator or bGH polyA from a gene encoding bovine growth hormone). The composition may contain more than one plasmid or vector, whereby each vector contains and expresses a different T-cell epitope, but preferably all epitopes are encoded by a single nucleic acid (e.g., the vector). Also mentioned are Wasmoen U.S. Patent No. 5,849,303 and Dale U.S. Patent No. 5,811,104. DNA or DNA plasmid formulations can be formulated with or within cationic lipids; regarding cationic lipids and adjuvants, Loosmore U.S. Patent Application 2003 / 0104008 is also mentioned. Furthermore, the teachings in Audonnet U.S. Patent Nos. 6,228,846 and 6,159,477 can be relied upon to obtain DNA plasmid teachings that can be used to construct and use DNA plasmids containing and expressing in vivo sequences encoding T-cell epitopes.

[0085] Nucleic acids can also be delivered in combination with cationic compounds such as cationic lipids. Lipid-mediated gene delivery methods are described, for example, in WO1996 / 18372; WO1993 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Patent No. 5,279,833; WO1991 / 06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987). RNA encoding the peptide of interest (e.g., mRNA) can also be used for delivery (see, for example, Kiken et al, 2011; Su et al, 2011; see also US 8278036; Halabi et al. J Clin Oncol (2003) 21: 1232-1237; Petsch et al, Nature Biotechnology 2012 Dec 7;30(12): 1210-6).

[0086] Nucleic acids can be administered as part of a viral vector, such as adenovirus, lentivirus, or AAV vector, or any other viral vector known in the art.

[0087] The composition can also be administered via microspheres, liposomes, other particulate delivery systems, or as a sustained-release formulation placed in certain tissues, including blood. Suitable examples of sustained-release carriers include semi-permeable polymer matrices in the form of shared articles, such as suppositories or microcapsules. Implantable or microcapsule-based sustained-release matrices include polylactide (US Patent No. 3,773,919; EPA0058481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, and poly(2-hydroxyethyl methacrylate). Liposomes containing peptides are prepared by well-known methods: EP A 0052522; EP A 0036676; EP A0088046; EP A 0143949; EP A 0142541; JP A 83 11808; US Patent Nos. 4,485,045 and 4,544,545. Typically, liposomes are small (approximately 200-800 Å) monolayers containing a lipid content greater than approximately 30 mol.% cholesterol, with the selected ratio adjusted for optimal peptide leakage rate. The composition can be applied topically to tumor sites or other desired sites, or delivered in a manner that targets tumors or other cells.

[0088] This invention also relates to the use of constructs in the form of plasmids, vectors, transcription or expression cassettes, said constructs comprising the nucleic acids described above. This invention also provides a recombinant host cell comprising one or more constructs as described above. Systems for cloning and creating suitable constructs in a variety of 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 promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other appropriate sequences. Vectors can be plasmids, viruses such as bacteriophages or phage particles, as appropriate. Many known techniques and methods for nucleic acid manipulation (e.g., in nucleic acid construct preparation, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis) are described in detail in Ausubel et al., 1992.

[0089] Nucleic acids can be introduced into host cells. This introduction can be performed using any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-glucan, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia virus, or, for insect cells, baculovirus). For bacterial cells, suitable techniques may include calcium chloride conversion, electroporation, and phage transfection. After introduction, nucleic acid expression may be induced or permitted, for example, by culturing host cells under conditions suitable for gene expression. In one embodiment, the nucleic acids of the present invention are integrated into the genome (e.g., chromosome) of a host cell. Integration can be facilitated by including sequences that promote recombination with the genome, according to standard techniques. The present invention also provides a method comprising using the constructs described above in an expression system to express the aforementioned T-cell epitopes.

[0090] One or more nucleic acids in a vaccine composition may encode an antibody comprising a heavy chain and a light chain into which a T-cell epitope is inserted or substituted. In this respect, the T-cell epitope is heterologous to the antibody. The term “insertion or substitution” does not require a physical cloning step of the substituted sequence. These terms are used only to indicate that the T-cell epitope sequence is heterologously present in the antibody sequence. Such antibodies engineered to contain heterologous T-cell epitopes for stimulating an immune response are referred to in the art as “immunoantibodies.” Suitable antibodies are described in WO2008 / 116937, which is incorporated herein by reference. A suitable antibody that the nucleic acid in the vaccine composition may encode is the antibody referred to as “DCIB68” in WO2008 / 116937.

[0091] The antibody comprises a heavy chain and a light chain, each preferably containing a variable region and a constant region. Suitable antibody sequences, and the structure and location of the immunoglobulin domains therein, are well known in the art and can be determined with reference to http: / / www.imgt.org / . Throughout this specification, residue numbering refers to the standardized IMGT system used for antibody sequence numbering, as disclosed in Lefranc et al., 2009. Other suitable numbering systems are known to those skilled in the art.

[0092] Antibodies can be monoclonal or polyclonal, and can be IgA, IgD, IgE, IgG, or IgM, but IgG is preferred. IgG antibodies can be any IgG subclass, such as human IgG1, IgG2, IgG3, or IgG4, or mouse IgG1, IgG2a, IgG2b, or IgG3. Human IgG1 antibodies are preferred. Antibodies may have a constant region of a human antibody, and a variable or hypervariable region of a mouse or human monoclonal antibody in which T-cell epitopes have been inserted. The variable region other than the hypervariable region can also be derived from the variable region of a human antibody. Such antibodies are referred to as humanized. Methods for preparing humanized antibodies are known in the art. For example, a method is described in Winter U.S. Patent No. 5,225,539. The variable region of an antibody other than the mouse hypervariable region can also be derived from a mouse monoclonal antibody. In this case, the entire variable region is derived from a mouse monoclonal antibody, which is referred to as chimeric. Methods for preparing chimeric antibodies are known in the art. Such methods include those described in, for example, U.S. patents of Boss (Celltech) and Cabilly (Genentech). See U.S. Patent Nos. 4,816,397 and 4,816,567, respectively.

[0093] In certain aspects of this invention, nucleic acids that are expressed by the heavy chain, light chain, or antibody possess at least one heterologous T-cell epitope, such that the heavy chain, light chain, or immunoglobulin molecule may not exhibit its native conformation. The T-cell epitope may disrupt the expressed protein, for example, preventing the heavy chain or immunoglobulin molecule from binding to its antigen, preventing (if present) heavy and light chains from associating, or preventing the heavy chain or immunoglobulin molecule from being secreted normally. Disruption may occur in the tertiary structure of the immunoglobulin molecule, potentially preventing disulfide bond formation.

[0094] T-cell epitopes can be inserted into or replace the CDR region of an antibody. CDR1 and CDR2 form part of the antibody's β-sheet conformation and are partially embedded within the folded molecule. Any alteration to their length, amino acid composition, or charge can disrupt this structure and prevent heavy and light chain folding and association. CDRH3 is exposed on the surface of the immunoglobulin molecule and is therefore more tolerant to alterations. In this invention, it is preferred that at least CDR1 and / or CDR2 be replaced by T-cell epitopes. In practice, in some embodiments, the loss of the framework region at the CDRH junction completely disrupts antibody folding; however, epitope insertion in these regions produces a good T-cell response. Incorporation of any epitope into CDRH1 (5 amino acids long) or CDRH2 (17 amino acids long) causes sufficient disruption to allow heavy chain secretion, but the amount of intact antibody is very low, even if the light chain has its native sequence. This indicates that secondary structure is important for heavy and light chain pairing. Incorporating any epitope into CDRL1 of the light chain results in low levels of light chain secretion, as does incorporating only a single epitope into CDRH3 of the heavy chain.

[0095] "Heterologous T-cell epitope" refers to a T-cell epitope that is heterologous to the antibody. For example, a heterologous T-cell epitope can be an epitope that was not previously present in the antibody. Heterologous T-cell epitopes can be inserted as a whole, although they may consist of the inserted amino acid sequence along with flanking amino acids from a second part. This is to ensure that the inserted epitope has similar processing characteristics to the original antigen in the heterologous nucleic acid. One or more CTL / helper epitopes can be inserted within the same variable region.

[0096] T-cell epitopes can be inserted at any location in the heavy or light chain. Preferably, the epitope, or each epitope, is inserted into the variable region of the heavy and / or light chain; however, nucleic acids encoding the heavy chain, or nucleic acids encoding antibodies that have the inserted T-cell epitope only in the constant region, or in both the constant and variable regions of the heavy and / or light chain, are also included in this invention. In the nucleic acids of this invention, the sequence encoding the T-cell epitope can be inserted (i.e., added to) the sequence encoding the heavy and / or light chain, or can be substituted into the sequence encoding the heavy and / or light chain.

[0097] In the variable region, T-cell epitopes can be inserted into or replace any one or more CDRs (i.e., L1, L2, L3, H1, H2, or H3) of the heavy and / or light chains. Currently, L1, H1, and H2 are preferred. In some embodiments, T-cell epitopes are inserted into or replace CDRs L1 and / or H1 and / or H2. Preferably, the incorporated T-cell epitopes differ in size and charge from the original CDR amino acids of the antibody, causing the antibody to not exhibit its native conformation, such as misfolding and missecretion. Optionally or additionally, they can be inserted into or replace the frame region surrounding the CDR. Epitopes can be inserted using complementary oligonucleotides encoding antigenic epitopes, which are annealed and cloned into specific locations within the antibody frame where the CDR (or other regions) has been replaced by unique restriction enzyme sites. The ability of recombinant antibodies to stimulate helper and cytotoxic T cell responses can be screened according to the examples in this article.

[0098] Various combinations are possible within this invention. In some embodiments, one or more CD8 epitopes are inserted into, and / or replace, the CDRs H1 and / or H2 of the heavy chain of the antibody, or are inserted into, and / or replace, a non-CDR variable region. Additionally or alternatively, one or more CD4 epitopes may be inserted into, and / or replace, the CDR L1 of the light chain of the antibody, or be inserted into, and / or replace a non-CDR variable region. When multiple T-cell epitopes are present, these T-cell epitopes may be the same or different. Those skilled in the art will understand that many combinations are possible, including:

[0099] • CD8 epitope in CDR H1 and CD4 epitope in CDR L1;

[0100] • CD8 epitope in CDR H2 and CD4 epitope in CDR L1;

[0101] • CD8 tabletops in CDR H1 and CDR H2, and CD4 tabletops in CDR L1;

[0102] • Two CD8 tabletops in CDR H1 and a CD4 tabletop in CDR L1;

[0103] • Two CD8 tabletops in CDR H2 and a CD4 tabletop in CDR L1; etc.

[0104] The nucleic acids of the present invention may incorporate multiple T-cell epitopes from a single target antigen, which can bind to most class I and class II MHC molecules. This can produce a vaccine that can be used for vaccination of a broad population. Alternatively, the nucleic acids used in the present invention may incorporate multiple T-cell epitopes from multiple target antigens, which can bind to the most common class I and class II phenotypes. This can produce a vaccine that prevents selection to antigen-loss variants. The target antigen may be derived from a single pathogen or tumor type, or may be selected to generate an immune response against multiple pathogens or cancers. Numerous T-cell epitopes from multiple cancers and / or pathogens may be incorporated into the nucleic acids used in the present invention that target specific common HLA phenotypes, providing a single vaccine for disease prevention.

[0105] The nucleic acid-encoded antibody in the vaccine composition may include:

[0106] (i) T cell epitope GTGRAMLGTHTMEVTVYH in CDR-H1 (SEQ ID NO: 1);

[0107] (ii) T cell epitope SVYDFFVWL in CDR-H2 (SEQ ID NO: 2); and

[0108] (iii) T cell epitope WNRQLYPEWTEAQRLD in CDR-L1 (SEQ ID NO: 3).

[0109] Preferably, the antibody comprises:

[0110] (i) T cell epitopes GTGRAMLGTHTMEVTVYH in CDR-H1 and CDR-L3 respectively (SEQ ID NO: 1);

[0111] (ii) T cell epitope SVYDFFVWL in CDR-H2 (SEQ ID NO: 2); and

[0112] (iii) T cell epitopes WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-H3 and CDR-L1 respectively.

[0113] Another preferred antibody includes:

[0114] (i) T cell epitope GTGRAMLGTHTMEVTVYH in CDR-H1 (SEQ ID NO: 1);

[0115] (ii) T cell epitope SVYDFFVWL in CDR-H2 (SEQ ID NO: 2);

[0116] (iii) T cell epitope VPLDCVLYRYGSFSVTLDIVQG in CDR-H3 (SEQ ID NO: 4);

[0117] (iii) T cell epitope WNRQLYPEWTEAQRLD in CDR-L1 (SEQ ID NO: 3);

[0118] (iv) T cell epitopes ANCSVYDFFVWLHYYSVRDTLLGPGRPYR in CDR-L2 (SEQ ID NO: 5); and

[0119] (v) T cell epitopes QCTEVRADTRPWSGPYILRNQDDRELWPRKFF in CDR-L3 (SEQ ID NO: 6).

[0120] The antibody may include: VH, which comprises the amino acid sequence provided 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 provided in SEQ ID NO: 7 or 9; and VL, which comprises the amino acid sequence provided 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 provided in SEQ ID NO: 8 or 10.

[0121] The antibody may include: a heavy chain comprising the amino acid sequence provided in SEQ ID NO: 11 or 13, 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 provided in SEQ ID NO: 11 or 13; and a light chain comprising the amino acid sequence provided in SEQ ID NO: 12 or 14, 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 provided in SEQ ID NO: 12 or 14.

[0122] Preferred antibodies encoded by nucleic acids in vaccine compositions include the antibody referred to as “DCIB68” in WO2008 / 116937 and the antibodies encoded by DNA vectors referred to as “SCIB1” and iSCIB1+ in WO2022 / 043400. The entire contents of WO2008 / 116937 and WO2022 / 043400 are incorporated herein by reference.

[0123] The antibody sequence described above may include a leader sequence (i.e., a “signal peptide” or “segment signal”) for promoting antibody secretion after expression of one or more nucleic acids in the vaccine composition. The leader sequence may be located at the N-terminus of the antibody heavy chain and / or the N-terminus of the light chain. Preferably, the leader sequence comprises the amino acid sequence provided in SEQ ID NO: 16 or a functional variant thereof.

[0124] Preferably, the vaccine composition comprises a single nucleic acid encoding multiple tumor antigen T-cell epitopes. Preferably, the nucleic acid is a DNA vector. As described herein, the DNA vector may comprise a first nonspecific promoter sequence operatively linked to a sequence encoding an antibody heavy chain and a second nonspecific promoter sequence operatively linked to a sequence encoding an antibody light chain, wherein the T-cell epitopes are inserted into or substituted into one or more CDRs of the antibody. The first and second promoter sequences may be identical or different. Both the heavy and light chain sequences may contain a leader sequence (secretion signal) at their respective N-termini. This sequence is arranged in... Figure 1 The feature map of the exemplary carrier "SCIB1" and Figure 3 The feature map of another exemplary carrier, “iSCIB1+”, is shown.

[0125] The preferred DNA sequences are those provided herein as “SCIB1” and “iSCIB1+” (also referred to herein as “SCIB1plus iV1”) as SEQ ID NO: 17 and 18, respectively. Therefore, the nucleic acid can be SCIB1 or iSCIB1+ as described in WO2022 / 043400, or any other suitable nucleic acid described in WO2022 / 043400.

[0126] The vaccine composition described herein is administered in combination with a programmed death-1 (PD-1) inhibitor and a cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitor. The PD-1 inhibitor and CTLA-4 inhibitor act as immune checkpoint inhibitors.

[0127] Immune checkpoints are crucial signaling pathways in the immune system, maintaining self-tolerance and regulating the duration and magnitude of physiological immune responses. Under normal circumstances, these pathways prevent excessive effector activity of T cells. Two important examples of this pathway are the cell surface receptors CTLA-4 and PD-1 (Teft, 2006; Keir, 2008). In some cases, tumor expression or overexpression of inhibitory immune checkpoint pathways serves as a major mechanism for immune escape. Because many immune checkpoints are initiated by ligand-receptor interactions, these signals can be readily blocked by antibodies or regulated by recombinant forms of ligands or receptors (Pardoll, 2012).

[0128] Immune checkpoint proteins are important targets for drug blockade (Teft, 2006; Keir, 2008). Significant clinical responses have been observed after antibody therapy that blocks PD-1 and CTLA-4 (see, for example, Brahmer, 2010; Robert, 2011; Topalian, 2012; Powles, 2014; Topalian, 2014; Brahmer, 2015; Le, 2015; Robert, 2015; Reck, 2016; Langer, 2017). Therefore, in exemplary embodiments, the present invention provides novel combinations of nucleic acid-based vaccines with one or more CTLA-4 and PD-1 inhibitors.

[0129] CTLA-4 inhibitors can be anti-CTLA-4 antibodies. Anti-CTLA-4 antibodies can be, for example, ipilimumab or trimemumab. Anti-CTLA-4 antibodies bind to CTLA-4 and block the interaction between CTLA-4 and its ligands CD80 / CD86. Blocking CTLA-4 has been shown to enhance T cell activation and proliferation, including the activation and proliferation of tumor-infiltrating effector T cells. Inhibition of CTLA-4 signaling can also reduce regulatory T cell function, which may contribute to a general increase in T cell reactivity, including anti-tumor immune responses. Ipilimumab is an IgG1 kappa immunoglobulin with a molecular weight of approximately 148 kDa. Ipilimumab (Yervoy®, Bristol-Meyers Squibb, New York, NY) is a recombinant human mAh produced in mammalian (Chinese hamster ovary) cell cultures.

[0130] CTLA-4 regulates early T cell activation, while programmed death-1 (PD-1) signaling modulates T cell activation in peripheral tissues. The PD-1 receptor is an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed in various cell types, including Tregs, activated B cells, and natural killer (NK) cells, and is primarily expressed on previously activated T cells in vivo, binding to two ligands, PD-L1 and PD-L2. The endogenous ligands PD-L1 and PD-L2 are expressed on activated immune cells and non-hematopoietic cells, including tumor cells. The PD-1 used in this article is intended to include human PD-1 (hPD-1), variants, isotypes, and species homologs of hPD-1, as well as analogs having at least one epitope shared with hPD-1. The complete hPD-1 sequence can be found under GENBANK accession number U64863. Programmed death-ligand-1 (PD-L1) is one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. The PD-L3 used in this study includes human PD-L1 (hPD-L1), variants, isotypes, and species homologs of hPD-L1, as well as analogs with at least one epitope co-occurring with hPD-L1. The complete hPD-L1 sequence can be found under GENBAN accession number Q9NZQ7. Tumors have been shown to evade immune surveillance by expressing PD-L1 / L2, thereby suppressing tumor-infiltrating lymphocytes through the PD-1 / PD-L1,2 interaction (Dong et al. Nat. Med. 8:793-800. 2002).

[0131] PD-1 inhibitors can be anti-PD-1 antibodies. Anti-PD-1 antibodies can be, for example, nivolumab, pembrolizumab, or pildizumab. Nivolumab (Opdivo®, Bristol-Myers Squibb Company, NY) is a human immunoglobulin G4 (IgG4) mAb that binds to the programmed death 1 (PD-1) receptor and blocks its interaction with PD-L1 and programmed death ligand 2 (PD-L2), reversing the inhibition of PD-1 pathway-mediated immune responses, including anti-tumor immune responses. PD-L1 and PD-L2 bind to the PD-1 receptor expressed on T cells, inhibiting T cell proliferation and cytokine production. Upregulation of PD-1 ligands occurs in certain tumors, and signaling through this pathway can help suppress the immune surveillance of tumors by active T cells. The antibodies of this invention include, but are not limited to, all anti-PD-1 and anti-PD-L1 antibodies disclosed in U.S. Patent Nos. 8,008,449 and 7,943,743, respectively. Other anti-PD-1 mAbs have been described, for example, in U.S. Patent Nos. 7,488,802 and 8,168,757, and anti-PD-LlmAbs have been described, for example, in U.S. Patent Nos. 7,635,757 and 8,217,149, and U.S. Publication No. 2009 / 0317368. U.S. Patent No. 8,008,449 exemplifies seven anti-PD-1 HuMAbs: 1708, 2D3, 4M, 5C4 (also referred to herein as nivolumab or BMS-936558), 4A11, 7D3, and 5F4.

[0132] Nivolumab (Opdivo®) has been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for use in combination with ipilimumab (Yervoy®) for the treatment of melanoma, and vice versa. The Opdivo® EMA product information (incorporated hereby by reference) obtained from the EMA website (https: / / www.ema.europa.eu / documents / product-information / opdivo-epar-product-information_en.pdf) outlines the following recommended dosing:

[0133] "In adults and adolescents aged 12 years and older weighing at least 50 kg, the recommended dose is 1 mg / kg nivolumab intravenously every three weeks in combination with 3 mg / kg ipilimumab for the first four doses. This is followed by a second phase of nivolumab monotherapy, administered intravenously at 240 mg every two weeks or 480 mg every four weeks (see Sections 5.1 and 5.2), as shown in Table 2. For the monotherapy phase, the first dose of nivolumab should be administered as follows:"

[0134] - If using 240 mg every two weeks, administer 3 weeks after the last dose of the nivolumab and ipilimumab combination; or

[0135] - If using 480 mg every four weeks, administer 6 weeks after the last dose of nivolumab and ipilimumab in combination.

[0136] In adolescents aged 12 years and older and weighing less than 50 kg, the recommended dose is 1 mg / kg nivolumab intravenously every three weeks in combination with 3 mg / kg ipilimumab for the first four doses. This is followed by a second phase of nivolumab monotherapy, administered intravenously every two weeks at 3 mg / kg or every four weeks at 6 mg / kg (see Sections 5.1 and 5.2), as shown in Table 2. For the monotherapy phase, the first dose of nivolumab should be administered as follows:

[0137] - If using 3 mg / kg every two weeks, administer 3 weeks after the last dose of the nivolumab and ipilimumab combination; or

[0138] - If using 6 mg / kg every four weeks, administer 6 weeks after the last dose of the nivolumab and ipilimumab combination.

[0139] The table below is reproduced from the Opdivo® EMA product information mentioned above, and provides the recommended dosage and infusion time for intravenous administration of nivolumab and ipilimumab in combination for the treatment of melanoma:

[0140]

[0141] The recommended dosing regimen of nivolumab in combination with ipilimumab is also specified in the Yervoy® EMA product information obtained from the EMA website (https: / / www.ema.europa.eu / en / documents / product-information / yervoy-epar-product-information_en.pdf) (incorporated hereby by reference).

[0142] For the purposes of this invention, when the PD-1 inhibitor is nivolumab and the CTLA-4 inhibitor is ipilimumab, these antibodies may be administered to the subject according to the recommended dosing regimen in the Yervoy® EMA product information (also known as the “product label”) or the Opdivo® EMA product information (for combination therapy against melanoma). Preferably, ipilimumab and nivolumab are administered to the subject according to the dosing regimen described in the clinical trial protocol (“SCOPE” trial”) provided in Example 3 of this document.

[0143] Preferably, each dose of a CTLA-4 inhibitor (e.g., ipilimumab) is administered on the same day as a dose of a PD-1 inhibitor (e.g., nivolumab). Therefore, the CTLA-4 inhibitor (e.g., ipilimumab) and the PD-1 inhibitor (e.g., nivolumab) can be administered as separate compositions or as the same composition. The first dose of both the CTLA-4 inhibitor and the PD-1 inhibitor is preferably administered to the subject approximately one week after the first dose of the vaccine composition.

[0144] As demonstrated by the interim clinical trial results described in Example 4, the vaccine composition described herein, in combination with CTLA-4 inhibitors and PD-1 inhibitors, is particularly suitable for the treatment of melanoma, especially advanced melanoma. Specifically, the melanoma can be stage III or IV melanoma. The melanoma can be metastatic melanoma. In this regard, "metastatic" melanoma refers to primary melanoma cells that have spread to organs far from the original tumor site, such as lymph nodes, lungs, liver, brain, and bone. The melanoma can be unresectable (i.e., cannot be surgically removed). For example, the melanoma can be unresectable stage III or IV melanoma. Preferably, the subject has not previously received systemic therapy for advanced melanoma.

