Combination therapy for treating brain cancer

Abstract

Provided herein are nucleic acid molecules, proteins, compositions, and methods for treating brain cancer in a subject. In some embodiments, the composition comprises cancer antigens hTERT, WT-1, and PSMA. In some embodiments, the composition also comprises an adjuvant. The method comprises administering the cancer antigen to a subject in need thereof. According to certain embodiments, the method further comprises administering an adjuvant and an anti-PD-1 antibody. In certain embodiments, the method further comprises administering radiation therapy and / or a chemotherapeutic agent. In certain embodiments, the method has been clinically proven safe, clinically proven effective, or both.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. applications No. 63 / 070,987 filed on 27 August 2020, No. 63 / 018,060 filed on 30 April 2020, No. 62 / 988,102 filed on 11 March 2020, and No. 62 / 930,417 filed on 4 November 2019. Each of these applications is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes an array listing submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy was created on 4 November 2020, is named 104409_000581_SL.txt, and is 89,861 bytes in size.

[0003] The present invention relates to combination therapy and methods for treating brain cancer. [Background technology]

[0004] Despite advances in therapy, glioblastoma (GBM) remains one of the deadliest cancers. The current standard of care for GBM is surgery followed by radiotherapy (RT), with daily temozolomide (TMZ) chemotherapy during RT, and then, for selected patients, 6 to 12 cycles of maintenance (adjuvant) after the completion of RT [Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005, 352:987-996].

[0005] While checkpoint inhibitors such as programmed cell death-1 (PD-1) inhibitors have shown increased response rates in many cancers, their clinical efficacy in GBM has not yet been demonstrated.

[0006] Therefore, there is a need to identify and develop treatments for GBM to facilitate the clinical management and progression of the disease. Furthermore, more effective treatments are needed to slow disease progression and / or reduce mortality in patients with cancer. [Overview of the Initiative]

[0007] Vaccines for preventing or treating cancer and methods of using them are provided herein. Cancer may be brain cancer, for example, glioblastoma. The vaccine preferably comprises at least three cancer antigens, hTERT, WT-1, and PSMA. In certain embodiments, the vaccine also comprises an adjuvant such as IL-12 and an anti-PD-1 antibody. The method comprises administering the cancer antigens hTERT, WT-1, and PSMA, an adjuvant, and a programmed death receptor-1 (PD-1) checkpoint inhibitor such as an anti-PD-1 antibody to a subject diagnosed with cancer. In some embodiments, the method prevents tumor growth. In some embodiments, the method can reduce tumor growth and / or tumor volume. In some embodiments, the method can prevent metastasis of tumor cells. In some embodiments, the method can increase the cellular immune response in the subject. In some embodiments, the method increases tumor-free survival, progression-free survival, overall survival, or any combination thereof in the subject.

[0008] In certain embodiments, IL-12 is encoded by a DNA plasmid, e.g., INO-9012 or its biosimilar or bioequivalent. In certain embodiments, hTERT, WT-1, and PSMA are encoded by one or more DNA plasmids, e.g., INO-5401 or its biosimilar or bioequivalent. In certain embodiments, the anti-PD-1 antibody is cemiprimab or its biosimilar or bioequivalent. In certain embodiments, the method further comprises administering a radiotherapy and / or chemotherapeutic agent, e.g., temozolomide or its bioequivalent.

[0009] In certain embodiments, the method is either proven clinically safe, proven clinically effective, or both.

[0010] The overview and the following detailed description will be better understood when read in conjunction with the attached drawings. For illustrative purposes, exemplary embodiments of the disclosed method are shown in the drawings; however, the method is not limited to the specific embodiments disclosed. Drawings: [Brief explanation of the drawing]

[0011] [Figure 1] The design of the example is shown.

[0012] [Figure 2] The demographics of the study population in the example are shown.

[0013] [Figure 3]Representative MRI images of two patients showing increased MRI signals suggestive of edema or tumor at the time of initial administration of INO-5401+INO-9012 and cemiprimab-rwlc are shown. Biopsies of several patients showed treatment-related changes with necrosis and mixed inflammation, absence of mitotic activity, and no evidence of viable tumor. The subject represented by the MRI images in the lower panel showed evidence of disease progression at week 9 but regressed at week 21. A subject with similar findings on resected MRI showed only immune infiltration in the absence of viable tumor.

[0014] [Figure 4] ELISpot results support the immunogenicity of the INO-5401 and semiprimab-rwlc combination, which exhibits above-baseline IFN-g magnitudes for all three antigens in 5 out of 11 subjects and at least one antigen in 9 subjects, based on data cutoff data at 12 months.

[0015] [Figure 5A] Figure 5A shows the results of a lysic granule loading assay, indicating the frequency of live, antigen-specific, activated (CD38+) CD3+CD8+ T cells with lysis potential (expressing granzyme A and perforin) obtained at a 12-month data cutoff. Figure 5A shows the frequency of live, antigen-specific, activated (CD38+) CD3+CD8+ T cells from pre-treatment (pre) and post-treatment peaks with INO-5401 and semiprimab-rwlc. Each subject is represented by a white circle, and the bar represents the mean value. The graphs for each antigen, as well as the difference between pre-treatment and the peak (delta), are shown for the eight subjects assayed together (Figure 5B) and the five subjects for whom samples were available up to week 12 (Figure 5C). INO-5401 is the sum of WT1, PSMA, and hTERT. The box plots extend from the 25th to the 75th percentile, with a horizontal line at the median and a "+" at the mean value. [Figure 5B] Same as above. [Figure 5C] Same as above.

[0016] [Figure 6] Figure 1 shows the visual representation of the Kaplan-Meier estimates of the 6-month progression-free survival (PFS6) for cohort A, which consists of patients having an unmethylated O6-methylguanine methyltransferase gene promoter in tumor cells. The curve indicates the probability of an event at regular time intervals. The probability of the event is represented numerically on the y-axis, and the time intervals are represented on the x-axis. The event shown is progression-free survival. Progression-free survival is the absence of disease progression at a given point in time for a given subject.

[0017] [Figure 7] Figure 2 shows the visual representation of the Kaplan-Meier estimates of the 6-month progression-free survival (PFS6) for cohort B, which consists of patients having a methylated O6-methylguanine methyltransferase gene promoter in tumor cells. The curve indicates the probability of an event at regular time intervals. The probability of the event is represented numerically on the y-axis, and the time intervals are represented on the x-axis. The event shown is progression-free survival. Progression-free survival is the absence of disease progression at a given point in time for a given subject.

[0018] [Figure 8] Figure 3 shows the visual representation of the Kaplan-Meier estimates of the 6-month progression-free survival (PFS6) for cohorts A and B, which consist of patients having either an unmethylated or methylated O6-methylguanine methyltransferase gene promoter in tumor cells. The curve indicates the probability of an event at regular time intervals. The probability of the event is represented numerically on the y-axis, and the time intervals are represented on the x-axis. The event shown is progression-free survival. Progression-free survival is the absence of disease progression at a given point in time for a given subject.

[0019] [Figure 9]This shows a visual representation of the Kaplan-Meier estimates of 6-month progression-free survival (PFS6) for Cohort A, Cohort B, and both cohorts combined. The total number of subjects per cohort, the number of events, the estimated number of events (PFS6), and the 95% confidence interval (CI) for which the numerical estimate of events (PFS6) exists are all provided.

[0020] [Figure 10A] This diagram shows a visual representation of Kaplan-Meier estimates of 12-month overall survival for Cohort A, patients with the unmethylated O6-methylguanine methyltransferase gene promoter in tumor cells. The stepped curves represent the probability of surviving to and beyond specific points in time. Survival probabilities are represented numerically on the y-axis, and survival time is represented in days on the x-axis. [Figure 10B] This diagram shows a visual representation of Kaplan-Meier estimates of 18-month overall survival for Cohort A, patients with the unmethylated O6-methylguanine methyltransferase gene promoter in tumor cells. The stepped curves represent the probability of surviving to and beyond specific time points. Survival probabilities are expressed numerically on the y-axis, and survival time is expressed in days on the x-axis. The median follow-up period in Cohort A is 17.8 months. mITT includes any subjects who received one or more study doses of research therapy. Shades represent confidence intervals for the point estimate of survival at that time point.

[0021] [Figure 11A] This diagram shows a visual representation of Kaplan-Meier estimates of 12-month overall survival for Cohort B, patients with the O6-methylguanine methyltransferase gene promoter methylated in tumor cells. The stepped curves represent the probability of surviving to and beyond specific points in time. Survival probabilities are represented numerically on the y-axis, and survival time is represented in days on the x-axis. [Figure 11B]This diagram shows a visual representation of Kaplan-Meier estimates of 18-month overall survival for Cohort B, patients with the O6-methylguanine methyltransferase gene promoter methylated in tumor cells. The stepped curves represent the probability of surviving to and beyond specific points in time. Survival probabilities are expressed numerically on the y-axis, and survival time is expressed in days on the x-axis. The median follow-up period in Cohort B is 15.6 months. Censored, two subjects in Cohort B withdrew their consent for follow-up at week 3. mITT includes any subjects who received one or more study doses of the therapy. Shades represent confidence intervals for the point estimate of survival at that point in time.

[0022] [Figure 12] This diagram shows a visual representation of the Kaplan-Meier estimates of overall survival probability over 12 months for the combined cohorts A and B. The stepped curves represent the probability of surviving up to and beyond a specific point in time. Survival probabilities are represented numerically on the y-axis, and survival time is represented in days on the x-axis.

[0023] [Figure 13] The figures show efficacy data for overall survival at 12 and 18 months for cohort A, cohort B, and combinations. The figures show the total number of subjects who reported survival at 12 and 18 months. The total number of subjects, the estimated event (OS12 or OS18), and the 95% confidence interval (CI) for which the numerical estimate of the event (OS12 or OS18) exists are all provided. The 95% CI is calculated using the Clopper-Pearson exact confidence interval method.

[0024] [Figure 14] The clinical trial protocol from the examples shows all adverse events defined by NCI CTCAE Grade 3. [Figure 15-1] All adverse events defined by the clinical trial protocol from the examples are shown. [Figure 15-2] Same as above.

[0025] [Figure 16A] This report provides ELISpot results for cohorts with an 18-month data cutoff. In Cohort A, 19 out of 22 subjects (86%) tested to date had elevated IFN-g levels for one or more antigens of INO-5401 (Figure 16A). In Cohort B, 16 out of 17 subjects (94%) tested to date had elevated IFN-g levels for one or more antigens of INO-5401 (Figure 16B). Baseline values ​​from the peak time after treatment are plotted. Samples were collected four times at week Q3, followed by week Q12. [Figure 16B] Same as above.

[0026] [Figure 17A] This report presents the results of cohort-based evaluation of peripheral immune responses to INO-5401 using flow cytometry (enlargement of antigen-specific CD8+ T cells with lysis potential) at an 18-month data cutoff. In Cohort A, 13 out of 19 (68%) of subjects tested to date had above-baseline CD38+GrzA+Prf+CD8+ T cell frequencies for one or more antigens with INO-5401 (Figure 17A). In Cohort B, 8 out of 10 (80%) of subjects tested to date had above-baseline CD38+GrzA+Prf+CD8+ T cell frequencies for one or more antigens with INO-5401 (Figure 17B). Baseline values ​​from the peak time after treatment are plotted. Samples were collected four times at week Q3, followed by week Q12. [Figure 17B] Same as above. [Modes for carrying out the invention]

[0027] The disclosed nucleic acid molecules, proteins, vaccines, and methods may be more readily understood by referring to the following detailed description taken in conjunction with the accompanying drawings that form part of this disclosure. It will be understood that the disclosed nucleic acid molecules, proteins, vaccines, and methods are not limited to the specific nucleic acid molecules, proteins, vaccines, and methods described and / or shown herein, and that the terms used herein are for illustrative purposes to illustrate specific embodiments and are not intended to limit the claimed nucleic acid molecules, proteins, vaccines, and methods.

[0028] Unless otherwise specified, any description of possible mechanisms, modes of action, or reasons for improvement is for illustrative purposes only, and the disclosed nucleic acid molecules, proteins, vaccines, and methods should not be limited by the accuracy or inaccuracy of any such proposed mechanism, mode of action, or reasons for improvement.

[0029] Throughout this document, descriptions refer to compositions and methods of using such compositions. Where this disclosure describes or claims features or embodiments relating to a composition, such features or embodiments are equally applicable to methods of using such composition. Similarly, where this disclosure describes or claims features or embodiments relating to methods of using a composition, such features or embodiments are equally applicable to the composition.

[0030] Certain features of the disclosed nucleic acid molecules, proteins, vaccines, and methods, which are described herein in the context of separate embodiments, should be understood, for clarity, to be provided in combination in a single embodiment.

[0031] Conversely, various features of the disclosed nucleic acid molecules, proteins, vaccines, and methods, which are described in the context of a single embodiment, may be provided separately or in any subcombination for the sake of brevity.

[0032] Vaccines for preventing or treating cancer and methods of using them are provided herein. Cancer may be brain cancer, for example, glioblastoma. The vaccine preferably comprises at least three cancer antigens, hTERT, WT-1, and PSMA. In certain embodiments, the vaccine also comprises an adjuvant such as IL-12 and an anti-PD-1 antibody. The method comprises administering the cancer antigens hTERT, WT-1, and PSMA, an adjuvant, and a programmed death receptor-1 (PD-1) checkpoint inhibitor such as an anti-PD-1 antibody to a subject in need. In some embodiments, the method prevents tumor growth. In some embodiments, the method can reduce tumor growth and / or tumor volume. In some embodiments, the method can prevent metastasis of tumor cells. In some embodiments, the method can increase the cellular immune response in the subject. In some embodiments, the method increases tumor-free survival, progression-free survival, overall survival, or any combination thereof in the subject.

[0033] In certain embodiments, IL-12 is encoded by a DNA plasmid, e.g., INO-9012 or its biosimilar or bioequivalent. In certain embodiments, hTERT, WT-1, and PSMA are encoded by one or more DNA plasmids, e.g., INO-5401 or its biosimilar or bioequivalent. In certain embodiments, the anti-PD-1 antibody is cemiprimab or its biosimilar or bioequivalent. In certain embodiments, the method further comprises administering a radiotherapy and / or chemotherapeutic agent, e.g., temozolomide or its bioequivalent.

[0034] In certain embodiments, the method is either proven clinically safe, proven clinically effective, or both.

[0035] Recombinant cancer antigens induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or triggering an immune response directed towards or responsive to the cancer or tumor expressing the antigen. In some embodiments, the induced or triggered immune response may be a cellular immune response, a humoral immune response, or both. In some embodiments, the induced or triggered cellular immune response may include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or induced immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of antigen-expressing tumors or cancers, such as, but not limited to, factors that downregulate MHC presentation; antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In case of any conflict, this specification, including its definitions, shall prevail. Exemplary methods and materials are described below, but similar or equivalent methods and materials may be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are for illustrative purposes only and are not intended to limit the scope of the invention. The terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the invention.

[0037] The terms “comprise,” “include,” “having,” “has,” “can,” and “contain,” and their variations, when used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include multiple references unless otherwise indicated by the context. This disclosure also contemplates other embodiments “including,” “consisting of,” and “essentially consisting of,” the embodiments or elements presented herein, whether expressly described or not.

[0038] In the enumeration of numerical ranges as described herein, each number intervening between them is explicitly intended with the same precision. For example, in the range 6–9, the numbers 6 and 9 are intended, as are 7 and 8, and in the range 6.0–7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0039] Some of the quantitative expressions given herein are not limited by the term “approximately.” Whether the term “approximately” is used expressly or not, all quantities given are intended to refer to actual given values, and are understood to also refer to approximations to such given values ​​that would be reasonably inferred on the basis of the general art of a person skilled in the art, including approximations by experimental and / or measurement conditions to such values.

[0040] As used herein, “adjuvant” means any molecule added to the immunogenic composition described herein to enhance the immunogenicity of the nucleic acid molecules and coding nucleic acid sequences described below.

[0041] A “biosimilar” (of an approved reference product / biological drug, i.e., a reference-listed drug) refers to a biological product that is highly similar to the reference product in terms of safety, purity, and efficacy, and is clinically meaningful, despite minor differences in clinically inactive components, based on data from (a) analytical studies showing that the biological product is highly similar to the reference product despite minor differences in clinically inactive components, (b) animal studies (including toxicity assessments), and / or (c) clinical studies or studies (including immunogenicity and pharmacokinetic or pharmacodynamic assessments) sufficient to demonstrate safety, purity, and efficacy under one or more appropriate conditions of use, where the reference product is approved and intended for use and a license for the biosimilar is required. A biosimilar may be an interchangeable product that can be substituted for the reference product in a pharmacy without intervention from a prescribing healthcare professional. To meet the additional criterion of “interchangeability,” the biosimilar is expected to produce the same clinical outcomes as the reference product in any given patient, and if the biosimilar is administered to an individual more than once, the risk in terms of reduced safety or efficacy of alternating or switching between the use of the biosimilar and the reference product is not greater than the risk of using the reference product without such alternation or switching. The biosimilar utilizes the same mechanism of action for the proposed conditions of use, insofar as the mechanism is known for the reference product. The conditions or conditions of use specified, recommended or proposed on the label to which the biosimilar is proposed have been previously approved for the reference product. The route of administration, dosage form, and / or potency of the biosimilar are the same as those of the reference product, and the biosimilar is manufactured, processed, packaged or stored in a facility that meets standards designed to ensure that the biosimilar remains safe, pure, and potent. The biosimilar may contain minor modifications to the amino acid sequence compared to the reference product, such as N-terminal or C-terminal cleavage, which are not expected to alter the biosimilar performance.

