Compositions and methods for using antigen-specific apoptotic DNA immunotherapy to prevent and treat side effects caused by the administration of immune checkpoint inhibitors.

Abstract

This disclosure provides therapeutic compositions comprising immune checkpoint inhibitors and methods for using such compositions to prevent or treat adverse events resulting from apoptotic DNA immunotherapy and administration of immune checkpoint inhibitors.

Description

[Technical Field]

[0001] background Autoimmune diseases arise from an immune response to self-antigens, while cancer develops when the immune system fails to respond to malignant cells. For many years, autoimmunity and cancer were considered two separate research areas with little in common. However, the discovery of immune checkpoints and the development of immune checkpoint inhibitors (ICIs) targeting pathways such as programmed cell death receptor (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) have revealed that studying autoimmune diseases can be extremely useful in developing novel anticancer drugs and improved treatments. Thus, autoimmunity and cancer may be two sides of the same coin. (Sakowska J, Arcimowicz L, Jankowiak M, et al. Autoimmunity and Cancer—Two Sides of the Same Coin. Front Immunol. 2022;13:793234. Published May 13, 2022. doi:10.3389 / fimmu.2022.793234.)

[0002] The use of ICIs to treat cancer is known to present a risk of the emergence of pre-existing autoimmune disorders and the development of novel autoimmune manifestations in patients with pre-existing autoimmunity (even if this manifests only biologically, e.g., as positive autoantibodies). For this reason, patients with pre-existing autoimmune manifestations have traditionally been excluded from clinical trials if they are expected to have a severe autoimmune manifestation that may outweigh the potential benefits of tumor control. This subgroup of cancer patients with autoimmune manifestations is not insignificant. See Coureau M, Meert AP, Berghmans T, Grigoriu B. Efficacy and Toxicity of Immune-Checkpoint Inhibitors in Patients with Preexisting Autoimmune Disorders. Front Med (Lausanne). 2020 May 7;7:137. doi: 10.3389 / fmed.2020.00137. PMID: 32457912; PMCID: PMC7220995. For example, depending on the definition used, a large register-based analysis (SEER) including patients with lung cancer identified 13.5% (more restrictive definition) to 24.6% (more flexible definition) of cancer patients with any type of autoimmune disease. (Ibid., internal citations removed).

[0003] Cancer treatment with intracytoplasmic receptive infusions (ICIs) has been linked to the development of type 1 diabetes mellitus (T1DM) in some patients. T1DM is an autoimmune disease in which insulin-producing B cells in the pancreatic islets are destroyed by an autoimmune attack regulated by autoantigen-specific polyclonal T lymphocytes that have escaped the control of immune tolerance. T1DM is a rare but potentially life-threatening and irreversible immune-related adverse event that occurs in 0.6–1.4% of patients who receive ICIs. Xuan Chen, Alison H. Affinati, Yungchun Lee, Adina F. Turcu, Norah Lynn Henry, Elena Schiopu, Angel Qin, Megan Othus, Dan Clauw, Nithya Ramnath, Lili Zhao; Immune Checkpoint Inhibitors and Risk of Type 1 Diabetes. Diabetes Care 1 May 2022; 45 (5): 1170-1176. doi.org / 10.2337 / dc21-2213.

[0004] The field of immunotherapy addresses lost tolerance processes with vaccine-like immunotherapies that avoid the undesirable effects inherent in broad-acting immunosuppressive therapies. A promising category of immunotherapy utilizes apoptosis, a natural cell death process that is a native non-inflammatory tolerance-inducing pathway. Antigen-presenting cells (APCs), such as dendritic cells (DCs), become immune-tolerating after engulfing apoptotic cells; this allows for the presentation of processed apoptotic cell autoantigens (without co-stimulation) to regulatory T cells (Tregs) for stimulation or to autoreactive memory effector T cells (Teffs) for inactivation.

[0005] Developing and building our improved understanding of how immune responses are modified in autoimmunity and cancer is crucial for the appropriate design of novel, selective immunotherapies. There is a need for novel approaches to mitigate undesirable side effects resulting from the use of ICIs, as well as for better applying available existing treatments to patients with pre-existing autoimmune diseases and / or those developing novel autoimmune manifestations in response to ICIs. Specifically, the use of novel therapies that promote antigen-specific immune tolerance to combat harmful autoimmune responses, particularly in individuals receiving or potentially receiving ICIs for cancer treatment, may be used to prevent the development of ICI-related adverse immunological side effects, complement, support, maintain or extend the continuity of treatment, and / or significantly reduce potential risks and adverse side effects associated with ICI therapy. [Brief explanation of the drawing]