[0145] Preferably, the melanoma is an unresectable stage III or IV melanoma, and the object:

[0146] (a) At least 18 years old;

[0147] (b) Has not received prior systemic treatment for advanced disease; and

[0148] (c) No brain metastases, ocular melanoma, mucosal melanoma, or autoimmune disease.

[0149] Preferably, the object also includes:

[0150] (d) HLA serotype I with HLA-A*02 (i.e., HLA-A2); and

[0151] (e) Having a class II HLA serotype selected from HLA-DR4, HLA-DR7, HLA-DR53 and HLA-DQ6.

[0152] The prior art referenced herein is incorporated to the fullest extent permitted by law. Other features or advantages of the invention will become apparent from the following drawings, embodiments, numbered implementations, and appended claims.

[0153] Sequence Description

[0154] T cell epitopes

[0155]

[0156] [Example antibody sequence]

[0157] SEQ ID NO: 7 - SCIB1 antibody heavy chain variable region (VH):

[0158] QVQLVETGGGLIQPGGSLRMSCGTGRAMLGTHTMEVTVYHWVRQAPGKGLEWIAYIGSGGSVYDFFVWLRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARWNRQLYPEWTEAQRLDWGQGTTVTVSS

[0159] SEQ ID NO: 8 - SCIB1 antibody light chain variable region (VL):

[0160] DVLMTQSPLSLPVTPGEPASISCWNRQLYPEWTEAQRLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDTGVYYCGTGRAMLGTHTMEVTVYHFGGGTKVEIK

[0161] SEQ ID NO: 9 - SCIB1+ Antibody Heavy Chain Variable Region (VH):

[0162] QVQLVETGGGLIQPGGSLRMSCGTGRAMLGTHTMEVTVYHWVRQAPGKGLEWIAYIGSGGSVYDFFVWLRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARVPLDCVLYRYGSFSVTLDIVQGWGQGTTVTVSS

[0163] SEQ ID NO: 10 - SCIB1+ Antibody Light Chain Variable Region (VL):

[0164] DVLMTQSPLSLPVTPGEPASISCWNRQLYPEWTEAQRLDWYLQKPGQSPQLLIYANCSVYDFFVWLHYYSVRDTLLGPGRPYRGVPDRFSGSGSGTDFTLKISRVEAEDTGVYYCQCTEVRADTRPWSGPYILRNQDDRELWPRKFFFGGGTKVEIK

[0165] SEQ ID NO: 11 - SCIB1 Antibody Full Heavy Chain:

[0166] QVQLVETGGGLIQPGGSLRMSCGTGRAMLGTHTMEVTVYHWVRQAPGKGLEWIAYIGSGGSVYDFFVWLRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARWNRQLYPEWTEAQRLDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0167] SEQ ID NO: 12 - Full light chain of SCIB1 antibody:

[0168] DVLMTQSPLSLPVTPGEPASISCWNRQLYPEWTEAQRLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDTGVYYCGTGRAMLGTHTMEVTVYHFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0169] SEQ ID NO: 13 - Full heavy chain of SCIB1+ antibody:

[0170] QVQLVETGGGLIQPGGSLRMSCGTGRAMLGTHTMEVTVYHWVRQAPGKGLEWIAYIGSGGSVYDFFVWLRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARVPLDCVLYRYGSFSVTLDIVQGWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHTAWTQPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0171] SEQ ID NO: 14 - SCIB1+ antibody full light chain:

[0172] DVLMTQSPLSLPVTPGEPASISCWNRQLYPEWTEAQRLDWYLQKPGQSPQLLIYANCSVYDFFVWLHYYSVRDTLLGPGRPYRGVPDRFSGSGSGTDFTLKISRVEAEDTGVYYCQCTEVRADTRPWSGPYILRNQDDRELWPRKFFFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0173] SEQ ID NO: 15 – CMV IE promoter sequence:

[0174] GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTC

[0175] SEQ ID NO: 16 - Leader sequence: MGWSCIILFLVATATGVHS

[0176] SEQ ID NO: 19 - Human Kappa constant region Acc No: P01834 (amino acids, 1 - 107):

[0177] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0178] SEQ ID NO: 20 - HuIgG1 CH1 - hinge - CH2 - CH3 sequence with "iV1" murine IgG3 23 AA substitutions (bolded), Acc No: P01857 (amino acids, 1 - 330):

[0179] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHTAWTQPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0180] Exemplary whole DNA vector sequence

[0181] Complete SCIB1 DNA plasmid sequence (SEQ ID NO: 17 - also see Example 1):

[0182]

[0183] Complete iSCIB1+ DNA plasmid sequence (SEQ ID NO: 18 – see also Example 2):

[0184]

[0185] SEQ ID NO: 21 – gp100 178-186: MLGTHTMEV

[0186] SEQ ID NO: 22 – gp100 174-190: TGRAMLGTHT MEVTVYH

[0187] SEQ ID NO: 23 – gp100 175-189: GRAMLGTHTMEVTVY

[0188] SEQ ID NO: 24 – gp100 476-490: VLYRYGSFSVTLDIV

[0189] SEQ ID NO: 25 – TRP2 178-192: NCSVYDFFVWLHYYS

[0190] SEQ ID NO: 26 – TRP2 60-81: QCTEVRADTRPWSGPYILRNQD

[0191] SEQ ID NO: 27 – TRP2 71-91: WSGPYILRNQDDRELWPRKFF

[0192] SEQ ID NO: 28 – TRP2 67-83: DTRPWSGPYILRNQDDR

[0193] SEQ ID NO: 29 – TRP2 69-77: RPWSGPYIL

[0194] SEQ ID NO: 30 - Amino acid sequence of pSCIB1 - light chain with leader:

[0195] MGWSCIILFLVATATGVHSDVLMTQSPLSLPVTPGEPASISCWNRQLYPEWTEAQRLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDTGVYYCGTGRAMLGTHTMEVTVYHFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0196] SEQ ID NO: 31 – pSCIB1 amino acid sequence – heavy chain with leader:

[0197] MGWSCIILFLVATATGVHSQVQLVETGGGLIQPGGSLRMSCGTGRAMLGTHTMEVTVYHWVRQAPGKGLEWIAYIGSGGSVYDFFVWLRFTISRDNSKNTLYLQLNSLRAEDTAVYYCA RWNRQLYPEWTEAQRLDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0198] Example

[0199] The invention will now be further described with reference to the following embodiments and accompanying drawings. Attached Figure Description

[0200] Figure 1 : Plasmid map of SCIB1.

[0201] Figure 2 Schematic diagram of engineered IgG encoded by SCIB1.

[0202] Figure 3 Plasmid map of iSCIB1+.

[0203] Figure 4 Schematic diagram of engineered IgG encoded by SCIB1 and iSCIB1+.

[0204] Figure 5 Simon Phase I and Simon Phase II study schemas of SCIB1 in patients with advanced unresectable melanoma receiving nivolumab and ipilimumab or pembrolizumab.

[0205] Figure 6 The waterfall plot shows the maximum change from baseline in the total reference diameter of target lesions in patients receiving SCIB1 in combination with nivolumab and ipilimumab. Data shown represent all patients assessed at baseline by target lesion evaluation and who reached the first imaging time point (week 13). Bars in negative intervals indicate tumor shrinkage, and bars in positive intervals indicate tumor size increase. Horizontal lines represent a 30% reduction or a 20% increase in target lesion tumor burden, which are thresholds for response or progression. Change in tumor burden is defined as the percentage decrease in the total reference diameter of target lesions from baseline to the lowest point (observation up to September 2023).

[0206] Figure 7 Spider plot, which shows the persistence of tumor response over time. The data shown are for all patients assessed by target lesion at baseline and who reached the first imaging time point (week 13). Change in tumor burden is defined as the percentage decrease in the sum of reference diameters of target lesions from baseline to the lowest point (observation up to September 2023).

[0207] Figure 8 C57Bl / 6 mice (n≥5) were implanted with B16F1 tumor cells, then administered checkpoint inhibitor antibodies, and tumor growth and survival were monitored.

[0208] Figure 9 (A) HLA-DR4 transgenic mice implanted with B16 DR4 tumor cells or (B) transgenic HLA-DP4 mice implanted with B16F1 HHDII / DP4 tumor cells, then treated with checkpoint inhibitor antibodies and tumor growth and survival were monitored (n≥5).

[0209] Figure 10 In vitro immunoreactivity was measured by IFNγ ELISpot assay in C57Bl / 6 mice (A), HLA-DR4 transgenic mice (B), or HHDII / DR1 transgenic mice (C) following immunization with iSCIB1+ (the epitope CDR position shown). Data represent the mean of at least two independent experiments, where n=3. Error bars indicate standard deviation.

[0210] Figure 11 (A) C57Bl / 6 mice implanted with B16F1 tumor cells or (B) transgenic HHDII / DP4 mice implanted with B16 F1 HHDII / DP4 tumor cells, then immunized with iSCIB1+ and monitored tumor growth and survival (n=10).

[0211] Figure 12Splenocytes from C57Bl / 6 mice immunized with iSCIb1+ alone or in combination with dual checkpoint inhibitors (anti-PD1 and anti-CTLA4 antibodies) were expanded 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 mean values ​​for n=3, and error bars indicate standard deviation.

[0212] Figure 13 C57Bl / 6 mice implanted with b16F1 tumor cells were then immunized with iSCIB1+ alone, iSCIB1+ combined with dual checkpoint (CPI) therapy (anti-PD1 and anti-CTLA4 antibodies), or dual checkpoint therapy alone, and tumor growth and survival were monitored (n=10).

[0213] Methods and Materials (Preclinical Study)

[0214] peptides

[0215] Peptides were selected based on published sequences, binding predictions from the IEDB database (http: / / www.iedb.org / ), and binding predictions from SYFPEITHI (http: / / www.syfpeithi.de). Peptides (see table below) were synthesized with >90% purity (Genscript), aliquoted into single-use vials, lyophilized at -80°C, and then reconstituted in PBS on the day of use.

[0216]

[0217] Animals and cell lines

[0218] Mice were used in groups ranging from 8 to 16 weeks of age, including C57Bl / 6J (Charles River), HLA-DR4 mice (model #4149, Taconic), HHDII / DP4 mice (EM:02221, European Mouse Mutant Archive), or HHDII / DR1 mice (Pasteur Institute). All work was conducted with ethical approval under a Home Office-approved project license. Mice were randomly assigned to different groups for all studies, and the researchers were not blinded.

[0219] Cells (including B16 melanoma cells expressing relevant MHC I and MHC II alleles (previously described [Pudney et al 2010; Brentville et al 2016; Brentville et al 2019])) were cultured in RPMI 1640 medium containing 10% FCS and appropriate antibiotics, with L-glutamine (2 mmol / L) to maintain plasmids. Mouse spleen cells were cultured in RPMI 1640 medium containing 10% FBS (Sigma), 2 mM glutamine, 20 mM HEPES buffer, 100 units / ml penicillin, 100 mg / ml streptomycin, and 10 mg / ml glutamine. -5 Cells were cultured in RPMI-1640 medium containing M 2-mercaptoethanol. The cell lines used were mycoplasma-free, vendor-certified (STR-typed), and used within ten passages.

[0220] Immunization regimen

[0221] Animal experiments were conducted with ethical approval under a Home Office-approved project license. Eight to sixteen-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) mice were used. For all studies, mice were randomly assigned to different groups without blinding the researchers. 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 days 1, 8, and 15, and spleens were harvested on day 21, and spleen cells were collected for analysis (unless otherwise specified). For the tumor attack experiment, on day 1, 2.5 x 10⁻⁶ cells were used. 4 One B16F1, 2.5x10 4 One B16 DR4 or 1x10 5Mice were subcutaneously challenged with B16 HHDII / DP4 tumor cells in the right flank, followed by immunization with DNA bullets injected via gene gun on days 4, 11, and 18. In a combined study with anti-PD-1 antibody clone RMPI-14 (250 µg / dose) and anti-CTLA-4 clone 9D9 (100 µg / dose), DNA was administered via gene gun on days 8, 15, and 22, followed by intraperitoneal administration of antibody-containing saline solutions. Tumor size was measured with calipers, and tumor growth was monitored every 3–4 days. Mice were euthanized humanely once the tumor diameter approached 15 mm.

[0222] Spleen cell culture

[0223] Spleen cells harvested in vitro were divided into 5 x 10⁻⁶ cells. 6 Each well contains 10 cells / well in 2 ml of complete culture medium (containing 10 cells / well). -5 The cell pool was established using M 2-mercaptoethanol and 20 IU recombinant mouse IL-2 (Miltenyi Biotech). Peptides were added to the pool to a final concentration of 10 µg / ml for each peptide. Cells were incubated at 37˚C / 5% CO2 for up to 8 days.

[0224] Elispot assay

[0225] Elispot assays were performed using the mouse IFNγ capture and detection reagent, following the manufacturer's instructions (Mabtech). In short, IFNγ-specific antibodies were coated onto the wells of 96-well Immobilin-P plates. 10 µg / ml of the synthetic peptide (Genescript) was diluted in RPMI 1640 (GIBCO / BRL) medium supplemented with 10% FCS (Sigma), 2 ml glutamine (Sigma), and sodium bicarbonate (with an additional 20 mM HEPES (Sigma) buffer) and 50 µM 2-mercaptoethanol (Thermofisher) and directly applied to each well in vitro at 5 x 10⁻⁶ cells / well. 5 Four splenocytes were added to each well in a quadruplicate and incubated at 37˚C / 5% CO2 for 40 hours. Cells containing only culture medium were added as a negative control, and 5 µg / ml lipopolysaccharide (LPS, Sigma) was added as a positive control.

[0226] For the assay of cultured spleen cells, cells were counted and divided into 3 x 10⁻⁶ cells. 4 / hole to 1x10 5 / Well plated. Peptides were added as described above, with 5 µg / ml anti-mouse CD3 antibody (clone 145-2C11) used as a positive control. Plates were incubated for 20 hours. After incubation, captured IFNγ was detected by biotinylated specific IFNγ antibody and developed using streptavidin alkaline phosphatase and chromogenic substrate. Spots were analyzed and counted using an automated plate reader (Cellular Technologies Ltd).

[0227] statistics

[0228] The ELISpot results were compared and analyzed by applying appropriate paired or unpaired ANOVA or Student's t-test, and the p-values ​​were calculated accordingly. Survival analysis was performed using the rank test with Graphpad Prism software.

[0229] References:

[0230] Pudney, VA, et al., DNA vaccination with T-cell epitopes encodedwithin Ab molecules induces high-avidity anti-tumor CD8+ T cells. Eur JImmunol, 2010. 40(3): p. 899-910

[0231] Brentville, VA, et al., Citrullinated Vimentin Presented on MHC-IIin Tumor Cells Is a Target for CD4+ T-Cell-Mediated Antitumor Immunity. Cancer Res, 2016. 76(3): p. 548-60

[0232] 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 tumourtherapy. Oncoimmunology, 2019. 8(5): p. e1576490.

[0233] Example 1 – SCIB1

[0234] An exemplary 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 encoding the light and heavy chains of the expression product):

[0235]

[0236]

[0237]

[0238]

[0239] Figure 1 Displaying the plasmid map of SCIB1. Figure 2 A schematic diagram showing engineered IgG encoded by SCIB1.

[0240] The amino acid sequence of the modified IgG1 light chain (SEQ ID NO: 12) encoded by pSCIB1 (with a leader peptide (SEQ ID NO: 16) at the N-terminus) is shown below:

[0241] SEQ ID NO: 30:

[0242]

[0243] The melanoma T-cell epitopes contained in the light chain sequence are color-coded as follows:

[0244]

[0245] The amino acid sequence of the modified IgG1 heavy chain (SEQ ID NO: 11) encoded by pSCIB1 (with a leader peptide (SEQ ID NO: 16) at the N-terminus) is shown below:

[0246] SEQ ID NO: 31:

[0247]

[0248] The melanoma T-cell epitopes contained in the heavy chain sequence are color-coded as follows:

[0249]

[0250] Example 2 – iSCIB1+

[0251] Another exemplary nucleic acid used in this invention is iSCIB1+. The DNA sequence of iSCIB1+ (SEQ ID NO: 18) is shown below (including the amino acid sequences encoding the light and heavy chains of the expression product):

[0252]

[0253]

[0254]

[0255]

[0256]

[0257] Figure 3 Displaying the plasmid map of iSCIB1+. Figure 4 A schematic diagram showing engineered IgG encoded by SCIB1 and iSCIB1+.

[0258] Table 1 below shows the amino acid sequences of the heavy and light chains within the iSCIB+ inner chain.

[0259]

[0260] Example 3 – Clinical Study Details

[0261] Table of contents

[0262] List of abbreviations

[0263] Terminology Definition

[0264] 1. Solution Overview

[0265] 2. Introduction and Background Information

[0266] 2.1. Introduction

[0267] 2.2. Results of non-clinical and clinical studies

[0268] 2.2.1. Non-clinical studies

[0269] 2.2.2. Clinical Research

[0270] 2.3. Reasons for Dosage Selection

[0271] 2.4. Known and potential benefits and risks for patients

[0272] 3. Research Objectives and Endpoints

[0273] 3.1. Main purpose and endpoint

[0274] 3.1.1. Importing Subqueues

[0275] 3.1.2. Main Research

[0276] 3.2. Secondary Objectives and Endpoints

[0277] 3.2.1. Main Research

[0278] 3.3. Exploratory Objectives and Endpoints

[0279] 4. Research Design

[0280] 4.1. Overall Research Design

[0281] 4.2. Planned number of patients and countries

[0282] 4.3. Duration of the study

[0283] 4.4. Patient Number Allocation

[0284] 4.5. Assume blinding and randomization

[0285] 4.6. Rationale for Study Design and Patient Population Selection

[0286] 4.7. Periodic review of safety and efficacy

[0287] 4.8. Standards for Research Completion

[0288] 4.9. Procedures and Evaluation Schedule

[0289] 4.10. Post-treatment follow-up

[0290] 5. Patient selection and withdrawal

[0291] 5.1 Selection Criteria

[0292] 5.2. Exclusion Criteria

[0293] 5.3. Treatment Discontinuation

[0294] 5.4. Exit Criteria

[0295] 5.4.1. Alternative Patient

[0296] 5.4.2. Filter again

[0297] 5.5. Research terminated

[0298] 6. Treatment

[0299] 6.1. Research drugs

[0300] 6.1.1. Non-investigation drug: Nivolumab in combination with ipilimumab

[0301] 6.1.2. Non-investigation drug: Pembrolizumab

[0302] 6.2. Preparation, processing, storage, inventory, and disposal of research drugs.

[0303] 6.2.1.SCIB1

[0304] 6.2.2. Checkpoint Inhibitors

[0305] 6.2.3. Drug inventory

[0306] 6.3. SCIB1 Application Procedure

[0307] 6.4. Checkpoint Inhibitors: Usage and Precautions

[0308] 6.5. Treatment Schedule

[0309] 6.6. Dosage Adjustment, Modification, and Delay

[0310] 6.6.1. Infusion reaction

[0311] 6.6.2. Dosage Adjustment and Modification

[0312] 6.6.3. Delay and Discontinuation of Checkpoint Inhibitor Therapy

[0313] 6.7. Previous and concomitant medications and treatments

[0314] 6.7.1. Medication and treatment are prohibited.

[0315] 6.7.2. Permitted medication, pre-medication, and supportive care

[0316] 6.8. Patient compliance monitoring

[0317] 7. Efficacy evaluation

[0318] 7.1. Tumor imaging and RECIST assessment

[0319] 7.2. Progression-free survival and overall survival

[0320] 8. Pharmacodynamic and biomarker evaluation

[0321] 8.1. Blood sample

[0322] 8.1.1. Sample processing and transportation

[0323] 8.1.2. Evaluation and analysis of specific blood samples.

[0324] 8.2. Tumor Samples

[0325] 8.2.1. Evaluation and analysis of specific tumor samples.

[0326] 9. Security Assessment

[0327] 9.1. Adverse Events

[0328] 9.1.1. Definition of Adverse Events

[0329] 9.1.2. Recording and Reporting of Adverse Events

[0330] 9.1.3. Severity of Adverse Events

[0331] 9.1.4. Relationship between adverse events and study treatments or devices

[0332] 9.1.5. Deviation from the plan due to adverse events

[0333] 9.2. Serious Adverse Events

[0334] 9.2.1. Definition of serious adverse events

[0335] 9.2.2. Anticipation

[0336] 9.2.3. Reporting of serious adverse events

[0337] 9.3. Pregnancy

[0338] 9.4. Administration and medication errors related to research treatment

[0339] 9.5. Clinical and laboratory assessments and examinations

[0340] 9.5.1. Serum chemistry, hematology and urine analysis

[0341] 9.5.2. Other clinical laboratory tests

[0342] 9.5.3. Medical History

[0343] 9.5.4. Physical Examination

[0344] 9.5.5. Injection Site Reaction

[0345] 9.5.6. Vital signs

[0346] 9.5.7. Electrocardiogram

[0347] 9.5.8. Ophthalmological examination

[0348] 9.5.9. Physical fitness assessment

[0349] 10. Statistics

[0350] 10.1. Sample size and power considerations

[0351] 10.1.1. Importing Subqueues

[0352] 10.1.2. Cohort 1: Nivolumab in combination with ipilimumab

[0353] 10.1.3. Cohort 2: Pembrolizumab

[0354] 10.2. Analysis Set

[0355] 10.2.1. Full Analysis Set

[0356] 10.2.2. Security Analysis Set

[0357] 10.2.3. Compliant with the set of schemes

[0358] 10.3. Data Processing Conventions

[0359] 10.4. Mid-term Analysis

[0360] 10.5. Study population

[0361] 10.5.1. Patient Management

[0362] 10.5.2. Demographic and Baseline Characteristics

[0363] 10.6. Therapeutic Effect Analysis

[0364] 10.6.1. Therapeutic endpoint

[0365] 10.6.2. Methods for Analyzing Plans

[0366] 10.7. Security Analysis

[0367] 10.7.1. Adverse Events

[0368] 10.7.2. Laboratory Parameters

[0369] 10.7.3. Physical Examination

[0370] 10.7.4. Vital Signs

[0371] 10.7.5.12-lead electrocardiogram

[0372] 10.7.6. Injection Site Reaction

[0373] 10.7.7. Concomitant medications

[0374] 10.8. Pharmacodynamic and Biomarker Analysis

[0375] 10.9. Deviation between the report and the statistical analysis plan

[0376] 11. Quality Control and Quality Assurance

[0377] 12. Legal and ethical obligations

[0378] 12.1. Adhere to good clinical practices

[0379] 12.2. Informed Consent

[0380] 12.3. Contact your primary care physician

[0381] 12.4. Research Ethics Committee

[0382] 12.5. Regulatory Approval

[0383] 12.6. Periodic review of safety and efficacy

[0384] 12.7. Register the clinical study on the trial registry website.

[0385] 12.8. Patient confidentiality

[0386] 12.9. Data Protection

[0387] 12.10. Future Use of Stored Specimens

[0388] 13. Administrative and legal obligations

[0389] 13.1. Researcher Obligations

[0390] 13.2. Accessing Source Data and Files

[0391] 13.3. Insurance

[0392] 13.4. Amendments to the Plan and Informed Consent Form

[0393] 13.5. Test document recording and data storage

[0394] 13.6. Availability and Retention of Research Records

[0395] 13.7. Data Collection

[0396] 13.8. Language

[0397] 13.9. Clinical monitoring, source data verification and auditing

[0398] 13.10. Deviation and violation of the plan

[0399] 14. References

[0400] Abbreviation list

[0401]

[0402]

[0403]

[0404]

[0405] Terminology Definition

[0406]

[0407] 1. Solution Overview

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420] 2. Introduction and Background Information

[0421] 2.1 Introduction

[0422] Malignant melanoma is an aggressive skin tumor. When melanoma is diagnosed in its early stages (stages I and II), a large proportion of patients can be cured by surgery alone. However, once the tumor begins to spread, the disease becomes difficult to treat, and the mortality rate is high. It is estimated that in the United States in 2016, more than 76,000 people were diagnosed with melanoma, and approximately 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 approximately 15,700 deaths (European Cancer Watch Database [EUCAN], 2012).