[0042] As used herein, the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains, as well as their polymers (e.g., IgM). Each heavy chain comprises a heavy chain variable region (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (hereinafter abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the antibody (or its antigen-binding portion) may be identical to those of the human germline sequence, or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0043] As used herein, the term “antibody” also includes the antigen-binding fragment of a complete antibody molecule. Terms such as “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, as used herein, include naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from a complete antibody molecule using any suitable standard technique, such as proteolytic digestion techniques or recombinant genetic engineering techniques, which are involved in the manipulation and expression of DNA encoding the antibody variable domain and optionally the antibody constant domain. Such DNA is known and / or readily available, for example, from commercial suppliers, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biological techniques to, for example, position one or more variable domains and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0044] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv(scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide), or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other manipulated molecules such as shark variable IgNAR domains are also included in the expression “antigen-binding fragment” as used herein.

[0045] Antibody antigen-binding fragments typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be positioned relative to each other in any preferred configuration. For example, the variable region may be dimer and may contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, antibody antigen-binding fragments may contain monomeric VH or VL domains.

[0046] In certain embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently bound to at least one constant domain. Non-limiting exemplary stereochemistry of variable and constant domains that may be found within the antigen-binding fragment of the antibody of the present invention includes (i)VH-CH1, (ii)VH-CH2, (iii)VH-CH3, (iv)VH-CH1-CH2, (V)VH-CH1-CH2-CH3, VH-CH2-CH3, (vii)VH-CL, (Viii)VL-CH1, (ix)VL-CH2, (x)VL-CH3, (xi)VL-CH1-CH2, (xii)VL-CH2-CH2-CH3, (xiii)VL-CH2-CH3, and (xiv)VL-CL. In any configuration of the variable domain and constant domain, including any of the exemplary configurations listed above, the variable domain and constant domain may be directly linked to each other or linked by a complete or partial hinge region or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that result in a mobile or semi-mobile linkage between adjacent variable domains and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may include a homodimer or heterodimer (or other polymer) of any of the variable domain configurations and constant domain configurations listed above, in non-covalent bonds (e.g., by disulfide bonds) to each other and / or to one or more monomeric VH or VL domains.

[0047] As used herein, “coding sequence” or “coding nucleic acid” means a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence that codes for a protein. The coding sequence may further include start and terminate signals operably linked to a regulatory element, including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0048] As used herein, “complement” or “complementary” means that nucleic acids may refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairs between nucleotides or between nucleotide analogs of nucleic acid molecules.

[0049] As used herein, "consensus" or "consensus sequence" refers to a polypeptide sequence based on the analysis of alignments of multiple sequences for the same gene from different organisms. Nucleic acid sequences encoding consensus polypeptide sequences can be prepared. A broad immune response to an antigen can be induced using an immunogenic composition containing a protein with a consensus sequence and / or a nucleic acid molecule encoding such a protein.

[0050] As used interchangeably herein, “electroporation,” “electrical permeability,” or “electrokinetic enhancement” (“EP”) refers to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes, which in turn allow biomolecules such as plasmids, oligonucleotides, siRNAs, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0051] As used herein with respect to nucleic acid sequences, “fragment” means a nucleic acid sequence or portion thereof that encodes a polypeptide capable of inducing an immune response in mammals that cross-react with the antigens disclosed herein. A fragment may be a DNA fragment selected from at least one of the various nucleotide sequences encoding the protein fragments shown below. A fragment may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more nucleic acid sequences shown below. In some embodiments, the fragment may contain at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 550 nucleotides, at least 600 nucleotides, at least 650 nucleotides, at least 700 nucleotides, at least 750 nucleotides, at least 800 nucleotides, at least 850 nucleotides, at least 900 nucleotides, at least 950 nucleotides, or at least 1000 nucleotides of at least one nucleic acid sequence as shown below.

[0052] With respect to polypeptide sequences, “fragments” or “immunogenic fragments” means polypeptides capable of inducing an immune response in mammals that cross-react with the antigens disclosed herein. Fragments may be polypeptide fragments selected from at least one of the following diverse amino acid sequences. A consensus protein fragment may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the consensus protein. In some embodiments, the consensus protein fragment may include at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids, at least 110 amino acids, at least 120 amino acids, at least 130 amino acids, at least 140 amino acids, at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, or at least 180 amino acids of the protein sequence disclosed herein.

[0053] As used herein, the term “gene construct” refers to a DNA or RNA molecule containing a nucleotide sequence that codes for a protein. The coding sequence includes start and termination signals operably ligated to a regulatory element including a promoter, as well as polyadenylation signals that can direct the expression of the nucleic acid molecule in the cells of an individual to which it is administered. As used herein, the term “expressible form” refers to a gene construct containing the necessary regulatory elements operably ligated to the coding sequence that codes for a protein, so that the coding sequence is expressed if present in the cells of an individual.

[0054] As used herein, the term “homology” refers to some degree of complementarity. There may be partial homology or complete homology (i.e., identity). Partially complementary sequences that at least partially inhibit a fully complementary sequence from hybridizing to a target nucleic acid are referred to using the functional term “substantially homologous.” When used in relation to double-stranded nucleic acid sequences such as cDNA or genomic clones, the term “substantially homologous” as used herein refers to a probe that can hybridize to a strand of the double-stranded nucleic acid sequence under low stringency conditions. When used in relation to single-stranded nucleic acid sequences, the term “substantially homologous” as used herein refers to a probe that can hybridize to (i.e., is its complement to) a single-stranded nucleic acid template sequence under low stringency conditions.

[0055] As used herein in the context of two or more nucleic acid or polypeptide sequences, “identical” or “sequence” means that the sequences have a certain proportion of residues that are the same across a particular region. The proportion can be calculated by optimally aligning the two sequences, comparing them across the identified region, determining the number of positions where identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the identified region, and multiplying the result by 100 to obtain the proportion of sequence identity. If the two sequences are of different lengths, or if the alignment produces one or more staggered ends and the identified region of comparison contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity can be performed manually or using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0056] As used herein, “substantially complementary” means that the first sequence spans a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids. The complement of the second sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or the two sequences hybridize under stringent hybridization conditions.

[0057] As used herein, “substantially identical” means that the first sequence is substantially complementary to the complement of the second sequence, and the first and second sequences are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270 This means that the nucleic acids are identical by at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0058] The term "therapeutic dose" refers to the therapeutic dose of a biological, compound, or composition that can produce a therapeutic effect in a human subject. A therapeutic dose is the amount that can treat, improve, or prevent a specific disease or condition, or that can produce a detectable therapeutic effect. A therapeutic dose is the amount that results in one or more of the following: (a) a reduction in the severity or duration of symptoms, or signs of cancer, e.g., glioblastoma; (b) inhibition of tumor growth, or an increase in tumor necrosis, tumor shrinkage, and / or tumor disappearance; (c) delay of tumor growth and development; (d) inhibition of tumor metastasis; (e) prevention of recurrence of tumor growth; (f) an increase in the survival rate of a subject with cancer; and / or (g) a reduction in the use or need for conventional anticancer therapy (e.g., reduction or elimination of the use of chemotherapeutic agents or cytotoxic agents) compared to an untreated subject or a subject treated with anticancer therapy as monotherapy. The exact effective dose for a subject depends on the subject's weight, size, and health status, as well as the nature and extent of the subject, and the chosen treatment for administration. The effective therapeutic dose for a given situation can be determined through routine experiments, within the scope of the clinician's skills and judgment.

[0059] As used herein, “therapeutic effect” is the result of any type of medical treatment in which the outcome is deemed desirable and beneficial. This is true whether the outcome is expected, unexpected, or an unintended consequence of the treatment. A therapeutic effect may be an objectively identifiable improvement, such as one noted by a clinician or other qualified observer.

[0060] As used herein with respect to nucleic acids, “variant” means (i) a portion or fragment of a referenced nucleotide sequence, (ii) a complement of a referenced nucleotide sequence or a portion thereof, (iii) a nucleic acid substantially identical to a referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to a referenced nucleic acid, its complement, or a sequence substantially identical thereto. A variant may be a nucleic acid sequence substantially identical over the full length of a complete gene sequence or a fragment thereof. A nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of a gene sequence or a fragment thereof.

[0061] A "variant" of a polypeptide is one that differs in its amino acid sequence from a reference polypeptide in terms of amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity of the reference polypeptide. A variant may also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. A variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or a fragment thereof. An amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0062] As used herein, “vector” means a nucleic acid sequence containing an origin of replication. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector, and in one embodiment, it may be an expression plasmid. A vector may contain, or may contain, one or more heterologous nucleic acid sequences.

[0063] As used herein, “immune response” means the activation of the host’s immune system, such as the mammalian immune system, in response to the introduction of an antigen. The immune response may take the form of a cellular response, a humoral response, or both.

[0064] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” means at least two nucleotides covalently linked to one another. A single-stranded description also defines the sequence of a complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the single-stranded description. Many variants of a nucleic acid can be used for the same purposes as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0065] Nucleic acids can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, RNA, or hybrids of both genome and cDNA, and may contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or by recombinant methods.

[0066] As used herein, “operably linked” means that the expression of a gene is under the control of a promoter to which it is spatially connected. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter originates. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

[0067] As used herein, “peptide,” “protein,” or “polypeptide” may mean a linked sequence of amino acids, which may be natural, synthetic, or a modified or combined form of natural and synthetic amino acids.

[0068] As used herein, “promoter” means a synthetic or naturally occurring molecule that can confer, activate, or enhance the expression of nucleic acids in cells. A promoter may include one or more specific transcriptional regulatory sequences for further enhancing its expression and / or for modifying spatial and / or temporal expression. A promoter may also include distal enhancer or repressor elements that may be located several thousand base pairs away from the transcription start site. Promoters may originate from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively, differentially, or differentially in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers, in relation to cells, tissues or organs where expression occurs, or developmental stages where expression occurs. Typical examples of promotors include the bacteriophage T7 promotor, bacteriophage T3 promotor, SP6 promotor, lac operator-promotor, tac promotor, SV40 late promotor, SV40 early promotor, RSV-LTR promotor, CMV IE promotor, SV40 early promotor, or SV40 late promotor, and CMV IE promotor.

[0069] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to amino acid sequences that can be ligated at the amino terminus of proteins described herein. Signal peptides / leader sequences typically direct the localization of a protein. As used herein, signal peptides / leader sequences can facilitate the secretion of proteins from the cell from which they are produced. Signal peptides / leader sequences are often cleaved from the remainder of the protein and are often referred to as mature proteins upon secretion from the cell. Signal peptides / leader sequences are ligated at the amino terminus (i.e., the N terminus) of a protein.

[0070] As used herein, the expression “subject in need of it” means a human or non-human mammal exhibiting one or more symptoms or signs of brain cancer and / or being diagnosed with brain cancer, including, for example, glioblastoma, and requiring treatment for it. In many embodiments, the term “subject” may be used interchangeably with the term “patient.” For example, a human subject may be diagnosed with primary or metastatic tumor, and / or one or more symptoms or signs including, but not limited to, unexplained weight loss, general weakness, persistent fatigue, loss of appetite, fever, nocturnal sweating, bone pain, shortness of breath, abdominal distension, chest pain / pressure, splenomegaly, and elevated levels of cancer-related biomarkers (e.g., CA125). This expression includes subjects with primary or established tumors. This term includes subjects with primary or metastatic tumors (advanced malignant tumors). For example, this expression includes newly diagnosed subjects. In some embodiments, the expression includes subjects for whom treatment by the disclosed method is initial treatment (e.g., “first-line” treatment in which the patient has not received prior systemic treatment for cancer). In certain embodiments, the expression includes subjects for whom treatment by the disclosed method is a “second-line” treatment, and the patient has previously been treated with “standard treatment” therapies, including but not limited to chemotherapy, surgery, and radiation.

[0071] As used herein, terms such as “treat” and “treating” mean to alleviate symptoms, to temporarily or permanently eliminate the cause of symptoms, to delay or inhibit tumor growth, to reduce tumor cell load or tumor load, to promote tumor regression, to cause tumor shrinkage, necrosis and / or disappearance, to prevent tumor recurrence, to prevent or inhibit metastasis, to inhibit metastatic tumor growth, and / or to increase the survival of the subject.

[0072] As used herein, the phrase “in combination with” means that the cancer antigens hTERT, PSMA, and WT-1 are administered to the subject concurrently with, immediately before, or immediately after the administration of an adjuvant, programmed death receptor-1 (PD-1) checkpoint inhibitor, radiotherapy, and / or chemotherapeutic agent. In certain embodiments, the cancer antigens are administered as a co-formulation with the adjuvant.

[0073] Where used herein, unless otherwise stated, the term “clinically proven” (used independently or to modify terms such as “safe” and / or “effective”) means that it has been proven by a clinical trial that meets the approval criteria of the U.S. Food and Drug Administration, the EMA, or the corresponding national regulatory authority. For example, proof may be provided by a clinical trial described in the examples provided herein.

[0074] The term "clinically proven safety" refers to a dose, administration regimen, treatment, or method of combining cancer antigens such as hTERT, PSMA, and WT1 (e.g., administered as INO-5401 or its biosimilar or bioequivalent), adjuvants such as IL-12 (e.g., administered as INO-9012 or its biosimilar or bioequivalent), and programmed death receptor-1 (PD-1) checkpoint inhibitors such as anti-PD-1 antibodies (e.g., anti-PD-1 antibody REGN2810 or its biosimilar or bioequivalent), with a favorable risk-benefit ratio having an acceptable frequency and / or a favorable severity of adverse events (referred to as AEs or TEAEs) occurring under acceptable treatment conditions, compared to standard treatment or another control drug. Adverse events are inappropriate medical events that occur in a patient receiving a drug. One indicator of safety is the incidence of adverse events (AEs) graded according to the National Cancer Institute (NCI) General Toxicity Criteria for Adverse Events (CTCAE v4.03).

[0075] As used herein in the context of dosage, administration plan, treatment or method, the terms “clinically proven efficacy” and “clinically proven effective” refer to the efficacy of a particular dose, administration or treatment plan. Efficacy can be measured based on changes in the course of the disease in response to the agents of the present invention. For example, a combination of the cancer antigen hTERT, PSMA, WT1, and an adjuvant (e.g., INO-5401 or its biosimilar or bioequivalent combined with INO-9012 or its biosimilar or bioequivalent) and a PD-1 checkpoint inhibitor such as an anti-PD-1 antibody (e.g., anti-PD-1 antibody semiprimab or its biosimilar or bioequivalent) is administered to the patient in an amount and time sufficient to induce improvement, preferably sustained improvement, in at least one index reflecting the severity of the disorder being treated. Various indexes reflecting the extent of the disease, disorder or condition in question may be evaluated to determine whether the amount and time of treatment are sufficient. Such indicators include, for example, clinically recognized indicators of disease severity, symptoms, or manifestation of the disorder in question. The degree of improvement is generally determined by a physician, who may use questionnaires administered to the subject, such as quality of life questionnaires developed for a given disease, and this determination may be made based on signs, symptoms, biopsies, or other test results. For example, a combination of cancer antigens hTERT, PSMA, WT1, and an adjuvant (e.g., INO-5401 or its biosimilar or bioequivalent combined with INO-9012 or its biosimilar or bioequivalent) and an anti-PD-1 antibody (e.g., anti-PD-1 antibody cemiprimab or its biosimilar or bioequivalent) may be administered to achieve improvement in the condition of patients associated with brain cancer such as glioblastoma (GBM). Improvement may be indicated by improvement in indicators of disease activity, improvement in clinical symptoms, or by any other measure of disease activity.

[0076] As used herein, "INO-5401" refers to an immunological composition of three DNA plasmids: a DNA plasmid containing an insert encoding hTERT operably controlled by a promoter, a DNA plasmid containing an insert encoding WT1 operably controlled by a promoter, and a DNA plasmid containing an insert encoding PSMA operably controlled by a promoter.

[0077] As used herein, the term “radiotherapy,” also referred to as “XRT,” generally means the use of ionizing radiation to kill cancer cells as part of anti-cancer therapy. Ionizing radiation is generated using X-rays, gamma rays, or charged particles (e.g., protons or electrons).

[0078] Radiotherapy can be delivered by a machine placed outside the patient's body (external beam radiation therapy), by a source placed inside the patient's body (internal or near-brightness radiation therapy), or via a whole-body radioisotope delivered intravenously or orally (whole-body radioisotope therapy). Radiotherapy can be planned and administered in combination with imaging-based techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) to precisely determine the dose and location of the radiation to be administered. In various embodiments, radiotherapy is selected from the group consisting of whole-body radiotherapy, conventional external beam radiation therapy, stereotactic radiosurgery, stereotactic body radiotherapy, 3D conformal radiotherapy, intensity-modulated radiotherapy, image-guided radiotherapy, tomotherapy, near-brightness radiation therapy, and whole-body radiation therapy. Depending on the intent, in certain embodiments, radiotherapy can be curative, adjunctive, or palliative. In certain embodiments, the term “radiotherapy” refers to low-fractionation radiotherapy. Low-fractionation radiotherapy refers to a radiotherapy schedule in which the total dose of radiation is divided into larger doses and administered at a frequency of once a day or less. Low-fractionation radiotherapy can deliver more radiation per session with a lower dose than standard radiotherapy. In various embodiments, each fraction contains 2 to 20 Gy. For example, a radiation dose of 50 Gy can be divided into 10 fractions, each containing 5 Gy. In certain embodiments, two or more fractions are administered consecutively or on consecutive days. In certain other embodiments, two or more fractions are administered every two days, every three days, every four days, every five days, every six days, every seven days, or in combination thereof.