[0006] Brief explanation of the drawing [Figure 1] Figure 1 shows the initial study design and time course for the studies described in Example 1 and Example 2. However, the study design was modified in that the weekly administration (QW) of the test substance (TA) was stopped at 5 weeks instead of 8 weeks. Eight-week-old female NOD / ShiLtj mice (commonly referred to as NOD mice) providing a polygene model of autoimmune type 1 diabetes (T1D) and characterized by hyperglycemia and insulinitis were obtained and acclimatized for 1 week. After random assignment of 10(10) mice to one of four groups, each animal was given either an anti-PD1 antibody or IgG as a negative control to be started on day 0 (D0). Treatment with vehicle control or ADI-100 was started on day 0 (D0) and administered once a week for several weeks. Blood glucose levels were monitored during treatment administration and for several weeks after treatment discontinuation. The initial study design was scheduled to end 84 days (D84) or 12(12) weeks after the start of treatment on day 0 (D0). [Figure 2]Figures 2A, 2B, 2C, and 2D provide preliminary data in the form of absolute glucose readings obtained from test subjects included in the study shown in Figure 1 and described in Example 1. Control group 1 mice (subject numbers 203, 204, 207, 212, 216, 220, 226, 233, 239, and 244, also referred to as 003, 004, 007, 012, 016, 020, 026, 033, 039, and 044 in later figures) (Figure 2A) showed relatively stable blood glucose levels over a 25-day observation period, with absolute blood glucose measurements in the range of approximately 80 mg / dL to 160 mg / dL. Most of the mice in vehicle group 2 that received anti-PD1 antibody and vehicle (9 / 10) (Figure 2B) (subject numbers 202, 208, 209, 215, 217, 218, 232, 234, 241, and 248, also referred to as 002, 008, 009, 015, 017, 018, 032, 034, 041, and 048 in later figures) showed clear hyperglycemia within 11 days. Most (7 / 10) (Figure 2C) mice treated with low-dose ADI-100 (1 μg / μL with a BAX / msGAD ratio of 1:2 (BAX 17 μg + msGAD 33 μg)) in group 3 showed hyperglycemia within the first 8 days. (Subject numbers 201, 219, 224, 227, 229, 230, 231, 235, 237, and 243, also referred to as 002, 001, 019, 024, 027, 029, 030, 031, 035, 037, and 043 in later figures) In contrast, seven out of ten mice (7 / 10) (Figure 2D) treated with high-dose ADI-100 (2 μg / μL (BAX 34 μg + msGAD 66 μg) in a 1:2 BAX / msGAD ratio) in group 4 showed normal blood glucose levels at the end of the 25-day observation period. These data support the efficacy of ADI-100 in controlling the effects of anti-PD1 antibodies that promote autoimmune diabetes in NOD mice. These data also demonstrate that the efficacy of ADI-100 is dose-dependent. [Figure 3]Figures 3A, 3B, 3C, and 3D provide preliminary data in the form of log-glucose readings obtained from test subjects included in the tests generally shown in Figure 1 and described in Example 2. Nine out of ten mice (9 / 10) in control group 1 (Figure 3A) showed relatively stable blood glucose levels during the 45-day observation period, most mice (9 / 10) in vehicle group 2 that received anti-PD1 antibody and vehicle (Figure 3B) showed obvious hyperglycemia within 11 days, and one mouse (mouse 018) did not. Most mice (7 / 10) in group 3 treated with a low-dose ADI-100 (Figure 3C) also showed hyperglycemia within the first eight days, and some mice (3 / 10) showed normal blood glucose levels during the 45-day observation period. In contrast, 7 out of 10 mice treated with high-dose ADI-100 in group 4 (7 / 10) (Figure 3D) showed normal blood glucose levels during the 45-day observation period. These data support the efficacy of ADI-100 in controlling the effects of anti-PD-1 antibodies in promoting autoimmune diabetes in NOD mice. These data also demonstrate that the efficacy of ADI-100 is dose-dependent and can be extended for at least 6(6) weeks. [Figure 4] Figures 4A, 4B, 4C, and 4D provide preliminary data in the form of absolute glucose readings obtained from test subjects included in the tests generally shown in Figure 1, described in Example 2, and corresponding to Figures 3A, 3B, 3C, and 3D. [Figure 5]Figure 5 shows the revised and extended study design and time course for the studies shown in Figure 1 and described in Examples 1 and 2, to investigate the effectiveness of resumed treatment to restore any mice showing evidence of ADI-100 tolerance and further evidence of loss of tolerance. This extended study and its results are further described in Example 3. The change in study design is shown on day 29 (D29) to indicate that administration was stopped for all groups after the fifth dose on day 28 (D28), instead of being carried out weekly for 8 (8) weeks and ending the study on day 84 (D84). As shown in Figure 5, only the remaining control group 1 mice and the remaining high-dose ADI-100 group 4 mice continued the Phase 2 extension after day 84 (D84) of the study. [Figure 6] Figures 6A and 6B provide data for the extension study generally shown in Figure 5 and described in Example 3. Figure 6A shows absolute glucose readings obtained from 7(7) control group mice remaining at day 84 (D84) of the study. Note that of these remaining 7(7) mice, 4(4) died before day 112 (D112) of the study, and only 3(3) survived to day 301 (D301) of the study. Thus, Figure 6A shows that 70% of the control mice developed diabetes 14 weeks prior. Figure 6B shows absolute glucose readings obtained from 7(7) high-dose ADI-100 group mice remaining at day 84 (D84) of the study. Note that of these remaining 7(7) high-dose ADI-100 mice, 1(1) (subject number 247) died around day 196 (D196) of the study, while the remaining 6(6) survived to day 301 (D301) of the study. Also note that 2(2) mice (subject numbers 214 and 247) showed evidence of loss of tolerance in the form of elevated absolute glucose readings. Of these two mice, one (subject number 214) was successfully treated and recovered, and the resumed treatment resulted in a decrease or normalization of absolute glucose readings. [Figure 7]Figures 7A, 7B, and 7C show the results of a study conducted in a Hepa1-6 liver model using C57BL / 6 mice to test the antitumor efficacy of anti-PD-1 in combination with vehicle alone, anti-PD-1 alone, and high-dose ADI-100 test substance (2 μg / μL with a BAX / msGAD ratio of 1:2 (BAX 34 μg + msGAD 66 μg)). Anti-PD-1 is reported to be highly effective in the Hepa1-6 mouse tumor model. Thirty (30) mice were enrolled in the study. All animals were randomly assigned to three (3) different test groups of 10 mice per group. Figure 7A shows tumor volume measurements (mm3) over time (days in the study) with mean absolute tumor volume ± standard error of measurement (SEM). Figure 7B shows body weight measurements (g) over time (days in the study) as mean absolute body weight ± SEM. Figure 7C shows the percentage change in body weight over time (number of days in the study) as mean percentage change ± SEM. [Modes for carrying out the invention]