[0423] Until recently, treatment for unresectable stage IV melanoma included chemotherapy (commonly dacarbazine) or cytokine therapy (interferon [IFN]α or interleukin-2). The objective response rate (ORR) for these treatments was low, with a 2-year overall survival (OS) of less than 20% (Keilholz et al. 2005). Two new classes of therapies have significantly improved treatment and outcomes for patients with stage IV disease. For patients with BRAF gene mutations, combination therapy with BRAF and MEK kinase inhibitors resulted in an ORR exceeding 60% and a 2-year OS of 50% (Long et al. 2015, Ascierto et al. 2016).

[0424] Another new class of therapies for advanced melanoma is checkpoint inhibition. These treatments significantly improve clinical outcomes by blocking immune checkpoints (such as PD-1 and CTLA-4) that melanoma cells have used to evade the immune system. In a randomized trial in patients with previously untreated BRAF V600 wild-type melanoma, the anti-PD-1 antibody nivolumab was compared with dacarbazine (Robert et al. 2015a). The results for nivolumab and dacarbazine were: ORR 40% vs. 13.9%, median PFS 5.1 months vs. 2.2 months, and significantly improved OS (hazard ratio for death 0.42; p<0.001). In a randomized trial in patients with advanced melanoma, the anti-PD-1 antibody pembrolizumab was compared with the CTLA-4 checkpoint protein antibody ipilimumab (Robert et al. 2015b). The results for pembrolizumab and ipilimumab were as follows: ORR 33% vs. 12%, median PFS 4.1 months vs. 2.8 months, and OS significantly improved (hazard ratio for death 0.69; p=0.004).

[0425] Combination therapy with anti-PD-1 and anti-CTLA-4 antibodies has been used to provide improved efficacy. The results of nivolumab combined with ipilimumab compared to nivolumab alone were: ORR 50% vs. 40%, median PFS 11.5 months vs. 6.9 months, and 2-year OS rate 64% vs. 59% (Larkin et al. 2017). Although combination therapy improved response rate and PFS compared to nivolumab alone, it 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 combination therapy due to AEs (Larkin et al. 2015). In another study, the ORR of nivolumab 3 mg / kg combined with 1 mg / kg ipilimumab was 45.6%, while the ORR of 1 mg / kg nivolumab combined with 3 mg / kg ipilimumab was 50.6%. PFS and OS were similar for both regimens. However, nivolumab 1 mg / kg combined with ipilimumab 3 mg / kg had more grade 3 / 4 adverse events (48.3% vs. 33.9%) compared to nivolumab 3 mg / kg combined with ipilimumab 1 mg / kg (Lebbé 2019).

[0426] Although immune checkpoint blockade has been successful in the treatment of melanoma, a significant proportion of patients do not respond. One reason for this lack of response may be an insufficient initial immune response to the developing tumor. Therefore, therapies that enhance anti-tumor immune activity as companion therapies to checkpoint inhibitors have attracted considerable attention.

[0427] SCIB1 is a plasmid DNA designed to express an engineered human immunoglobulin-1 (IgG1) antibody molecule containing a CTL epitope derived from the TRP-2 melanoma antigen plus two helper T cell sequences derived from the melanoma gp100 protein. One of the gp100 sequences also contains nested CTL epitopes. SCIB1 is administered via needle-free injection and is designed to be taken up by antigen-presenting cells that synthesize antibody constructs, process the antigen, and present epitopes on MHC class I or MHC class II molecules. T cells that recognize these complexes act as amplifiers of the immune response (cluster 4-positive [CD4+] helper T cells) or as direct effectors of tumor cell attack (cluster 8-positive [CD8+] CTLs) (Metheringham et al. 2009). In addition to this direct presentation process, there is evidence that SCIB1 antibodies are secreted, and that this secretion product binds to the CD64 Fc-γ receptor (FcgR) on dendritic cells via the heavy chain Fc region, leading to epitope uptake and cross-presentation to CD8+ and CD4+ cells (Pudney et al. 2010; Durrant et al. 2010). Both processes result in a high-frequency and high-affinity immune response to melanoma-associated antigens.

[0428] 2.2 Results of non-clinical and clinical studies

[0429] 2.2.1 Non-clinical studies

[0430] Main pharmacodynamics

[0431] The TRP-2 CD8+ CTL epitope encoded by SCIB1 has been recognized in certain mouse strains, enabling the assessment of immune responses induced by SCIB1 administration in animal models. Preliminary studies using intradermal administration of the SCIB1 plasmid using a gene gun have shown that high-frequency and high-affinity responses were induced in mice, and these responses were associated 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, indicating that the sequence is correctly processed and presented.

[0432] Intramuscular delivery of SCIB1 using the TDS-IM electroporation device also induced a high-frequency and high-affinity T-cell immune response in mice and has been shown to elicit a favorable memory response. Evaluation of dose-response in mice showed that the dose selected for the first clinical study, whether per unit body weight or calculated human equivalent, was within the logarithmic range of the effective dose in mice; furthermore, the 3-week interval selected for clinical administration of SCIB1 using the TDS-IM device produced a positive response in animal studies.

[0433] In mouse studies, IFNg ELISpot assays showed that repeated administration at 2- or 3-week intervals produced a stronger immune response than a single administration, and the affinity of the response was higher after repeated SCIB1 administration. Assessment after 3 weekly administrations at 10-week intervals indicated the presence of a memory response. Fluorescently activated cell sorting analysis of TRP-2-specific CD8+ cell phenotypes showed that they were double-positive for CF62L and CD127, indicating the presence of central memory.

[0434] In a dose-response study of SCIB1 administered to C57BL / 6 mice at 3-week intervals, a dose of 10 µg / mouse showed superior immune response frequency and affinity compared to doses of 3 µg / mouse and 1 µg / mouse. A dose of 10 µg / mouse is approximately equivalent to a human dose of 2.5 mg.

[0435] Compared with treatment with the control plasmid, treatment of HLA-DR4 transgenic mice subcutaneously injected with B16 mouse melanoma cells with SCIB1 resulted in a significant delay in tumor growth (p=0.0027). Treatment was administered via intramuscular injection and electroporation using a TDS-IM device on days 0, 6, and 13 post-tumor cell injection.

[0436] In the experiment designed to support the combination therapy used in this study, SCIB1 was administered intradermally at a dose of 1 µg / mouse using a gene gun device on days 4, 8, and 11, and an anti-PD-1 blocking mAb was injected intraperitoneally on days 4 and 11 at a dose of 250 µg / mouse. HLA-DR4 transgenic mice were injected with B16F1 DR4 mouse melanoma tumor cells (2.5 x 10⁻⁶) on day 0. 4 The results showed that both SCIB1 and anti-PD-1 alone were effective in this mouse cancer model, but the combination therapy was significantly superior to either single agent. In mice treated with SCIB1 alone, 40% showed long-term survival, while the survival rate in the control group was less than 20% (p=0.022). PD-1 blockade also induced a similar anti-tumor response, resulting in long-term survival in 50% of the mice (p=0.0047). Combining PD-1 blockade with SCIB1 significantly enhanced this anti-tumor response, increasing the survival rate of treated animals to 80% compared to the control group (p<0.0001), SCIB1 alone (p=0.0066), or PD-1 blockade alone (p=0.0234). When using six times the high dose of B16F1 DR4 tumor cells (1.5 x 10⁻⁶ mcg), the results were significantly improved. 5 When the number of cells was 1, only the combination therapy showed a significant survival advantage relative to the control group (p=0.0126).

[0437] Studies of infiltrating lymphocytes in these experimental tumors showed that in animals treated with SCIB1, differentiation cluster 3 (CD3) were positive. + CD8 + The proportion of CD3 cells increased significantly, while the increase was more moderate in animals treated with PD-1 blockade alone. However, the combination therapy significantly increased CD3 cell count. + CD8 + T cell infiltration was significantly higher with SCIB1 alone (p=0.0004) or PD-1 blockade alone (p=0.0026). T cell proliferation, as measured by Ki67, was also increased with both SCIB1 and PD-1 blockade alone, and the combination therapy further increased T cell proliferation relative to each agent alone (p=0.0373 and p=0.0167, respectively). In these studies, CD4+ infiltrating tumor sites... + The proportion of T cells was also increased by using SCIB1 alone (p=0.0086) or in combination with PD-1 blockade (p=0.0321). These treatments expressed regulatory CD4 in tumors that did not induce invasive forkhead box P3 expression. + Significant differences in T cell numbers were observed. Finally, SCIB1 (rather than PD-1 blockade) was shown to upregulate PD-L1 expression (Xue et al. 2016).

[0438] Toxicological studies

[0439] A single-dose toxicology study of SCIB1 via electroporation immediacy was conducted in C57BL / 6 mice (Report LHP0002). SCIB1 plasmids in tissue samples collected up to day 90 were analyzed using a validated quantitative polymerase chain reaction (qPCR) assay (Report LHP0005). A single dose of 40 μg / mouse did not produce any treatment-related significant adverse effects. qPCR analysis showed high copy numbers of DNA plasmids at the injection site in all mice on day 3, with mean values ​​of 653,000 and 18,700,000 copies / μg genomic DNA in males and females, respectively. By day 90, the copy number at the injection site was less than 100 copies / μg genomic DNA in males and 207 copies / μg genomic DNA in females, indicating effective clearance of the DNA plasmid from the injection site. On day 3, one ovarian sample showed a copy number of 21,773. However, all other gonadal tissues were below quantitative levels on days 3, 35, and 90, except for a testicular sample on day 90 that showed a very low but positive signal (greater than 11 copies / μg genomic DNA).

[0440] Preliminary studies (Report LHP0003) and Good Laboratory Practice (GLP)-compliant repeated-dose studies (Report LHP0001) of SCIB1 have been conducted in C57BL / 6 mice. In the preliminary study, a dose of 40 µg / mouse was administered on days 1, 8, and 15, and no adverse findings were observed. Increased IFNg responses were observed in four out of five female mice treated with SCIB1, but not in control female mice. In the GLP-compliant study, mice were administered a dose of 40 µg / mouse of SCIB1 via intramuscular injection and electroporation on days 1, 22, 43, 64, and 85. A control group was included, which was administered phosphate-buffered saline. One control female mouse and one control male mouse died shortly after the fourth and fifth doses, respectively. No treatment-related clinical signs were observed, and no effects on body weight, food consumption, ophthalmological examination, hematology, blood chemistry, or organ weight were noted. In both control and SCIB1-treated mice, dark areas, myofibril necrosis, hemorrhage, inflammatory responses, and muscle regeneration were observed at the injection sites in both tibialis anterior muscles. These local tolerance effects were stronger in SCIB1-treated mice compared to control mice and were almost completely reversible after a 4-week recovery period. No significant histopathological findings were observed in the testes of male mice or the uterus of female mice, indicating that these organs were not adversely affected by SCIB1 administration. Histopathological evaluation of the ovaries in female mice was not performed; therefore, the findings in the biodistribution study (Report LHP0002) regarding the potential delivery and incorporation of SCIB1 into the ovaries could not be confirmed from the histopathological results of this repeated-dose toxicology study.

[0441] The study concluded that, based on the results of various parameters assessed when SCIB1 was administered intramuscularly at a dose of 40 µg / mouse every three weeks in combination with electroporation for a total of five doses, no signs of systemic toxicity were observed. Compared to control mice, SCIB1-treated mice showed higher morbidity and severity at the injection site; these changes were almost entirely reversible within a 4-week recovery period. Furthermore, an increased IFNg response was observed in SCIB1-treated mice as measured by ELISpot assay, indicating that SCIB1 produced the expected immune system response.

[0442] 2.2.2 Clinical Research

[0443] SCIB1 has been investigated in a single-center, open-label, non-randomized, phase 1 / 2 study (SCIB1-001) in UK patients with melanoma. The study aimed to determine the safety and tolerability of four dose levels of SCIB1 administered via the immediacy route using a TDS-IM electroporation device, and to assess its immune effects and antitumor activity. In the first part of the study, patients were enrolled in dose cohorts with at least three patients per cohort. The SCIB1 dose was escalated from 0.4 mg to 2 mg, then to 4 mg, and finally, with a protocol revision, to 8 mg. Treatment regimens included dosing at weeks 0, 3, 6, 12, and 24. The second part of the study was an extension phase where patients were treated with a 4 mg dose. Additional patients were enrolled in the second part at an 8 mg dose following the protocol revision. For more details on the study design and evaluation, please refer to SCIB1 IB.

[0444] patient population

[0445] For cohorts 1 through 3 in Part I, patients must have histologically confirmed AJCC stage III or IV melanoma, with or without tumor at screening; patients with tumor must have measurable disease according to RECIST. Patients in cohort 4 (8 mg dose) in Part I and cohort 2a (8 mg dose) in Part II must have measurable disease at screening. Patients in cohorts 1 (4 mg dose) and 2b (8 mg dose) in Part II must have resected disease. Patients must be HLA-A2 positive and also positive for HLA-DR4, HLA-DR7, HLA-DR53, or HLA-DQ6.

[0446] A total of 35 patients (18 men and 17 women) were enrolled in the study, with a median age of 60 years (range 25 to 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 prior chemotherapy, one had received prior 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 prior treatment (vaccine). No DLT was observed during the dose escalation phase of Part I (n=11), and a 4 mg dose was selected for Part II extension (n=14). An 8 mg dose was subsequently studied in Part I (n=5) and Part II (n=5).

[0447] Security

[0448] Overall, 23 patients completed all planned injections for the main phase of the study (up to 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 time for both main and continuation treatment was 170 days, with a maximum of 1107 days (16 doses).

[0449] The most common adverse events (AEs) during the primary study period (>10%) were injection site hematoma (77%), injection site pain (37%), fatigue (26%), headache (20%), procedure pain (17%), arthralgia, blurred vision, constipation, limb pain (14%), depressed mood, dizziness, dyspnea, nausea, and rash (11%). Overall, 28 patients reported AEs related to the study drug. The most common events considered related to the study drug were injection site hematoma (37%), injection site pain (20%), fatigue (14%), blurred vision, headache, and procedure pain (11%). A total of 32 patients (91%) experienced AEs related to the electroporation device and procedure. SAEs were most commonly associated with the progression of malignant melanoma, with none considered potentially related to the study drug or its administration. Serious and life-threatening (Grade 3 and 4) AEs were uncommon; in the primary study, 14 events meeting these criteria occurred in five patients. Two grade 3 / 4 events were considered related to SCIB1: one patient experienced a grade 3 injection site hematoma, and the other experienced grade 3 anxiety; both patients were treated with 4 mg SCIB1. Five grade 3 / 4 events in the two patients were considered related to the electroporation device or procedure: one grade 3 injection site hematoma event, and two grade 3 anxiety events, injection site pain, and anticipatory anxiety events. The injection site pain and anticipatory anxiety events led to the patient discontinuing SCIB1 treatment. The second patient refused to continue treatment due to pain and discomfort during the electroporation procedure.

[0450] Three deaths were recorded in the primary study phase database, although they occurred after patients withdrew from the primary study and were documented as follow-up information; two were disease-related (one patient treated with 0.4 mg; one patient treated with 4 mg), and one was due to community-acquired pneumonia (8 mg). Two additional deaths were recorded after patients withdrew from continued administration, both disease-related: one patient treated with 4 mg and one patient treated with 8 mg. All of these patients had tumors at screening.

[0451] therapeutic effect

[0452] In immunoreactivity assays, including the primary study period plus the continuation phase, 88% of patients responded in the ELISpot assay and 82% responded in the proliferation assay. With continued SCIB1 administration, the number of patients responding to individual melanoma-specific peptides in the ELISpot assay increased. Comparison of ELISpot responses between dose groups showed a stronger response at 8 mg than at 4 mg. With continued SCIB1 administration, the number of patients responding to individual peptides in the proliferation assay also increased. Patients without tumors at screening who received the 8 mg dose showed a significantly stronger proliferative response to one peptide compared to patients receiving the 2 / 4 mg dose. Patients without tumors at screening also showed a significantly stronger proliferative response to any peptide compared to patients with tumors at screening.

[0453] Of the 15 patients with tumors at enrollment, one patient treated with an 8 mg dose achieved a partial response (PR) that met RECIST criteria for 204 days (29 weeks). Another patient treated with a 4 mg dose experienced a reduction of more than 30% in target lesions but progress in non-target lesions.

[0454] Patients with no detectable tumors at enrollment and treated with SCIB1 in an adjuvant setting were followed for disease-free survival. The longest follow-up was achieved in 16 patients who received 2 mg and 4 mg SCIB1. As of the cutoff date of October 29, 2016, all 16 patients were alive, with a median observation time of 39 months (range 33 to 52 months). At 2 years, 12 of these patients (75%) remained disease-free without receiving additional treatment besides continued SCIB1 therapy.

[0455] 2.3 Reasons for Dosage Selection

[0456] Based on the safety results of the SCIB1-001 study and the observed stronger immune response at an 8 mg dose compared to a 4 mg dose, the 8 mg dose was selected for this study. The SCIB1 dose will be administered via immediacy using the PharmaJet Stratis® needle-free injection device. In patients, an 8 mg dose corresponds to approximately 0.114 mg / kg or 4.44 mg / m². 2 The dosage was [not specified]. In contrast, the dose administered to mice in toxicology studies was 40 µg / mouse, equivalent to 2.0 mg / kg or 6.0 mg / m². 2 .

[0457] 2.4 Known and potential benefits and risks to patients

[0458] The most common adverse events (AEs) attributed to SCIB1 were fatigue (14%), blurred vision, and headache (11%). Injection site hematoma and injection site pain were attributed to the electroporation procedure used in SCIB1-001 and also to SCIB1, with overall incidence rates of 77% and 37%, respectively. SCIB1 has been shown to induce an immune response in melanoma patients (Patel et al. 2018).

[0459] 3. Research Objectives and Endpoints

[0460] 3.1 Main Objectives and End Points

[0461] 3.1.1 Importing Subqueues

[0462] Purpose

[0463] To evaluate the safety and tolerability of SCIB1 administration in patients receiving nivolumab in combination with ipilimumab or pembrolizumab.

[0464] end

[0465] Measurements were taken by recording adverse events (CTCAE v5.0), vital signs, physical examination, serum chemistry and hematology, thyroid function tests, injection site assessment, urinalysis, 12-lead ECG, performance status, and concomitant medications. The target SCIB1-related DLT rate was 0 or 1 in six evaluable patients.

[0466] 3.1.2 Main Research

[0467] Purpose

[0468] In this patient population, the aim was to determine whether adding SCIB1 to nivolumab in combination with ipilimumab or pembrolizumab alone improved the ORR, relative to historical data from nivolumab in combination with ipilimumab or pembrolizumab alone.

[0469] end

[0470] Total response rate as measured by RECIST 1.1 standard.

[0471] 3.2 Secondary Objectives and Endpoints

[0472] 3.2.1 Main Research

[0473] Purpose

[0474] In this patient population, compared with historical data on nivolumab in combination with ipilimumab or pembrolizumab alone, we determined whether the addition of SCIB1 to nivolumab in combination with ipilimumab or pembrolizumab improved duration of response.

[0475] end

[0476] Duration of response is measured from the first response (CR or PR) to the date of disease progression (according to RECIST 1.1) or death from any cause.

[0477] Purpose

[0478] Determine the ORR using the iRECIST standard (Seymour et al. 2017).

[0479] end

[0480] ORR determined by iRECIST.

[0481] Purpose

[0482] Assess the PFS rate for all patients at week 49 (day 344) and week 97 (day 680).

[0483] end

[0484] PFS rate is defined as the proportion of patients who have not progressed (according to RECIST 1.1 and iRECIST), or have not started new anti-cancer treatment, or have not died at each specified time point.

[0485] Purpose

[0486] Assess overall survival (OS) rates for all patients at week 49 (day 344) and week 97 (day 680).

[0487] end

[0488] OS rate is defined as the proportion of patients who are still alive at each specified time point.

[0489] Purpose

[0490] Further evaluate the safety and tolerability of SCIB1 in patients receiving nivolumab in combination with ipilimumab or pembrolizumab.

[0491] end

[0492] Safety and tolerability are evaluated using the measurements defined above for the imported sub-queue.

[0493] 3.3 Exploratory Objectives and Endpoints

[0494] Purpose

[0495] To explore the relationship between immune response and clinical outcomes.

[0496] end

[0497] Immunological response assessment (ELISpot and other assays) of peripheral blood samples from patients at a limited number of sites.

[0498] 4. Research Design

[0499] 4.1 Overall Research Design

[0500] This is a phase 2, multicenter, open-label study of SCIB1 in patients with advanced unresectable melanoma receiving nivolumab in combination with ipilimumab or pembrolizumab. There will be two cohorts, depending on whether patients are receiving standard-of-care dual therapy (nivolumab in combination with ipilimumab) or monotherapy (pembrolizumab) with a checkpoint inhibitor. In the introductory cohort, six patients will receive SCIB1 and nivolumab in combination with ipilimumab or pembrolizumab concurrently to ensure no unexpected serious toxicity would prevent further testing. The first two patients in these cohorts will be enrolled sequentially, at least 14 days apart, to minimize the risk of unexpected serious toxicity. If no more than one of the six evaluable patients experiences SCIB1-related DLT before day 35, the remaining patients will be enrolled using the Simon optimal two-stage design (Simon 1989) in the cohort. Figure 5 ).

[0501] Cohort 1: Nivolumab combined with ipilimumab

[0502] In Phase 1, 15 patients will be enrolled (including six from the introductory sub-cohort). This cohort will be discontinued if eight or fewer patients achieve a RECIST 1.1 objective response [CR or PR] within 25 weeks of the first dose of SCIB1. Otherwise, an additional 28 patients will be enrolled in Phase 2, for a total of 43 patients. Recruiting will continue until a decision on Phase 1 is made.

[0503] Cohort 2: Pembrolizumab

[0504] In Phase 1, 14 patients will be enrolled (including six from the introductory sub-cohort). This cohort will be discontinued if five or fewer patients achieve a RECIST 1.1 objective response [CR or PR] within 25 weeks of the first dose of SCIB1. Otherwise, an additional 30 patients will be enrolled in Phase 2, for a total of 44 patients. Recruiting will continue until a decision on Phase 1 is made.

[0505] If a queue cannot complete recruitment within a reasonable timeframe after another queue has fully enrolled, enrollment will be halted. Furthermore, as part of the interim analysis, a feasibility assessment will be conducted on further enrollment of the queue that has not reached its first-stage quota when the other queue reaches its first-stage quota.

[0506] DLT is defined as follows:

[0507] CTCAE v5.0 ≥ grade 3 drug-related non-hematological AEs, but the following conditions except :

[0508] • Grade 3 gastrointestinal toxicity (nausea, vomiting, diarrhea, constipation) that can be controlled by antiemetics and other therapies and lasts for no more than 48 hours

[0509] • Asymptomatic laboratory changes (excluding kidney and liver laboratory values ​​and grade 4 lipase / amylase) that can be successfully corrected within 72 hours (e.g., electrolyte replacement).

[0510] • Level 3 fatigue lasting less than 72 hours

[0511] • For patients with liver metastases, an elevation of ALT or AST of grade ≥3 is observed, which decreases to grade ≤2 within 7 days.

[0512] CTCAE v5.0 grade 4 drug-related hematological AEs, grade 3 thrombocytopenia with bleeding (platelet count decrease <50,000-25,000 / mm³) 3 Grade 3 hemolysis (requiring blood transfusion or medical intervention, such as steroids) and Grade 3 febrile neutropenia (absolute neutrophil count <1000 / mm³) 3 A single body temperature of 38.3°C [101°F] or a sustained body temperature of ≥38°C [100.4°F] for more than one hour.

[0513] Toxicity meeting these criteria and deemed relevant to the administration of the study drug will be considered a DLT. Grade 3 events clearly associated with tumor eruption (local pain, irritation, local rash) will not be considered a DLT. DLTs in the ingress subcohort will be assessed before day 35. Patients who withdraw from the study before week 5 (day 36) in the ingress subcohort for reasons other than DLT will be replaced.