[0079] According to certain embodiments, methods for treating cancers such as brain cancer (e.g., glioblastoma) in a subject are provided herein. The disclosed methods include administering to a subject an immunogenic composition of the cancer antigen human telomerase reverse transcriptase (hTERT), Wilms tumor-1 (WT-1), and prostate-specific membrane antigen (PSMA), an adjuvant, and an anti-programmed cell death receptor 1 (PD-1) antibody or an antibody-conjugated fragment thereof.

[0080] Disclosed herein are optimized consensus sequences for the cancer antigens hTERT, WT-1, and PSMA. In one embodiment, the antigen encoded by the optimized consensus sequence is capable of inducing an immune response in mammals. In one embodiment, the antigen encoded by the optimized consensus sequence may include an epitope that is particularly effective as an immunogen in which an immune response can be induced.

[0081] In one embodiment, an optimized consensus PSMA is provided that is designed to break tolerance to natural human PSMA. In one embodiment, the human optimized consensus PSMA code sequence is sequence number 11, sequence number 12, sequence number 21, or sequence number 12. 29 As shown below, in one embodiment, the human optimized consensus PSMA coding antigen has the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 28.

[0082] In one embodiment, the optimized consensus WT-1 is designed to break the tolerance of natural human WT-1. In one embodiment, the human optimized consensus WT-1 coding sequence is as described in SEQ ID NO: 15 or SEQ ID NO: 27. In one embodiment, the human optimized consensus WT-1 coding antigen has the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 26.

[0083] In one embodiment, the optimized consensus TERT is designed to break the tolerance of natural human TERT. In one embodiment, the human optimized consensus TERT coding sequence is as described in SEQ ID NO: 17 or SEQ ID NO: 19. In one embodiment, the human optimized consensus TERT coding antigen has the amino acid sequences shown in SEQ ID NO: 18 and SEQ ID NO: 20.

[0084] The disclosed vaccine may further include an adjuvant. In certain embodiments, the disclosed method of treatment further includes administering the adjuvant to a subject. In certain embodiments, the adjuvant is IL12. IL12 may be included in the vaccine in the form of its p35 and p40 subunits. Adjuvant IL12 may be administered to a subject as its p35 and p40 subunits. The IL12 p35 and p40 subunits may be encoded by the same expression vector or separate expression vectors. In one embodiment, the IL12 p35 coding sequence is as shown in SEQ ID NO: 22. In one embodiment, the IL12 p35 subunit has the amino acid sequence shown in SEQ ID NO: 23. In one embodiment, the IL12 p40 coding sequence is as shown in SEQ ID NO: 24. In one embodiment, the IL12 p40 subunit has the amino acid sequence shown in SEQ ID NO: 25.

[0085] Cancer antigens TERT, WT-1, PSMA, and / or adjuvants may be present in the vaccine or administered to a subject as polypeptides, fragments thereof, variants thereof, nucleic acid sequences encoding polypeptides, fragments or variants thereof, or any combination thereof. Cancer antigens may be in any form that induces an immune response in a subject. Nucleic acid sequences may be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. Nucleic acid sequences may also include additional sequences encoding linker or tag sequences linked to the antigen by peptide bonds. Amino acid sequences may be proteins, peptides, variants thereof, fragments thereof, or combinations thereof.

[0086] Cancer antigens TERT, WT-1, PSMA, and / or adjuvants may be included in a vaccine or administered to a subject as polypeptides, fragments thereof, variants thereof, nucleic acid sequences encoding polypeptides, fragments or variants thereof, or any combination thereof. Cancer antigens may be in any form that induces an immune response in a subject. Nucleic acid sequences may be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. Nucleic acid sequences may also include additional sequences encoding linker or tag sequences linked to the antigen by peptide bonds. Amino acid sequences may be proteins, peptides, variants thereof, fragments thereof, or combinations thereof.

[0087] Cancer antigens TERT, WT-1, PSMA, and / or IL-12 may be included in a vaccine or administered as one or more nucleic acid molecules, e.g., an expression vector, but not limited to these. An expression vector may be a circular plasmid or a linear nucleic acid. An expression vector can induce the expression of a specific nucleotide sequence in appropriate target cells. An expression vector may have a promoter operably linked to the nucleotide sequence encoding the antigen, and may be operably linked to a termination signal. An expression vector may also contain sequences necessary for the proper translation of the nucleotide sequence. An expression vector containing the nucleotide sequence of interest may be a chimeric vector, meaning that at least one of its components is heterogeneous to at least one of the other components. Expression of the nucleotide sequence in an expression cassette may be under the control of a constitutive or inductive promoter, which initiates transcription only when the host cell is exposed to certain specific external stimuli. In multicellular organisms, the promoter may also be specific to a particular tissue or organ or developmental stage.

[0088] In one embodiment, the nucleic acid is an RNA molecule. Therefore, in one embodiment, the present invention provides an RNA molecule encoding one or more target polypeptides. The RNA may be a positive-straight strand. Therefore, in some embodiments, the RNA molecule can be translated by a cell without requiring any intervening replication steps such as reverse transcription. RNA molecules useful in the present invention may have a 5' cap (e.g., 7-methylguanosine). This cap can improve in vivo translation of the RNA. The 5' nucleotide of the RNA molecule useful in the present invention may have a 5' triphosphate group. In capped RNA, this may be linked to 7-methylguanosine via a 5'-5' crosslink. The RNA molecule may have a 3' poly-A tail. It may also contain a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. RNA molecules useful in the present invention may be single-stranded. In some embodiments, the RNA molecule is a naked RNA molecule. In one embodiment, the RNA molecule is contained within a vector.

[0089] In one embodiment, the RNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is 0 to 3000 nucleotides long. The lengths of the 5' and 3' UTR sequences appended to the coding region are not limited to these but can be modified by different methods, including designing PCR primers that anneal to different regions of the UTR. Using this approach, those skilled in the art can modify the 5' and 3' UTR lengths necessary to achieve optimal translation efficiency after transfection of the transfected RNA.

[0090] The 5' and 3' UTRs may be naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequence into forward and reverse primers, or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest may be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-enriched elements in 3' UTR sequences can increase RNA stability. Therefore, 3' UTRs can be selected or designed to increase the stability of transcribed RNA based on UTR properties that are well known in the art.

[0091] In one embodiment, the 5'UTR may contain the Kozak sequence of an endogenous gene. Alternatively, if a non-endogenous 5'UTR has been added to the gene of interest by the PCR described above, the consensus Kozak sequence can be redesigned by adding the 5'UTR sequence. While the Kozak sequence can increase the translation efficiency of some RNA transcripts, it does not appear to be necessary to enable efficient transcription for all RNAs. The requirements for Kozak sequences of many RNAs are known in the art. In other embodiments, the 5'UTR may be derived from an RNA virus, whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5'UTR to prevent exonuclease degradation of RNA.

[0092] In one embodiment, the RNA has both a 5' cap and a 3' poly(A) tail, which determines ribosome binding, translation initiation, and RNA stability in cells.

[0093] In one embodiment, the RNA is nucleoside-modified RNA. Nucleoside-modified RNA has certain advantages over unmodified RNA, including, for example, increased stability, low or absent innate immunogenicity, and enhanced translation.

[0094] Expression vectors may transform target cells by being incorporated into the cell genome, or they may be circular plasmids that exist extrachromosomally (e.g., self-replicating plasmids with an origin of replication). The vector may be pVAX, pcDNA3.0, or provax, or any other expression vector that can express antigen-coding DNA and allow cells to translate the sequence into an antigen recognized by the immune system.

[0095] Also provided herein are linear nucleic acid immunological compositions or linear expression cassettes ("LECs") that can be efficiently delivered to a target via electroporation and express one or more desired antigens. An LEC may be any linear DNA lacking any phosphate backbone. The DNA may encode one or more antigens. An LEC may contain promoters, introns, stop codons, and / or polyadenylation signals. Antigen expression may be controlled by the promoter. An LEC may not contain any antibiotic resistance genes and / or phosphate backbone. An LEC may not contain other nucleotide sequences irrelevant to the expression of the desired antigen gene. An LEC may originate from any plasmid that can be linearized. A plasmid may be capable of expressing an antigen. A plasmid may be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid may be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector that can express antigen-coding DNA and allow cells to translate the sequence into an antigen recognized by the immune system. The LEC may be pcrM2. The LEC may be pcrNP. pcrNP and pcrMR may be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

[0096] The vector may contain heterologous nucleic acids encoding the above-mentioned antigens, and may further contain start codons that may be upstream of one or more cancer antigen coding sequences, and stop codons that may be downstream of the coding sequences of the above-mentioned antigens.

[0097] A vector may contain a promoter. The promoter can be any promoter capable of driving gene expression and controlling the expression of an isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription via DNA-dependent RNA polymerase transcribing the antigen sequence described herein. The choice of promoter used to direct the expression of a heterologous nucleic acid depends on the specific application. The promoter may be located at approximately the same distance from the transcription start site in the vector as it is at its native transcription start site. However, variations in this distance can be accommodated without loss of promoter function.

[0098] The start and stop codons may be in-frame with the coding sequence of the antigen described above. The vector may also include a promoter operably ligated to the coding sequence of the antigen described above. The promoter operably ligated to the coding sequence of the antigen described above may be a Simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as a bovine immunodeficiency virus (BIV) long-chain terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukemia virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as a CMV early-stage promoter, an Epstein-Barr virus (EBV) promoter, or a Roussarcoma virus (RSV) promoter. The promoter may also be a human gene-derived promoter such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a tissue-specific promoter such as a natural or synthetic muscle or skin-specific promoter. An example of such a promoter is described in U.S. Patent Application Publication No. 2004 / 0175727, the contents of which are incorporated herein by reference in their entirety.

[0099] The vector may also contain a polyadenylation signal that may be downstream of the coding sequences of the antigen and / or antibody described above. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal derived from the pCEP4 vector (Invitrogen, San Diego, CA).

[0100] The vector may also contain an enhancer upstream of the antigen mentioned above.

[0101] Enhancers may be necessary for expression. Enhancers can be human actin, human myosin, human hemoglobin, human muscle creatine, or viral enhancers such as those derived from CMV, HA, RSV, or EBV.

[0102] The vector may contain enhancers and introns with functional splice donor and acceptor sites. The vector may also contain a transcription termination region downstream of the structural gene to provide efficient termination. The termination region may be derived from the same gene as the promoter sequence or from a different gene.

[0103] The disclosed methods may include the administration of multiple copies of a single nucleic acid molecule, such as a single plasmid, or multiple copies of two or more different nucleic acid molecules, such as two or more different plasmids. For example, the methods may include the administration of two, three, four, five, six, seven, eight, nine, or ten or more different nucleic acid molecules.

[0104] Nucleic acid molecules used in accordance with disclosed methods, such as plasmids, may collectively contain coding sequences for a single antigen or multiple antigens. For example, in one embodiment, the antigen is TERT and multiple antigens selected from one or more additional cancer antigens. In an exemplary embodiment, the antigen is TERT and WT-1. In another exemplary embodiment, the antigen is TERT and PSMA. In an exemplary embodiment, the antigen is PSMA and one or more additional cancer antigens. In an exemplary embodiment, the antigen is PSMA and WT-1. In yet another exemplary embodiment, the antigen is TERT, WT-1, and PSMA.

[0105] The vector may further contain elements or reagents that inhibit its integration into chromosomes. The vector may contain a mammalian origin of replication to maintain the vector outside the chromosome and produce multiple copies of the vector within the cell. The vector may be pVAX1, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which may contain an Epstein-Barr virus origin of replication and a nuclear antigen EBNA-1 coding region, which may produce high-copy episomal replication without integration. The vector may be pVAX1 or a pVax1 variant having changes such as the variant plasmid described herein. The variant pVax1 plasmid is a 2998 base pair variant of the skeletal vector plasmid pVAX1 (Invitrogen, Carlsbad CA). The CMV promoter is located at bases 137–724. The T7 promoter / priming site is located at bases 664–683. The multicloning site is located at bases 696–811.

[0106] The bovine GH polyadenylation signal is located at bases 829-1053. The kanamycin resistance gene is located at bases 1226-2020. The starting point of pUC is at bases 2320-2993.

[0107] Based on the pVAX1 sequences available from Invitrogen, the following mutations were found in the pVAX1 sequences used as the backbone of plasmids 1–6 described herein: C > G241 C>T 1942 skeleton, downstream of bovine growth hormone polyadenylation signal (bGH polyA) A>-2876 skeleton, downstream of the kanamycin gene pUC replication origin (Ori) high copy number mutation C>T 3277 (see Nucleic Acid Research 1985) G>C 3753 at the outermost end of the pUC Ori upstream of the RNASeH site Base pairs 2, 3, and 4 are changed from ACT to CTG in the upstream skeleton of the CMV promoter. The vector skeleton may be pAV0242. The vector may be a replication-deficient adenovirus type 5 (Ad5) vector.

[0108] Vectors may also contain regulatory sequences that may be well-suited for gene expression in mammalian or human cells to which the vector is administered. One or more cancer antigens disclosed herein may contain codons that can enable more efficient transcription of coding sequences in host cells.

[0109] The vector may be pSE420 (Invitrogen, San Diego, Calif.), which can be used for protein production in Escherichia coli (E. coli). The vector may also be pYES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in the yeast strain Saccharomyces cerevisiae. The vector may also be from the MAXBAC® complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The vector may also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells. The vector may be an expression vector or system for producing a protein using routine techniques and readily available starting materials, including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is fully incorporated herein by reference.

[0110] Exemplary DNA plasmids encoding the cancer antigens hTERT, WT-1, and / or PSMA are disclosed in U.S. Application No. 62 / 899,543, filed September 12, 2019, the entire contents of which are disclosed herein by reference.

[0111] According to the disclosed method, a subject may be administered nucleic acid molecules encoding an antigen(s) in an amount of about 5 nanograms to about 20 mg. In some embodiments, a subject may be administered nucleic acid molecules encoding an antigen(s) in an amount of about 5 mg to about 15 mg. In some embodiments, a subject may be administered nucleic acid molecules encoding an antigen(s) in an amount of about 9 mg to about 11 mg. In some embodiments, a subject may be administered nucleic acid molecules encoding an antigen(s) in an amount of about 10 mg.

[0112] DNA plasmids can be delivered via various routes. Typical delivery routes include parenteral administration, such as intradermal, intramuscular, or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. In particular, for vaccine DNA, the vaccine can be delivered into the interstitial space of the individual's tissues (Felgner et al., U.S. Patent Nos. 5,580,859 and 5,703,055, the entire contents of which are incorporated herein by reference). DNA plasmids can also be administered intramuscularly, or percutaneously via intradermal or subcutaneous injection, or by means of ion electrophoresis. Epidermal delivery of DNA plasmids is also possible. Epidermal delivery may involve mechanical or chemical stimulation of the outermost layer of the epidermis to stimulate an immune response to an irritant (Carson et al., U.S. Patent No. 5,679,647, the entire contents of which are incorporated herein by reference).

[0113] DNA plasmids can be liquid preparations such as suspensions, syrups, or elixirs. Vaccines can also be preparations for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions.

[0114] DNA plasmids can be incorporated into liposomes, microspheres, or other polymer matrices (Felgner et al., U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III (2nd ed. 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes may be composed of phospholipids or other lipids and can be non-toxic, physiologically acceptable, and metabolizable carriers that can be constructed and administered relatively simply.

[0115] DNA plasmids can be administered via electroporation, such as the method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. Electroporation may be performed by the methods and / or apparatus described in U.S. Patents 6,302,874, 5,676,646, 6,241,701, 6,233,482, 6,216,034, 6,208,893, 6,192,270, 6,181,964, 6,150,148, 6,120,493, 6,096,020, 6,068,650, and 5,702,359, the contents of which are incorporated herein by reference in their entirety. Electroporation may be performed via minimally invasive devices.

[0116] Minimally invasive electroporation devices ("MIDs") may be devices for injecting the above-mentioned vaccines and associated fluids into body tissues. The device may include a hollow needle, a DNA cassette, and a fluid delivery means, which is adapted to actuate the fluid delivery means in use, simultaneously (e.g., automatically) injecting the DNA into the body tissue while the needle is inserted into the body tissue. This has the advantage that the ability to gradually inject the DNA and associated fluids while the needle is inserted leads to a more uniform distribution of the fluids through the body tissue. The pain experienced during injection may be reduced due to the distribution of the DNA injected over a larger area.

[0117] MID allows for the injection of DNA plasmids into tissue without the use of needles. MID can inject vaccines as small streams or jets with a force that allows the vaccine to penetrate the surface of the tissue and enter the underlying tissue and / or muscle. The force behind the small streams or jets may be provided by a compressed gas, such as carbon dioxide, expanding through a micro-orifice within seconds. Examples of minimally invasive electroporation devices and methods of using them are described in published U.S. Patent Applications 20080234655, U.S. Patents 6,520,950, 7,171,264, 6,208,893, 6,009,347, 6,120,493, 7,245,963, 7,328,064, and 6,763,264, the contents of each of these are incorporated herein by reference.