[0007] Detailed explanation Overview The use of checkpoint inhibitors to treat cancer presents a risk of sudden onset of pre-existing autoimmune disorders and the development of new autoimmune manifestations in patients with pre-existing autoimmunity, even if it exists only as a biological expression (e.g., as a positive autoantibody), or in patients without known autoimmune disorders.

[0008] Disclosed herein are antigen-specific treatments using antigen-specific nucleoside apoptotic DNA immunotherapy technology to counter actual or potential onsets of autoimmune disease and novel autoimmune manifestations. Administration of this antigen-specific treatment using nucleoside apoptotic DNA immunotherapy technology is intended to result in targeted upregulation of regulatory T-cell therapy, addressing specific aspects of unhelpful autoimmune responses induced by immune checkpoint drug therapy without impairing the tumor-killing activity of other effector T cells. Treated patients or subjects are intended to be tested before, during, or after treatment to assess actual or potential onsets of autoimmune disease and novel autoimmune manifestations. Such testing may facilitate optimal patient selection, optimal dose selection, and / or optimal administration regimen selection.

[0009] Antigen-specific treatment of possible or actual ICI adverse effects Immunotherapy using ICIs has opened the door to novel approaches for treating certain types of cancer by enabling effector T cells to discover and destroy otherwise undetectable tumor cells. However, it is well known that the use of checkpoint inhibitors has broad effects, as the removal of the "brakes" from these T cells is not tumor-specific, and can lead to immunological adverse effects. For example, a nonspecific increase in effector T cell activity can lead to autoimmunity in certain individuals receiving these novel treatments. TM Antigen-specific treatment of these possible or actual ICI adverse effects using ) or ADI-100 as described herein may result in targeted upregulation of regulatory T cells, thereby addressing certain aspects of a useless autoimmune response without impairing the tumor-killing activity of effector T cells.

[0010] Novel compositions and methods for preventing, mitigating, reducing, decreasing, restoring, reversing, or eliminating inappropriate antigen-specific or autoimmune-inducing side effects resulting from the use of ICIs are described herein. The compositions and methods described herein are extrapolate and constructed based on previously described compositions and methods for treating or reversing hyperglycemia and suppressing the onset of diabetes in patients at risk of developing T1DM by administering a nucleic acid technology antigen-specific apoptotic DNA immunotherapy vector system comprising (a) a first expression cassette encoding a BCL2-related X apoptotic regulator (BAX); and (b) a permethylated second expression cassette encoding a secreted form of glutamate decarboxylase 65 (e.g., sGAD55).