[0514] If more than one of the six patients in any introductory sub-cohort experiences SCIB1-related DLT, the investigator and sponsor will formally review the existing safety data and decide whether to enroll more patients to assess the safety of the combination therapy. This step is necessary because some events occurring in the approved use of checkpoint inhibitors meet the definition of DLT and require evaluation of the contribution of SCIB1 (if any). In 277 melanoma patients receiving pembrolizumab alone at the recommended dose and regimen, the incidence of associated CTCAE ≥ grade 3 events was 10% (Robert et al. 2015b). For nivolumab in combination with ipilimumab, the incidence of grade 3-4 toxicities was 33.9% in 180 patients receiving 3 mg / kg nivolumab in combination with 1 mg / kg ipilimumab, and 48.3% in 178 patients receiving 1 mg / kg nivolumab in combination with 3 mg / kg ipilimumab (Lebbé 2019). In the SCIB1-001 monotherapy study in melanoma patients (n=35), only two grade 3 / 4 events were considered SCIB1-related: one patient experienced a grade 3 injection site hematoma, and the other experienced a grade 3 anxiety, both related to the electroporation procedure used in that study. In this study, the PharmaJet Stratis® device will be used, which was only associated with mild discomfort in an ongoing study. Any patients enrolled under a previous protocol and receiving SCIB1 with the TDS-IM electroporation device will be re-informed and consent will be obtained to continue administration of the PharmaJet Stratis® device. Decisions to expand enrollment in this case require investigator and sponsor review and approval. Further evaluation for excessive serious toxicities will continue for the remainder of the study.

[0515] During the 28-day screening period, patients will complete procedures and assessments to confirm eligibility. Written informed consent must be obtained before any protocol-specific procedures (procedures and assessments performed for research purposes and not as part of routine medical care). Test results and tumor imaging from routine clinical management (including those performed prior to written informed consent) may be used for screening if available within the designated screening time window. HLA testing will be performed using a separate consent form; HLA consent must be obtained prior to screening.

[0516] During the study, patient safety, tumor response and disease progression, as well as survival and the initiation of any new anti-cancer treatments will be monitored.

[0517] 4.2 Number of patients and countries in the plan

[0518] The plan is to enroll 87 patients from up to 20 clinical oncology centers worldwide. Additional patients will be enrolled to replace those in the import sub-cohort who are not evaluable for DLT or for the RECIST 1.1 ORR primary endpoint (Section 5.4.1).

[0519] 4.3 Study Duration

[0520] The study was delayed due to COVID-19, recruitment began in 2021, and it is expected to be completed in 2025.

[0521] 4.4 Patient Number Allocation

[0522] After patients sign the HLA informed consent form and agree to participate in the study, they will be assigned a unique 3-digit identification number. This patient identification number will serve as the primary method for identifying each patient in the CRF and site source files throughout the study.

[0523] 4.5 Blinding and Randomization

[0524] This is an open-label, two-cohort, non-randomized study, so blinding or randomization is not required.

[0525] 4.6 Reasons for Study Design and Patient Population Selection

[0526] Pembrolizumab has been shown to be effective in patients with advanced melanoma. However, only about one-third of patients treated respond to this checkpoint protein antibody (Robert et al. 2015b). The reasons for the lack of clinical response in the remaining patients are unclear, but may be partly related to the immune system's failure to recognize the patient's melanoma. Combination therapy with antibodies targeting PD-1 and CTLA-4 has been shown to increase response rates to approximately 50% in previously untreated melanoma patients, but this combination therapy is associated with severe and life-threatening toxicities and a high rate of treatment discontinuation due to such toxicities (Larkin et al. 2015).

[0527] Vaccination offers a way to increase the immune system's recognition of a patient's tumor and may be associated with improved response rates without the risk of significant toxicity. SCIB1 has been shown to elicit a cellular immune response in over 85% of treated melanoma patients and has demonstrated indications of single-drug clinical activity.

[0528] The current design of an open-label, non-randomized phase 2 study in previously untreated, unresectable melanoma patients is reasonable, as these patients will receive current standard-of-care checkpoint inhibitor therapy, i.e., nivolumab in combination with ipilimumab or pembrolizumab. SCIB1 was well-tolerated in its phase 1 / 2 trials, and adding SCIB1 should not interfere with the activity of nivolumab in combination with ipilimumab or pembrolizumab in this population. The sample size of 87 patients is sufficient to determine whether SCIB1 has the potential to improve RECIST 1.1 response rates (compared to historical rates for nivolumab in combination with ipilimumab or pembrolizumab alone in this population), justifying the rationale for a larger randomized study.

[0529] 4.7 Periodic review of safety and efficacy

[0530] Safety and efficacy data from the studies will be formally reviewed frequently and periodically, including at the end of the lead-in phase and at the end of Phase 1 of the Simon Phase 2 design. The review committee will include the investigator, the sponsor's medical monitor, and the sponsor's head of clinical operations or their designated representative. A lead-in sub-cohort review plan, reflecting the safety reviews conducted during lead-in, will be developed prior to the enrollment of the first patient in the study. A complete safety and efficacy review charter will be finalized at the end of the lead-in phase and before further patient enrollment in the study after the lead-in phase.

[0531] 4.8 Standards for Research Completion

[0532] Each patient's study treatment period consists of ten doses of SCIB1. Patients completing these will have finished 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 assessed for tumor response, disease progression, initiation of new cancer treatment, and survival for 97 weeks from the first dose of SCIB1.

[0533] The study will be completed when all patients complete their post-treatment follow-up period or withdraw from the study.

[0534] 4.9 Procedures and Evaluation Schedule

[0535] The research procedures and assessments required for all patients are summarized in Table 2 below. Detailed information on the procedures and assessments is provided in Sections 7–9. Prior to the screening period, a pre-screening assessment to determine the patient's HLA histological type may be performed after the patient has given separate, specific consent for this assessment. Optionally, HLA consent may be obtained at the investigator's discretion during the screening visit.

[0536] Table 2: Procedures and Evaluation Timeline

[0537]

[0538] Notes to Table 2:

[0539] aPTT = Activated Partial Thromboplastin Time, CPI = Checkpoint Inhibitor; CT = Computed Tomography, ECG = Electrocardiogram, ECOG = Eastern Cooperative Oncology 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

[0540] a) If appropriate, checkpoint inhibitor (CPI) administration can be concurrent with SCIB1 administration to minimize the number of visits. If CPI has been suspended or discontinued, a CPI visit is not required.

[0541] (b) The patient will receive up to five doses of SCIB1, administered at weeks 37, 49, 61, 73, and 85 (days 260, 344, 428, 512, and 596, respectively). If a dose of SCIB1 is missed, for example due to an adverse event resulting from administration of nivolumab in combination with ipilimumab or pembrolizumab, the missed dose may be administered after week 13, with a minimum interval of at least 15 days, but preferably maximizing the dose interval to 6 weeks. During the administration of missed doses, all procedures listed at the 12-week follow-up visit should be performed, except for assessment of tumor and skin lesions.

[0542] c) Three weeks after each 12-week SCIB1 dose, i.e., 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 assessment visit will be replaced by an EOT visit (30 ± 10 days after the last dose of SCIB1). If the patient decides to discontinue SCIB1 at the 12+3 assessment visit, all examinations of the EOT visit may be performed at the 12+3 assessment visit if possible; otherwise, the EOT visit will be performed 30 (± 10) days after the last dose of SCIB1, or as soon as possible according to the schedule.

[0543] d) For all patients, the EOT visit should be completed 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 who discontinue SCIB1 at the study evaluation visit, the EOT visit may replace 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 until the next dose of a checkpoint inhibitor, whichever is longer. Concomitant medications taken within 30 days after the last dose of SCIB1 must be recorded in the CRF.

[0544] e) Post-treatment follow-up will be conducted for all patients who have received at least two doses of SCIB1 (day 1 and week 4) and have not withdrawn from the study. Post-treatment follow-up will be conducted every 12 weeks (±2 weeks) after the last dose of SCIB1 until week 97, and may be conducted in person, by telephone / video conferencing, or by review of patient medical records as needed. For patients who have not recorded confirmed disease progression (according to RECIST 1.1 and iRECIST) or have not started new anticancer therapy, tumor and skin lesion assessments will be conducted every 12 weeks (±2 weeks) after the last dose of SCIB1.

[0545] f) The first dose of the CPI (nivolumab in combination with ipilimumab or pembrolizumab) will be administered in week 1, following the standard treatment dosing regimen. On days when both CPI and SCIB1 are administered, SCIB1 should be administered first. When CPI is administered alone, the standard treatment procedure should be followed. On days when CPI is administered alone, AEs and concomitant medication monitoring should be performed, and blood assessments and clinical examinations should be based on standard treatment. Continuation of CPI (as administered according to standard treatment) will be determined at the investigator's discretion. If the CPI dose is delayed or the CPI is discontinued, SCIB1 administration will continue as planned.

[0546] (g) Written informed consent must be obtained before any protocol-specific procedure is performed. HLA and primary study written informed consent may be obtained simultaneously, but ideally, the primary study consent should be obtained in one go after HLA eligibility has been determined. Results of tests and tumor imaging from routine clinical management (including those performed prior to written informed consent) may be used for screening if available within the specified time window.

[0547] h) Medical history includes the initial diagnosis of melanoma, previous surgeries, previous adjuvant chemotherapy and adjuvant biotherapy, previous radiotherapy, and the start and stop dates of adjuvant therapy. Baseline medication history will also be collected.

[0548] i) For women of childbearing potential (including those ≤12 months from their last menstrual period), a urine pregnancy test will be performed within 72 hours prior to the first dose. If the urine pregnancy result is not confirmable as negative, a serum pregnancy test will be required. Pregnancy tests (serum and / or urine) will be repeated during the shown visit.

[0549] j) For any patient who does not have sufficient archived tumor tissue samples (<5 years) for PD-L1 assessment, a fresh tumor biopsy should be performed before the first dose of the investigational drug.

[0550] k) Assess or sample before administration.

[0551] (l) A limited physical examination (cardiovascular system, lungs, abdomen, skin, weight, and areas with signs and symptoms of disease) may be performed as needed during checkpoint inhibitor visits only. Height is measured only at screening.

[0552] m) Vital signs should be measured approximately 10 to 15 minutes before administration, and 5 minutes, 30 minutes, and 1 hour after SCIB1 dose administration. Patients should be observed for at least 1 hour after receiving at least the first two doses of CPI. Vital signs include body temperature, pulse, respiratory rate, and blood pressure.

[0553] n) Serum chemistry will include albumin, ALT, alkaline phosphatase, AST, bicarbonate or carbon dioxide, urea, calcium, chloride, creatinine, total bilirubin (and direct bilirubin (if clinically indicated)), gamma-glutamyl transferase, LDH, non-fasting blood glucose, phosphorus (measured as phosphate), potassium, sodium, uric acid (urate), and total protein.

[0554] o) Screening period serum chemistry, hematology, urine analysis and thyroid examination performed within 48 hours after the first dose of study drug do not need to be repeated on day 1.

[0555] p) Hematology will include hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, red blood cell (RBC) count, white blood cell (WBC) count, and WBC differential (basophils, eosinophils, lymphocytes, monocytes, neutrophils).

[0556] q) Urine analysis (test strips) will record protein, glucose, ketone bodies, blood, nitrite, white blood cells, bilirubin, pH, and specific gravity. Microscopic examination will be performed only when clinically indicated: bacteria, RBCs, WBCs, casts, and crystals.

[0557] r) On days when SCIB1 is administered, the injection site will be assessed 1 hour after the SCIB1 dose. If any moderate or severe injection site reaction is observed, digital photography of the injection site should be taken at the designated time point or, if necessary, outside of these time points. Anonymous copies of the digital photographs will be transferred to a dedicated database via secure file transfer.

[0558] s) If SCIB1 was not administered at the last study visit, no evaluation is required.

[0559] t) Tumor imaging (chest, abdomen, pelvis, brain, and any areas with known disease) will 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 contrast agent allergy) that makes CT scans unsafe, or the disease is better recorded by MRI. The same technique must be used throughout the study period for each individual patient. Baseline brain MRI (preferably) or CT scans are required to determine the presence of brain metastases. Repeat brain imaging is only required if the patient has brain metastases at baseline or new brain metastases are suspected. Patients with an objective tumor response or disease progression (PD) should be repeated scans no earlier than 4 weeks and no later than 8 weeks later to confirm the response and rule out the possibility of a tumor outbreak response, respectively. The investigator will assess the response status (according to RECIST 1.1 and iRECIST). Anonymous copies of imaging data will be collected for use in the study database. The imaging window is ±7 days, and the timing of imaging should follow the calendar and not be adjusted due to treatment delays.

[0560] u) For all patients who have not recorded disease progression or have not started new anticancer therapy, tumor assessment should be performed at the indicated time points during treatment or post-treatment follow-up, even if they discontinue SCIB1 treatment before week 13 (day 92).

[0561] v) Skin lesions will be documented through digital photography. Anonymous digital copies of the photographs will be collected for use in a research database.

[0562] w) At a limited number of sites, blood samples will be collected for cellular immune response analysis and delivered to the Scancell laboratory via express at ambient temperature, arriving within 8 hours of collection. Immunological blood samples collected within 7 days of the first dose of SCIB1 do not need to be repeated on day 1 (it is recommended that immunological samples collected during screening be collected at least 2 days prior to day 1 so that day 1 samples can still be collected if there are any issues with the sample). Lymphocytes and plasma will be prepared and tested immediately or stored in a freezer until analysis. Samples from the day of dosing should be collected prior to dosing.

[0563] x) If no assessment requiring a clinical visit is scheduled, the assessment will be conducted remotely by a healthcare professional via telephone or video conference.

[0564] 4.10 Post-treatment follow-up

[0565] Post-treatment follow-up will be conducted for all patients who have received at least two doses of SCIB1 (day 1 and week 4) and have not withdrawn from the study. Patients will attend clinical visits every 12 weeks (±2 weeks) after the last dose of SCIB1 for tumor evaluation (including CT / MRI scans) and skin lesion evaluation, including patients who received the last dose of SCIB1 at week 85 (day 596), unless disease progression occurs or a new anticancer therapy is started. If disease progression occurs or a new anticancer therapy is started, subsequent follow-up may be conducted by a healthcare professional via telephone / video conferencing during a routine clinical visit or by reviewing the patient's medical records. Therefore, all patients will be evaluated 97 weeks from the first dose of SCIB1. The following information will be collected:

[0566] • Dates of clinical visits / follow-up phone calls / video conference calls

[0567] • The date of onset of any new cancer, including its type and diagnostic method.

[0568] • For all patients who have not recorded confirmed disease progression or have not started new anticancer therapy, tumor assessment and skin lesion assessment shall be performed using RECIST 1.1 and iRECIST criteria (tumor assessment shall be performed during treatment or during follow-up after treatment at the time points shown in Table 2: Procedures and Assessment Schedule).

[0569] • Disease progression date and determination method

[0570] • New cancer treatment start date and generic name of treatment agent

[0571] • Date of death and primary cause of death

[0572] 5. Patient selection and withdrawal

[0573] 5.1 Selection Criteria

[0574] 1) The patient has a histologically confirmed, unresectable stage III or IV melanoma, as defined by the AJCC (8th edition; Gershenwald et al. 2017). Patients diagnosed with melanoma of unknown primary origin are also eligible.

[0575] 2) The patient has not received prior systemic treatment for advanced disease. Prior adjuvant therapy is permitted, defined as treatment following resection of all detectable disease; the last dose must be administered at least 4 weeks prior to the first dose of SCIB1.

[0576] 3) The patient has undergone clinical evaluation and checkpoint inhibition (nivolumab in combination with ipilimumab or pembrolizumab) has been determined to be the appropriate treatment for their advanced disease.

[0577] 4) The patient’s BRAF status must be known; at the investigator’s discretion, patients with BRAF mutation-positive disease may be enrolled without receiving BRAF inhibitor therapy, provided that they do not have evidence of rapidly progressive disease (PD).

[0578] 5) The patient has at least one measurable lesion according to RECIST 1.1 criteria via CT or MRI.

[0579] 6) The patient is HLA-A2 positive.

[0580] 7) The patient is HLA-DR4, HLA-DR7, or HLA-DR53. or HLA-DQ6 positive.

[0581] 8) The patient must be at least 18 years old.

[0582] 9) The patient’s life expectancy exceeds 3 months.

[0583] 10) The patient’s ECOG performance status score is 0 or 1.

[0584] 11) The patient has adequate organ function, as determined by the following laboratory values:

[0585]

[0586] 12) Patients must be able and willing to provide written, REC-approved informed consent before undertaking any study-related procedures. (A new REC-approved ICF must be signed if a patient is rescreened for a study or if a protocol revision changes the care of an ongoing patient.)

[0587] 13) Women of childbearing potential (including those ≤12 months from their last menstrual period) must have a negative serum pregnancy test during the screening period (72 hours prior to the planned administration of the first dose of the study drug on day 1), and must not be breastfeeding or intend to become pregnant during study participation. They should also be informed of the potential risks to the fetus from nivolumab in combination with ipilimumab or pembrolizumab. Women of childbearing potential must agree to use highly effective contraception (Appendix E) before study enrollment, throughout the study treatment period, and for 120 days after discontinuation of SCIB1, nivolumab in combination with ipilimumab, or pembrolizumab.

[0588] 14) Men who are of childbearing potential with a fertile partner must agree to use highly effective contraception throughout the study treatment and for 120 days after discontinuation of SCIB1, nivolumab in combination with ipilimumab or pembrolizumab (whichever is later) (Appendix E).

[0589] 15) Patients must be willing and able to comply with the scheduled visits, treatment plans, laboratory tests and other research procedures.

[0590] * HLA testing will be performed in an accredited laboratory using molecular (DNA) typing.

[0591] 5.2 Exclusion Criteria

[0592] 1) The patient was diagnosed with mucosal melanoma or ocular melanoma.

[0593] Patients with active central nervous system metastases or carcinomatous meningitis are eligible. (Patients who have responded to previous treatment for brain metastases are eligible if they have been stable for at least 4 weeks prior to the first dose of the study drug, have no evidence of MRI progression, and have discontinued systemic steroids at least 1 week prior to the first dose of the study drug. Patients who have recently undergone radiosurgery for brain metastases are eligible if they have fully recovered from postoperative sequelae, the screening scan must be at least 4 weeks after radiosurgery, and systemic steroids must have been discontinued at least 1 week prior to the first dose of the study drug.)

[0594] Patients who have previously received treatment to block CTLA-4, PD-1, PD-L1, or PD-L2 are eligible, except in the following cases: patients who have received these treatments as adjuvant therapy are eligible.

[0595] Patients are expected to require any other form of systemic or local anticancer therapy during the study treatment.

[0596] The patient is not currently taking any systemic steroid treatment or receiving any other form of immunosuppressant within one week of receiving the first dose of the study drug. Physiological doses of systemic steroids, such as those used to manage adrenal insufficiency, and topical and inhaled steroids, such as those used to manage asthma, are permitted.

[0597] Patients who received any investigational product within 28 days prior to the first dose of study treatment (or within 5 half-lives of the relevant treatment) must have received treatment with any investigational product.

[0598] The patient has a history (within 5 years) or currently has a malignant tumor other than melanoma, as well as a cured local tumor, such as breast carcinoma in situ, cervical carcinoma in situ, basal cell or squamous cell carcinoma of the skin, or prostate cancer with Gleason <6 and prostate-specific antigen within the normal range.

[0599] Patients with comorbidities that could impede the study and evaluation are not eligible, including but not limited to uncontrolled medical conditions, uncontrolled and active infections (considered opportunistic, life-threatening, or clinically significant), uncontrolled bleeding risk, uncontrolled diabetes, or lung disease (including obstructive pulmonary disease, pulmonary fibrosis, and a history of symptomatic bronchospasm), or alcoholic liver disease, or primary biliary cirrhosis. Caution should be exercised in patients suspected of or diagnosed with epilepsy.

[0600] Patients with NYHA class III or IV heart disease, a myocardial infarction within the past 6 months, a heart rate ≤50 bpm, a history of significant cardiac abnormalities and / or clinically significant abnormal baseline ECG readings, active ischemia, or any other uncontrolled cardiac condition such as angina, clinically significant arrhythmias requiring treatment (including anticoagulants), uncontrolled hypertension (>140 / 90 mmHg), significant cerebrovascular disease, or congestive heart failure are eligible for the following criteria:

[0601] The patient has a history of severe hypersensitivity reactions to mAb treatment.

[0602] Patients must have an active autoimmune disease or a documented history of an autoimmune disease or syndrome requiring systemic steroids or immunosuppressants (except for patients with vitiligo or cured childhood asthma / atopic syndrome, who are not excluded for these reasons). The following patients are not excluded from the study: those requiring 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, such as for managing adrenal insufficiency. In these cases, the recruiting investigator should discuss the patient's eligibility with the study medical monitor prior to enrollment.

[0603] The patient received the vaccine within 28 days of receiving the first dose of the study treatment.

[0604] The patient has a known history of HIV, or has tested positive for any HBV or HCV, indicating an active acute or chronic infection.

[0605] The patient has a known history of current or recent (within the past year) substance abuse, including illicit drugs or alcohol.

[0606] 5.3 Treatment Discontinuation

[0607] SCIB1 treatment will be permanently discontinued in the following circumstances:

[0608] 1) Any vaccine-related DLT

[0609] 2) Confirmed disease progression and / or initiation of new anti-cancer treatment

[0610] 3) Pregnancy

[0611] 4) The sponsor requests to discontinue treatment.

[0612] 5) The patient (or their legal representative) requests to discontinue treatment.

[0613] 6) If a serious or life-threatening adverse event (AE) occurs, treatment must be permanently discontinued, according to the investigator's opinion.

[0614] 7) Failure to comply with the study's treatment, procedures, and assessments based on the researcher's opinion; or

[0615] 8) Diseases that may interfere with continued SCIB1 treatment.

[0616] When treatment is discontinued, researchers will record the reason for discontinuation in the patient's medical records and CRF. Patients can choose to discontinue treatment but will continue to be followed up and monitored for disease progression, initiation of new cancer treatments, and survival.

[0617] In the event of discontinuation of SCIB1 treatment, the patient will complete the end-of-treatment visit and enter the post-treatment follow-up.

[0618] As described in relevant SmPC and clinical guidelines, treatment with nivolumab in combination with ipilimumab or pembrolizumab will be permanently discontinued in the event of a serious or life-threatening adverse reaction.

[0619] 5.4 Exit Criteria

[0620] Patients will withdraw from the study in the following circumstances:

[0621] They (or their legal representative) withdrew their informed consent.

[0622] They requested a permanent halt to treatment and all subsequent research-related activities, including the collection of follow-up information from patients / caregivers, relatives, medical records, or healthcare providers; or

[0623] The sponsor decided to terminate the research.

[0624] Patients who permanently discontinue treatment for any other reason (as defined in Section 5.3) are considered to have withdrawn from the study if follow-up information is not available from the sources described above.

[0625] Upon withdrawal, the researchers will record the reason for discontinuation in the patient's medical records and CRF. Patients may discontinue treatment without withdrawing from the study as described in Section 5.3.

[0626] 5.4.1 Alternative Patients

[0627] In the inbound subcohort, patients who withdrew before week 5 (day 36) for reasons other than DLT or failed to complete a safety assessment to the point that the overall safety of the vaccine could not be assessed will be replaced. Patients whose primary endpoint of ORR was not evaluable according to RECIST 1.1 will be replaced as needed to ensure that there are 43 evaluable patients in cohort 1 and 44 evaluable patients in cohort 2. Patients will be considered evaluable for the primary endpoint if they have received at least one dose of SCIB1 and completed at least one post-baseline tumor assessment before week 25 (day 176), or withdrew from the study due to disease progression or SCIB1 treatment-related toxicities, or died due to melanoma-related complications or SCIB1-related toxicities.

[0628] 5.4.2 Filtering again

[0629] Patients who previously failed screening may only be re-screened after consultation with the sponsor's medical monitor. Screening procedures that have exceeded the screening time window must be repeated.