[0118] MID may include syringes that produce a high-speed jet of fluid that painlessly penetrates tissue. Such needleless syringes are commercially available. Examples of needleless syringes that may be used herein include those described in U.S. Patents 3,805,783, 4,447,223, 5,505,697, and 4,342,310, the contents of which are incorporated herein by reference.

[0119] A desired vaccine in a form suitable for direct or indirect electrical transport can typically be introduced (e.g., injected) into the tissue to be treated using a needleless syringe with sufficient force to cause the vaccine to penetrate the tissue by bringing the tissue surface into contact with the syringe to activate the spraying of the drug. For example, if the tissue to be treated is mucous membrane, skin, or muscle, the drug is projected with sufficient force toward the mucous membrane or skin surface, and the drug penetrates through the stratum corneum into the skin layers, or into the underlying tissues and muscles, respectively.

[0120] Needle-free syringes are suitable for delivering DNA plasmids to all types of tissues, particularly skin and mucous membranes. In some embodiments, needle-free syringes can be used to propel a liquid containing DNA plasmids into the surface and the skin or mucous membrane of the target. Representative examples of various types of tissues that can be treated using the methods of the present invention include the pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lips, throat, lungs, heart, kidneys, muscles, breasts, colon, prostate, thymus, testes, skin, mucous membranes, ovaries, blood vessels, or any combination thereof.

[0121] MID may have needle electrodes for electroporating tissue. For example, by pulsing between multiple pairs of electrodes in multiple electrode arrays set in a rectangular or square pattern, it provides improved results compared to pulsing between pairs of electrodes. For example, disclosed in U.S. Patent No. 5,702,359, titled "Needle Electrodes for Mediated Delivery of Drugs and Genes," is an array of needles in which multiple pairs of needles can be pulsed during a therapeutic procedure. In that application, which is incorporated herein by reference as if fully described, the needles are arranged in a circular array but have connectors and switching devices that enable pulses between opposing pairs of needle electrodes. A pair of needle electrodes may be used to deliver recombinant expression vectors to cells. Such devices and systems are described in U.S. Patent No. 6,763,264, the contents of which are incorporated herein by reference. Alternatively, a single-needle device may be used that enables injection of DNA and electroporation with a single needle similar to a conventional injection needle, applying pulses of a lower voltage than those delivered by currently used devices, and thus reducing the electrical sensation experienced by the patient.

[0122] A MID may include one or more electrode arrays. An array may include two or more needles of the same or different diameters. The needles may be spaced evenly or unevenly. The needles may be 0.005 inches to 0.03 inches, 0.01 inches to 0.025 inches, or 0.015 inches to 0.020 inches in diameter. The needles may be 0.0175 inches in diameter. The needles may be spaced 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm or more.

[0123] A MID (Multi-Injector Device) may consist of a pulse generator and two or more needle syringes that deliver DNA plasmids and electroporation pulses in a single step. The pulse generator can be flexibly programmed with pulses and injection parameters via a flashcard-operated personal computer, as well as enabling comprehensive recording and storage of electroporation and patient data. The pulse generator can deliver pulses of various volts in a short period of time. For example, the pulse generator may deliver three 15-volt pulses with a duration of 100 ms. An example of such a MID is the Elgen 1000 system by Inovio Biomedical Corporation, which is manufactured in the United States. It is described in publication No. 7,328,064, the contents of which are incorporated herein by reference.

[0124] MID may be CELLECTRA® (Inovio Pharmaceuticals, Blue Bell Pa.) devices and systems for facilitating the introduction of macromolecules, such as DNA, into cells of selected tissues in the body or plants. The modular electrode system may include multiple needle electrodes; subcutaneous needles; electrical connectors providing conductive links from a programmable constant-current pulse controller to the multiple needle electrodes; and a power supply. An operator can grasp the multiple needle electrodes, mounted on a support structure, and firmly insert them into selected tissue in the body or plants. The macromolecule is then delivered to the selected tissue via the subcutaneous needles. The programmable constant-current pulse controller is activated, applying constant-current electrical pulses to the multiple needle electrodes. The applied constant-current electrical pulses facilitate the introduction of macromolecules into cells between the multiple electrodes. Cell death due to overheating of cells is minimized by limiting power dissipation within the tissue with the constant-current pulses. CELLECTRA® devices and systems are described in U.S. Patent No. 7,245,963, which is incorporated herein by reference.

[0125] The MID may be the Elgen 1000 system (Inovio Pharmaceuticals). The Elgen 1000 system may include a device providing a hollow needle and a fluid delivery means, the device being adapted to actuate the fluid delivery means in use, and simultaneously (e.g., automatically) injecting the fluid, the DNA plasmid described herein, into the body tissue during insertion of the needle into the body tissue. The advantage is that the fluid can be injected gradually while the needle is inserted, resulting in a more uniform distribution of the fluid through the body tissue. Also, the pain experienced during injection is thought to be reduced because the volume of the injected fluid is distributed over a larger area.

[0126] In addition, automatic fluid injection facilitates the automatic monitoring and registration of the actual amount of fluid injected. This data can be saved by the control unit for documentation purposes, if necessary.

[0127] It will be understood that the injection speed may be linear or nonlinear, and that the injection may be performed after the needle has been inserted through the skin of the target to be treated, and while it is further inserted into the body tissue.

[0128] Suitable tissues into which fluid can be injected by the apparatus of the present invention include tumor tissue, skin, or liver tissue, but muscle tissue may also be used.

[0129] The device further includes needle insertion means for guiding the insertion of the needle into body tissue. The fluid injection rate is controlled by the needle insertion rate. This has the advantage that both the insertion and injection of fluid into the needle can be controlled so that the insertion speed matches the injection speed as desired. This also makes the device easier to operate for the user. If desired, means for automatically inserting the needle into body tissue can be provided.

[0130] The user can choose when to begin injecting the fluid. However, ideally, the injection should begin when the tip of the needle reaches the muscle tissue, and the device may include means for sensing when the needle has been inserted deep enough to begin injecting the fluid. This means that the device can prompt the system to automatically begin injecting the fluid when the needle reaches a desired depth (typically the depth to which muscle tissue begins). The depth to which muscle tissue begins can be a preset needle insertion depth, such as 4 mm, which is considered sufficient for the needle to pass through the skin layer.

[0131] The sensing means may include an ultrasonic probe. The sensing means may include means for sensing changes in impedance or resistance. In this case, such means may be adapted not to record the depth of the needle in the body tissue, but rather to sense changes in impedance or resistance as the needle moves from different types of body tissue to muscle. Any of these alternatives offers relatively accurate and easy operation of the sensing means that may initiate injection. If necessary, the needle insertion depth can be further recorded and used to control the fluid injection so that the volume of fluid to be injected is determined as the needle insertion depth is recorded.

[0132] The device may further include a base for supporting a needle and a housing for receiving the base, the base being movable relative to the housing such that it is housed within the housing when the needle is in a first rearward position relative to the housing and extends from the housing when the needle is in a second forward position within the housing. This is advantageous to the user because the housing can be aligned with the patient's skin and the needle can then be inserted into the patient's skin by moving the housing relative to the base.

[0133] As described above, it is desirable to achieve a controlled fluid injection rate so that the fluid is evenly distributed over the length of the needle as it is inserted into the skin. The fluid delivery means may include a piston drive means adapted to inject fluid at a controlled rate. The piston drive means can be actuated, for example, by a servo motor. However, the piston drive means can also be actuated by the base being moved axially relative to the housing. It will be understood that alternative means for fluid delivery may be provided. For example, a sealed container that can be compressed for fluid delivery at a controlled or uncontrolled rate may be provided instead of a syringe and piston system.

[0134] The above apparatus can be used for any type of injection. However, it is assumed to be particularly useful in the field of electroporation, and therefore it may further include means for applying voltage to the needle. This allows the needle to be used not only for injection but also as an electrode in electroporation. This is particularly advantageous because it means that the electric field is applied to the same area as the injected fluid. Conventionally, electroporation has been problematic in that it is very difficult to precisely align the electrode with the previously injected fluid, so users have tended to inject a larger amount of fluid than required over a larger area and apply the electric field over a larger area to ensure overlap between the injected substance and the electric field. Using the present invention, both the volume of the injected fluid and the size of the applied electric field can be reduced while achieving a good fit between the electric field and the fluid.

[0135] When a target is administered the nucleic acid molecular-coding cancer antigens hTERT, PSMA, and WT-1, the transfected cells express and secrete one or more of the cancer antigens. These secreted proteins or synthetic antigens are recognized as foreign by the immune system, which initiates an immune response that may include antibodies produced against one or more cancer antigens and a T-cell response specific to one or more cancer antigens. In some cases, mammals administered with the immunogenic compositions considered herein have an antigen-stimulated immune system, and when inoculated with one or more cancer antigens disclosed herein, the antigen-stimulated immune system can rapidly eliminate the subsequent cancer antigens disclosed herein, whether by humoral, cellular, or both cellular and humoral immune responses.

[0136] Recombinant cancer antigens induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or triggering an immune response directed towards or responsive to the cancer or tumor expressing the antigen. In some embodiments, the induced or triggered immune response may be a cellular immune response, a humoral immune response, or both. In some embodiments, the induced or triggered cellular immune response may include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or induced immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of antigen-expressing tumors or cancers, such as, but not limited to, factors that downregulate MHC presentation; antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules.

[0137] The disclosed vaccine may further contain an anti-PD-1 antibody. The disclosed therapeutic method may further include administering the anti-PD-1 antibody as a target. According to certain embodiments of the present invention, an anti-PD-1 antibody comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity-determining region (CDR) containing any of the amino acid sequences of the anti-PD-1 antibodies shown in U.S. Patent Publication No. 20150203579, the whole of which is incorporated herein. In certain exemplary embodiments, an anti-PD-1 antibody that may be used in the context of the disclosed method comprises a heavy chain complementarity-determining region (HCDR) of a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 1, and a light chain complementarity-determining region (LCDR) of a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 2. According to one particular embodiment, the anti-PD-1 antibody comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), where HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 6, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 8. In yet another embodiment, the anti-PD-1 antibody comprises an HCVR containing SEQ ID NO: 1 and an LCVR containing SEQ ID NO: 2. In one particular embodiment, the method of the present invention comprises the use of an anti-PD-1 antibody, the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-PD-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 10. An exemplary antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO: 10 is a fully human anti-PD-1 antibody known as REGN2810, also known as semiprimab or semiprimab-rwlc.

[0138] According to certain exemplary embodiments, the methods of the present invention involve the use of REGN2810 or its biosimilar or bioequivalent. As used herein, the term “bioequivalent” refers to an anti-PD-1 antibody or PD-1 binding protein or fragment thereof that, when administered in the same molar amount under similar experimental conditions, whether single or multiple doses, does not show a significant difference from the absorption rate and / or absorption range of REGN2810. In the context of the present invention, this term refers to an antigen-binding protein that binds to PD-1 and does not have a clinically significant difference from REGN2810 in terms of safety, purity, and / or potency.

[0139] According to a particular embodiment of the present invention, the anti-human PD-1 antibody comprises an HCVR having 90%, 95%, 98%, or 99% sequence identity with respect to SEQ ID NO: 1.

[0140] According to a particular embodiment of the present invention, the anti-human PD-1 antibody comprises an LCVR having 90%, 95%, 98%, or 99% sequence identity with respect to SEQ ID NO: 2.

[0141] According to a particular embodiment of the present invention, the anti-human PD-1 antibody comprises an HCVR containing the amino acid sequence of SEQ ID NO: 1 having five or fewer amino acid substitutions. According to a particular embodiment of the present invention, the anti-human PD-1 antibody comprises an LCVR containing the amino acid sequence of SEQ ID NO: 2 having two or fewer amino acid substitutions.

[0142] Sequence identity can be measured by any method known in the art (e.g., GAP, BESTFIT, and BLAST).

[0143] The present invention includes the use of an anti-PD-1 antibody in a method for treating cancer, wherein the anti-PD-1 antibody comprises a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative amino acid substitutions. For example, the present invention includes the use of an anti-PD-1 antibody having an HCVR, LCVR, and / or CDR amino acid sequence having any of the conservative amino acid substitutions, such as 10 or less, 8 or less, 6 or less, or 4 or less, relative to the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0144] The amount of anti-PD-1 antibody administered to a subject according to the disclosed method may be a therapeutically effective dose. As used herein, the term “therapeutically effective dose” of anti-PD-1 antibody means an amount that results in one or more of the following: (a) a reduction in the severity or duration of symptoms, or signs of cancer, e.g., glioblastoma; (b) inhibition of tumor growth, or an increase in tumor necrosis, tumor shrinkage, and / or tumor disappearance; (c) delay of tumor growth and development; (d) inhibition of tumor metastasis; (e) prevention of recurrence of tumor growth; (f) an increase in the survival rate of a subject with cancer; and / or (g) a reduction in the use or need for conventional anticancer therapy (e.g., reduction or elimination of the use of chemotherapeutic agents or cytotoxic agents) compared to an untreated subject or a subject treated with the antibody as monotherapy.

[0145] In the case of anti-PD-1 antibodies or their antigen-binding fragments, the therapeutically effective dose may be approximately 0.05 mg to 600 mg, 1 mg to 500 mg, 10 mg to 450 mg, 50 mg to 400 mg, 75 mg to 350 mg, or 100 mg to 300 mg of the antibody. For example, in various embodiments, the amount of anti-PD-1 antibody can be approximately 0.05 mg, 0.1 mg, 1.0 mg, 1.5 mg, 2.0 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 2 The anti-PD-1 antibody is present in doses of 80 mg, approximately 290 mg, approximately 300 mg, approximately 310 mg, approximately 320 mg, approximately 330 mg, approximately 340 mg, approximately 350 mg, approximately 360 mg, approximately 370 mg, approximately 380 mg, approximately 390 mg, approximately 400 mg, approximately 410 mg, approximately 420 mg, approximately 430 mg, approximately 440 mg, approximately 450 mg, approximately 460 mg, approximately 470 mg, approximately 480 mg, approximately 490 mg, approximately 500 mg, approximately 510 mg, approximately 520 mg, approximately 530 mg, approximately 540 mg, approximately 550 mg, approximately 560 mg, approximately 570 mg, approximately 580 mg, approximately 590 mg, or approximately 600 mg. In one embodiment, 250 mg of anti-PD-1 antibody is administered according to the method of the present invention. In one embodiment, 200 mg of anti-PD-1 antibody is administered according to the method of the present invention. In another embodiment, 350 mg of anti-PD-1 antibody is administered according to the method of the present invention.

[0146] Anti-PD-1 antibodies may be administered to a subject in multiple doses, for example, as part of a specific therapeutic dosing regimen. For example, a therapeutic dosing regimen may include administering one or more doses of anti-PD-1 antibodies to a subject at a frequency of approximately once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every week, once every two weeks, once every three weeks, once every four weeks, once every month, once every two months, once every three months, once every four months, or less.

[0147] In some embodiments, anti-PD-1 antibodies are included in the pharmaceutical composition. The pharmaceutical compositions of the present invention can be formulated with suitable carriers, excipients, and other agents that provide suitable mobility, delivery, resistance, etc. Many suitable formulations can be found in the prescription collection known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Examples of these formulations include powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN®), DNA complexes, anhydrous absorbent pastes, oil-in-water emulsions and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0148] Various delivery systems are known and can be used to administer anti-PD-1 antibodies, such as liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and encapsulation via receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents.

[0149] Anti-PD-1 antibodies can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, for subcutaneous delivery, pen-type delivery devices facilitate application when delivering anti-PD-1 antibodies. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition of the anti-PD-1 antibody. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and easily replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. Disposable pen-type delivery devices do not have replaceable cartridges. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0150] In certain circumstances, anti-PD-1 antibodies can be delivered via a sustained-release system. In one embodiment, a pump may be used. In another embodiment, a polymer material may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, the sustained-release system may be placed near the target, thereby requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, Medical Applications of Controlled Release, supra, vol.2, pp.115-138). Other sustained-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0151] Injectable preparations of anti-PD-1 antibodies may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, as well as intravenous infusion. These injectable preparations may be prepared by known methods. For example, an injectable preparation may be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, glucose-containing isotonic solutions, and other adjuvants, which may be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection solutions thus prepared are preferably filled into appropriate ampoules.

[0152] In certain embodiments, the anti-PD-1 antibody is formulated into a pharmaceutical composition for use in intravenous administration.

[0153] In certain embodiments, the method further includes administering radiotherapy to a subject. In certain embodiments, one or more doses of radiotherapy are administered to the subject at intervals of approximately once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once every week, once every two weeks, once every three weeks, once every four weeks, once every month, once every two months, once every three months, once every four months, or less.

[0154] In certain embodiments, the radiotherapy is low-fractionation radiotherapy. In some embodiments, the subject is administered 20-60 Gy in 2-20 fractions. In certain embodiments, the low-fractionation radiotherapy consists of 15 fractions. In certain embodiments, the 15 fractions are administered over 15-25 consecutive days. In certain embodiments, the 15 fractions are administered over 21 consecutive days.