[0011] It should be noted that varying the degree of methylation can be achieved, for example, by using bacterial or enzymatic methylation. Enzymatic methylation can be achieved, for example, using the methods and techniques disclosed in the published international patent application WO 2023034727A1, titled Enzymatically methylated DNA and methods of production and therapeutic use.

[0012] When this apoptotic DNA immunotherapy is administered to patients, the therapy can induce an immune tolerance-inducing response, resulting in an increase in immune tolerance-inducing dendritic cell populations and an increase in the number of GAD-specific regulatory T cells in the influx region lymph nodes. Specifically, this immunotherapy and ADI-100 have been shown to be effective in reversing hyperglycemia and suppressing the onset of type 1 diabetes in non-obese diabetic (NOD) mice. (Alleva DG, Rezaee M, Yip L, Ren G, Rosenberg J, Concepcion W, Escher A, Shabahang S, Thakor AS. Reversal of Hyperglycemia and Suppression of Type 1 Diabetes in the NOD Mouse with Apoptotic DNA Immunotherapy) TM (ADi TM ), ADi-100. Biomedicines. 2020 Mar 4;8(3):53. doi: 10.3390 / biomedicines8030053.

[0013] Without interference, NOD / ShiLtJ mice are characterized by hyperglycemia and insulinitis, and islet lymphocyte infiltration, typically developing diabetes by 30 weeks of age (86% of females; 48% of males), with a median age of onset at 18 weeks in females. Diabetes in NOD mice is characterized by hyperglycemia and insulinitis, and islet lymphocyte infiltration. A marked decrease in pancreatic insulin content occurs around 12 weeks of age in females and several weeks later in males. The immunophenotype in NOD background consists of deficiencies in antigen presentation, T lymphocyte repertoire, NK cell function, macrophage cytokine production, wound healing, and C5 complement. These deficiencies make NOD background a common choice for immunodeficient mouse strains. See The Jackson Laboratory website, available at: 001976 - NOD Strain Details (jax.org).

[0014] The promotion of diabetic status in the NOD / ShiLtJ mouse model can be achieved by inhibiting PD1-PDL1 signaling in NOD mice to promote the onset of type 1 diabetes, indicating the involvement of this pathway in suppressing the emergence of pancreatic β-cell-reactive T cells. Kochupurakkal NM, Kruger AJ, Tripathi S, Zhu B, Adams LT, Rainbow DB, Rossini A, Greiner DL, Sayegh MH, Wicker LS, Guleria I. Blockade of the programmed death-1 (PD1) pathway undermines potent genetic protection from type 1 diabetes. PLoS One. 2014 Feb 28;9(2):e89561. doi: 10.1371 / journal.pone.0089561. PMID: 24586872; PMCID: PMC3938467. In the NOD mouse model, it was determined that anti-PD-L1, rather than anti-CTLA-4, rapidly induced diabetes.Perdigoto AL, Deng S, Du KC, Kuchroo M, Burkhardt DB, Tong A, Israel G, Robert ME, Weisberg SP, Kirkiles-Smith N, Stamatouli AM, Kluger HM, Quandt Z, Young A, Yang ML, Mamula MJ, Pober JS, Anderson MS, Krishnaswamy S, Herold KC. Immune cells and their inflammatory mediators modify β cells and cause checkpoint inhibitor-induced diabetes. JCI Insight 2022;7(17):e156330 https: / / doi.org / 10.1172 / jci.insight.156330. Furthermore, in these enhanced NOD mouse models, it was found that ICIs targeting the PD-1 / PD-L1 pathway resulted in transcriptional changes in β cells and immune infiltration that could lead to the development of diabetes, and that immune cells and their inflammatory mediators modified β cells to cause ICI-induced diabetes. Ibid. Treatment with anti-IFNγ and anti-TNFα was found to prevent TIDM in anti-PD-L1 treated NOD mice, suggesting that inhibition of inflammatory cytokines may be a clinically applicable strategy for preventing this complication. Ibid.

[0015] First, as described herein, the inventors demonstrate that undesirable immunological side effects resulting from the use of ICIs can be effectively blocked by the use of antigen-specific apoptotic DNA immunotherapy. Furthermore, the use of antigen-specific apoptotic DNA immunotherapy can be used to prevent, mitigate, reduce, decrease, restore, reverse, or eliminate inappropriate antigen-specific side effects resulting from the use of ICIs. Specifically, the development of T1DM is shown to be blocked in a regulated polygene NOD / ShiLtJ mouse model for autoimmune type 1 diabetes, including both anti-PD1 antibodies and antigen-specific apoptotic DNA immunotherapy.