[0630] 5.5 Research terminated

[0631] Investigators and sponsors will periodically review ≥ Grade 3 adverse events (AEs), laboratory values, and SAEs. The study will be suspended if a death causally related to SCIB1 occurs, or if the cumulative total of three Grade 4 events causally related to SCIB1 exceeds three. The study may be restarted if deemed appropriate by the investigators and sponsors after an assessment of the cause of toxicity and implementation of a corrective plan (if necessary). The study may be terminated in whole or in part if at any point in time the toxicity of SCIB1 in combination with nivolumab and ipilimumab or pembrolizumab makes the risk / benefit ratio no longer support continuing the study, and further patient recruitment is deemed unreasonable for medical or ethical reasons. This decision may be made jointly by the investigator and sponsor, or by the REC or national regulatory agency. The sponsor may also choose to terminate the study if enrollment is slow enough to prevent the study from being completed within an acceptable timeframe, or if development of SCIB1 is discontinued. The study may also be terminated if five or fewer clinical responses (RECIST 1.1 objective response [CR or PR]) are observed within 25 weeks after the first dose of SCIB1.

[0632] If the study is terminated early, the sponsor will notify the researchers and national health authorities. The researchers must immediately notify all enrolled patients and the termination of the REC study.

[0633] 6. Treatment

[0634] 6.1 Research drugs

[0635] The IMP used in this study was SCIB1, a plasmid DNA (laboratory name pVaxDCIB68) designed to express an engineered human IgG1 antibody molecule carrying a CTL epitope derived from the TRP-2 melanoma antigen, plus two helper T cell sequences derived from the melanoma gp100 protein. One of the gp100 sequences also contained nested CTL epitopes. SCIB1 was prepared at a concentration of 4 mg / mL in phosphate-buffered saline. Each 2 mL glass vial was sealed with a FluroTec® stopper and a Flip-off cap, containing a 1.0 mL extractable volume.

[0636] 6.1.1 Non-investigation drugs

[0637] 6.1.1 Nivolumab in combination with ipilimumab

[0638] Nivolumab in combination with ipilimumab is indicated for the treatment of adult patients with unresectable or metastatic melanoma. This combination therapy has since been adopted and reimbursed by international healthcare payers, including the UK's National Health Service (NHS). Nivolumab in combination with ipilimumab will be available as standard treatment through hospital pharmacies and will be used in accordance with prescribing information and local guidelines.

[0639] 6.1.2 Pembrolizumab

[0640] Pembrolizumab is approved in the EU, US, Australia, and UK for the treatment of patients with unresectable or metastatic melanoma. Pembrolizumab will be available as standard treatment through hospital pharmacies and will be used according to prescribing information and local guidelines.

[0641] 6.2 Preparation, processing, storage, inventory and disposal of research drugs

[0642] 6.2.1 SCIB1

[0643] SCIB1 must be stored frozen at −20°C (±5 °C) / −5°F (±10 °F) and thawed before use. See IMP handling instructions for more details.

[0644] 6.2.2 Checkpoint Inhibitors

[0645] The use of nivolumab in combination with ipilimumab or pembrolizumab will be determined by the investigator at their discretion based on standard treatment, as described in the relevant SmPC and clinical guidelines.

[0646] The first dose of a checkpoint inhibitor should be administered one week after the first SCIB1 dose to ensure a sufficient primary immune response following the first SCIB1 dose (Massarelli et al 2019). Checkpoint inhibitor treatment beyond the 85-week study duration will be determined by the investigator at their discretion, based on standard of care. The storage, packaging, and handling of checkpoint inhibitors will follow SmPC guidelines on a case-by-case basis.

[0647] 6.2.3 Drug Inventory

[0648] The drug inventory records will include the quantity of SCIB1s received and will maintain records of all SCIB1 dispensings. These records will include the patient study identification number; the date, time, and number of vials dispensed; the date and quantity administered; and the date and number of vials of any drugs returned to the pharmacy for destruction or disposal according to local standard operating procedures (SOPs). These records must be available for periodic review by the study monitor. Investigators are responsible for ensuring the inventory of all used and unused investigational supplies. Unused SCIB1 vials must not be destroyed or returned to the central warehouse before the investigational drug inventory is conducted.

[0649] IMP (SCIB1) is only used in patients who have given informed consent and have been enrolled in this study.

[0650] 6.3 SCIB1 Application Procedure

[0651] SCIB1 will be administered to the patient using the PharmaJet Stratis® needle-free injection device. The administration procedure will be performed by a field personnel qualified for intramuscular injection and who have completed a device training workshop. Each administration will consist of four injections at separate sites. Eligible injection sites include the left and right upper arm lateral (middle deltoid) or the left and right thigh lateral (vastus lateralis). Device setup and SCIB1 administration to the patient will be performed as described in the PharmaJet Stratis® needle-free injection instructions. Standard intramuscular injection techniques and precautions will be used in site preparation, SCIB1 administration, and medical waste disposal to ensure the safety of both the patient and researchers.

[0652] Any patient recruited under the previous protocol who has received SCIB1 using the TDS-IM electroporation device will be given informed consent again to continue administration of the PharmaJet Stratis® device.

[0653] 6.4 Checkpoint Inhibitors: Usage and Precautions

[0654] The instructions in the SmPC regarding the reconstitution, preparation, and administration of checkpoint inhibitors should be followed. Management of adverse events and modifications to the treatment regimen are described in Section 6.6.

[0655] 6.5 Treatment Schedule

[0656] The patient will receive four intramuscular injections of 2 mg SCIB1 each, using the PharmaJet Stratis® needle-free injection device, at weeks 0, 4, 7, 13, and 25 (days 1, 29, 50, 92, and 176, respectively), and every 12 weeks thereafter until week 85 (day 596). On day 1, patient eligibility must be confirmed before administering the first dose of SCIB1. On days when SCIB1 is being administered concurrently with nivolumab in combination with ipilimumab or pembrolizumab, SCIB1 will be administered before nivolumab in combination with ipilimumab or pembrolizumab.

[0657] 6.6 Dosage adjustment, modification and delay

[0658] 6.6.1 Infusion reaction

[0659] Management of infusion reactions associated with checkpoint inhibitor administration should follow standard treatment guidelines, as described in relevant SmPC and clinical guidelines. Patients should be monitored for infusion reactions for at least 1 hour after their last dose of the checkpoint inhibitor of the day.

[0660] 6.6.2 Dosage Adjustment and Modification

[0661] Reducing the SCIB1 dose is not expected to be of value in this study. Toxicity management will be carried out through the treatment delay described in the next section.

[0662] 6.6.3 Treatment delay and checkpoint inhibitor discontinuation

[0663] In the event of an adverse event, investigators should consider pausing, discontinuing, or resuming checkpoint inhibitors in accordance with relevant SmPC and national or local clinical treatment guidelines.

[0664] If nivolumab in combination with ipilimumab or pembrolizumab has been paused or discontinued, SCIB1 may continue if the investigator deems it to be in the patient's best interest. Furthermore, the patient must have no evidence of clinical disease progression, and the adverse events leading to checkpoint inhibitor discontinuation must have improved by the next SCIB1 dose. Patients with BRAF mutation-positive disease must be aware of available BRAF-targeted therapies and must re-obtain informed consent to continue receiving SCIB1 alone. SCIB1 administration will continue according to the regimens in Table 2.

[0665] If a dose of SCIB1 is missed, for example due to an adverse event, the missed dose may be administered after week 13, with a minimum interval of at least 15 days, but preferably maximized to 6 weeks according to the regimen in Table 2.

[0666] Special recommendations for immune-mediated adverse events

[0667] Immune-mediated toxicity can be managed with corticosteroid immunosuppression. Management of specific immune-mediated toxicities should follow the guidelines in the checkpoint inhibitor SmPC.

[0668] 6.7 Previous and concomitant medications and treatments

[0669] Detailed information on all previous medications received by the patient within 30 days prior to Day 1 will be collected. All concomitant medications administered to the patient during the study treatment and within 30 days after the last dose of SCIB1 must be recorded in the CRF. Concomitant medication records must include the generic name, indication, dosage, start and end dates, and route of administration.

[0670] 6.7.1 Medication and treatment are prohibited.

[0671] During treatment under this protocol, patients are not permitted to receive other forms of systemic melanoma therapy. This includes chemotherapy, biotherapy, other forms of immunotherapy, and gene therapy. Surgery and localized radiation therapy to skin lesions are not permitted and should be discussed in advance with the study medical monitor. Systemic corticosteroids and other immunosuppressants are not permitted except as described in Section 6.7.2.

[0672] 6.7.2 Permitted medication, pre-medication, and supportive care

[0673] Systemic corticosteroids are permitted only for the management of immune-mediated adverse events (AEs), as described in Section 6.6 (excluding physiological replacement therapy). For patients with immune-related adverse events that cannot be controlled with corticosteroids, the administration of other systemic immunosuppressants may be considered. Physiological doses of systemic steroids are permitted, such as those used to manage adrenal insufficiency. as well as Topical and inhaled steroids, such as those used to manage chronic conditions like asthma. Vaccinations (including influenza and COVID-19 vaccines, provided they are not live vaccines) are permitted, but should be scheduled after week 7 (day 50) between SCIB1 doses to allow a 28-day interval before the next SCIB1 dose. Live vaccines, including nasal influenza vaccines, are not permitted.

[0674] 6.8 Patient compliance monitoring

[0675] Researchers and clinical field staff will be responsible for monitoring patient adherence. Adherence will be checked at each visit following the initial dispensing of the investigational drug, and investigational drug inventory records will be completed. Patients who do not adhere to the study's treatment, procedures, and assessments should withdraw after discussion between the investigator and the sponsor, and the REC should be notified.

[0676] 7. Efficacy evaluation

[0677] 7.1 Tumor imaging and RECIST assessment

[0678] The primary efficacy endpoint of the study was tumor response as measured according to RECIST 1.1 criteria (Eisenhauer et al. 2009, Appendix A). Response as determined according to iRECIST (Seymour et al. 2017, Appendix B) will also be measured as a secondary efficacy endpoint.

[0679] Tumor imaging (chest, abdomen, pelvis, brain, and any areas with known disease) will 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 contrast agent allergy) that makes CT scans unsafe, or the disease is better recorded by MRI. The same technique must be used throughout the study period for each individual patient. Baseline brain MRI (preferably) or CT scans are required to determine the presence of brain metastases. Repeat brain imaging is only required if the patient has brain metastases at baseline or new brain metastases are suspected. Patients with an objective tumor response or disease progression (PD) should be repeated scans no earlier than 4 weeks and no later than 8 weeks later to confirm the response and rule out the possibility of a tumor outbreak response, respectively. The investigator will assess the response status (according to RECIST 1.1 and iRECIST). Anonymous copies of imaging data will be collected for use in the study database. The imaging window is ±7 days, and the timing of imaging should follow the calendar and not be adjusted due to treatment delays. Tumor assessments should be performed during treatment or during post-treatment follow-up at the time points shown in Table 2: Procedures and Assessment Schedule.

[0680] At least one measurable lesion according to RECIST 1.1 criteria must be present at the baseline. A measurable lesion is defined as a lesion with a longest diameter of at least 10 mm on CT scan, or a lymph node with a short axis of at least 15 mm (CT scan slice thickness 5 mm). Skin lesions will be documented digitally. Copies of the digital photographs will be collected for the research database.

[0681] The assessment conducted using iRECIST will be used to determine patient management and termination decisions within the study. If a patient is diagnosed with iRECIST PD, this needs to be confirmed in a second study treatment scan. The second scan should be conducted no earlier than 4 weeks and no later than 8 weeks afterward. During this period, if the patient's performance status does not deteriorate, there is no clinically significant increase in disease-related symptoms, intensive management of disease-related symptoms is not required, and the patient remains adequately able to tolerate study treatment, the investigator may decide, at their discretion, to continue the study treatment. Detailed information on the criteria for determining and confirming disease progression using iRECIST is provided in Appendix B.

[0682] 7.2 Progression-free survival and overall survival

[0683] Patients will undergo a progression and survival assessment 97 weeks from the first dose of SCIB1. The following will be recorded:

[0684] • Disease status and assessment date

[0685] • Date of disease progression determined by RECIST 1.1 and iRECIST

[0686] • The start date of any new cancer treatment and the generic name of the treatment drug

[0687] • Date of death and primary cause of death, regardless of cause.

[0688] 8. Pharmacodynamic and biomarker evaluation

[0689] 8.1 Blood Sample

[0690] At a limited number of sites, blood samples (60 mL) will be collected for analysis of cellular immune responses and liquid biomarkers.

[0691] 8.1.1 Sample processing and transportation

[0692] Blood samples will be delivered to the Scancell laboratory via express at ambient temperature, arriving within 8 hours of collection. Lymphocytes and plasma will be prepared and either immediately tested or frozen until analysis.

[0693] 8.1.2 Evaluation and analysis of specific blood samples

[0694] Cellular immunity to TRP-2 and gp100 peptides in samples will be assessed by antigen-specific T cell activation of lymphocytes, measured using an IFNγ ELISpot assay. Immune response data, including frequency and affinity (if applicable), will be tabulated and plotted to descriptively assess response patterns throughout the study.

[0695] The frequency of T cells against each antigen will be assessed before and after treatment using ELISpot assays. The criteria for designating patients as immune responders will be defined in the SAP.

[0696] Additional analyses can be performed on subsets of the sample, including: CFSE proliferation assay, CTL lysis assay, MHC multimer staining for memory cells, assays to assess epitope diffusion, Luminex assay for cytokine secretion, T cell subset analysis, and liquid biomarker analysis. Assays will be performed based on the observed response in the ELISpot assay and whether sufficient cells and plasma were recovered from the blood sample.

[0697] 8.2 Tumor Samples

[0698] Baseline formalin-fixed, paraffin-embedded (FFPE) archives (<5 years) or fresh tumor biopsies are required for PD-L1 expression analysis. PD-L1 analysis can be performed in an accredited laboratory.

[0699] 8.2.1 Evaluation and analysis of specific tumor samples

[0700] FFPE tumor tissue samples will be submitted for PD-L1 expression analysis using appropriate quantitative immunohistochemical assays.

[0701] 9. Security Assessment

[0702] 9.1 Adverse Events

[0703] 9.1.1 Definition of Adverse Events

[0704] An AE is any undesirable medical event or change in existing condition that occurs during or after administration of SCIB1, nivolumab in combination with ipilimumab, or pembrolizumab, whether or not it is considered related to these products or other trial procedures (21 CFR 312.32a, and in accordance with ICHE2A guidance). If the investigator believes that abnormal laboratory findings and other unusual study results (such as results on an ECG record) are medically significant, they should be reported as AEs.

[0705] In this study, disease progression is expected and will not be recorded as an AE. However, medical events and conditions associated with disease progression (such as respiratory failure, weight loss) will be recorded as AEs.

[0706] 9.1.2 Recording and Reporting of Adverse Events

[0707] AEs will be collected from the start of the first study-related procedure (including study-specific screening procedures) until 30 days after the last dose of SCIB1 or until the next dose of the checkpoint inhibitor (whichever is longer).

[0708] All adverse events (AEs) for patients who have signed primary study consent must be recorded on the patient's CRF and must include the following details: a concise medical terminology description of the event, date of onset, date of resolution or, if unresolved, status, severity, relationship to SCIB1 (IMP), relationship to needle-free injection procedures, relationship to nivolumab in combination with ipilimumab or pembrolizumab (non-IMP), actions taken, and outcomes. Investigators and clinical field personnel will collaborate with the sponsor to investigate and follow up on any concerning AEs.

[0709] All AEs must be evaluated to determine whether they meet the criteria for reporting as an SAE, as defined in Section 9.2.1.

[0710] 9.1.3 Severity of Adverse Events

[0711] The severity of adverse events (AEs) will be graded according to CTCAE v5.0. AEs not included in CTCAE will be graded according to the General CTCAE Grading Guidelines as follows:

[0712] Grade 1 Mild, asymptomatic or with mild symptoms; clinical or diagnostic observations only; no intervention required.

[0713] Level 2, moderate, requires minimal, localized, or non-invasive intervention; age-appropriate instrumental activities of daily living (ADL) are limited, such as preparing meals, purchasing groceries or clothing, using the telephone, and managing finances.

[0714] Level 3: Severe or medically significant but not immediately life-threatening; requires hospitalization or prolonged hospitalization; disabling; limits self-care activities such as bathing, dressing and undressing, feeding, toileting, and medication administration, and is not bedridden.

[0715] Level 4: Life-threatening consequences; requires emergency intervention.

[0716] Level 5 Deaths Related to Acute Effects

[0717] 9.1.4 Relationship between adverse events and study treatments or devices

[0718] Researchers will determine whether an AE is related to or unrelated to the following: IMP, non-IMP, or PharmaJet Stratis® needle-free injection procedures. For events considered related, there should be evidence of exposure to the product or procedure with a reasonable temporal relationship. AEs should be considered more likely to be explained by the product or procedure than by other causes; this includes events that are considered potentially related to the product or procedure. Events that do not meet these criteria should be reported as unrelated.

[0719] 9.1.5 Deviation from the plan due to adverse events

[0720] Protocol deviations may be permitted only on a case-by-case basis for patients experiencing adverse events (AEs) or medical emergencies. The attending investigator or other physician must contact the sponsor's medical experts as soon as possible to discuss the deviation and whether the patient should continue participating in the study.

[0721] 9.2 Serious Adverse Events

[0722] 9.2.1 Definition of serious adverse events

[0723] SAE refers to any adverse medical event that meets one or more of the following criteria:

[0724] • Leads to death, unless death is entirely caused by disease progression.

[0725] Life-threatening*

[0726] • Requires hospitalization or extension of existing hospital stay**

[0727] • Leads to significant disability / loss of ability (significant impairment in the ability to perform normal daily activities)

[0728] • It is a congenital abnormality / birth defect; and / or

[0729] • It is a significant medical event that may not result in death, endanger life, or require hospitalization, but may endanger the patient and may require medical intervention to prevent one of the outcomes listed in these criteria.

[0730] *This refers to the risk to the patient when the event occurs, excluding events that could be life-threatening if they are more severe.

[0731] Hospitalization for elective surgery, rehabilitation, social reasons, or other procedures planned before informed consent is obtained is not considered SAE.

[0732] 9.2.2 Anticipation

[0733] SAEs that are not included in the reference safety information in the current Investigator's Manual (IB) of the IMP or in the SmPC of non-IMP are considered unintended, based on their specificity, severity, outcome, or frequency.

[0734] 9.2.3 Reporting of serious adverse events

[0735] SAEs will be collected from the start of the first study-related procedure (including study-specific screening procedures) until 30 days after the last dose of SCIB1 or until the next dose of a checkpoint inhibitor (whichever is longer). Furthermore, any SAEs that the investigator or clinical field personnel become aware of and assess as being related to study participation (SCIB1 treatment or procedure) must be documented and include any follow-up contact until resolved or improved.

[0736] When a patient experiences a sarcastic emergency (SAE) during the study, it must be reported to the Safety Desk within 24 hours of becoming aware of the event on-site, even if the SAE appears unrelated to the trial. A copy of the SAE report form should be sent to the Safety Desk via email or fax. The completion and submission of the SAE report form should not be delayed, even if not all information is available. Contact details for SAE reporting will be provided before the first patient is enrolled.

[0737] Patients who experience a SAE should be closely monitored until the event is resolved or the investigator deems the patient stable. Investigators and clinical field personnel should collaborate with the sponsor's safety representative to determine the potential cause of the event. Any additional follow-up information related to the event should be reported to the safety management team as soon as it becomes available.

[0738] The sponsor will ensure compliance with all ICH GCP guidelines and relevant local and national regulatory requirements regarding the reporting of suspected unintended serious adverse events (SUSARs) and the preparation of periodic summary reports. The sponsor will notify investigators of all SUSARs, and investigators are required to comply with local reporting requirements and notify the REC.

[0739] 9.3 Pregnancy

[0740] Following administration of the study treatment, any pregnancy in a female patient or the female partner of a male patient will be followed up until the end of the pregnancy. A completed pregnancy report should be submitted to the Safety Management Team immediately within 24 hours of being notified of the event by telephone, email, or fax. The investigator will follow the patient until the end of the pregnancy and must assess the outcome as soon as possible (but no more than 30 days after delivery or termination). The investigator should notify the Safety Management Team of the pregnancy outcome by submitting a follow-up pregnancy report. For pregnant female patients, the continuation of one or both study treatments will be reviewed on a case-by-case basis by the investigator and the sponsor.

[0741] If the pregnancy outcome meets the criteria for immediate classification as SAE (spontaneous or treated miscarriage, stillbirth, neonatal death, or congenital abnormality), the investigator should also report the event by telephone and by email or fax within 24 hours of becoming aware of it. Any congenital abnormalities detected in the miscarried fetus should be documented.

[0742] 9.4 Administration and medication errors related to study treatment

[0743] Any instances of incorrect SCIB1 administration (exceeding the prescribed dose and timing) and any malfunctions of the PharmaJetStratis® needle-free injection device should be documented. Any overdose (single-day overdose or administration frequency less than prescribed) should be reported to the sponsor immediately. Dosing errors related to the administration of nivolumab in combination with ipilimumab or pembrolizumab during the SCIB1 treatment phase (up to EOT) should be documented and reported to the sponsor. Any adverse events (AEs) or special adverse events (SAEs) related to investigational drug errors should be reported as described in the preceding sections.

[0744] 9.5 Clinical and laboratory assessments and examinations

[0745] No study-specific procedures may be performed before the patient provides written informed consent. Clinical data from procedures performed during routine patient care may be used for screening purposes, provided they are within the specified screening time window. Laboratory values ​​and test results following adverse events (AEs) may be recorded.

[0746] 9.5.1 Serum chemistry, hematology and urine analysis

[0747] Serum chemistry, hematology, and urine samples will be collected according to the schedule detailed in Table 2. These samples should be collected prior to drug administration and will be analyzed by a local laboratory.

[0748] Serum chemistry will include albumin, ALT, alkaline phosphatase, AST, bicarbonate or carbon dioxide, urea, calcium, chloride, creatinine, total bilirubin (and direct bilirubin if clinically indicated), gamma-glutamyl transferase, LDH, non-fasting blood glucose, phosphorus (measured as phosphate), potassium, sodium, uric acid (urate), and total protein.

[0749] Hematology will include hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, RBC count, WBC count, and WBC differential (basophils, eosinophils, lymphocytes, monocytes, neutrophils).

[0750] Urine analysis (test strips) will record protein, glucose, ketone bodies, blood, nitrite, white blood cells, bilirubin, pH, and specific gravity. Microscopic examination is performed only when clinically indicated: bacteria, RBCs, WBCs, casts, and crystals.

[0751] As part of the screening procedure, the serum chemistry, hematology and urine analyses performed within 48 hours after the first dose of study treatment do not need to be repeated on day 1.

[0752] 9.5.2 Other clinical laboratory tests

[0753] HLA typing

[0754] HLA testing will be performed via molecular (DNA) typing in an accredited laboratory. HLA typing will be performed using a separate consent form to limit the number of invasive screening tests on patients who do not qualify for studies based on HLA type. Eligible patients must be HLA-A2 positive and positive for HLA-DR4, HLA-DR7, HLA-DR53, or HLA-DQ6.

[0755] Pregnancy test and confirmation of postmenopausal status

[0756] For women of childbearing potential (including those ≤12 months from 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 result is not confirmatory, a serum pregnancy test will be required. Pregnancy tests (serum and / or urine) will be performed at the time intervals shown in Table 2. Serum or urine pregnancy tests will also be performed during the EOT visit for these patients. The site may choose to use a serum pregnancy test throughout the entire course of treatment.

[0757] Hepatitis test

[0758] Blood samples will be obtained during screening for HBV and HCV testing.

[0759] Coagulation test

[0760] International Normalized Ratio (INR) and Activated Partial Thromboplastin Time (aPTT) will be measured during screening and EOT visits.

[0761] Thyroid function tests

[0762] Samples will be collected according to the schedule detailed in Table 2 for the determination of free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH). Samples should be collected prior to administration. As part of the screening procedure, thyroid function tests performed within 48 hours after the first dose of study treatment do not need to be repeated on day 1. If thyroid function tests are not available at the local laboratory at the study site, they may be performed at an accredited laboratory collaborating with the study site.

[0763] 9.5.3 Medical History

[0764] The patient's medical history will be recorded during screening and will include details of chronic diseases, major past illnesses, all current illnesses, and current concomitant medications.