[0155] In certain embodiments, this method further includes administering a chemotherapeutic agent, such as temozolomide (TMZ), to the subject. The chemotherapeutic agent may be administered in conjunction with radiotherapy. For example, TMZ may be administered in combination with low-fractionation radiotherapy at a dose of 75 mg / m². 2 It is administered at a daily dose of 50 mg / m². In some embodiments, subjects with tumors containing methylated MGMT promoters are given maintenance therapy with chemotherapeutic agents. For example, after radiotherapy, subjects with tumors containing methylated MGMT promoters are given 50 mg / m² per maintenance cycle, provided they do not have hematological toxicity. 2 Increase the dose to a maximum of 200 mg / m². 2 The dosage is 150 mg / m² over the first 5 days of a 28-day cycle (5 days "on", 23 days "off"). 2 Six cycles of TMZ can be received at a starting dose of / day. In some embodiments, maintenance therapy is initiated approximately 3–5 weeks after the last dose of radiotherapy, preferably approximately 4 weeks later.

[0156] In certain embodiments, the disclosed method can mediate tumor cell clearance or prevent its growth by inducing (1) humoral immunity via a B-cell response that blocks the production of monocyte chemotactic protein-1 (MCP-1), thereby delaying myeloid-derived suppressor cells (MDSCs) and suppressing tumor growth; (2) an increase in cytotoxic T lymphocytes (CTLs) such as CD8+ that attack and kill tumor cells; (3) an increase in the T helper cell response; (4) an increase in the inflammatory response mediated by IFN-γ and TFN-α; or (5) any combination of the foregoing. The method can increase progression-free survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45%. The method can reduce tumor volume by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60% after immunization. The method can prevent and block the increase of monocyte chemotactic protein 1 (MCP-1), a cytokine secreted from bone marrow-derived suppressor cells. The method can increase tumor survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60%.

[0157] The disclosed method can increase the cellular immune response in a subject by approximately 50 to 6000 times, 50 to 5500 times, 50 to 5000 times, 50 to 4500 times, 100 to 6000 times, 150 to 6000 times, 200 to 6000 times, 250 to 6000 times, or 300 to 6000 times compared to the cellular immune response in a subject that has not been administered the method or has been administered standard treatment. In some embodiments, the method increases the cellular immune response in a subject by approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2 It can be increased by 200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0158] In some embodiments, the method may increase tumor-free survival, reduce tumor load, increase progression-free survival, increase overall survival, or a combination thereof in subjects. The method can increase tumor-free survival in subjects by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60%. The method can reduce tumor volume in subjects by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, and 70%. The method can increase progression-free survival by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60% in the subjects. The method can increase overall survival in subjects by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60%.

[0159] In certain embodiments, the method is either proven clinically safe, proven clinically effective, or both. [Examples]

[0160] the purpose Main objective: To evaluate the safety and tolerability of intramuscular (IM) delivery of INO-5401 and INO-9012 followed by EP with CELLECTRA® 2000 in combination with semiprimab-rwlc in newly diagnosed adult patients with GBM.

[0161] Key endpoints and evaluation: ● The incidence of adverse events (AEs), graded according to the General Toxicity Criteria for Adverse Events (CTCAE) v4.03, is classified by system organ class, preferred terminology, severity, and association with the investigational treatment. ● Clinically significant changes in safety laboratory parameters from baseline.

[0162] Secondary purpose: ●In newly diagnosed adult patients with GBM, we will evaluate the preliminary clinical efficacy and immunogenicity of INO-5401 and INO-9012 delivered by intracellular injection, followed by EP with CELLECTRA® 2000 in combination with semiprimab-rwlc. ●In newly diagnosed adult patients with GBM, we will evaluate the preliminary immunogenicity of INO-5401 and INO-9012 delivered by IM injection, followed by EP with CELLECTRA® 2000 in combination with REGN2810.

[0163] Secondary endpoints and evaluation: ● Overall survival time at 18 months (OS18), ● Antigen-specific cellular immune responses are ○ Interferon-gamma secreting T lymphocytes in peripheral blood mononuclear cells (PBMCs) as measured by ELISpot. ○T cell phenotype in PBMCs by flow cytometry (e.g., activated and cytolytic cells, myeloid suppressor cell frequency (MDSC)), ○Diversity and putative antigen specificity are evaluated by T cell receptor (TCR) sequencing from PBMCs. ● Antigen-specific humoral response (e.g., B cell activation / antibody secretion).

[0164] Exploratory purpose: ● Explore the correlation between clinical efficacy and tumor genetics and / or biomarkers. ● Further evaluation of the efficacy of EP with CELLECTRA® 2000 in newly diagnosed adult patients with GBM, after delivery of INO-5401 and INO-9012 by IM injection, in combination with REGN2810 and low-fractionation radiotherapy. Exploratory endpoint: ●If possible, tumor-infiltrating lymphocytes (TILs) and immunosuppressive elements, ●Oncoprotein expression including, but not limited to, the onco-expression of hTERT, WT1, and PSMA by IHC, immunofluorescence (IF), or genome sequencing. ● MicroRNA signatures in plasma and / or serum, ●If possible, circulating tumor cells, circulating endothelial cells, and / or circulating cancer-associated macrophage-like cells from peripheral blood, ● Evaluation of tumor-associated antigen (TAA)-specific peripheral T cells by RNA sequencing. ● Evaluation of cytokine profiles from plasma and / or serum, ● Progression-free survival as evaluated by the RANO (Response Evaluation in Neuro-Oncology) criteria and the iRANO (Immunotherapy Response Evaluation in Neuro-Oncology) criteria, ●Overall survival (OS).

[0165] research design

[0166] The study described in this embodiment corresponds to ClinicalTrials_gov identifier NCT03491683. Data presented herein as relating to this study reflect the status of the study as of the filing date. In this study, the antigen-specific T-cell generation therapy INO-5401 was combined with INO-9012, followed by electroporation using the CELLECTRA® 2000 device with the PD-1 checkpoint inhibitor semiprimab-rwlc, in patients with newly diagnosed GBM, along with radiation and temozolomide, to evaluate the tolerability, immunogenicity, and antitumor activity of this combination. Ethical approval by the NYU Ethics Committee, approval number i17-00764.

[0167] This is a Phase 1 / 2, open-label, multicenter trial to evaluate the safety, immunogenicity, and preliminary efficacy of INO-5401 and INO-9012 in combination with cemiplimab (also known as REGN2810) in newly diagnosed patients with GBM. All patients received written informed consent.

[0168] Participants initiated immunotherapy with REGN2810 upon definitive histopathological diagnosis of GBM and appropriate recovery from surgical intervention. Participants were assigned to a cohort based on the results of MGMT gene methylation assays performed in CLIA-accredited laboratories that were available prior to the completion of RT. Immunotherapy initiation was designed as day 0. REGN2810 was administered intravenously (IV) every 3 weeks until disease progression, unacceptable toxicity, withdrawal of consent, or death, as defined by iRANO (Immunotherapy Response Assessment in Neuro-Oncology).

[0169] On day 0, subjects received INO-5401 and INO-9012 intramuscularly (IM), followed by electroporation (EP). INO-5401 and INO-9012 were administered, followed by EP in four doses every three weeks, and then every nine weeks until disease progression, unacceptable toxicity, withdrawal of consent, or death as defined by iRANO.

[0170] Temozolomide was administered to all subjects during radiotherapy, regardless of whether or not MGMT promoter methylation was present, unless clinically contraindicated. Radiotherapy (RT) was initiated within 42 days post-surgery. Radiotherapy was initiated approximately 1-2 weeks after day 0 and continued for approximately 3 weeks. Temozolomide (TMZ) was administered daily during radiotherapy (TMZ / RT). Subjects with MGMT promoter methylation received maintenance (adjuvant) TMZ for 6 cycles after recovery from TMZ / RT. Maintenance (adjuvant) TMZ was administered for the first 5 days of each 28-day cycle. This study consisted of two cohorts: Cohort A, consisting of subjects with unmethylated MGMT promoters, and Cohort B, consisting of subjects with methylated MGMT promoters.

[0171] Study group

[0172] Each potential subject met all of the following criteria for enrollment in the study: Patient Eligibility Summary

[0173] Newly diagnosed adults with GBM who have undergone definitive surgery and are eligible for standard therapy. Estimated number of subjects: 52. Cohort A: Non-methylated MGMT promoters (N=32, 30 evaluable subjects). Cohort B: Methylated MGMT promoters (N=20, 19 evaluable subjects).

[0174] Inclusion criteria: ●The relevant parties must provide written, IRB-approved informed consent in accordance with the facility's guidelines. ● You must be 18 years of age or older on the day you sign the informed consent form, be able to follow all clinical trial procedures, and be willing to participate. Newly diagnosed brain cancer accompanied by a histopathological diagnosis of glioblastoma (GBM), ● Baseline Karnovsky Performance Status (KPS) score ≥ 70, ● Administer dexamethasone equivalent dose ≤ 2 mg / day, and the condition is stable or decreasing from day 0 to ≥ 3 days prior. ● Recovery from the effects of previous GBM surgery as defined by the principal investigator. ● An electrocardiogram (ECG) with no clinically significant findings, as evaluated by the principal investigator within 28 days of signing the Informed Consent Form (ICF). ● Appropriate organ function demonstrated by hematological, renal, and hepatic parameters as defined in the table below, obtained 28 days prior to the first clinical trial treatment. [Table 1] ●During the trial, men agree not to become fathers, and women agree not to become pregnant if they are at risk of pregnancy. Participants must agree not to become pregnant (untreated induced amenorrhea of ​​12 months or more, as confirmed by follicle-stimulating hormone [FSH], if not receiving hormone replacement therapy), or be surgically infertile (vasectomy in men, or absence of ovaries and / or uterus in women), or agree to use a highly effective or combined method of contraception that results in an annual failure rate of <1% during the treatment period and for at least 12 weeks after the last dose. Regular abstinence (e.g., calendar, ovulation, symptomatic thermometer, or post-ovulation methods) and withdrawal are not acceptable methods of contraception. Examples of contraceptives expected to have an annual failure rate of less than <1% include male sterilization and hormonal implants. Alternatively, a suitable combination of oral or injectable hormonal contraceptives and certain intrauterine devices (IUDs), or a combination of two methods (e.g., two barrier methods such as condoms and a cervical cap), can achieve an annual failure rate of <1% (barrier methods must always be supplemented by the use of spermicides). ● Must be able to withstand magnetic resonance imaging (MRI).

[0175] Exclusion criteria: ● Postoperative MRI shows enhancement of residual tumor exceeding 1 cm x 1 cm. ●Multifocal disease or leptomeningeal disease (LM) on postoperative MRI, ● Radiation therapy cannot be started within 42 days of surgical removal of the tumor. ●Administer dexamethasone equivalent dose > 2 mg / day. ● Previous treatment with drugs that block the PD-1 / PD-L1 pathway at any point in the past, ● You must have received an approved or investigational immunomodulatory agent (e.g., anti-TNF, therapeutic anticancer vaccine, cytokine therapy (other than G-CSF or erythropoietin), or a drug targeting cytotoxic T lymphocyte antigen 4 (CTLA-4), 4-1BB (CD137), PI3K delta, or OX-40) within 28 days of the first dose. ● You have received treatment with idelalisib at any point in the past. ●Past, present, or planned treatments in the field of oncology (Optune, NovoTTF), past exposure to investigational drugs or devices including Gliadel wafer (Carmustine) implants for oncolytic virus therapy or chemotherapy, within 28 days of receiving the first dose. ● Allergy or hypersensitivity to REGN2810 or any of its excipients, ● A history of allergic reactions or acute hypersensitivity reactions caused by antibody therapy. ● Progressive or recent (within the last 5 years) evidence of an autoimmune disease requiring treatment with systemic immunosuppressive therapy that suggests a risk of immune-related adverse events (irAEs), with the exception of vitiligo, remission childhood asthma, type 1 diabetes, residual hypothyroidism requiring hormone replacement only, or psoriasis not requiring systemic treatment. ●As stated in the inclusion criteria, within 28 days prior to the first dose of the investigational treatment, the patient has received a diagnosis of immunodeficiency other than dexamethasone for the underlying disease under investigation, or has been treated with systemic immunosuppressive therapy. A positive serological test for human immunodeficiency virus (HIV), a history of HIV infection, or a positive test for hepatitis B virus surface antigen (HBV sAg) or hepatitis C virus ribonucleic acid (HCV RNA) indicating active or chronic infection may interfere with the ability of these infections to initiate an appropriate immune response to vaccination. ●Current malignant tumors in another site with no evidence of disease within the past three years, excluding appropriately treated basal cell or squamous cell carcinoma or cervical carcinoma. Cancer survivors who have received treatment for a previous malignant tumor, have had no evidence of disease for three years, and are considered to be at low risk of recurrence are eligible for the trial. ●Receive any vaccine, excluding inactivated influenza vaccines which may be administered up to two weeks prior, at least four weeks before the first dose of the investigational treatment. ● If the patient has a clinically significant and medically unstable medical history that, in the judgment of the principal investigator, could endanger the patient's safety, interfere with the evaluation of the study or endpoints, or affect the validity of the study results (e.g., chronic renal failure, angina pectoris, myocardial infarction, New York Heart Association (NYHA) class III / IV heart disease), or has a prodromal cardiac condition (e.g., Wolff-Parkinson-White, cardiomyopathy, or clinically significant arrhythmia). ● History of pneumonia within the past 5 years, ● If you have acute or chronic bleeding or coagulation disorders that make the use of IM injection or anticoagulants (e.g., anticoagulants or antiplatelet agents, excluding nonsteroidal anti-inflammatory drugs such as commercially available aspirin or ibuprofen) contraindicated within two weeks of day 0, ●Considering the anterolateral aspect of the deltoid and quadriceps muscles, there are fewer than two acceptable sites for IM injection. The following sites are unacceptable: ○ Tattoos, keloids, or hypertrophic scars located within 2 cm of the intended treatment area. ○ A defibrillator or pacemaker located on the same side as the triangular injection site (unless deemed acceptable by a cardiologist) (to prevent life-threatening arrhythmias) ○ Metal implants or implantable medical devices within the intended treatment site (i.e., EP area), ● In the opinion of the principal investigator, there was active use or dependence on drugs or alcohol that interfered with compliance with the clinical trial requirements. ● Confinement or forced detention (involuntary detention) for the treatment of mental or physical illness (i.e., infectious disease), ●Pregnant or currently breastfeeding, ● If the principal investigator determines that a participant has a medical, psychological, or non-medical condition that could impair their ability to participate or affect their safety.

[0176] Dosage and administration

[0177] Investigational drug products [Table 2]

[0178] The active pharmaceutical ingredient (API) in INO-5401 is a DNA plasmid sequence designed and constructed using proprietary synthetic consensus (SynCon®) technology. This process involves synthetically inducing consensus genes across multiple strains and optimizing DNA insertion at the gene level to enable high expression in human cells. The INO-5401 plasmid is as follows: ● pGX1108 is a plasmid for the expression of prostate-specific membrane antigen (PSMA, SEQ ID NO: 28). 3 mg of pGX1108 is present in each 10 mg dose of the investigational treatment (INO-5401 + INO-9012). ● pGX1404 is a plasmid for the expression of Wilms tumor gene-1 (WT1) antigen (SEQ ID NO: 26). 3 mg of pGX1404 is present in each 10 mg dose of the investigational treatment. ● pGX1434 is a plasmid for the expression of human telomerase reverse transcriptase (hTERT) (SEQ ID NO: 20). 3 mg of pGX1434 is present in each 10 mg dose of the investigational treatment.

[0179] The API in drug product INO-9012 is pGX6001, a DNA plasmid for the expression of human IL-12 p35 and p40 subunit proteins. 1 mg of pGX6001 is present in each 10 mg dose of the investigational treatment. Both DNA plasmid products INO-5401 and INO-9012 are administered using syringes and the investigational CELLECTRA® 2000 electroporation (EP) device.

[0180] Semiprimab-rwlc(REGN2810) is a covalent heterotetramer consisting of two disulfide-bonded human heavy chains, each covalently linked to a human kappa light chain via disulfide bonds. The antibody has a molecular weight of approximately 143.6 kDa based on its primary sequence. Each heavy chain has a single N-linked glycosylation site located within the constant region of the Fc portion of the molecule.

[0181] The REGN2810 heavy chain contains a constant IgG4 isotype region. Variable domains of the heavy and light chains bind to form a PD-1 binding site within the antibody. Antibody production using VelocImmune® mice is performed using standard techniques after immunization with PD-1. Genes encoding the heavy and light chains of REGN2810 were introduced into CHO cells, and a stable expression cell line (cell line 2) with a higher titer was developed for this antibody. For both cell lines, recombinant CHO cells were grown in suspension culture and chemically induced to initiate antibody expression and secretion into cell culture medium. The antibody was recovered via filtration and purified through a series of preparation column chromatography and filtration steps to produce the active pharmaceutical ingredient (API). The API was then formulated and subjected to sterile filtration to produce the final API product.

[0182] REGN2810 (50 mg / mL) is formulated in a pH 6.0 buffered aqueous solution containing 10 mM histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline, and 0.2% (w / v) polysorbate 80. REGN2810 is supplied as 5.5 mL of sterile solution in 10 or 20 mL glass vials for intravenous administration. A maximum volume of 5.0 mL can be drawn from each vial containing 250 mg of REGN2810. Since 7 mL is required to provide a 350 mg dose of REGN2810, two vials should be used if supplied with 5 mL vials. REGN2810 (50 mg / mL) is also supplied as 7.44 mL of sterile solution in 10 or 20 mL glass vials for intravenous administration. A maximum volume of 7.0 mL can be drawn from each vial containing 350 mg of REGN2810.