[0016] First, as described herein, the inventors demonstrate that the use of antigen-specific apoptotic DNA immunotherapy does not interfere with the efficacy of ICI. Specifically, the antitumor efficacy of antigen-specific apoptotic DNA immunotherapy is tested in the C57BL / 6 mouse Hepa1-6 model, which has been reported to be highly effective in the Hepa1-6 mouse tumor model. Specifically and as further described herein, the inventors test the antitumor efficacy of anti-PD-1 in combination with the vehicle alone, anti-PD-1 alone, and high-dose ADI-100 test in the Hepa1-6 mouse tumor model. It was found that the anti-PD-1 alone group and the anti-PD-1 + ADI-100 group exhibited tumor inhibition of 80 percent or higher (>80%) compared to the vehicle alone control group. Thus, ADI-100 in the anti-PD-1 + ADI-100 group did not negatively interfere with the efficacy shown by anti-PD-1 compared to the anti-PD-1 alone group. Furthermore, note that ADI-100 showed no signs of toxicity whatsoever.

[0017] Through experiments, ADI-100, which contains two plasmids formulated in a 1:2 ratio of one hypermethylated plasmid encoding BAX and the other encoding the secretory form of GAD (sGAD55), has been shown to address anti-PD1-Ab-promoted autoimmune diabetes in the non-obese diabetic NOD-ShiLtj mouse model of type 1 diabetes. These findings indicate that the undesirable effects of checkpoint immunoinhibitors can be mitigated, reduced, or eliminated by using antigen-specific apoptotic DNA immunotherapy. Antigen-specific apoptotic DNA immunotherapy, here ADI-100, is thought to result in targeted upregulation of regulatory T cells, thereby addressing specific aspects of unwanted autoimmune responses without impairing the tumor-killing activity of effector T cells.

[0018] As described and exemplified herein, ADI-100 is used to address anti-PD1-Ab-promoted autoimmune diabetes in an animal model of type 1 diabetes and has been shown to suppress hyperglycemia and diabetes onset. The current findings are based on previous methods of using ADI-100 to treat type 1 diabetes. Alleva DG, Rezaee M, Yip L, Ren G, Rosenberg J, Concepcion W, Escher A, Shabahang S, Thakor AS. Reversal of Hyperglycemia and Suppression of Type 1 Diabetes in the NOD Mouse with Apoptotic DNA Immunotherapy TM (ADi TM ), ADi-100. Biomedicines. 2020 Mar 4;8(3):53. doi: 10.3390 / biomedicines8030053 and reference to published US patent application US 2024 / 0016905.

[0019] The ADI-100 vector system comprises or consists of (a) a first expression cassette encoding BCL2-related X apoptosis regulator (BAX); and (b) a second hypermethylated expression cassette encoding secreted glutamic acid decarboxylase 65 (e.g., sGAD55), and is administered to a patient to induce an immune tolerance-inducing response, which may include an increase in the population of immune tolerance-inducing dendritic cells in the draining regional lymph nodes and an increase in the number of GAD-specific regulatory T cells, and, as described above, the vector system may be in a pharmaceutically acceptable formulation.

[0020] In one aspect, compositions and methods are provided for preventing or reversing hyperglycemia in a patient at risk of developing type 1 diabetes, the compositions and methods comprising a checkpoint inhibitor, and administering a therapeutically effective amount of a vector system comprising (a) a first expression cassette comprising a polynucleotide encoding BAX; and (b) a hypermethylated second expression cassette comprising a polynucleotide encoding a secreted form of glutamic acid decarboxylase 65 (GAD65), which may be in a pharmaceutically acceptable formulation.

[0021] In another aspect, compositions and methods are provided for suppressing the onset of diabetes in a patient at risk of developing type 1 diabetes, the compositions and methods comprising a checkpoint inhibitor, and administering a therapeutically effective amount of a vector system comprising (a) a first expression cassette comprising a polynucleotide encoding BAX; and (b) a hypermethylated second expression cassette comprising a polynucleotide encoding a secreted form of glutamic acid decarboxylase 65 (GAD65).