[0765] 9.5.4 Physical Examination

[0766] A complete physical examination will include assessment of the eyes, nervous and cardiovascular system, lungs, abdomen, head, neck, ears, nose, mouth, larynx, thyroid gland, lymph nodes, extremities, skin, and other areas with signs and symptoms of disease, performed at the time points detailed in Table 2. Height and weight will be recorded at screening; at other time points, only weight will be recorded. A “limited” physical examination (cardiovascular system, lungs, abdomen, skin, weight, and areas with signs and symptoms of disease) may be performed as needed during checkpoint inhibitor visits only, as shown in Table 2. The physical examination should be performed prior to medication administration.

[0767] Skin lesions will be assessed according to the schedule in Table 2. Photographs should be taken to document changes in existing and new skin lesions.

[0768] 9.5.5 Injection Site Reaction

[0769] Assessment of each injection site will be performed approximately one hour after SCIB1 administration. As described in Table 2, each injection site will also be assessed during some study visits where the study drug was not administered. Pain, tenderness, erythema, and swelling will be assessed on a scale of 0 to 3, where 0 is none, 1 is mild, 2 is moderate, and 3 is severe (Appendix C). If any moderate or severe injection site reaction is observed, digital photography of the injection site should be taken at the time points specified in Table 2 or, if necessary, outside of these time points. Anonymous copies of the digital photographs will be transferred to a dedicated database via secure file transfer.

[0770] 9.5.6 Vital Signs

[0771] Vital signs will be measured approximately 10 to 15 minutes before administration, and 5 minutes, 30 minutes, and 1 hour after SCIB1 dose administration. Patients should be observed for at least 1 hour after receiving nivolumab in combination with ipilimumab or pembrolizumab (at least the first two doses). Vital signs will also be measured during EOT visits. Vital signs include temperature, pulse, respiratory rate, and blood pressure.

[0772] 9.5.7 Electrocardiogram

[0773] 12-lead ECGs will be performed at the time points detailed in Table 2. ECG results will be interpreted by an eligible physician. Any clinically significant findings on the screening ECGs will be recorded in the patient's medical history. Clinically significant findings on ECGs during the study period will be recorded as adverse events (AEs).

[0774] 9.5.8 Ophthalmological examination

[0775] Patients need to undergo funduscopy by a healthcare professional trained in funduscopy (such as an ophthalmologist, optometrist, or other medical professional), which is performed during the screening period and, if the patient experiences any visual impairment during the study, as clinically necessary.

[0776] 9.5.9 Physical fitness assessment

[0777] ECOG performance status will be assessed at the time points detailed in Table 2. ECOG performance status criteria are provided in Appendix D. ECOG performance status should be assessed before administration.

[0778] 10. Statistics

[0779] The following is a summary of the statistical analyses planned for the research. Further details will be provided in the Statistical Analysis Plan (SAP).

[0780] 10.1 Sample size and power considerations

[0781] 10.1.1 Importing Subqueues

[0782] Introducing subcohorts were used to determine the safety and tolerability of SCIB1 treatment in patients receiving nivolumab in combination with ipilimumab or pembrolizumab, to a level sufficient to support continued recruitment of subjects in the full study. For each cohort, an introducing subcohort of six patients was planned to identify DLTs. Each cohort would only proceed if no more than one of the six patients in the introducing subcohort developed SCIB1-related DLTs. Patients who withdrew from the study before week 5 (day 36) for reasons other than DLTs in the introducing subcohorts would be replaced. If more than one of the six patients in the introducing subcohort developed SCIB1-related DLTs, the investigator and sponsor would formally review the data and decide whether it was appropriate to recruit an additional six patients for the introducing subcohort at a reduced SCIB1 dose.

[0783] 10.1.2 Cohort 1: Nivolumab in combination with ipilimumab

[0784] Forty-three patients will be enrolled using the Simon optimal two-stage design (Simon 1989). Six of these patients will form an introductory sub-cohort for formal evaluation of DLT in combination therapy.

[0785] Patients who have received at least one dose of SCIB1 and completed at least one post-baseline tumor assessment before week 25 (day 176), or who withdrew from the study due to disease progression or SCIB1 treatment-related toxicities, or who died due to melanoma-related complications or SCIB1-related toxicities, will be considered evaluable for the primary endpoint. Unevaluable patients will be replaced. This sample size is determined based on the Simon two-stage design. The assumption of a 50% ORR in the sample size calculation is not significant, as this is close to the historical observed ORR of nivolumab plus ipilimumab in this group (Lebbé 2019).

[0786] Conversely, an ORR of 70% would be considered a positive signal for further development of SCIB1 in combination with nivolumab and ipilimumab. The null hypothesis for the trial design is an ORR of 50%. In Phase 1, 15 patients will be enrolled, and further recruitment of this cohort will be stopped if 8 or fewer clinical responses (RECIST 1.1 objective response [CR or PR] within 25 weeks after the first dose of SCIB1) are observed. 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 has a Type I error rate of 5% and a power of 80% when the true response rate is 70%.

[0787] 10.1.3 Cohort 2: Pembrolizumab

[0788] The full cohort will enroll a total of 44 evaluable patients (including six patients from the import sub-cohort) using the Simon optimal two-stage design (Simon 1989). Patients deemed evaluable for the primary endpoint will be those who have received at least one dose of SCIB1 and completed at least one post-baseline tumor assessment by week 25 (day 176), or withdrawn from the study due to disease progression or SCIB1 treatment-related toxicities, or die from melanoma-related complications or SCIB1-related toxicities. Unevaluable patients will be replaced.

[0789] The sample size was determined based on Simon's two-phase design. The sample size calculation assumed an ORR of 30%, which is meaningless because it is close to the historical observed ORR of pembrolizumab alone in this group of patients (Robert et al. 2015b). Conversely, an ORR of 55% would be considered a positive signal for further development of combination therapy. The null hypothesis of the trial design is an ORR of 35%. In Phase I, 14 patients will be enrolled, and further recruitment of this cohort will be stopped if 5 or fewer clinical responses occur (RECIST 1.1 objective response [CR or PR] within 25 weeks after the first dose of SCIB1). Recruitment will continue until a Phase I decision is made. If 21 or more responses are observed in 44 patients at the end of the study, the null hypothesis will be rejected. With a true response rate of 55%, this design has a Type I error rate of 5% and a power of 80%.

[0790] If the proportion of patients receiving PD-1 inhibitors in the adjuvant setting is higher than expected, these assumptions will be adjusted to reflect the lower expected response rate (ORR estimated to be less than 10%) of these patients to pembrolizumab alone.

[0791] 10.2 Analysis Set

[0792] 10.2.1 Full Analysis Set

[0793] The FAS will consist of all enrolled patients who have received at least one dose of SCIB1, nivolumab in combination with ipilimumab or pembrolizumab.

[0794] 10.2.2 Security Analysis Set

[0795] SAF will consist of all enrolled patients who have received at least one dose of SCIB1, nivolumab in combination with ipilimumab or pembrolizumab.

[0796] 10.2.3 Compliant Scheme Set

[0797] The PPS will consist of all enrolled patients whose primary endpoint of tumor response is evaluable according to RECIST 1.1 criteria. Patients will be included in the PPS if they meet the following criteria:

[0798] • Has received at least one dose of SCIB1 and completed at least one post-baseline tumor assessment before week 25 (day 176).

[0799] • Death due to melanoma-related complications or SCIB1-related toxicity; or

[0800] • Withdrawal from the study due to disease progression or SCIB1 treatment-related toxicity.

[0801] • No significant protocol deviations were considered to have a serious impact on safety or efficacy outcomes.

[0802] 10.3 Data Processing Conventions

[0803] For all variables, statistical analysis will be performed using only observational data from patients. Missing data imputation is not planned unless otherwise specified in SAP.

[0804] 10.4 Mid-term Analysis

[0805] Safety data will be reviewed once the six-patient introductory subcohort has been enrolled and assessed up to day 35.

[0806] When 15 (nivolumab plus ipilimumab cohort) or 14 (pembrolizumab cohort) evaluable patients complete enrollment and follow-up for response, an interim analysis will be performed at the end of Phase I. If 8 or fewer of the 15 patients in the nivolumab plus ipilimumab cohort or 5 or fewer of the 14 patients in the pembrolizumab cohort respond, the trial will be terminated. Otherwise, the trial will proceed to Phase II, recruiting an additional 28 (nivolumab plus ipilimumab cohort) or 30 (pembrolizumab) patients.

[0807] As part of the interim analysis, when another cohort reaches Phase 1, a feasibility assessment will be conducted for further enrollment in the cohort that did not reach its Phase 1 quota. A Clinical Research Report (CSR) will be compiled when all patients complete study treatment. A CSR appendix will be created when all patients complete post-treatment follow-up.

[0808] 10.5 Study population

[0809] The FAS (Factors Assay) will be used to present baseline and efficacy parameters. Additionally, the PPS (Prognostics Per Parameter) will be used to assess efficacy parameters. The SAF (Safety Assay) will be used to present safety parameters.

[0810] 10.5.1 Patient Management

[0811] For the introductory and complete portions of the study (i.e., all patients), the number of patients screened, enrolled, and in each analysis population will be summarized separately. Additionally, the number of patients who completed / did not complete the study and the main reasons for withdrawal will be presented. Withdrawal will be identified as either withdrawal from treatment only or withdrawal from both treatment and the study (e.g., post-treatment follow-up). The number of patients who completed the study treatment (ten doses of SCIB1) will be summarized.

[0812] 10.5.2 Demographic and Baseline Characteristics

[0813] Demographic variables and baseline characteristics at the time of screening for patients in the import group and all patients in the FAS will be summarized separately.

[0814] The medical history information collected during screening will be included in the patient list.

[0815] 10.6 Efficacy Analysis

[0816] Efficacy analysis will be performed on enrolled patients based on FAS and PPS. No efficacy analysis will be performed at the end of the import sub-cohort.

[0817] 10.6.1 Therapeutic endpoint

[0818] Objective response rate as measured by RECIST 1.1 standard

[0819] The primary efficacy endpoint was based on ORR as measured using RECIST 1.1 criteria. It corresponds to whether a patient achieved PR or CR at any time after treatment initiation. ORR will be summarized for all patients in both FAS and PPS. Each patient will be summarized based on their best treatment response. Patients who were evaluable for the primary endpoint but did not complete at least one post-baseline tumor assessment before week 25 (day 176) were counted as non-responders.

[0820] reaction duration

[0821] Duration of response will be measured from the point at which CR or PR is met (as defined by ORR) until the date of first recorded PD (according to RECIST 1.1) or death from any cause, whichever occurs first. Patients who neither progress nor die will be censored at their last assessment date. Total duration of response will be estimated using the Kaplan-Meier method, and data from all patients in the FAS and PPS will be summarized. Kaplan-Meier survival function estimation curves will be plotted.

[0822] Immune-related ORR as measured by iRECIST criteria

[0823] The reporting method for this endpoint will be similar to that for the primary endpoint (see above), except that the determination of ORR will be based on the use of the iRECIST criteria (Seymour et al. 2017).

[0824] Progression-free survival

[0825] The PFS rate at week 49 (day 344) and week 97 (day 680) was defined as the proportion of patients who had not progressed (according to RECIST 1.1 and iRECIST) or had not started new anticancer therapy or had not died at each time point. PFS for all patients in FAS and PPS will be summarized.

[0826] Patients who neither progressed nor died will be censored based on the date of their last disease assessment. Patients who started new anticancer therapy without reporting disease progression will be censored based on the date of their last disease assessment prior to the start of new therapy. Patients who did not complete any tumor assessments and did not die will be censored based on the date of their first dose of SCIB1. PFS will be estimated using the Kaplan-Meier method.

[0827] Cancer treatment is defined as any systemic treatment for malignant tumors, including melanoma.

[0828] Overall survival rate

[0829] The overall survival (OS) rate at week 49 (day 344) and week 97 (day 680) was defined as the proportion of patients still alive at each time point. OS for all patients in the FAS and PPS will be summarized and estimated using the Kaplan-Meier method.

[0830] 10.6.2 Analysis Methods for Planning

[0831] No statistical tests are planned because there is only one treatment group and no hypothesis testing was set for this study. Where deemed appropriate, 95% confidence intervals and summary statistics will be presented. Further details will be provided in the SAP. For all continuous data endpoints, descriptive statistics will present the following summary statistics: number of non-missing observations, mean, standard deviation, median, lower quartile, upper quartile, minimum, and maximum. Categorical variables will be summarized using proportions (counts and percentages). For time-event variables, the following will be presented: number of events, number of censored observations, median (50%) estimated by Kaplan-Meier. th ), 25 th and 75 th Percentiles, and the estimated event rate at a given time point.

[0832] 10.7 Security Analysis

[0833] Safety reports will be provided for patients in the import group and all patients enrolled in the study based on SAF.

[0834] 10.7.1 Adverse Events

[0835] AEs will be coded using the current version of MedDRA at the start of the study. If an AE occurs on or after the date of the first dose of study treatment (SCIB1), it is classified as a treatment-emergent AE. Any AE occurring before the first dose of study treatment will be classified as a pre-treatment AE. If a patient experiences more than one AE with the same preferred term, that preferred term will be counted only once. It will be assigned the greatest observed severity and the strongest relationship to the study treatment.

[0836] The report will include the number and percentage of patients who experienced treatment-emergent adverse events (TEAEs), the severity of the TEAEs, TEAEs categorized by relationship (showing their relationship to SCIB1, nivolumab combined with ipilimumab, pembrolizumab, or the PharmaJet Stratis® needle-free injection procedure), and a summary of treatment-emergent adverse events (SAEs). Additionally, a summary of TEAEs of grade ≥3 will be presented. SAFs will be presented separately for (i) patients enrolled during the implementation period and (ii) all patients.

[0837] The SAEs and AEs that directly led to the withdrawal from the study will be listed.

[0838] 10.7.2 Laboratory Parameters

[0839] Scheduled visits will be conducted to summarize laboratory parameters (hematology, chemistry, urinalysis, and other laboratory parameters [Section 9.5.2]). Where appropriate, the corresponding CTCAE grading will be calculated based on the reported values. The baseline grading and the worst-case grading after baseline will be summarized.

[0840] Laboratory values ​​that are outside the reference range will be identified in the patient list.

[0841] 10.7.3 Physical Examination

[0842] Physical examination data will be included in the patient list.

[0843] 10.7.4 Vital Signs

[0844] The actual values ​​of vital signs measurements and their changes relative to baseline will be summarized from planned visits (and assessments at each time point within each visit).

[0845] 10.7.5 12-lead electrocardiogram

[0846] 12-lead ECG data will be included in the patient list.

[0847] 10.7.6 Injection Site Reaction

[0848] Injection site reactions will be summarized as planned. Since injection site reactions will be recorded at four sites, the most severe reaction observed at each site will be summarized.

[0849] 10.7.7 Concomitant medications

[0850] Concomitant treatments will be administered using the current version of the World Health Organization (WHO) Drug Dictionary at the start of the study, categorized by their generic name and Anatomical Therapeutic Chemical Classification (ATC) code. The medication will be assigned as either pre-study treatment or concomitant study treatment. If the medication discontinuation date is earlier than the study treatment start date (SCIB1), the medication will be assigned as pre-study treatment. In all other cases, the medication will be assigned as concomitant study treatment.

[0851] Concomitant medications will be summarized separately by ATC classification and generic (preferred) name. Previous medications will be identified in the patient list. If a patient has a separate period of time during which a particular medication was administered, that medication will only be counted once within that specific observation period (i.e., previous or concomitant).

[0852] 10.8 Pharmacodynamic and Biomarker Analysis

[0853] Immune responses will be assessed using ELISpot assays on peripheral blood samples from a limited number of sites. Additional analyses may be performed on subsets of these samples, including CFSE proliferation assays, CTL lysis assays, MHC multimer staining for memory cells, assays to assess epitope diffusion, Luminex assays for cytokine secretion, T cell subset analysis, and liquid biomarker analysis.

[0854] The expression of PD-L1 in tumor samples will be evaluated.

[0855] 10.9 Deviation between the report and the statistical analysis plan

[0856] The handling of deviations from SAP will be described in the final CSR.

[0857] 11 Quality Control and Quality Assurance

[0858] This study will be conducted in accordance with SOPs that comply with ICH and national clinical trial guidelines.

[0859] 12 Legal and ethical obligations

[0860] 12.1 Adhere to good clinical practices

[0861] This study will be conducted in accordance with the ICH GCP guidelines.

[0862] 12.2 Informed Consent

[0863] Researchers are responsible for obtaining written informed consent from patients after fully explaining the purpose, methods, expected benefits, and potential harms of the study. Researchers will also explain to patients that they are free to refuse participation in the study and can withdraw from it at any time without affecting future treatment. A signed informed consent form must be obtained before a patient receives any study treatment or undergoes any study-specific procedures or assessments (procedures and assessments performed for study purposes and not as part of routine medical care). The signed ICF must be the current version and include any relevant information regarding protocol revisions. When the revision to the ICF is substantial and includes changes indicating that study treatment or study participation alters the benefit / risk relationship, informed consent from the patient in the study should be obtained again.

[0864] Before proceeding with further screening procedures, a separate ICF will be signed to allow for the collection of blood samples for HLA histological typing. The HLA ICF can be obtained from a trained healthcare professional designated by the investigator. The signed ICF must be the current version and contain any relevant information regarding protocol revisions.

[0865] An original copy of the signed and dated ICF must be kept in the investigator's file and made available for inspection by the research monitor, sponsor representative, or representative of the relevant national regulatory agency.

[0866] 12.3 Contact your primary care physician

[0867] The ICH GCP guidelines recommend that if a patient has a primary care physician and the patient agrees to inform the primary care physician, the investigator should inform the patient's primary care physician about the patient's participation in the trial.

[0868] 12.4 Research Ethics Committee

[0869] The protocol, the proposed patient information form, and a copy of the ICF will be submitted to REC. Written approval of the protocol and ICF is required before patients are enrolled in the study and before the study treatments are transported to clinical sites.

[0870] Researchers must notify the REC and obtain its approval for all protocol revisions, as well as all revisions to the patient information form and ICF. All SUSARs that may affect patient safety or the progress of the study will be sent to the REC. A copy of the REC notification and confirmation must be provided to the sponsor.

[0871] 12.5 Regulatory Approval

[0872] Research may not commence until regulatory approval is obtained from the relevant national regulatory authorities.

[0873] 12.6 Periodic review of safety and efficacy

[0874] Researchers and sponsors will conduct frequent and regular formal reviews of safety and efficacy data. The review committee will include the investigator, the sponsor's medical monitor, and the sponsor's head of clinical operations or their designee.

[0875] 12.7 Register clinical studies on the trial registry website

[0876] Information regarding the study objectives and design, as well as patient eligibility criteria, will be published on the public clinical trial registry website prior to enrollment.

[0877] 12.8 Patient confidentiality

[0878] To comply with ICH GCP guidelines, investigators and their institutions are required to allow authorized representatives of sponsors, regulatory agencies, and RECs to directly access patients' original medical records on-site to verify trial-related procedures and data.

[0879] Confidentiality measures include: patients will be identified only by study number, patient number, and date of birth in the CRF or other documents submitted to the sponsor. This information will be used for patient identification in the database. Patient names or addresses will not be entered into the CRF or database. The sponsor will not retain materials containing patient names. Patients will be informed of their rights in the ICF.

[0880] 12.9 Data Protection

[0881] All personnel involved in the research will comply with national guidelines on data protection.

[0882] 12.10 Future Use of Stored Specimens

[0883] All biological specimens were collected solely for the research purposes of this clinical study. Blood samples will be destroyed 12 months after the completion of the CSR. Tumor tissue blocks will be returned from the analytical laboratory to the original site using local procedures.

[0884] If a patient withdraws informed consent after sample collection, the sample will still be tested, and the results will be used as detailed in the protocol. No further samples will be obtained upon withdrawal of consent.

[0885] 13 Administrative and legal obligations

[0886] 13.1 Researcher Obligations

[0887] All researchers will ensure they comply with ICH GCP and will be provided with training documentation at the start of their research.

[0888] 13.2 Accessing Source Data and Files

[0889] Researchers should allow authorized representatives of sponsors and national regulatory agencies direct access to patients' medical records relevant to the study. As part of the informed consent requirement, patients must be informed that their medical records will be reviewed for this purpose.

[0890] 13.3 Insurance

[0891] Before the trial begins, the sponsor will ensure that adequate insurance is provided for the trial to proceed.

[0892] 13.4 Revision of the Plan and Informed Consent Form

[0893] Unless it addresses a direct safety concern for patients or involves only non-material logistical or administrative changes, no changes to the final approved (signed) protocol may be implemented without prior written approval or favorable comments from the REC and national regulatory authorities (if applicable). The principal investigator and sponsor of each clinical site will sign off on the protocol revisions.

[0894] 13.5 Storage of test documents and data

[0895] Researchers must maintain a comprehensive and centralized archiving system for all trial-related documents, which requires inspection by sponsor representatives and national regulatory agencies.

[0896] 13.6 Availability and Retention of Research Records

[0897] Researchers must retain essential documents in accordance with applicable regulations and based on the future development of the research product, as follows:

[0898] • At least 15 years after the completion or termination of the experiment

[0899] • At least two years after obtaining final approval for listing in the United States or the European Community; or

[0900] • At least two years after the formal termination of clinical development of the investigational product.

[0901] The sponsor will determine the minimum retention period and notify the researchers when the documents can be destroyed.

[0902] Patient records and other source data (including copies of protocols, CRFs, original test results reports, drug dispensing logs, communications, informed consent records, and other documents related to the trial) must be retained for the longest period permitted by the clinical facility.

[0903] No trial records may be destroyed without the prior written consent of the sponsor and the investigator. If an investigator wishes to distribute trial records to another party or move them to another location, the sponsor's written consent must be obtained.

[0904] 13.7 Data Collection

[0905] Data will be collected using a study-specific electronic CRF. Data collected from the CRF will be captured in a clinical data management system that meets the technical requirements described in 21 CFR Part 11 (USA) and other relevant regulatory requirements. Data will be collected at clinical sites by designated and trained personnel. A CRF must be completed for each patient who provides informed consent for the primary study. Patient identification should not be performed from the data provided in the CRF.

[0906] Data processed from other sources (such as central laboratories or bioanalytical laboratories) will be sent to clinical sites, where they will be retained but not transcribed to the CRF unless otherwise specified in the protocol. This data may also be sent electronically to the sponsor (or the data management contract research organization).

[0907] Before being transcribed into the CRF, all patient data must have supporting original source documentation in medical records or equivalent documents. Post-treatment follow-up data also requires source documentation. Data must not be directly recorded on the CRF and treated as source data.

[0908] 13.8 Language

[0909] The CRF should be completed in English. The generic names of accompanying medications should be recorded in the CRF whenever possible. All written materials for patient use must be in a language that the patient can read and understand.

[0910] 13.9 Clinical monitoring, source data verification and auditing

[0911] The responsibilities of the study monitor will be assumed by experienced clinical study monitors. The clinical study monitor will be responsible for ensuring that patients have signed their ICF (Independent Clinical Decision Form) and that the study is conducted in accordance with applicable SOPs, protocols, and other written instructions and regulatory guidelines, and complies with GCP (Good Clinical Practice) guidelines.

[0912] The primary responsibility of a study monitor is to conduct pre-, during-, and post-study visits with participating investigators and clinical field personnel. This is to ensure protocol compliance and that all data is correctly and completely documented and reported. The study monitor must also verify that signed informed consent has been obtained before any study-specific procedures or study treatments are performed. The monitor should also verify that the signed ICF is the current version and complies with any protocol revisions and REC approvals.

[0913] Clinical site monitoring, source data verification, and auditing will be conducted as outlined in the clinical monitoring plan.

[0914] 13.10 Deviation and violation of the plan

[0915] Deviation of plan

[0916] Deviation from the plan refers to any behavior that fails to comply with the procedures, assessments, or timelines specified in the plan.

[0917] The plan violates

[0918] Protocol violation refers to any failure to comply with protocol inclusion / exclusion criteria, or other serious non-compliance with the procedures, assessments, or schedules specified in the protocol, that affects the primary research objective or patient safety. For the purposes of this study, protocol violation will be considered a serious protocol deviation.