[0183] treatment

[0184] Subjects meeting all inclusion criteria and none of the exclusion criteria were initiated with immunotherapy using REGN2810 and INO-5401+INO-9012 on day 0. REGN2810 was administered intravenously every 3 weeks at a dose of 350 mg per dose, without dose retention, until disease progression, unacceptable toxicity, withdrawal of consent, or death, as defined by iRANO. INO-5401 and INO-9012 IM, followed by EP, were administered four times every 3 weeks, and then every 9 weeks, without dose retention, at a dose of 10 mg / DNA per dose, until disease progression, unacceptable toxicity, withdrawal of consent, or death, as defined by iRANO. RT was initiated by 42 days after surgical intervention, approximately 1-2 weeks after day 0. RT lasted approximately 3 weeks. The total dose of RT was 40 Gy over 3 weeks.

[0185] Daily TMZ with radiotherapy (TMZ / RT) was initiated 42 days after surgical intervention and approximately two weeks after day 0. TMZ / RT continued for approximately three weeks. The TMZ dose was 75 mg / m² if there was no dose reduction. 2The drug was administered at the specified dose. Subsequently, the subjects should receive an additional 6 cycles of maintenance (adjuvant) TMZ. Cohort B received TMZ after up to 6 cycles of radiotherapy. Maintenance (adjuvant) TMZ was administered at 150-200 mg / m² for the first 5 days of each 28-day cycle, after peripheral blood counts had recovered from TMZ / RT according to standard guideline TMZ treatment. 2 The drug was administered to subjects in cohort B at the specified dose.

[0186] Day 0 (first dose of INO5401, INO9012, and REGN2810) was at least 14 days after the completion of primary tumor resection, and subjects recovered from surgery by 28 days post-operatively.

[0187] For patients who discontinued one treatment (either INO-5401 + INO-9012 or REGN2810) for reasons other than disease progression, they were able to continue other treatments after consulting with a medical monitor.

[0188] Figure 1 illustrates the trial designs for cohorts A and B. ●INO-5401 (3 mg each of hTERT, WT-1, and PSMA plasmids) was administered via intramuscular (IM) injection, followed by electroporation (EP) using a CELLECTRA® 2000 device, for a total of 10 mg of DNA, delivered in four doses every three weeks, and then every nine weeks. ● Chemoradiotherapy: Radiation (RT) delivered in a low-fractionation schedule (40 Gy over 3 weeks) ● Temozolomide (TMZ) (Cohorts A and B) administered concurrently with radiation, followed by 6 maintenance (adjuvant) cycles (Cohort B only).

[0189] Semiprimab-rwlc (REGN2810) was administered intravenously at a dose of 350 mg over approximately 30 minutes every three weeks (Q3W), starting on day 0, and continued until disease progression, unacceptable toxicity, withdrawal of consent, or death as defined by iRANO.

[0190] INO-5401 is a mixture of three separate synthetic plasmids targeting WT1, PSMA, and hTERT proteins. Each plasmid was dosed with 3 mg of DNA, for a total of 9 mg of DNA per dose of INO-5401. INO-5401 was administered IM on days 0, 3, 6, and 9 weeks, then every 9 weeks thereafter, continuing until disease progression defined by iRANO, unacceptable toxicity, withdrawal of consent, or death. INO-9012 is a synthetic plasmid expressing human IL-12, which was administered at 1 mg of DNA and co-administered IM with INO-5401. The total dose of DNA in each dose of INO-5401 + INO-9012 when administered together was 10 mg. INO-5401 (3 mg each of hTERT, WT-1, and PSMA plasmids) was combined with 1 mg of INO-9012 (IL-) to a total of 10 mg of DNA, and administered via intramuscular (IM) injection followed by electroporation (EP) with a CELLECTRA® 2000 device, with 4 doses delivered every 3 weeks, then every 9 weeks.

[0191] All subjects received a total of 40 Gy in 15 fractions (3 weeks).

[0192] Hypofractionated radiation therapy (hfRT) was initiated within 42 days after surgery. Radiation therapy was performed for 3 weeks.

[0193] [[ID=一三]] Unless clinically contraindicated, all patients were administered TMZ regardless of MGMT methylation status during radiation therapy. TMZ was administered orally at 75 mg / m 2 per day for 21 days (7 days per week, 3 weeks) with hfRT. ​​​​​​​2 TMZ maintenance therapy was continued for 6 cycles at the starting dose of / day. TMZ maintenance (adjuvant) was initiated approximately 4 weeks after the last dose (±3 days) of RT, following recovery of peripheral blood counts, in accordance with TMZ treatment guidelines. The dose was determined using the actual body surface area (BSA) calculated in square meters at the start of each treatment cycle.

[0195] The daily dose was rounded to the nearest 5 mg.

[0196] Efficacy evaluation / endpoint ELISpot

[0197] ELISpot was used to qualitatively measure the presence of antigen-specific T cells in peripheral blood mononuclear cell (PBMC) samples. PBMCs were collected from subjects at weeks 0, 3, 6, 9, 12, and 24 of the study and assayed with IFN-g ELISpot. At the 12-month data cutoff, antigen-specific IFNg spot-forming units (SFUs) per million PBMCs are shown from the highest pre-treatment (pre) and post-treatment (peak) values ​​for INO-5401 and semiprimab-rwlc from eight subjects with samples available up to week 24. Each subject is represented by a white circle, and the bar represents the mean value. The graph for each antigen, as well as the difference between pre-treatment and the peak (delta), is shown for the 11 subjects assayed together and the 8 subjects with available samples up to week 24. At the 18-month data cutoff, the antigen-specific IFNg spot-forming units (SFUs) per million PBMCs are shown from the highest pre-treatment (pre) and post-treatment (peak) values ​​for INO-5401 and semiprimab-rwlc in 39 subjects. Each subject is represented by a white circle, and the bar represents the mean value. The graphs for each antigen, and for all 39 subjects, show the difference between pre-treatment and the peak, or delta. INO-5401 is the sum of WT1, PSMA, and hTERT. The box plots extend from the 25th to the 75th percentile, with a horizontal line at the median and a "+" at the mean.

[0198] Soluble granule loading

[0199] A lysic granule loading assay was performed to explore the activation state and lysis potential of antigen-specific T cells present in PBMC samples collected from subjects at weeks 0, 3, 6, 9, 12, and 24 of the study. PBMCs were stimulated with overlapping peptide libraries for INO-5401 antigens (hTERT, PSMA, and WT1) or relevant controls in the absence of any exogenous cytokines. After 5 days, cells were stained with antibodies and evaluated by flow cytometry. The frequency of live, antigen-specific, activated (CD38+) CD3+CD8+ T cells with lysis potentials (expressing granzyme A, perforin) from pre-treatment (pre) and post-treatment (peak) with INO-5401 and semiprimab-rwlc was determined from 8 subjects at a 12-month data cutoff and from 29 subjects at an 18-month data cutoff.

[0200] Safety evaluation

[0201] Adverse events (AEs), graded according to the General Toxicity Criteria for Adverse Events (CTCAE) v4.03, are classified by system organ class, preferred terminology, severity, and relevance to the investigational treatment, as well as clinically significant changes in safety laboratory parameters from baseline.

[0202] The safety trial was conducted using a modified rolling 6 design, enrolling up to 6 subjects in each cohort (up to 12 subjects in total for cohorts A and B). Enrollment was staggered in each cohort, with a one-week waiting period between the enrollment of the first and second subjects and between the second and third subjects. Each subject was evaluated for up to 9 weeks. By week 9, subjects had completed RT, having received three doses of REGN2810 and three doses of INO-5401 + INO-9012.

[0203] Enrollment in each cohort was fully initiated after the first three subjects of the six subjects in each cohort completed the 9-week evaluation without dose-limiting toxicity (DLT), following a review of all available safety data from these patients by medical monitors from the sponsor and Regeneron Pharmaceuticals, as well as the lead investigator. However, if a single subject from the first six subjects of a cohort experienced DLT before the first three subjects of that cohort completed the 9-week evaluation, enrollment was limited to the six subjects in that cohort until all six subjects reached the 9-week evaluation and were assessed for DLT.

[0204] If a second participant within the initial six experiences a DLT in the same cohort within the first nine weeks, enrollment in that cohort will be stopped, and the principal investigator (PI) of the participating site, along with the sponsor's medical monitor, will discuss the case and decide whether to change the dosage of the investigational drug or concomitant medication (RT / TMZ) in that cohort, or to stop further enrollment in that cohort. If a protocol change is necessary, enrollment will only be resumed after the protocol has been amended and approved by the IRB.

[0205] DLT is defined as follows: Non-hematological toxicity ● Uveitis of Grade ≥ 2. ● Non-hematological toxicity of grade ≥ 3, except for the following: ● Except for Grade 3 nausea, vomiting, diarrhea that persists despite maximum supportive care prescribed by a treating physician (duration >7 days). ●Abnormal laboratory findings of grade ≥ 3 that are considered clinically insignificant and do not meet the criteria for adverse events (AEs). ● Grade 3 infusion-related reaction in response to medical management. ● A grade 3 immune-related adverse event (IRAE), excluding uveitis, that improves to grade ≤ 2 within 14 days with medical management (including steroid treatment). hematological toxicity ● Grade 4 neutropenia > 7 days. ● Grade 4 thrombocytopenia with bleeding, or grade 3 thrombocytopenia. ● Febrile neutropenia of grade ≥ 3 (fever ≥ 38.5°C, absolute neutrophil count [ANC] < 1000 / mm3), or neutropenia of grade ≥ 3 with a recorded infection.

[0206] Events that the principal investigator considered to be related to the underlying tumor, concomitant medication, or comorbidity, as well as events considered unlikely to be related to the study therapy (INO-5401, INO-9012, or REGN2810), were not considered DLTs if they were considered to be at least likely related to either temozolomide or RT. Patients who experienced a DLT discontinued further study therapy and entered the post-trial follow-up phase of the study. Adverse events that met the DLT criteria but occurred outside the DLT window were classified as unacceptable AEs, and study therapy was discontinued.

[0207] If, after the safety adjustment period, more than 30% of subjects in either cohort experienced dose-limiting toxicity deemed to be related to the investigational drug at any point in the course of this clinical trial, enrollment in one or both cohorts was to be stopped, and the principal investigator (PI) of the relevant site, in addition to the sponsor's medical monitor, would discuss the safety profile of the investigational drug and decide whether to change the dose of the investigational drug or the combination drug (RT / TMZ) in one or both cohorts, or to stop further enrollment in one or both cohorts. If a change in the protocol was necessary, enrollment would only be resumed after the protocol was amended and approved by the IRB.

[0208] Medical and clinical evaluation

[0209] From the moment the ICF was signed, concomitant medications were collected throughout the entire period, from subsequent clinical trial visits to discharge from the clinical trial.

[0210] All adverse event (AE) assessments were collected from the time of ICF signing until 30 days after the last dose of INO-5401+INO-9012 or REGN2810, whichever was later. However, AESI and SAEs as defined in this protocol were planned to be collected after 6 months after the last dose of INO-5401+INO-9012 or REGN2810, whichever was later. AEs were assessed using NCI CTCAE v4.03.

[0211] Physical evaluation

[0212] A complete neurological examination (cranial nerve assessment, deep tendon reflexes, muscle strength and sensation), and a complete assessment including the Karnovski Performance Status (KPS) were performed every three weeks.

[0213] Evaluation of response after medical treatment

[0214] On day 0, vital signs were collected before both REGN2810 and INO-5401+INO-9012 infusions, at the end of the REGN2810 infusion, every 30 minutes for the first 4 hours after the REGN2810 infusion, and 30 minutes after the INO-5401+INO-9012 infusion by EP. At all other visits, the principal investigator assessed local and systemic responses for 30 minutes after each treatment (REGN2810 and / or INO-5401+INO-9012) and at visits designated according to the event schedule.

[0215] Vital signs

[0216] During all clinical trial treatment visits, vital signs, including body temperature, respiratory rate, blood pressure, and heart rate, were measured.

[0217] Weight, height, weight index

[0218] Weight (kg) and height (cm) were collected during the screening visit. Weight was collected at each additional treatment visit from day 0 until the end of treatment.

[0219] Body weight index was assessed at day 0, week 3, week 6, week 9, week 18, and every 9 weeks thereafter, as needed, while the subjects were receiving EP treatment, to evaluate the needle gauge.

[0220] 12-lead electrocardiogram

[0221] To determine eligibility, electrocardiograms (ECGs) were performed at screening up to 28 days prior to day 0. Abnormal ECGs were interpreted as either clinically significant or not clinically significant. Abnormal ECGs at screening were to be discussed with the clinical trial monitor to determine eligibility.

[0222] Pregnancy test For women of reproductive capacity, a negative result on a serum pregnancy test (the test must have a sensitivity of at least 25 mIU / mL) was obtained at the time of the screening visit and before administration of INO-5401+INO-9012 and REGN2810. If a β-HCG (pregnancy) test was positive at any point in time, indicating that the subject was pregnant, no additional investigational treatment was administered, but the subject needed to be followed up during and after the trial to determine the outcome of the pregnancy (with the subject's consent).

[0223] Laboratory evaluation

[0224] Blood samples were collected according to the clinical trial schedule for the event.

[0225] Screening tests can be used during visit #1 (day 0) if they are taken within 7 days of day 0. Otherwise, all tests related to the treatment visit (CBC and chemicals) were collected within 72 hours of treatment and reviewed / evaluated by the principal investigator before treatment. Complete blood count (CBC) includes the following: ● White blood cell count (WBC) with differential ● Red blood cell (RBC) count ● Hemoglobin, hematocrit ● Platelet count Serum chemicals include the following: ● Glucose ● Albumin ●Total protein ●SGPT (Serum Glutamate Pyruvic Acid Transaminase, ALT) ●SGOT (Serum glutamate oxaloacetate transaminase, AST) ● Alkaline phosphatase ● Bilirubin (total) ● Direct bilirubin ●BUN (Blood Urea Nitrogen) ●Calcium ●Creatinine ●Electrolytes (sodium, potassium, chloride, carbon dioxide, or bicarbonate) ●Lipase ● Amylase ●CPK Assessments performed only during screening: ● HIV screening tests (antibody immunoassay tests), or documentation of these results in medical records. ● Hepatitis B serology: HBsAg (Hepatitis B surface antigen), or documentation of these results in medical records. ●Hepatitis C antibody immunoassay, or documentation of these results in medical records. ●Specific gravity, glucose, blood and urine tests including ketones, ●Activated partial thromboplastin time (aPTT), INR.

[0226] Peripheral blood immunogenicity assessment

[0227] Whole blood and serum samples were obtained. Immunological samples were collected at screening, on day 0, week 3, week 6, week 9, week 12, and then every 12 weeks (weeks 24, 36, 48, etc.).

[0228] T cell responses were evaluated using an antigen-specific IFN-γ ELISpot assay with a duplicate peptide library covering the INO-5401 antigen (hTERT, WT-1, and PSMA). Furthermore, to track general cellular immune capacity during the trial, the PBMC response to a pool of known antigen epitopes combined from cytomegalovirus, Epstein-Barr virus and influenza (CEF) was evaluated.

[0229] T cell responses were evaluated by flow cytometry via an overlapping peptide library covering the INO-5401 antigen. The flow cytometry assay may include examination of the effect of immunotherapy on the ability of target T cells to show phenotypic markers associated with cytolytic ability, activation or exhaustion after stimulation with peptides corresponding to the INO-5401 antigen. Markers used for this purpose include CD3, CD4, CD8, CD137, CD69, CD38, PD-1, granzyme, granzyme A, granzyme B and perforin. These markers may change as new data useful for this evaluation become available.

[0230] Assessment of the presence of cells known to play a role in immunosuppression can be performed via the application of flow cytometry. The flow cytometry assay may include examination of the effect of these cells on the induction or expansion of the immune response after immunotherapy. Markers used for this purpose include CD3, CD16, CD19, CD20, CD56, CD11b, CD14, CD15, CD33 and HLA-DR. These markers may change as new data useful for this evaluation become available.

[0231] TCR sequencing from PBMCs to assess diversity and putative antigen specificity was performed on total PBMCs, with or without prior in vitro stimulation.

[0232] The humoral response was evaluated through the application of enzyme-linked immunosorbent assay (ELISA) or other methods for the detection of antigen-specific antibody secretion and / or the use of flow cytometry for B cell phenotypes. Analysis of TAA-specific T cells can be performed through the isolation of these cells based on the expression of markers such as CD137. At the time of isolation, RNAseq can be performed to understand the unique transcriptome of these cells. Analysis of cytokine profiles from peripheral blood can be performed using evaluation platforms such as Luminex.