[0022] In yet another aspect, a composition and method are provided to increase the number of immune tolerance-inducing dendritic cells and GAD-specific regulatory T cells in patients at risk of developing type 1 diabetes, the composition and method comprising administering an effective amount of a vector system comprising a checkpoint inhibitor and a first expression cassette comprising a polynucleotide encoding BCL2-related X apoptosis regulator (BAX) and a second expression cassette comprising a permethylated polynucleotide encoding the secreted form of glutamate decarboxylase 65 (e.g., sGAD55). In any of the above embodiments, the first expression cassette may further comprise a promoter operably ligated to the polynucleotide encoding BAX, and the second expression cassette may further comprise a promoter operably ligated to a polynucleotide encoding the secreted form of GAD65. In one embodiment, the first expression cassette comprises a CMV promoter or an SV-40 promoter operably ligated to the polynucleotide encoding BAX. In one embodiment, the second expression cassette comprises an SV-40 promoter operably ligated to a polynucleotide encoding the secreted form of GAD65.

[0023] In any of the embodiments described above, the secreted form of GAD65 may be encoded as msGAD55.

[0024] In any of the embodiments described above, the vector system may include (a) a first vector comprising a first expression cassette expressing BAX; and (b) a permethylated second vector comprising a second expression cassette expressing the secreted form of GAD65. In some embodiments, the first and second vectors are administered in a ratio ranging from 1:1 to 1:8, such as any ratio within this range, e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8. In some embodiments, the first and second vectors are administered in a ratio of 1:2.

[0025] In any of the embodiments described above, the patient may have mild, moderate, or severe hyperglycemia. In one embodiment, the patient has severe hyperglycemia, and the first vector and the second vector are administered in a 1:2 ratio.

[0026] In any of the embodiments described above, the patient may have a quantity of insulin-producing pancreatic β-cells less than 50%, less than 60%, less than 70%, or less than 80% of the reference quantity of β-cells for a non-diabetic subject. In some embodiments, the patient has lost any quantity within this range, such as 50% to 80% of β-cells, for example, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of β-cells.

[0027] In any of the embodiments described above, the patient may be human or non-human.

[0028] In another context, a composition and method are provided for increasing the number of immune tolerance-inducing dendritic cells and GAD-specific regulatory T cells in patients at risk of developing type 1 diabetes, the composition and method comprising administering an effective amount of a vector system comprising a checkpoint inhibitor and a first expression cassette comprising a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX) and a second expression cassette comprising a permethylated polynucleotide encoding a secreted form of glutamate decarboxylase 65 (e.g., sGAD55).

[0029] While not limited to ICIs, examples include CTLA-4 (ipilimumab); PD-1 (cimiplimab; nivolumab; pembrolizumab); PD-L1 (atezolizumab; avelumab; durvalumab); LAG-3 (LAG525-IMP701, REGN3767-R3767), also known as CD223, BI 754,091, tebotelimab-MGD013, eftilagimod alpha-IMP321, FS118; TIM-3 (MBG453, Sym023, TSR-022); by-h3, b7-h4 (MGC018, FPA150); A2aR (EOS100850, AB928); CD73 (CPI-006); NKG2A (Monalizumab); PVRIG / PVRL2 (COM701);CEACAM1 (CM24);CEACAM 5 / 6 (NEO-201);FAK (Defactinib);CCL2 / CCR2 (PF-04136309);LIF (MSC-1);CD47 / SIRPα (Hu5F9-G4 (5F9), ALX148, TTI-662, RRx-001);CSF-1, also known as M-CSF / CSF-1R (Lacnotuzumab-MCS110, LY3022855, SNDX-6352, emactuzumab-RG7155), pexidartinib-PLX3397);IL-1 and IL-1R3, also known as IL-1RAP (CAN04, canakinumab-ACZ885);IL-8 Examples include (BMS-986253); SEMA4D (Pepinemab-VX15 / 2503); Ang-2 (Trebananib); CLEVER-1 (FP-1305); Axl (Enapotamab vedotin-EnaV); and those targeting phosphatidylserine (Bavituximab).

[0030] Before describing the compositions, methods, and kits, it is understood that the present invention is not limited to the specific methods or compositions described and is therefore naturally subject to change. Since the scope of the present invention is limited solely by the appended claims, it is also understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them.

[0031] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that the values ​​between the upper and lower limits of such range, up to one-tenth of the lower limit unit, are also specifically disclosed. Each smaller range between any stated values, or any other stated values ​​or values ​​within the stated range or between the stated values, are included in the invention. The upper and lower limits of these smaller ranges may or may not be included in the range, and each range that includes either, either, or both of the limits in a smaller range is also included in the invention and belongs to any specifically excluded limit within the stated range. Where a stated range includes one or both of the limits, the ranges excluding either or both of these limits are also included in the invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, but several potential and preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and describe such methods and / or materials in the context in which such publications are referenced. This disclosure is understood to override any disclosures of the referenced publications to the extent that they are inconsistent.