[0919] Protocol deviations will be defined in the study monitoring plan and documented in the source documentation by clinical field personnel.

[0920] When a patient violates the reporting protocol, the sponsor will decide whether to withdraw the patient from the study or allow them to continue, subject to written approval from a medical expert. This decision will be based on ensuring patient safety and maintaining the integrity of the study.

[0921] Sponsors will not be permitted to deviate from the protocol's inclusion and exclusion criteria, or any requests for protocol timelines, mandatory procedures, or evaluations. If clinical field personnel become aware that a patient who does not meet the protocol's inclusion and exclusion criteria has been included in the study, they must immediately notify the sponsor or sponsor representative and the REC (if required) of the protocol violation.

[0922] 14 References

[0923] Ascierto PA, McArthur GA, Dréno B, et al. Cobimetinib combined with vemurafenib in advanced BRAF(V600)-mutant melanoma (coBRIM): updated efficacyresults from a randomized, double-blind, phase 3 trial. Lancet Oncol. 2016Sep;17(9):1248–60.

[0924] Durrant LG, Pudney V, Spendlove I, et al. Vaccines as earlytherapeutic interventions for cancer therapy: neutralising theimmunosuppressive tumour environment and increasing T cell avidity may leadto improved responses. Expert Opin Biol Ther. 2010 May;10(5):735–48.

[0925] Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluationcriteria in solid tumours: Revised RECIST guideline (version 1.1). Eur JCancer. 2009;45:228–47.

[0926] EUCAN. Cancer factsheets. Available at: https: / / ecis.jrc.ec.europa.eu / .

[0927] Gershenwald JE, Scolyer RA, Hess KR, et al. Melanoma staging:evidence-based changes in the American Joint Committee on Cancer eighthedition Cancer Staging Manual. CA Cancer J Clin. 2017;67(6):472–492.

[0928] Gubin MM, Zhang X, Schuster H, et al. Checkpoint blockade cancerimmunotherapy targets tumour-specific mutant antigens. Nature. 2014 Nov 27;515(7528):577–81.

[0929] Keilholz U, Punt CJ, Gore M, et al. Dacarbazine, cisplatin, andinterferon-alfa-2b with or without interleukin-2 in metastatic melanoma: arandomized phase III trial (18951) of the European Organisation for Researchand Treatment of Cancer Melanoma Group. J Clin Oncol. 2005 Sep 20;23(27):6747–55.

[0930] Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined nivolumaband ipilimumab or monotherapy in untreated melanoma. N Engl J Med. 2015 Jul2;373(1):23–34.

[0931] Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Overall survivalresults from a phase III trial of nivolumab combined with ipilimumab intreatment-naive patients with advanced melanoma (CheckMate 067). 2017 AACRAnnual Meeting. Abstract CT075. Presented April 3, 2017.

[0932] Lebbé C, Meyer N, Mortier L, et al. Evaluation of two dosing regimensfor nivolumab in combination with ipilimumab in patients with advancedmelanoma: results from the phase IIIb / IV CheckMate 511 trial. J Cln Oncol2019;37:867–875.

[0933] Long GV, Stroyakovskiy D, Gogas H, et al. Dabrafenib and trametinibversus 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.

[0934] Massarelli E, William W, Johnson F, et al. Combining immunecheckpoint blockade and tumor-specific vaccine for patients with incurablehuman papillomavirus 16-related cancer. A Phase 2 clinical trial. JAMAOncology 2019;5:67–73.

[0935] Metheringham RL, Pudney VA, Gunn B, et al. Antibodies designed aseffective cancer vaccines. MAbs. 2009 Jan-Feb;1(1):71–85.

[0936] Oken MM, Creech RH, Tormey DC, et al. Toxicity and response criteriaof the Eastern Cooperative Oncology Group. Am J Clin Oncol. 1982;5:649–655.

[0937] Patel PM, Ottensmeier CH, Mulatero C, et al. Targeting gp100 and TRP-2 with a DNA vaccine: Incorporating T cell epitopes with a human IgG1antibody induces potent T cell responses that are associated with favourableclinical outcome in a phase I / II trial. Oncoimmunology. 2018;7(6):e1433516.

[0938] 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.

[0939] Ribas A, Hamid O, Daud A, et al. Association of pembrolizumab withtumor response and survival among patients with advanced melanoma. JAMA. 2016Apr 19;315(15):1600–9.

[0940] Robert C, Ribas A, Wolchok JD, et al. Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: arandomised dose-comparison cohort of a phase 1 trial. Lancet. 2014 Sep 20;384(9948):1109–17.

[0941] Robert C, Long GV, Brady B, et al. Nivolumab in previously untreatedmelanoma without BRAF mutation. N Engl J Med. 2015a Jan 22;372(4):320–30.

[0942] Robert C, Schachter J, Long GV, et al. Pembrolizumab versusipilimumab in advanced melanoma. N Engl J Med. 2015b Jun 25;372(26):2521–32.

[0943] Seymour L, Bogaerts J, Perrone A, et al. iRECIST: guidelines forresponse criteria for use in trials testing immunotherapeutics. Lancet Oncol.2017 Mar;18(3):e143–e152.

[0944] Simon R. Optimal two-stage designs for Phase II clinical trials.Controlled Clinical Trials. 1989; 10:1–10.

[0945] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer JClin. 2016 Jan-Feb;66(1):7–30.

[0946] 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.

[0947] Xue W, Brentville VA, Symonds P, et al. SCIB1, a huIgG1 antibody DNAvaccination, combined with PD-1 blockade induced efficient therapy of poorlyimmunogenic tumors. Oncotarget. 2016 Dec 13;7(50):83088–83100.

[0948] Example 4 – Clinical Study Results

[0949] This example illustrates the results of a clinical study conducted essentially according to Example 3.

[0950] introduction

[0951] Immunotherapy using checkpoint inhibitors (CPIs), including monotherapy with programmed death-1 (PD-1) inhibitors and in combination with cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitors, has altered first-line treatment outcomes for patients with unresectable or metastatic advanced melanoma. 1,2 However, at 5 years, the overall survival rate was 52%. 1 This means that approximately half of the patients did not derive a meaningful benefit from the treatment. Furthermore, in the pivotal Checkmate-067 trial, surrogate survival endpoints such as overall response rate (ORR) and 3-year progression-free survival (PFS) were 58% and 34%, respectively. 3 In real-world settings, this combination therapy achieved an ORR close to 50%. 4 Furthermore, in real-world environments, some individuals skilled in the art have reported 28% [of something unspecified]. 5Recently, a nucleic acid therapeutic vaccine, in combination with pembrolizumab, has shown efficacy in delaying relapse in the unresectable stage of melanoma. 6 However, in the unresectable stage of the disease, nucleic acid vaccines have not yet shown meaningful effects in reducing tumor burden.

[0952] method

[0953] patient

[0954] In the SCOPE clinical trial, which examined the addition of SCIB1 to standard checkpoint inhibitor regimens, eligible patients had histologically confirmed stage III (unresectable) or IV melanoma and had not received prior systemic therapy for advanced disease, although prior therapy in adjuvant melanoma therapy was permitted. Other eligibility criteria included age at least 18 years; an Eastern Cooperative Oncology Group (ECOG) performance status score of 0 (indicating asymptomatic) or 1 (indicating mild symptoms); measurable disease as assessed by computed tomography or magnetic resonance imaging according to the Recognition of Efficacy in Solid Tumors (RECIST) version 1.1; availability of tissue (archived or most recent biopsy sample) from metastatic or unresectable tumors for PD-L1 status assessment; and a known BRAF V600 mutation status. Patients must also be HLA-A2 positive (HLA type I) and positive for at least one of the following HLA types II: HLA-DR4, HLA-DR7, HLA-DR53, or HLA-DQ6.

[0955] Key exclusion criteria included the presence of active brain metastases, ocular melanoma, mucosal melanoma, or autoimmune disease. Patients who received steroid treatment at doses higher than physiological levels within one week prior to the first dose of SCIB1 were excluded from the study. Patients with BRAF mutation-positive disease and evidence of rapidly progressive disease were also excluded from SCIB1 vaccination.

[0956] Trial design and treatment

[0957] Cohort 1: Nivolumab combined with ipilimumab

[0958] In the first phase (see Figure 5 The first phase will enroll 15 patients (including six from the introductory sub-cohort), and will be discontinued if eight or fewer patients achieve a RECIST 1.1 objective response (CR or PR) within 25 weeks of the first dose of SCIB1. Otherwise, an additional 28 patients will be enrolled in the second phase, for a total of 43 patients. Recruiting will continue until a decision is made on the first phase. Patients in this cohort will receive the following treatments:

[0959] Four intramuscular injections (2 mg SCIB1 each) were administered using the PharmaJet Stratis needle-free injection device at weeks 0, 4, 7, 13, 25, and every 12 weeks thereafter up to week 85, for a total dose of 8 mg of SCIB1 per administration.

[0960] For the first four doses, nivolumab 1 mg / kg was administered intravenously every three weeks (30 minutes) in combination with ipilimumab 3 mg / kg intravenously (30 minutes). This was followed by the second phase, in which nivolumab 240 mg was administered intravenously every two weeks (30 minutes) or nivolumab 480 mg was administered intravenously every four weeks (60 minutes).

[0961] For the monotherapy phase, the first dose of nivolumab should be administered as follows:

[0962] - If using 240 mg every two weeks, administer 3 weeks after the last dose of nivolumab and ipilimumab combination therapy; or

[0963] - If using 480 mg every four weeks, administer 6 weeks after the last dose of nivolumab and ipilimumab combination therapy.

[0964] The first dose of the checkpoint inhibitor is administered one week after the first dose of SCIB1, and the total duration of treatment is 97 weeks or until disease progression or dose-limiting toxicity occurs.

[0965] Cohort 2: Pembrolizumab

[0966] In Phase 1, 14 patients will be enrolled (including six from the introductory sub-cohort). This cohort will be closed if five or fewer patients achieve a RECIST 1.1 objective response [CR or PR] within 25 weeks of the first dose of SCIB1. Otherwise, an additional 30 patients will be enrolled in Phase 2, for a total of 44 patients. Recruitment will continue until a decision on Phase 1 is made. Patients in this cohort will receive the following treatments:

[0967] Four intramuscular injections (2 mg SCIB1 each) were administered using the PharmaJet Stratis needle-free injection device at weeks 0, 4, 7, 13, 25, and every 12 weeks thereafter up to week 85, for a total dose of 8 mg of SCIB1 per administration.

[0968] Administer 200 mg every three weeks or 400 mg every six weeks via intravenous infusion over 30 minutes.

[0969] The total duration of treatment is 97 weeks or until disease progression or dose-limiting toxicity occurs. The first dose of the checkpoint inhibitor is administered 1 week after the first dose of SCIB1.

[0970] This study employed a Simon two-stage design, with a power of 80% and an overall Type I error rate of 5% when the true response rate was 70% (cohort 1) and 55% (cohort 2). The null hypothesis in the trial design was an ORR of 50% (cohort 1) and 30% (cohort 2). The null hypothesis values ​​are comparable to real-world outcomes in patients with unresectable melanoma who received treatment.

[0971] The primary endpoint of this study is the overall response rate, measured according to RECIST 1.1 criteria, and compared with historical data from nivolumab in combination with ipilimumab or pembrolizumab alone in these different patient populations. The secondary endpoint is to determine whether the addition of SCIB1 to nivolumab in combination with ipilimumab or pembrolizumab improves duration of response relative to historical data from nivolumab in combination with ipilimumab or pembrolizumab alone in this patient population. Progression-free survival, defined as the proportion of patients who have not progressed (according to RECIST 1.1 and iRECIST), started new anticancer therapy, or died at each specified time point, will be analyzed, as will overall survival, defined as the proportion of patients still alive at each specified time point. Safety and tolerability data from both cohorts will also be summarized. Furthermore, exploratory endpoints will be assessed using ELISpot and other assays on peripheral blood samples from patients to evaluate the relationship between immune response and clinical outcomes.

[0972] result

[0973] Patients and treatment

[0974] From October 2019 to September 2024, cohort 1 enrolled 17 patients, and cohort 2 enrolled 3 patients. In cohort 1, the baseline characteristics of the patients were 12 males and 5 females, and all 17 patients had metastatic disease in the lungs, liver, and / or brain. Eight patients had Braf mutations, and nine were wild-type. Eight patients had lactate dehydrogenase (LDH) levels above the upper limit of normal, and nine had levels below the upper limit of normal. LDH is an important prognostic factor, and elevated LDH indicates a poor prognosis. Twelve patients were under 65 years old, two were between 65 and 74 years old, and only three were 75 years old or older. In cohort 2, one patient was male and two were female. All three patients had metastatic disease in the lungs or liver. Only one patient had a Braf mutation, and one patient had LDH levels above the upper limit of normal. All three patients in cohort 2 were over 75 years old. The total tumor burden of all patients in both cohorts was greater than 20 cm. See Table 3:

[0975]

[0976] Table 3: Baseline characteristics of patients

[0977] Data were truncated in September 2023. In cohort 1, 11 patients had reached the time point of their first computed tomography or magnetic resonance imaging (MRI) scan at 13 weeks post-SCIB1 vaccination. Six other patients were recently enrolled and therefore had not yet reached their first imaging time point. For efficacy purposes, this article summarizes only patients from cohort 1. Of these 11 patients, 9 (81.8%) showed an overall response rate (ORR) at week 13 as measured by RECIST 1.1. Of these, 5 patients reached week 19, 4 reached week 25, and 2 reached week 37. Responses in these patients were maintained, showing an overall reduction in total tumor burden. Among patients who reached the week 25 diagnostic imaging time point, the greatest overall reduction in total tumor burden (-69% to -94%) was observed, see [link to relevant documentation]. Figure 6 and Figure 7 :

[0978] Waterfall picture ( Figure 6 This data shows the maximum change from baseline in the total reference diameter of target lesions in patients receiving SCIB1 in combination with nivolumab and ipilimumab. The data shown represent all patients assessed by target lesion at baseline and who reached the first imaging time point (week 13). Bars in negative intervals indicate tumor shrinkage, and bars in positive intervals indicate tumor size increase. Horizontal lines represent a 30% decrease or a 20% increase in target lesion tumor burden; these are thresholds for response or progression. Change in tumor burden is defined as the percentage decrease in the total reference diameter of target lesions from baseline to the lowest point (observation up to September 2023). Figure 6 As can be seen, only one patient experienced disease progression, while the total tumor burden shrank in all other 10 patients. Of the 10 patients who showed a response, only one had not yet reached the threshold for partial response at the time of data truncation. However, it is expected that this patient will also become a responder if the tumor continues to shrink at the next scan, further demonstrating the significant therapeutic effect of SCIB1 combined with checkpoint inhibitors.

[0979] Adverse events

[0980] All patients receiving SCIB1 in combination with ipilimumab and nivolumab experienced treatment-related adverse events of any grade (see Table 4). The most common adverse event reported to date was injection site reaction associated with SCIB1. These reactions were mild and resolved over time. Patients with the greatest overall reduction in tumor burden (-94%) also experienced vitiligo, characterized by hyperpigmentation / depigmentation covering >10% of body surface area. This adverse event was still ongoing at the last evaluation and was reported to be causally related to SCIB1.

[0981] According to reports, 16 patients experienced treatment-related adverse events of CTCAE grade 3 or 4 severity. Eight of these events were causally related to combination therapy with checkpoint inhibitors, while only one event was considered related to SCIB1. One patient died of pneumonia during the study, which was not considered to be related to metastatic melanoma or the treatment regimen including SCIB1 in combination with ipilimumab and navomucumab.

[0982] No serious adverse events causally related to SCIB1 were reported (see Table 4). The reported serious adverse events causally related to checkpoint inhibitors are consistent with the product characteristics summary. Adding SCIB1 to checkpoint inhibitors did not show an increase in the toxicity of the checkpoint inhibitors.

[0983]

[0984] Table 4: Summary of Serious Adverse Events (Cohort 1)

[0985]

[0986] discuss

[0987] In this open-label phase II study involving patients in a first-line treatment setting for advanced / metastatic melanoma, the addition of SCIB1 to ipilimumab in combination with nivolumab resulted in a significant overall response rate of 81.8%. Study participants' baseline characteristics were consistent with typical characteristics of patients with advanced / metastatic melanoma and reflected a real-world setting, although patients were selected based on their HLA type. The ORR reported in the pivotal clinical trial was 54-58%. 1,2,3 In contrast, the ORR reported in studies conducted in real-world environments ranged from 28% to 48%. 4,5 No waterfall plot has previously been shown in this treatment setting in any approved combination immunotherapy or currently under development. Figure 6 Spider diagram Figure 7 The ORR data shown in the image represent the degree of tumor shrinkage. Those experienced in the field use RECIST ORR as a surrogate marker of survival because by shrinking the tumor and halting its growth, these patients are expected to have their lives extended. Figure 7This demonstrates the durability of tumor response, which is significant in the field because durability of response is associated with survival, as seen in long-term outcome studies.2,3 When SCIB1 is administered in combination with ipilimumab and nivolumab, it has a meaningful impact on tumor shrinkage and life prolongation in a treatment setting where 50% of patients with melanoma still die even with immune checkpoint inhibitor therapy. It is also hypothesized that SCIB1 can be added to any combination of checkpoint inhibitor therapy, including but not limited to the combination of renalalimab and nivolumab, to achieve similar therapeutic effects as shown when ipilimumab is combined with nivolumab. In conclusion, when SCIB1 is added to ipilimumab and nivolumab and continued after ipilimumab discontinuation or nivolumab discontinuation, it has a profound impact on the overall tumor response rate, to the extent that it can improve overall survival to more than the 52% shown in long-term outcome studies of ipilimumab plus nivolumab. 1 .

[0988] References:

[0989] 1. J.Larkin et al. Five-Year Survival with Combined Nivolumab andIpilimumab in Advanced Melanoma N Engl J Med 2019; 381:1535-1546

[0990] 2. Jedd D. Wolchok et al. Long-Term Outcomes With Nivolumab PlusIpilimumab or Nivolumab Alone Versus Ipilimumab in Patients With AdvancedMelanoma Journal of Clinical Oncology 2022 40:2, 127-137

[0991] 3. Errico, A. CheckMate 067—frontline nivolumab improves PFS alone or in combination with ipilimumab. Nat Rev Clin Oncol 12, 435 (2015).

[0992] 4. P. Serra-Bellver et al. Real-world outcomes with ipilimumab andnivolumab in advanced melanoma: a multicentre retrospective study EuropeanJournal of Cancer 176 (2022) 121e132

[0993] 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 JClin Oncol. 2019 Feb;15(1):26-30

[0994] 6. LJ Albrecht et al., The Latest Option: Nivolumab and Relatlimab in Advanced Melanoma. Curr Oncol Rep 25, 647–657 (2023).

[0995] Example 5 – Evidence of checkpoint inhibition efficacy in a preclinical mouse melanoma model

[0996] Based on existing literature, mouse B16F1 melanoma is known to be unresponsive to checkpoint therapy. C57Bl / 6 mice were subcutaneously injected with 2.5 x 10⁻⁶ ppm of the drug. 4 B16F1 melanoma cells were implanted, and then treated intraperitoneally on days 8, 15, and 22 post-tumor implantation with either 100 µg of anti-mouse CTLA4 antibody (clone 9D9) or 250 µg of anti-mouse PD1 antibody (clone RMP1-14) and anti-CTLA4 antibody. Tumor growth was monitored, and mice were sacrificed when the tumor reached a diameter of 15 mm. Neither checkpoint antibody therapy showed any significant effect on tumor growth or survival. Figure 8 ).

[0997] Nevertheless, some tumor models are known to respond to checkpoint therapy. The B16F1 melanoma cell line has been engineered to express human HLA alleles (HLA-DR4 or HLA-DP4) for use in human HLA transgenic mouse models (Brentville et al. 2016, Cancer Research 76:548-60; Brentville et al. 2019, Oncoimmunology 8:e1576490). HLA-DR4 mice (Taconic model 4149) have a C57Bl / 6 background, but the mouse MHC class II allele has been replaced with human HLA-DR4. For this model, the B16F1 cell line has been engineered to express human HLA-DR4 (B16DR4). HHDII / DP4 mice (HLA-A2.1+ / + HLADP4+ / + hCD4+ / +, EM:02221, European Mouse Mutant Archive) also have a C57Bl / 6 background, in which the mouse class I allele has been replaced with chimeric human HLA-A2 (HHDII), and the class II allele has been replaced with HLA-DP4. For this model, B16F1 tumor cells were engineered to not express mouse MHC class I or II, but to express human HLA-A2 and HLA-DP4 (B16 HHDII / DP4). B16 DR4 or B16 HHDII / DP4 melanoma cells were implanted into HLA-DR4 or HHDII / DP4 transgenic mice, respectively. HLA-DR4 transgenic mice were subcutaneously injected with 2.5 x 10⁻⁶ mcg cells. 4 B16 DR4 cells were then intraperitoneally treated on days 8 and 11, or days 4 and 11, with 250 µg anti-PD1 (clone RMP1-14), 250 µg anti-PD1 and anti-PDL1 (clone 10F.9G2), or 250 µg anti-PD1, anti-PDL1, and 100 µg anti-CTLA4 (clone 9D9) antibodies. All checkpoint antibody combinations showed significantly improved survival rates compared to the control group. Figure 9 A).

[0998] Subcutaneous injection of 1x10 into HLA-DP4 transgenic mice 5 B16 HHDII / DP4 cells were administered intraperitoneally on days 10, 14, and 17 with 250 µg anti-PD1 (clone RMP1-14), and on days 8, 15, and 22 with 250 µg anti-PD1 and 100 µg anti-CTLA4 (clone 9D9) or 100 µg anti-CTLA4 antibody. Anti-PD1 antibody administered at later time points showed no effect on tumor growth or survival. Figure 9B), but anti-CTLA4 or anti-PD1 and anti-CTLA4 in combination showed evidence of tumor treatment efficacy in this HLA-DP4 transgenic mouse model, with a survival rate of 100% at day 30. Figure 9 C).

[0999] These data demonstrate the efficacy of checkpoint therapy in mouse models where the expression of certain HLA alleles makes them more immunogenic.

[1000] References

[1001] 1.Brentville VA, Metheringham RL, Gunn B, et al. Citrullinatedvimentin presented on MHC-II

[1002] in tumor cells is a target for CD4+ T-cell-mediatedantitumorimmunity. Cancer Res 2016;76:548–60.

[1003] 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-DRSE alleles; targeting of citrullinated self-peptides presented by HLA-DP4 fortumour therapy. Oncoimmunology 2019;8:e1576490.

[1004] Example 6 – iSCIB1+ DNA vaccine stimulates immune response in mouse model

[1005] The immunogenicity of the iSCIB1+ DNA vaccine in C57Bl / 6 mice was evaluated. C57Bl / 6 mice responded to the TRP-2 aa180-188 sequence, present in both SCIB1 and iSCIB1+, a response restricted by mouse H-2Kb (MHC class I) alleles. However, iSCIB1+ also includes three sequences not present in SCIB1 (TRP-2 aa60-91 inserted into CDR L3; TRP-2 aa177-205 inserted into CDR L2; and gp100 aa471-492 inserted into CDR H3). When C57Bl / 6 mice were immunized with 1 µg iSCIB1+ using a gene gun device on days 1, 8, and 15, and T cell responses to specific peptides were monitored by IFNγ ELISpot assay, high-frequency responses were observed against TRP-2 aa180-188 and TRP-2 aa178-198 peptides (containing the aa180-188 sequence), while response frequencies were lower for peptides covering the other two sequences. Figure 10 A).

[1006] The response observed in C57Bl / 6 mice was restricted by mouse MHC alleles. To confirm that iSCIB1+ induces an immune response restricted by human HLA alleles, HLA-DR4 transgenic mice were immunized with a 1 µg dose of iSCIB1+ using a gene gun device on days 1, 8, and 15, and T-cell responses to specific peptides were monitored by IFNγ ELISpot assay. HLA-DR4 mice had a C57Bl / 6 background, but the mouse MHC class II allele had been replaced with human HLA-DR4; therefore, they still possessed the mouse H-2Kb (MHC class I) allele that binds the TRP-2 aa180-188 peptide.