[0233] Tissue immunology

[0234] Before and after INO-5401 administration, the immune infiltration and the presence of immunomodulatory factors in tumor tissues were examined through a number of evaluations, including the following: ● Expression of PD-L1 and tumor oncoproteins in tumors and infiltrating immune cells by IHC, immunofluorescence (IF), or genomic sequencing ● Characterization of TIL infiltration (CD3, CD8, CD4) by RNAScope ● Evaluation of tumor Treg, myeloid immunosuppressive populations, and T cell immune checkpoint expression by RNAScope ● Correlate any existing clonal T cell populations with treatment-related changes in T cell diversity in peripheral blood by T cell receptor sequencing in resected tumor tissues

[0235] Biomarker

[0236] Biomarker evaluations were performed on the peripheral and tissue samples referenced above and collected according to the test instructions. Immunohistochemical evaluations of the expression of hTERT, WT1, and / or PSMA proteins in tissue samples from enrolled subjects occurred according to the continuing relevance supported by the presence of sufficient sample volume and available data. IDH-1 status was to be performed on tumor tissue if possible. MicroRNA signatures in plasma and / or serum should be evaluated to determine disease and / or therapy-specific signatures that predict the disease course and / or response to treatment with INO-5401 and INO-5401-driven alterations. RNA sequencing can be used in this method.

[0237] Evaluation of circulating tumor cells, circulating endothelial cells, and / or circulating cancer-associated macrophage-like cells derived from peripheral blood may occur. The use of size exclusion-based filters may be employed for this purpose, and markers may include GFAP, CD45, vimentin, PD-L1, CD146, TIE-2, and possibly others.

[0238] Clinical evaluation

[0239] Clinical assessment of disease response was performed at every clinical trial visit (assessed by clinical signs and symptoms of disease progression). MRI for disease progression was obtained for all subjects 9 weeks (±3 days) after day 0 and every 3 months thereafter, unless pseudo-progression was deemed to have occurred by iRANO assessment. In such cases, a repeat confirmatory MRI scan was performed at 3 months if pseudo-progression was suspected.

[0240] overall survival

[0241] All subjects were tracked for survival and return. After the completion of the treatment completion visit, documentation of survival status was required every six months and at 18 months following day zero. To document survival, the following methods were acceptable: telephone, personal contact, certified letter, or documentation of visits confirmed in medical records.

[0242] progress

[0243] Disease progression was assessed using both RANO and iRANO. Patients who discontinued treatment before progression were followed up for progression and survival.

[0244] RANO and IRANO: Evaluation of (immunotherapy) response in neuro-oncology

[0245] In this trial, both RANO and iRANO criteria were used. The Radiographic Evaluation of Neurotumors (RANO) criteria were proposed in 2010 to improve the assessment of the evolving complexity of images in subjects with malignant gliomas [Wen, et al., J Clin Oncol 2010, 28:1963-1972.]. The RANO criteria provide guidance on the occurrence of pseudo-progression, which occurs in approximately 10–20% of subjects newly diagnosed with GBM after radiotherapy and TMZ therapy. Clinical benefits, including long-term survival and tumor regression, can still occur after initial disease progression or the appearance of new lesions. The iRANO criteria were developed by a multinational and multidisciplinary panel of experts in neuro-oncology immunotherapy (RANO Working Group) and established guidance for determining tumor progression in immunotherapy trials in neuro-oncology [Okada, et al. Lancet Oncol 2015,16:e534-542, Reardon, et al. Neuro Oncology 2014,16(Suppl 2)]. The iRANO Working Committee recommends that for patients with early signs of progression (treatment with immunotherapy for less than 6 months), including those who develop new lesions but do not have substantial neurological impairment, follow-up imaging should be requested 3 months after initial radiographic evidence of progression to confirm progression, in order to reduce the likelihood of prematurely declaring progressive disease in patients with pseudo-progression or delayed response. In such subjects, those who had further confirmation of radiographic progression based on comparison with the first scan showing evidence of disease progression, or who exhibited substantial clinical decline at any given time, should be classified as having a progressive disease with a disease progression date retroactive to the first date on which the subject met the criteria for radiographic progression.

[0246] In this study, researchers used the iRANO criteria to decide whether to discontinue the investigational drug if disease progression was suspected, but all subjects needed to be assessed for progression using both the RANO and iRANO criteria.

[0247] Nanoscale: Neurological evaluation in neuro-oncology

[0248] Nanoscale is used only in conjunction with iRANO and RANO standards.

[0249] While both the RANO and iRANO scales explicitly state the need to incorporate clinical status for overall assessment, neither scale provides specific parameters. An international group of neuro-oncologists was convened to draft nanocriteria as an objective and quantifiable indicator of neurological function that can be assessed in routine examinations. The nanoscale involves assessment of eight relevant neurological regions based on direct observation / examination performed during routine outpatient visits. The scores define criteria for region identification and overall score for response, progression, stable disease, and “not assessed.” These criteria provide detailed and objective measures of neurological function that can be assessed across clinical trials and therapeutic interventions [Nayak, et al., Neuro Oncol 2017, 19:625-635]. This trial included clinical assessments utilizing nanoscales when RANO and iRANO were assessed.

[0250] An AE is defined as any adverse medical event in a patient receiving a drug or in a clinical trial, which does not necessarily have to be causally related to the treatment. Therefore, an AE may be an undesirable, unintended sign (e.g., an abnormal laboratory finding), symptom, or illness that is time-related to the use of the drug, whether or not it is related to the drug.

[0251] In this study, AEs were monitored, classified, and summarized. Medical conditions / diseases that were present before the start of the investigational drug were considered AEs only if they deteriorated after the start of treatment with the investigational drug. Unanticipated AEs were those not identified in the reference safety document of the investigational drug or the corresponding section of the IB. Throughout the study, all AEs were monitored and reported on an AE CRF, including the severity, seriousness, treatment taken, and relationship to the IP. AEs were followed up until they resolved or stabilized, and the results were recorded on the appropriate CRF. All AEs were recorded in standard medical terms, not in the subject's own words.

[0252] AEs included the following: ● Pre- or post-treatment complications that occurred as a result of a protocol-required surgery. ● Pre-existing conditions that increased in severity or changed in nature during or as a result of the investigational drug phase of a human clinical trial. ● Complications of pregnancy. AEs did not include the following: ● Medical or surgical procedures (e.g., surgery, endoscopy, tooth extraction, blood transfusion) were performed, and the conditions resulting from the procedures could be considered AEs. ● Diseases, conditions, or test abnormalities that were present or detected before the screening visit and did not deteriorate. ● Situations where no unexpected events occurred (e.g., hospitalization for elective surgery, social and / or convenience hospitalization). ● Overdose of the investigational drug without clinical sequelae ● Medical conditions or clinically significant test abnormalities with an onset date before signing the ICF were not AEs unless they deteriorated. They were recorded in the medical history CRF. ● Pregnancy without complications. ● Induced elective abortion to terminate pregnancy without medical reason (recorded in the pregnancy CRF).

[0253] All AEs that occurred from the time of signing the consent form throughout the study period and up to 30 days after the last dose (6 months for immune-related AEs) were recorded.

[0254] A serious adverse event (SAE) is any AE that meets one of the following conditions: ● Deaths during the monitoring period as defined by the protocol (excluding deaths due to disease progression). ●Things that immediately threaten life (for example, in the opinion of the principal investigator, the subject was at risk of immediate death if the event occurred), During the monitoring period defined in the protocol, the target hospitalization or extension of an existing hospitalization is required (including any one- or two-day hospitalization regardless of the length of stay, even if the hospitalization is merely a precautionary measure to allow for continued observation). However, hospitalization for a pre-existing condition that has not worsened (including hospitalization for elective surgery) does not qualify as a SAE. ● Causing a persistent or significant disability / impairment (a significant impairment of the ability to perform normal daily living functions), ● As a result of congenital abnormalities or birth defects, ● A significant medical event that may not result in death, life-threatening complications, or hospitalization, but which, based on appropriate medical judgment, could endanger the subject and may require medical or surgical intervention to prevent one of the above outcomes. Examples of such medical events include: ○ Allergic bronchospasm requiring emergency or home-based intensive care. ○ Blood disorders or seizures that do not require hospitalization, ○Onset of drug addiction or drug abuse. SAEs are clarified as follows: ●Death is a result of AE, not AE itself. ● The target may not have received an IP address when the incident occurred. ● Medication may have been administered as part of a treatment cycle or temporarily discontinued before the onset of SAE, but it may have contributed to the event. ● "Life-threatening" means that, when the event occurred, there was a risk of immediate death as a result of that event. This does not include events that could lead to death in severe cases. ● Complications that occur during hospitalization are called AEs (Accidental Exposures). If the complication prolongs hospitalization, it becomes a SAE (Severe Adverse Event). ● Hospitalization means that the subject is formally hospitalized for a specified period of time due to medical reasons.

[0255] The principal investigator attempted to establish a diagnosis of the event based on signs, symptoms, and / or other clinical information. In such cases, the diagnosis had to be recorded as an AE and / or SAE, rather than as individual signs / symptoms.

[0256] Statistical analysis plan

[0257] This is a single-arm, open-label, multicenter clinical trial of newly diagnosed GBM patients with tumors having non-methylated MGMT promoters (Cohort A) and tumors having methylated MGMT promoters (Cohort B), using a combination of INO-5401 + INO9012 with REGN2810. The primary analysis of this trial is the safety and tolerability of INO-5401 and INO-9012 in combination with REGN2810. Secondary analyses of this trial evaluated the efficacy of INO-5401 and INO-9012 in combination with REGN2810 using OS18 and immunogenicity biomarkers (ELISpot / flow cytometry / TCR sequencing / antigen-specific humoral response).

[0258] Exploratory analyses focused on correlations between clinical response and tumor genetics and biomarkers. Progression-free survival and overall survival, as assessed by the RANO (Response Assessment in Neuro-Oncology) criteria, were also evaluated as exploratory endpoints.

[0259] Statistical hypothesis

[0260] Each cohort in this trial had a separate, independent hypothesis to evaluate the secondary endpoint of overall survival (OS18) at 18 months. The true therapeutic effect on OS18 was defined as p, where p represents the true population probability of OS18 in each cohort. Then, for the MGMT promoter unmethylated subjects (Cohort A), the significance hypothesis over history controls was H0:p ≤ 0.45 vs. H1:p > 0.45, and for the MGMT promoter methylated subjects (Cohort B), the significance hypothesis over history controls was H0:p ≤ 0.60 vs. H1:p > 0.60.

[0261] Analysis group / dataset

[0262] The analysis group was, ○ The modified intention-to-treat (mITT) population includes all subjects receiving at least one dose of the investigational treatment. The mITT population will be used to analyze the secondary endpoint OS18, as well as all exploratory efficacy endpoints, including OS and PFS. The per-protocol (PP) population consists of subjects who have received at least three of the first four doses of the investigational treatment and who have no protocol violations. Analysis of the PP population is considered to support the corresponding mITT population for efficacy analysis. Subjects excluded from the PP population are identified and recorded before locking the trial database. ○The safety analysis set will include all subjects who receive at least one dose of either INO-5401, INO-9012, or REGN2810 as investigational treatment. Subjects will be analyzed for the treatment they received.

[0263] Description of statistical methodological analysis of key safety endpoints

[0264] The primary analyses of this clinical trial are the safety analysis of treatment-induced adverse events (TEAEs) and clinically significant changes in safety laboratory parameters from baseline.

[0265] For the purposes of this study, a TEAE is defined as any AE occurring from day 0 onward up to 30 days after the last dose of the investigational treatment, while irAEs, AESIs, and SAEs may occur up to 6 months after the last dose of the investigational treatment. All TEAEs are summarized for the purposes of the safety analysis, with frequency, proportion, and 95% Clopper-Pearson confidence intervals set within each cohort and across both cohorts.

[0266] These frequencies are presented overall and separately by dose number, and overall, by system organ class and by preferred terminology, they represent the proportion of affected subjects. The frequency of additional events is presented in relation to maximum severity and the strongest relationship with the investigational treatment. Multiple occurrences of the same AE in a single subject are counted only once, following the worst-case approach in relation to severity and relationship with the investigational treatment. All serious TEAEs are also summarized as described above.

[0267] AEs with unknown or partial onset / cessation dates are included in the overall AE summary but are excluded from the calculation of AE duration. AE duration is calculated as AE cessation date to AE onset date + 1 day. TEAEs, non-severe TEAEs, irAEs, AESIs, and SAEs are presented in the list.

[0268] Test response variables are descriptively summarized as changes from baseline at each time point, including 95% confidence intervals. Shifts from baseline by CTCAE are also presented. Clinically significant laboratory values ​​are listed.

[0269] All safety analyses are performed on the subjects of the safety analysis set.

[0270] Analysis of secondary efficacy endpoints

[0271] Secondary endpoints of OS18 are summarized using the frequency, proportion, 95% Clopper-Pearson confidence interval, and p-value for each cohort. Participants are considered survivors if they are determined to be alive after 18 months (548 days).

[0272] A one-sided p-value of <0.025 indicates a significant difference. OS18 was analyzed for subjects in both the mITT and perprotocol populations, and all mITT / PP subjects were included in the OS18 denominator.

[0273] Cellular and humoral immune responses are presented using descriptive statistics at each time point and for changes from baseline.

[0274] Analysis of other safety data

[0275] The proportion of subjects with abnormal medical history findings is summarized for each cohort of subjects in the safety population by body system and preferred terminology.

[0276] Prior medications are those used before the start of the trial (within 28 days prior to day 0). Concomitant medications are those used during the course of the trial (from day 0 onward). The partial start date for prior and concomitant medications is assumed to be the earliest possible date coinciding with the partial start date. The partial discontinuation date for prior and concomitant medications is assumed to be the latest possible date coinciding with the partial discontinuation date. All prior and concomitant medication data are summarized as the proportion of each cohort in the safety population.

[0277] Vital signs measurements and changes from baseline are descriptively summarized at each time point for each cohort of subjects in the safety analysis set. The proportion of subjects with abnormal physical examination findings at each time point is descriptively summarized by physical system for the subjects in the safety analysis set overall and within each cohort.

[0278] The electrocardiogram and vir serology at screening, as well as serum pregnancy at each time point, will be descriptively summarized both overall and within each cohort.

[0279] disposal

[0280] Disciplinary actions for all enrolled subjects are summarized cohort-wise and overall, including the number and percentage of enrolled subjects, the number and percentage of subjects who received each planned dose, and the number of subjects who completed the trial. The number and percentage of subjects who discontinued treatment are summarized overall and by reason. The size of each analysis population is also presented.

[0281] Demographic and other baseline characteristics

[0282] Demographic and baseline characteristic data were descriptively summarized for each cohort of subjects within the safety analysis set.

[0283] Exploratory analysis

[0284] Progression-free survival (PFS) was assessed by RANO (defined as the period from day 0 to the date of death from any cause or the date of progression, whichever came first) and OS (defined as the period from day 0 to the date of death from any cause), and summarized using Kaplan-Meier statistical methods within each cohort and globally. PFS was censored for subjects who were deemed not to have progressed at the time of withdrawal of consent or the last date of progression assessment. For subjects not recorded as having died, OS was censored at the time of withdrawal of consent or the last date on which the subject was confirmed to be alive. Progression-free survival and OS were analyzed in mITT and per-protocol populations.

[0285] Exploratory tumor genetic and / or biomarker responses were presented as changes from baseline in the mITT population and per-protocol population, using descriptive statistics at each time point.

[0286] OS18, PFS, and OS were modeled for exploratory responses and their associations were examined using logistic regression and Cox PH models. Baseline variables such as patient demographics or patient disease characteristics were included in the models as potential confusion factors. Cellular and humoral immune responses were used separately as explanatory variables.

[0287] result

[0288] The demographics of the registered patients are shown in Figure 2 (Demographic Table). Evaluation of tumor-derived gene transcripts at the time of diagnosis confirmed the expression of antigens encoded by INO-5401 and diverse immunogene profiles. Of the 47 subjects with tissue available for evaluation, 5 (811%) showed transcriptional expression of one or more tumor-associated antigens (WT1, PSMA, and hTERT) encoded by INO-5401. Of the 47 subjects with tissue available for evaluation, 43 (89%) showed transcriptional expression of two or more tumor-associated antigens (WT1, PSMA, and hTERT) encoded by INO-5401. Of the 47 subjects, 19 (40%) showed transcriptional expression of all three tumor-associated antigens. No subjects showed PD-1 expression without PD-L1 expression. Of the 47 subjects, 27 (57%) showed PD-L1 expression without PD-1 expression. Of the 47 subjects, 20 (43%) showed co-expression of PD-1 and PD-L1. A normalized transcript read count > 1 was considered "positive".

[0289] MRI images

[0290] Some patients experience pseudo-progression, with radiographic evidence of progression on MRI but no evidence of tumor on repeated biopsies. Images from exemplary patients show increased MRI signaling at time after initial administration of INO-5401 + INO-9012 and cemiprimab-rwlc, suggesting edema or tumor. Biopsies from some patients have shown treatment-related changes with necrosis and mixed inflammation, absence of mitotic activity, and no evidence of viable tumor. Representative images from two patients are shown in Figure 3.

[0291] ELISpot

[0292] ELISpot was used to qualitatively measure the presence of antigen-specific T cells in peripheral blood mononuclear cell (PBMC) samples. PBMCs were collected from subjects at weeks 0, 3, 6, 9, 12, and 24 of the study and assayed with IFN-g ELISpot. At the 12-month data cutoff, the antigen-specific IFNg spot-forming units (SFUs) per million PBMCs are shown from the highest pre-treatment (pre) and post-treatment (peak) values ​​for INO-5401 and semiprimab-rwlc from eight subjects with samples up to week 24 (Figure 4). Each subject is represented by a white circle, and the bar represents the mean value. The graph for each antigen, as well as the difference between pre-treatment and the peak (delta), is shown for the 11 subjects assayed together and the 8 subjects with samples available up to week 24. INO-5401 is the sum of WT1, PSMA, and hTERT. The box plot extends from the 25th to the 75th percentile, with a horizontal line at the median and a "+" at the mean. ELISpot results support that the INO-5401 and semiprimab-rwlc combination is immunogenic, exhibiting above-baseline IFN-g magnitudes for all three antigens in 5 out of 11 subjects and for at least one antigen in 9 subjects, as shown in Figure 4.