[0033] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has other components and features that can be readily separated from or combined with features of any of several other embodiments without departing from the scope or spirit of the invention. Any described method may be carried out in the order of the described events or in any other logically possible order.

[0034] When used herein and in the appended claims, it should be noted that the singular forms "a," "an," and "the" refer to multiple objects unless the context explicitly indicates otherwise. Thus, for example, a reference to "a vector" includes multiple such vectors, and a reference to "the cell" includes references to one or more cells.

[0035] The publications discussed herein are provided only for disclosures prior to the filing date of this application. Nothing herein should be construed as an acceptance that the present invention is not granted prior rights to such publications by prior invention. Furthermore, the publication dates provided may differ from the actual publication dates which may need to be independently verified.

[0036] definition "Tolerogenic" refers to the ability to suppress or downmodulate the adaptive immune response.

[0037] The term "immune tolerance-inducing dendritic cells" refers to dendritic cells that have the ability to induce immune tolerance. Immune tolerance-inducing dendritic cells have a low ability to activate effector T cells, but a high ability to induce and activate regulatory T cells.

[0038] When used herein to describe nucleic acid molecules, “recombinant” means a polynucleotide of genomic, cDNA, viral, semi-synthetic, or synthetic origin that, by its origin or manipulation, is not related in nature to all or part of the polynucleotide to which it relates. When the term “recombinant” is used in relation to proteins or polypeptides, it means a polypeptide produced by the expression of a recombinant polynucleotide. Generally, the gene of interest is cloned and then expressed in a transformed organism as further described below. The host organism expresses the exogenous gene to produce the protein under expression conditions.

[0039] The term "transformation" refers to the insertion of exogenous polynucleotides into a host cell, regardless of the method used for insertion. Examples include direct incorporation, transduction, or f-mating. Exogenous polynucleotides may be maintained as non-integrated vectors, such as plasmids, or alternatively integrated into the host genome.

[0040] The terms “recombinant host cell,” “host cell,” “cell,” “cell line,” “cell culture,” and other such terms, referring to higher eukaryotic cell lines cultured as microorganisms or single-cell entities, mean cells that can or have been used as recipients for recombinant vectors or other transferred DNA, and include the original offspring of the transfected original cells.

[0041] A "coding sequence," or a sequence that "codes" a selected polypeptide, is a nucleic acid molecule that, under the control of an appropriate regulatory sequence (or "regulatory factor"), is transcribed (in the case of DNA) and translated in vivo into a polypeptide (in the case of mRNA). The boundaries of a coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, viral cDNA, prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from viral or prokaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence may be located 3' relative to the coding sequence.

[0042] Typical "regulatory factors" include, but are not limited to, transcription promoters, transcription enhancers, transcription termination signals, polyadenylation sequences (located 3' relative to the translation stop codon), sequences for optimizing translation initiation (located 5' relative to the coding sequence), and translation termination sequences.

[0043] "Operationally linked" refers to an alignment of factors configured such that the components described as such perform their normal functions. Therefore, a given promoter operably linked to a coding sequence can perform the expression of the coding sequence if the appropriate enzyme is present. The promoter does not need to be contiguous with the coding sequence, insofar as it functions to direct the expression of the coding sequence. Thus, for example, an untranslated but transcribed sequence may exist between the promoter sequence and the coding sequence, and the promoter sequence may still be considered "operationally linked" to the coding sequence.

[0044] "Encoded by" means a nucleic acid sequence that encodes a polypeptide sequence, wherein the polypeptide sequence or a portion thereof includes an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids, derived from the polypeptide encoded by the nucleic acid sequence.

[0045] An expression cassette or expression construct is a set that can direct the expression of one or more sequences or one or more genes of interest. An expression cassette generally includes regulatory factors such as promoters that are operablely linked to (one or more) sequences or one or more genes of interest (to direct their transcription), as described above, and often also includes polyadenylated sequences. In certain aspects of the present invention, the expression cassettes described herein may be included in a plasmid construct. In addition to the components of an expression cassette, a plasmid construct may also include one or more selectable markers, signals that enable the plasmid construct to exist as single-stranded DNA (e.g., an M13 origin), at least one multicloning site, and a "mammalian" origin (e.g., SV40 or an adenovirus origin).

[0046] "Purified polynucleotide" means a polynucleotide or fragment of which the polynucleotide essentially does not contain the naturally associated protein, for example, less than about 50%, preferably less than about 70%, and more preferably less than about 90% of the protein. Techniques for purifying the polynucleotide of choice are well known in the art and include, for example, disruption of cells containing the polynucleotide using chaotropic agents and separation of the polynucleotide(s) and protein by ion exchange chromatography, affinity chromatography, and density-based precipitation.