[1007] A strong response was observed to the TRP-2 aa180-188 and aa178-198 peptides. Furthermore, a strong response was also observed to the HLA-DR4-restricted gp100 aa44-59 peptide inserted into the iSCIB1+ plasmid L1 CDR. Figure 10 B).

[1008] Evidence for responses restricted by the human HLA-A2 allele was provided by immune responses detected in HHDII / DR1 mice, in which the class I allele was replaced with chimeric human HLA-A2 (HHDII) and the class II allele was replaced with HLA-DR1. Immunization of HHDII / DR1 mice and monitoring of T-cell responses to specific peptides by IFNγ ELISpot assay showed strong immune responses to the TRP-2 aa180-188 and TRP-2 aa178-198 peptides. Furthermore, lower responses were also observed to peptides within the TRP-2 aa60-91 sequence (L3) and the gp100 aa173-190 sequence (H1). Figure 10 C).

[1009] These data demonstrate the immunogenicity of iSCIB1+ through mouse and human MHC alleles.

[1010] Example 7 – B16F1 tumor model unresponsive to checkpoints and B16 HHDII / DP4 tumor model mediated by checkpoint response after iSCIB1+ DNA vaccination.

[1011] The mouse B16F1 melanoma model is widely used in tumor research for potential melanoma therapies. In C57Bl / 6 mice, it is highly aggressive and typically unresponsive to checkpoint blockade therapy (Example 5). Figure 8 (As demonstrated in [the study]). On day 1, C57Bl / 6 mice were subcutaneously injected with 2.5 x 10 [units of something]. 4 B16F1 tumor cells were then immunized with 1 µg iSCIB1+ DNA using a gene gun device on days 4, 11, and 18. The iSCIB1+ DNA vaccine was effective, resulting in significant survival in treated mice. Figure 11 A).

[1012] Similar effects were observed in HHDII / DP4 transgenic mice, where mice were implanted with 1x10⁻⁶ cells on day 1. 5 B16 HHDII / DP4 cells were used, and then the mice were immunized with 1 µg iSCIB1+ DNA using a gene gun device on days 4, 11, and 18. Significant survival was observed in immunized mice compared to the control group. Figure 11 B).

[1013] These data indicate that an immune response induced by vaccination with iSCIB1+ DNA can mediate tumor treatment in checkpoint-responsive B16 HHDII / DP4 tumor models, and that stimulating a tumor-specific response through vaccination can also mediate treatment in checkpoint-unresponsive B16F1 tumor models. Therefore, vaccination can be effective against tumors unresponsive to checkpoint blockade therapy.

[1014] Example 8 – Checkpoint Blockade Enhances Immune Response and Tumor Therapy of iSCIB1+ DNA Vaccine

[1015] The current standard of care for patients with metastatic melanoma is the use of checkpoint inhibitors, such as a combination of anti-PD-1 and anti-CTLA therapy. The iSCIB1+DNA product was evaluated in C57Bl / 6 mice in combination with two checkpoint inhibitors (anti-PD-1 plus anti-CTLA-4) to reproduce the dual regimen described in the SCIB1-002 Phase II clinical trial in Example 3. Immune responses were assessed by IFNγ ELISpot assay after injection of iSCIB1+DNA via gene gun on days 1, 8, and 15 with the dual checkpoint inhibitors. Cells were harvested on day 22 and further expanded in vitro for 6 days prior to analysis. Anti-PD-1 and anti-CTLA-4 antibodies (clones RMP1-14 and 9D9, respectively) were administered intraperitoneally at doses of 250 µg / mouse and 100 µg / mouse, respectively. Although checkpoint blockade had no significant effect on the dominant immune response against 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 a checkpoint inhibitor. Figure 12 (A and B). Because iSCIB1+ immunization produced a significant survival benefit in the C57Bl / 6 / B16F1 tumor model (Example 7), immunization was delayed after tumor implantation to observe differences between treatment groups. On day 1, C57Bl / 6 mice were implanted with 2.5 x 10⁻⁶ tumor cells. 4 B16F1 tumor cells were collected. Then, on days 8, 15, and 22, the vaccinated groups were immunized with iSCIB1+ alone, dual checkpoint therapy alone (250 µg anti-PD1 and 100 µg anti-CTLA4 antibody), or iSCIB1+ in combination with a checkpoint inhibitor. Compared with the control group, the tumor-free survival rate was significantly improved in the groups immunized with iSCIB1+ alone or with iSCIB1+ in combination with dual checkpoint therapy. Figure 13 Although dual therapy alone showed some improvement in survival compared to the control group, this was not significant. However, iSCIB1+ combined with dual therapy significantly improved survival compared to dual therapy alone (p=0.0265).

[1016] These data provide evidence for the enhanced T-cell response induced by the iSCIB1+ DNA vaccine in the presence of checkpoint inhibitor antibodies, and the additional benefit of the vaccine combined with checkpoint therapy for antitumor efficacy in a mouse melanoma model.

[1017] These data suggest that checkpoint blockade can enhance vaccine-induced responses, and combining vaccines that stimulate de novo responses with checkpoint blockade that modulates the tumor environment and protects T cells from depletion may improve anti-tumor efficacy in clinical studies.

[1018] The present invention will be further described in the following numbered paragraphs.

[1019] 1. A vaccine composition in a method for treating cancer in a subject, comprising one or more nucleic acids encoding a plurality of tumor antigen T-cell epitopes, wherein said T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3), and wherein said method comprises administering said composition in combination with the following substances to said subject:

[1020] (a) Programmed death-1 (PD-1) inhibitors; and

[1021] (b) Cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors.

[1022] 2. A composition comprising a programmed death-1 (PD-1) inhibitor in a method of treating a subject with cancer, wherein the method comprises administering the PD-1 inhibitor in combination with a substance thereof to the subject:

[1023] (a) A vaccine composition comprising one or more nucleic acids encoding multiple tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3); and

[1024] (b) Cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors.

[1025] 3. A composition comprising a cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitor in a method for treating a cancer subject, wherein the method comprises administering the CTLA-4 inhibitor in combination with the following substances to the subject:

[1026] (a) A vaccine composition comprising one or more nucleic acids encoding multiple tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3); and

[1027] (b) Programmed death-1 (PD-1) inhibitors.

[1028] 4. The composition for use according to any one of paragraphs 1 to 3, wherein the cancer is melanoma.

[1029] 5. The composition for use according to paragraph 4, wherein the melanoma is a stage III or IV melanoma.

[1030] 6. The composition for use according to paragraph 4 or paragraph 5, wherein the melanoma is a metastatic melanoma.

[1031] 7. The composition for use according to any one of paragraphs 4 to 6, wherein the melanoma is unresectable.

[1032] 8. The composition for use according to any one of paragraphs 1 to 7, wherein the subject has not received prior systemic treatment for advanced melanoma.

[1033] 9. The composition for use according to any one of paragraphs 4 to 8, wherein the melanoma is an unresectable stage III or IV melanoma, and the subject has not received prior systemic treatment for advanced disease.

[1034] 10. A composition for use according to any one of paragraphs 1 to 9, wherein the nucleic acid or each nucleic acid encodes the plurality of tumor antigen T-cell epitopes.

[1035] 11. A composition for use according to any one of paragraphs 1 to 10, wherein the one or more nucleic acids encode an antibody, the antibody comprising a heavy chain and a light chain, and the T cell epitope is inserted into or substituted into the antibody.

[1036] 12. A composition for use according to paragraph 11, wherein the T cell epitope is inserted into or substituted into the heavy chain variable region (VH) and / or the light chain variable region (VL) of the antibody.

[1037] 13. A composition for use according to paragraph 11 or paragraph 12, wherein the T cell epitope is inserted into or substituted into one or more CDRs of the antibody.

[1038] 14. The composition for use according to any one of paragraphs 11 to 13, wherein the antibody comprises:

[1039] (i) T cell epitope GTGRAMLGTHTMEVTVYH in CDR-H1 (SEQ ID NO: 1);

[1040] (ii) T cell epitope SVYDFFVWL in CDR-H2 (SEQ ID NO: 2); and

[1041] (iii) T cell epitope WNRQLYPEWTEAQRLD in CDR-L1 (SEQ ID NO: 3).

[1042] 14. The composition for use according to any one of paragraphs 11 to 13, wherein the antibody comprises:

[1043] (i) T cell epitopes GTGRAMLGTHTMEVTVYH in CDR-H1 and CDR-L3 respectively (SEQ ID NO: 1);

[1044] (ii) T cell epitope SVYDFFVWL in CDR-H2 (SEQ ID NO: 2); and

[1045] (iii) T cell epitopes WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-H3 and CDR-L1 respectively.

[1046] 15. The composition for use according to any one of paragraphs 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).

[1047] 16. The composition for use according to any one of paragraphs 1 to 15, wherein the antibody comprises:

[1048] (i) T cell epitope GTGRAMLGTHTMEVTVYH in CDR-H1 (SEQ ID NO: 1);

[1049] (ii) T cell epitope SVYDFFVWL in CDR-H2 (SEQ ID NO: 2);

[1050] (iii) T cell epitope VPLDCVLYRYGSFSVTLDIVQG in CDR-H3 (SEQ ID NO: 4);

[1051] (iv) T cell epitope WNRQLYPEWTEAQRLD in CDR-L1 (SEQ ID NO: 3);

[1052] (v) T-cell epitopes ANCSVYDFFVWLHYYSVRDTLLGPGRPYR in CDR-L2 (SEQ ID NO: 5); and

[1053] (vi) T cell epitopes QCTEVRADTRPWSGPYILRNQDDRELWPRKFF in CDR-L3 (SEQ ID NO:6).

[1054] 17. A composition for use according to any one of paragraphs 11 to 16, wherein the antibody comprises VH comprising the amino acid sequence provided in SEQ ID NO: 7 and VL comprising the amino acid sequence provided in SEQ ID NO: 8.

[1055] 18. A composition for use according to any one of paragraphs 11 to 16, wherein the antibody comprises VH comprising the amino acid sequence provided in SEQ ID NO: 9 and VL comprising the amino acid sequence provided in SEQ ID NO: 10.

[1056] 19. The composition for use according to any one of paragraphs 11 to 18, wherein the antibody is a human IgG1 antibody.

[1057] 20. A composition for use according to any one of paragraphs 11 to 19, wherein the antibody comprises a heavy chain comprising the amino acid sequence provided in SEQ ID NO: 11 and a light chain comprising the amino acid sequence provided in SEQ ID NO: 12.

[1058] 21. The composition for use according to any one of paragraphs 11 to 19, wherein the antibody comprises a heavy chain comprising the amino acid sequence provided in SEQ ID NO: 13 and a light chain comprising the amino acid sequence provided in SEQ ID NO: 14.

[1059] 22. A composition for use according to any one of paragraphs 11 to 21, wherein the antibody does not present its native conformation when expressed.

[1060] 23. The composition for use according to any one of paragraphs 1 to 22, wherein the one or more nucleic acids comprise one or more nonspecific promoters for promoting the expression of the T cell epitopes.

[1061] 24. The composition according to the use described in paragraph 23, wherein the promoter is a CMV-IE promoter.

[1062] 25. A composition for use according to paragraph 23 or paragraph 24, wherein the promoter comprises the nucleotide sequence provided in SEQ ID NO:15.

[1063] 26. A composition for use according to any one of paragraphs 1 to 25, wherein the antibody comprises a leader sequence for promoting antibody secretion after expression of the one or more nucleic acids.

[1064] 27. The composition for use according to paragraph 26, wherein the leader sequence is at the N-terminus of the heavy chain and / or the N-terminus of the light chain of the antibody.

[1065] 28. A composition for use according to paragraph 26 or 27, wherein the leader sequence comprises the amino acid sequence provided in SEQ ID NO: 16.

[1066] 29. A composition for use according to any one of paragraphs 1 to 28, wherein the one or more nucleic acids are DNA.

[1067] 30. A composition for use according to any one of paragraphs 1 to 29, wherein the one or more nucleic acids are plasmids and / or doggybone vectors.

[1068] 31. A composition for use according to any one of paragraphs 1 to 30, wherein the vaccine composition comprises a single nucleic acid encoding the plurality of tumor antigen T-cell epitopes.

[1069] 32. The composition according to the use described in paragraph 31, wherein the single nucleic acid is a DNA plasmid.

[1070] 33. A composition for use according to paragraph 31 or paragraph 32, wherein the nucleic acid comprises the nucleotide sequence provided in SEQ ID NO: 17.

[1071] 34. A composition for use according to paragraph 31 or paragraph 32, wherein the nucleic acid comprises the nucleotide sequence provided in SEQ ID NO: 18.

[1072] 35. The composition for use according to any one of paragraphs 1 to 34, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

[1073] 36. The composition for use according to any one of paragraphs 1 to 35, wherein the PD-1 inhibitor is nivolumab.

[1074] 37. The composition for use according to any one of paragraphs 1 to 36, wherein the CTLA-4 inhibitor is an anti-CTLA-4 antibody.

[1075] 38. The composition for use according to any one of paragraphs 1 to 37, wherein the CTLA-4 inhibitor is ipilimumab.

[1076] 39. A composition for use according to any one of paragraphs 1 to 38, wherein the vaccine composition is administered at a total amount of nucleic acid in the range of 6-10 mg per dose.

[1077] 40. A composition for use according to any one of paragraphs 1 to 39, wherein the vaccine composition is administered to the subject at a total nucleic acid amount of about 8 mg per dose.

[1078] 41. A composition for use according to any one of paragraphs 1 to 40, wherein each dose of the vaccine composition comprises four injections at a separate injection site.

[1079] 42. A composition for use according to any one of paragraphs 1 to 41, wherein a first dose of the vaccine composition is administered to the subject at week 0, followed by subsequent doses at weeks 4, 7, 13, 25, and every 12 weeks thereafter.

[1080] 43. The composition for use according to any one of paragraphs 1 to 42, wherein a first dose of the vaccine composition is administered to the subject on day 1, and subsequent doses are administered on days 29, 50, 92, 176 and every 84 days thereafter.

[1081] 44. A composition for use according to any one of paragraphs 1 to 43, wherein the vaccine composition is administered by needle-free injection.

[1082] 45. A composition for use according to any one of paragraphs 1 to 44, wherein the vaccine composition is administered intramuscularly.

[1083] 46. ​​A composition for use according to any one of paragraphs 1 to 45, wherein the vaccine composition comprises a concentration of about 4 mg / mL of nucleic acid.

[1084] 47. A composition for use according to any one of paragraphs 1 to 46, wherein the vaccine composition comprises about 8.1 mM of disodium hydrogen phosphate, about 1.5 mM of potassium dihydrogen phosphate, about 2.7 mM of potassium chloride and about 137 mM of sodium chloride, and wherein the pH of the vaccine composition is about 7.4.

[1085] 48. The composition for use according to any one of paragraphs 1 to 47, wherein in a first phase, the PD-1 inhibitor is administered to the subject at four doses of about 1 mg / kg every three weeks, followed by administration in a second phase:

[1086] (i) a dose of approximately 240 mg every two weeks, wherein the first dose of the second phase is administered approximately three weeks after the fourth dose of the first phase; or

[1087] (ii) A dose of approximately 480 mg every four weeks, wherein the first dose of the second phase is administered approximately six weeks after the fourth dose of the first phase.

[1088] 49. The composition for use according to any one of paragraphs 1 to 48, wherein the CTLA-4 inhibitor is administered to the subject at four doses of about 3 mg / kg every three weeks.

[1089] 50. The composition for use according to any one of paragraphs 1 to 49, wherein each dose of the CTLA-4 inhibitor and the PD-1 inhibitor are administered on the same day.

[1090] 51. A composition for use according to any one of paragraphs 1 to 50, wherein the CTLA-4 inhibitor and the PD-1 inhibitor are administered as separate compositions or as a single composition.

[1091] 52. The composition according to the use described in paragraph 49 of claim 48, wherein each dose of the CTLA-4 inhibitor is administered on the same day as each of the four doses of the PD-1 inhibitor of the first phase.

[1092] 53. The composition for use according to any one of paragraphs 1 to 52, wherein the first dose of the CTLA-4 inhibitor and the first dose of the PD-1 inhibitor are administered about one week after the first dose of the vaccine composition.

[1093] 54. The composition for use according to any one of paragraphs 1 to 53, wherein the CTLA-4 inhibitor and the PD-1 inhibitor are administered by intravenous infusion.

[1094] 55. The composition for use according to any one of paragraphs 1 to 54, wherein the object is a person who is at least 18 years old.

[1095] 56. The composition for use according to any one of paragraphs 1 to 55, wherein the object has HLA type I HLA serotype HLA-A*02.

[1096] 57. The composition for use according to any one of paragraphs 1 to 56, wherein the object has a class II HLA serotype selected from HLA-DR4, HLA-DR7, HLA-DR53 and HLA-DQ6.

[1097] 58. The composition for use according to any one of paragraphs 1 to 57, wherein the subject is free from brain metastases, ocular melanoma, mucosal melanoma, or autoimmune diseases.

[1098] 59. A composition for use according to any one of paragraphs 1 to 58, wherein the one or more nucleic acids are vectors, preferably DNA vectors.

[1099] 60. A vaccine composition in a method for treating unresectable stage III or IV melanoma, comprising a DNA molecule encoding an antibody, wherein a plurality of tumor antigen T-cell epitopes are inserted into or substituted into said antibody.

[1100] The antibody comprises: the T-cell epitope GTGRAMLGTHTMEVTVYH inserted or substituted into CDR-H1 (SEQ ID NO: 1), the T-cell epitope SVYDFFVWL inserted or substituted into CDR-H2 (SEQ ID NO: 2), and the T-cell epitope WNRQLYPEWTEAQRLD inserted or substituted into CDR-L1 (SEQ ID NO: 3).

[1101] The first dose of the vaccine composition is administered to the subject at week 0, followed by subsequent doses at weeks 4, 7, 13, 25, and every 12 weeks thereafter, and the vaccine composition is administered intramuscularly at a total DNA amount of approximately 8 mg per dose.

[1102] The vaccine composition is administered in combination with nivolumab and ipilimumab.

[1103] In the first phase, nivolumab was administered to the subjects at four doses of approximately 1 mg / kg every three weeks, followed by administration in the second phase:

[1104] (i) a dose of approximately 240 mg every two weeks, wherein the first dose of the second phase is administered approximately three weeks after the fourth dose of the first phase; or

[1105] (ii) A dose of approximately 480 mg every four weeks, wherein the first dose of the second phase is administered approximately six weeks after the fourth dose of the first phase.

[1106] Ipilimumab was administered to the subjects in four doses of approximately 3 mg / kg every three weeks, with each dose of ipilimumab being administered on the same day as each of the four doses of nivolumab in the first phase.

[1107] The first dose of nivolumab and the first dose of ipilimumab were administered approximately one week after the first dose of the vaccine composition.

[1108] 61. The composition for use according to paragraph 60, further characterized by one or more of the features defined in any one of paragraphs 2 to 59.

Claims

1. A composition in a method for treating cancer, wherein the composition: (i) is a vaccine composition comprising one or more nucleic acids encoding multiple tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3), and wherein the method comprises administering the composition in combination with the following substances to the subject: (a) Programmed death-1 (PD-1) inhibitors; and (b) Cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors; (ii) Containing a programmed death-1 (PD-1) inhibitor, and wherein the method comprises administering the PD-1 inhibitor in combination with the following substances to the subject: (a) A vaccine composition comprising one or more nucleic acids encoding multiple tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise 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) inhibitors; or (iii) Containing a cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitor, wherein the method comprises administering the CTLA-4 inhibitor in combination with the following substances to the subject: (a) A vaccine composition comprising one or more nucleic acids encoding multiple tumor antigen T-cell epitopes, wherein the T-cell epitopes comprise GTGRAMLGTHTMEVTVYH (SEQ ID NO: 1), SVYDFFVWL (SEQ ID NO: 2), and WNRQLYPEWTEAQRLD (SEQ ID NO: 3); and (b) Programmed death-1 (PD-1) inhibitors.

2. The composition for use according to claim 1, wherein the cancer is melanoma, optionally wherein the melanoma is stage III or IV melanoma, metastatic melanoma, and / or unresectable.

3. The composition for use according to any one of the preceding claims, wherein the subject has not received prior systemic treatment for advanced melanoma, optionally wherein the melanoma is an unresectable stage III or IV melanoma, and the subject has not received prior systemic treatment for advanced disease.

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

5. The composition for use according to claim 4, wherein the T-cell epitope is inserted into or substituted into one or more CDRs of the antibody.

6. The composition for use according to claim 4 or claim 5, wherein the antibody comprises: (i) T cell epitope GTGRAMLGTHTMEVTVYH in CDR-H1 (SEQ ID NO: 1); (ii) T cell epitope SVYDFFVWL in CDR-H2 (SEQ ID NO: 2); and (iii) T cell epitope WNRQLYPEWTEAQRLD in CDR-L1 (SEQ ID NO: 3). or (i) T cell epitopes GTGRAMLGTHTMEVTVYH in CDR-H1 and CDR-L3 respectively (SEQ ID NO: 1); (ii) T cell epitope SVYDFFVWL in CDR-H2 (SEQ ID NO: 2); and (iii) T cell epitopes WNRQLYPEWTEAQRLD (SEQ ID NO: 3) in CDR-H3 and CDR-L1 respectively.

7. The composition for use according to any one of the preceding claims, wherein the T cell epitopes further comprise VPLDCVLYRYGSFSVTLDIVQG (SEQ ID NO: 4), ANCSVYDFFVWLHYYSVRDTLLGPGRPYR (SEQ ID NO: 5), and QCTEVRADTRPWSGPYILRNQDDRELWPRKFF (SEQ ID NO: 6).

8. The composition according to the use of claim 7 when dependent on any one of claims 4 to 6, wherein the antibody comprises: (i) T cell epitope GTGRAMLGTHTMEVTVYH in CDR-H1 (SEQ ID NO: 1); (ii) T cell epitope SVYDFFVWL in CDR-H2 (SEQ ID NO: 2); (iii) T cell epitope VPLDCVLYRYGSFSVTLDIVQG in CDR-H3 (SEQ ID NO: 4); (iv) T cell epitope WNRQLYPEWTEAQRLD in CDR-L1 (SEQ ID NO: 3); (v) T-cell epitopes ANCSVYDFFVWLHYYSVRDTLLGPGRPYR in CDR-L2 (SEQ ID NO: 5); and (vi) T cell epitopes QCTEVRADTRPWSGPYILRNQDDRELWPRKFF in CDR-L3 (SEQ ID NO: 6).

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

10.

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

14.

11. The composition for use according to any one of the preceding claims, wherein the one or more nucleic acids comprise one or more nonspecific promoters for promoting the expression of the T cell epitopes, optionally wherein the promoter is a CMV-IE promoter, and further optionally wherein the promoter comprises the nucleotide sequence provided in SEQ ID NO:

15.

12. The composition for use according to any one of the preceding claims, wherein the antibody comprises a leader sequence for promoting antibody secretion after expression of the one or more nucleic acids, optionally wherein the leader sequence is at the N-terminus of the heavy chain and / or the N-terminus of the light chain of the antibody, and further optionally wherein the leader sequence comprises the amino acid sequence provided in SEQ ID NO:

16.

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

18.

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

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

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

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

18. The composition for use according to any one of the preceding claims, wherein in a first phase, the PD-1 inhibitor is administered to the subject at four doses of about 1 mg / kg every three weeks, followed by administration in a second phase: (i) a dose of approximately 240 mg every two weeks, wherein the first dose of the second phase is administered approximately three weeks after the fourth dose of the first phase; or (ii) A dose of approximately 480 mg every four weeks, wherein the first dose of the second phase is administered approximately six weeks after the fourth dose of the first phase.

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

20. A composition for use according to any one of the preceding claims, wherein each dose of the CTLA-4 inhibitor and the PD-1 inhibitor are administered on the same day, wherein the CTLA-4 inhibitor and the PD-1 inhibitor are administered in separate compositions or as a single composition, and / or 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.

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

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

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