[0293] Evaluation of peripheral immune responses after INO-5401 in cohorts with an 18-month data cutoff revealed antigen-specific T cell responses mediated by interferon-gamma ELISpot (cytokine production in response to each component of INO-5401). The results of the evaluation of peripheral immune responses after INO-5401 using ELISpot in cohorts are provided in Figures 16A and 16B. Baseline values ​​from the peak time after treatment are plotted. In Cohort A, 19 / 22 subjects (86%) tested so far had IFN-g magnitudes above baseline for one or more INO-5401 antigens (Figure 16A). In Cohort B, 16 / 17 subjects (94%) tested so far had IFN-g magnitudes above baseline for one or more INO-5401 antigens (Figure 16B).

[0294] Soluble granule loading

[0295] A lysic granule loading assay was performed to explore the activation status and lysis potential of antigen-specific T cells present in PBMC samples collected from subjects during weeks 0, 3, 6, 9, 12, and 24 of the study. PBMCs were stimulated with overlapping peptide libraries for INO-5401 antigens (hTERT, PSMA, and WT1) or relevant controls in the absence of any exogenous cytokines. After 5 days, cells were stained with antibodies and evaluated by flow cytometry. The frequency of live, antigen-specific, activated (CD38+) CD3+CD8+ T cells with lysis potential (expressing granzyme A, perforin) is shown for each antigen, pre-treatment (before) and post-treatment (peak) values ​​from 8 subjects with INO-5401 and semiprimab-rwlc at baseline (Figure 5A). Each subject is represented by a white circle, and the bar represents the mean value. For each antigen at the 12-month data cutoff, as well as for INO-5401, the difference between baseline and peak, and the delta, are shown for the 8 subjects assayed (Figure 5B) and 5 out of 8 subjects with available samples up to week 12 (Figure 5C). INO-5401 is the sum of WT1, PSMA, and hTERT. The box plots extend from the 25th to the 75th percentile, with a horizontal line at the median and a "+" at the mean. Five subjects had a frequency of activated CD8+ T cells with a lysis potential (CD38+Prf+GrzA+) above baseline for more than one antigen, and three subjects had a frequency of activated CD8+ T cells with a lysis potential (CD38+Prf+GrzA+) above baseline for all three antigens. Three subjects did not show an above-baseline response to any antigen at any time point.

[0296] The evaluation of peripheral immune responses to INO-5401 by cohort at the 18-month data cutoff revealed antigen-specific T cell responses by flow cytometry (magnification of antigen-specific CD8+ T cells with lysis potential). In Cohort A, 13 out of 19 subjects (68%) tested so far had a frequency of CD38+GrzA+Prf+CD8+ T cells above baseline for one or more INO-5401 antigens (Figure 17A). In Cohort B, 8 out of 10 subjects (80%) tested so far had a frequency of CD38+GrzA+Prf+CD8+ T cells above baseline for one or more INO-5401 antigens (Figure 17B). Samples were collected four times at week Q3, followed by week Q12. Baseline values ​​from the peak time after treatment are plotted.

[0297] Progression-free survival

[0298] Figure 6 shows a visual representation of the Kaplan-Meier estimate of 6-month progression-free survival (PFS6) for Cohort A, patients with the O6-methylguanine methyltransferase gene promoter in tumor cells. The curve shows the probability of an event at a given time interval. The probability of an event is represented numerically on the y-axis, and the time interval is represented on the x-axis. The event shown is progression-free survival. Progression-free survival is the absence of disease progression in a given subject at a given time point in time.

[0299] Figure 7 shows a visual representation of the Kaplan-Meier estimate of 6-month progression-free survival (PFS6) for Cohort B, patients with the O6-methylguanine methyltransferase gene promoter methylated in tumor cells. The curve shows the probability of an event at a given time interval. The probability of an event is represented numerically on the y-axis, and the time interval is represented on the x-axis. The event shown is progression-free survival. Progression-free survival is the absence of disease progression at a given time point for a given subject.

[0300] Figure 8 shows a visual representation of Kaplan-Meier estimates of 6-month progression-free survival (PFS6) for Cohorts A and B, which are patients with unmethylated or methylated O6-methylguanine methyltransferase gene promoters in tumor cells. The curves show the probability of an event at a given time interval. The probability of an event is represented numerically on the y-axis, and the time interval is represented on the x-axis. The event shown is progression-free survival. Progression-free survival is the absence of disease progression at a given time point for a given subject.

[0301] Figure 9 shows a visual representation of the Kaplan-Meier estimates of 6-month progression-free survival (PFS6) for cohort A, cohort B, and both cohorts combined. The total number of subjects per cohort, the number of events, the estimated number of events (PFS6), and the 95% confidence interval (CI) for which the numerical estimate of events (PFS6) exists are all provided.

[0302] Confirmed progressive disease (PD) is determined by confirmation through consecutive PD scans at least four weeks after the original PD event, or by confirmation of progression in response to biopsy. Subjects who terminated the study for any reason six months prior to the original PD event, including two subjects from Cohort B who dropped out at week 3, were included as confirmed progressive events and refused long-term follow-up.

[0303] overall survival

[0304] All efficacy analyses (OS12, OS18, and Kaplan-Meier) were analyzed for subjects in the modified intention-to-treatment (mITT) population, defined as all subjects who received at least one dose of the planned treatment. Overall survival at 12 months (OS12) was expressed as the proportion of subjects who were still alive at 12 months out of all subjects at risk of death at study baseline. Subjects who dropped out were considered failures (i.e., deceased). All subjects in Cohort A who dropped out before 12 months also died before the 12-month follow-up. 95% confidence intervals (CI) are calculated using the Clopper-Pearson exact confidence interval method. Overall survival at 18 months (OS18) was expressed as the proportion of subjects who were still alive at 18 months out of all subjects at risk of death at study baseline. 95% confidence intervals (CI) are calculated using the Clopper-Pearson exact confidence interval method.

[0305] Figure 10A shows a visual representation of the Kaplan-Meier estimate of overall survival over 12 months for Cohort A, patients with a non-methylated O6-methylguanine methyltransferase gene promoter in tumor cells. The stepped curves show the probability of surviving to and beyond a specific point in time. Survival probabilities are represented numerically on the y-axis, and survival time is represented in days on the x-axis. Figure 10B shows a visual representation of the Kaplan-Meier estimate of overall survival over 18 months for Cohort A, patients with a non-methylated O6-methylguanine methyltransferase gene promoter in tumor cells. The stepped curves show the probability of surviving to and beyond a specific point in time. Survival probabilities are represented numerically on the y-axis, and survival time is represented in days on the x-axis. The median follow-up period in Cohort A is 17.8 months. mITT includes any subjects who received one or more study doses of the therapy. Shading represents the confidence interval for the point estimate of survival at that point in time.

[0306] Figure 11A shows a visual representation of the Kaplan-Meier estimate of overall survival over 12 months for Cohort B, patients with the O6-methylguanine methyltransferase gene promoter methylated in tumor cells. The stepped curve shows the probability of surviving to and beyond a specific point in time. Survival probabilities are represented numerically on the y-axis, and survival time is represented in days on the x-axis. Figure 11B shows a visual representation of the Kaplan-Meier estimate of overall survival over 18 months for Cohort B, patients with the O6-methylguanine methyltransferase gene promoter methylated in tumor cells. The stepped curve shows the probability of surviving to and beyond a specific point in time. Survival probabilities are represented numerically on the y-axis, and survival time is represented in days on the x-axis. The median follow-up period in Cohort B was 15.6 months. Censored, two subjects in Cohort B withdrew their consent for follow-up at week 3. mITT includes any subjects who received one or more study doses of the therapy. The shading represents the confidence interval for the point estimate of survival at that point in time.

[0307] Figure 12 shows a visual representation of the Kaplan-Meier estimates of overall survival probability over 12 months for the combined cohorts A and B. The stepped curves represent the probability of surviving up to and beyond a specific point in time. Survival probabilities are represented numerically on the y-axis, and survival time is represented in days on the x-axis.

[0308] Figure 13 shows efficacy data for overall survival at 12 and 18 months for cohorts A, B, and combined cohorts. The figure shows the total number of subjects who reported survival at 12 and 18 months. The total number of subjects, the estimated event (OS12 or OS18), and the 95% confidence interval (CI) for which the numerical estimate of the event (OS12 or OS18) exists are all provided. The 95% CI is calculated using the Clopper-Pearson exact confidence interval method.

[0309] Safety data

[0310] Safety data were compiled from subjects who were members of the safety analysis population, defined as having received at least one dose of the investigational drug (IP).

[0311] Figure 14 shows all adverse events defined by the clinical trial protocol ≥ NCI CTCAE Grade 3. Figure 15 shows immune-related adverse events defined by the clinical trial protocol.

[0312] The most common grade ≥3 adverse events reported in the subjects were decreased platelet count (11.5%), decreased lymphocyte count (11.5%), tumor inflammation (7.7%), seizures (7.7%), and elevated ALT (7.7%). One grade 5 unrelated event, urinary tract sepsis, was reported. Only one SAE associated with INO-5401+INO-9012 was reported: fever. 48% of subjects reported irAEs, the most frequent being elevated ALT (9.6%), elevated AST (7.7%), diarrhea (7.7%), fever (7.7%), and tumor inflammation (7.7%). 71% of reported SAEs and irAEs occurred within the first 12 weeks of treatment.

[0313] conclusion

[0314] In newly diagnosed GBM patients, INO-5401 + INO-9012, in combination with radiation and cemiplimab-rwlc administered with temozolomide, has an acceptable safety profile, is immunogenic, and may be effective in newly diagnosed GBM patients. Common adverse events (AEs) included injection-site events, with grade ≥3 AEs primarily attributable to TMZ or radiation, and immune-related AEs were consistent with the profile of cemiplimab-rwlc. SAEs were consistent with SAEs observed in patients with GBM (seizures).

[0315] Antigen-specific T cell responses were observed in nearly all patients tested to date for one or more antigens included in INO-5401. PFS6 was higher than that of previous controls in this study, regardless of the presence or absence of MGMT promotermethylation, and OS12 was higher than that of previous controls in patients without MGMT promotermethylation [Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005, 352:987-996].

[0316] The above-mentioned detailed description and accompanying examples are merely illustrative and should not be construed as limiting the scope of the invention. The scope of the invention is defined solely by the appended claims and their equivalents.

[0317] Various changes and modifications to embodiments of this disclosure will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those relating to the chemical structure, substituents, derivatives, intermediates, compounds, compositions, formulations, or methods of use of the present invention, may be made without departing from the spirit and scope thereof.

[0318] Sequence List Sequence ID 1 R2810 HCVR [ka] Sequence ID 2 R2810 LCVR [ka] Sequence ID 3 R2810 HCDR1 [ka] Sequence ID 4 R2810 HCDR2 [ka] Sequence ID 5 R2810 HCDR3 [ka] Sequence ID 6 R2810 LCDR1 [ka] Sequence ID 7 R2810 LCDR2 [ka] Sequence ID 8 R2810 LCDR3 [ka] Sequence ID 9 R2810 HC [ka] [ka] Sequence ID 10 R2810 LC [ka] Sequence ID 11: PSMA lacking an IgE reader sequence but possessing an HA tag. [ka] PSMA having sequence number 12 IgE reader sequence (underlined) and HA tag [ka] [ka] Sequence ID 13: PSMA without IgE reader sequence [ka] [ka] PSMA containing sequence number 14 IgE reader sequence (underlined) [ka] [ka] Sequence ID 15: Consensus WT1-L with modified zinc finger nucleic acid sequence and IgE reader. [ka] Sequence ID 16: Con WT1-L protein having an IgE reader (underlined) in addition to a modified zinc finger protein sequence. [ka] [ka] Sequence ID No. 17: Synthetic consensus hTERT nucleic acid sequence linked to IgE (underlined) [ka] [ka] Sequence ID No. 18: Synthetic consensus hTERT amino acid sequence linked to IgE for action (underlined) [ka] [ka] Sequence ID 19: Synthetic Consensus hTERT Nucleic Acid Sequence [ka] [ka] Sequence ID No. 20 Synthetic Consensus hTERT Amino Acid Sequence [ka] Sequence ID 21: Nucleic acid sequence encoding PSMA with an IgE reader sequence. [ka] [ka] Nucleic acid sequence encoding SEQ ID NO: 22 IL12 p35 (pGX6001) [ka] Sequence ID 23 IL12 p35 amino acid sequence (pGX6001) [ka] Nucleic acid sequence encoding SEQ ID NO: 24 IL12 p40 (pGX6001) [ka] Sequence ID 25 IL12 p40 amino acid sequence (pGX6001) [ka] Sequence ID No. 26: Con WT1-L with modified zinc finger protein [ka] Sequence ID No. 27: Consensus WT1-L with modified zinc finger nucleic acid sequence. [ka] [ka] Sequence ID 28 PSMA [ka] Sequence ID 29: Nucleic acid sequence encoding PSMA [ka]

Claims

1. A vaccine comprising an immunogenic composition comprising a nucleic acid sequence encoding a human telomerase reverse transcriptase (hTERT) antigen including SEQ ID NO: 20, and a DNA plasmid comprising a nucleic acid sequence including SEQ ID NO: 19; a nucleic acid sequence encoding a Wilms tumor-1 (WT-1) antigen including SEQ ID NO: 26, and a DNA plasmid comprising a nucleic acid sequence including SEQ ID NO: 27; and a nucleic acid sequence encoding a prostate-specific membrane antigen (PSMA) including SEQ ID NO: 28, and a DNA plasmid comprising a nucleic acid sequence including SEQ ID NO:

29. A vaccine for use in combination with cemiplimab, a DNA plasmid containing nucleic acid sequences encoding the IL-12 p35 subunit and the IL-12 p40 subunit, one or more doses of radiotherapy, and one or more doses of chemotherapeutic agents, in the treatment of newly diagnosed glioblastoma in subjects.

2. The IL12 p35 subunit comprises the amino acid sequence of Sequence ID No. 23; The IL12 p40 subunit comprises the amino acid sequence of Sequence ID No. 25; The IL12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 23, and the IL12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 25; The nucleic acid sequence encoding the IL12 p35 subunit includes the amino acid sequence of SEQ ID NO: 22; The nucleic acid sequence encoding the IL12 p40 subunit includes the amino acid sequence of SEQ ID NO: 24; or The nucleic acid sequence encoding the IL12 p35 subunit includes the amino acid sequence of SEQ ID NO: 22, and the nucleic acid sequence encoding the IL12 p40 subunit includes the amino acid sequence of SEQ ID NO:

24. A vaccine for use as described in claim 1.

3. The vaccine or vaccine for use according to claim 1 or 2, comprising 3 mg of a DNA plasmid encoding the hTERT antigen, 3 mg of a DNA plasmid encoding the PSMA antigen, 3 mg of a DNA plasmid encoding the WT-1 antigen, and 1 mg of a DNA plasmid containing nucleic acid sequences encoding the IL12 p35 subunit and the IL12 p40 subunit.

4. The immunogenic composition is administered by intramuscular injection in four doses every three weeks, and then every nine weeks. A vaccine for use according to claim 2 or 3, co-administered with a DNA plasmid containing nucleic acid sequences encoding IL12 p35 subunit and IL12 p40 subunit.

5. The vaccine for use according to claim 4, further comprising electroporation after each intramuscular injection.

6. The vaccine for use according to any one of claims 1 to 5, wherein the glioblastoma is characterized by a non-methylated O6-methylguanine methyltransferase (MGMT) gene promoter or a methylated MGMT promoter.

7. The vaccine for use according to any one of claims 1 to 6, wherein the semiprimab is formulated for intravenous administration.

8. A vaccine for use according to any one of claims 1 to 7, wherein 350 mg of the semiprimab is formulated for administration every three weeks.

9. The vaccine or vaccine for use according to any one of claims 1 to 8, wherein the immunogenic composition is formulated for intramuscular injection.

10. The vaccine for use according to claim 9, wherein the vaccine is formulated to be administered in four doses every three weeks, and then every nine weeks.

11. The vaccine for use according to claim 9, wherein the vaccine is formulated for administration by intramuscular injection by electroporation.

12. A vaccine for use according to claims 1 to 11, wherein each dose of radiotherapy comprises 20 to 50 Gy.

13. The vaccine for use according to claims 1 to 12, wherein the radiotherapy is fractionated radiotherapy.

14. The vaccine for use according to claim 13, wherein the fractionated radiotherapy comprises 2 to 20 fractions.

15. The vaccine for use according to claim 14, wherein the fractionated radiotherapy comprises 40 Gy in 15 fractions.

16. The vaccine for use according to claim 15, wherein the fractionated radiotherapy is administered for 21 consecutive days.

17. The vaccine for use according to any one of claims 1 to 16, wherein the chemotherapeutic agent is temozolomide.

18. The chemotherapeutic agent is administered at a dose of 75 mg / m² per day for 21 consecutive days. ２ A vaccine for use according to claim 17, wherein temozolomide.

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