[0047] The term “transfection” is used to refer to the uptake of foreign DNA by a cell. A cell is “transfected” when exogenous DNA is introduced into the cell membrane. Several transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197. Such techniques may be used to introduce one or more exogenous DNA portions into a suitable host cell. The term refers to both the stable and transient uptake of genetic material, including the uptake of peptide or antibody-bound DNA.

[0048] A "vector" is a device capable of transferring a nucleic acid sequence to a target cell (e.g., viral vectors, non-viral vectors, granular carriers, and liposomes). Typically, "vector constructs," "expression vectors," and "transfer vectors" refer to any nucleic acid construct capable of directing the expression of a desired nucleic acid and transferring a nucleic acid sequence to a target cell. Therefore, the term includes cloning and expression vehicles as well as viral vectors.

[0049] "Genetic transfer" or "gene delivery" refers to a method or system for reliably inserting target DNA or RNA into a host cell. Such methods may result in transient expression of unintegrated transfer DNA, extrachromosomal replication and expression of transfer replicons (e.g., episomes), or integration of transfer gene material into the host cell's genomic DNA. Gene delivery expression vectors include, but are not limited to, bacterial plasmid vectors, viral vectors, non-viral vectors, and vectors derived from alphaviruses, poxviruses, and vaccinia viruses.

[0050] A polynucleotide "derived" from a given sequence means a polynucleotide sequence comprising a continuous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least 15-20 nucleotides, that corresponds to, i.e., is identical to or complementary to, a region of the given nucleotide sequence. A derived polynucleotide is not necessarily physically derived from the nucleotide sequence of interest, but can be prepared in any manner, such as chemical synthesis, replication, reverse transcription, or transcription, based on information provided by the sequence of bases in the region(s) from which the polynucleotide originates. Thus, it may present either the sense or antisense orientation of the original polynucleotide.

[0051] The “reference level” or “reference value” of a biomarker refers to the level of a biomarker (e.g., blood glucose level or the number of pancreatic β-islets) that indicates a specific disease state, phenotype, or predisposition or deficiency to develop a specific disease state or phenotype, and a combination of such conditions. The “positive” reference level of a biomarker refers to the level that indicates a specific disease state or phenotype. The “negative” reference level of a biomarker refers to the level that indicates a deficiency of a specific disease state or phenotype. The “reference level” of a biomarker may be the absolute or relative amount or concentration of the biomarker, the presence or absence of the biomarker, a range of amounts or concentrations of the biomarker, the minimum and / or maximum amounts or concentrations of the biomarker, the average amount or concentration of the biomarker and / or the median amount or concentration of the biomarker; furthermore, the “reference level” of a combination of biomarkers may also be the ratio of the absolute or relative amounts or concentrations of two or more biomarkers relative to each other. Appropriate positive and negative reference levels for a biomarker for a specific disease state, phenotype, or deficiency may be determined by measuring the level of the desired biomarker in one or more suitable subjects, and such reference levels may be adjusted for a specific population of subjects (e.g., reference levels may be age-matched or sex-matched so that biomarker levels in samples from subjects of a specific age or sex can be compared with reference levels for a specific disease state, phenotype, or deficiency in a specific age or sex group). Such reference levels may also be adjusted for specific techniques used to measure the level of the biomarker in a sample (e.g., fluorescence cell analysis (FACS), immunoassays (e.g., ELISA), mass spectrometry (e.g., LC-MS, GC-MS), tandem mass spectrometry, NMR, biochemical or enzymatic assays, PCR, microarray analysis, etc.) if the level of the biomarker may differ based on the specific technique used.

[0052] The terms “quantity,” “amount,” and “level” are used interchangeably herein and may refer to the absolute quantification of an analyte in a molecule, cell (e.g., pancreatic islet), or sample, or the relative quantification of an analyte in a molecule or sample, i.e., against another value, such as a reference value taught herein or a range of values ​​for a biomarker. These values ​​or ranges may be obtained from a single patient or a group of patients.

[0053] As used herein, “diagnosis” generally includes a determination of whether a subject is likely to develop a given disease, disorder, or dysfunction. Those skilled in the art often make a diagnosis based on one or more diagnostic indicators, i.e., biomarkers whose presence, absence, or quantity indicates the presence or absence of a disease, disorder, or dysfunction. As commonly used herein, “prognosis” means the expected course and outcome of a clinical condition or disease. A patient’s prognosis is usually made by evaluating the factors or symptoms of the disease that indicate a favorable or unfavorable course or outcome of the disease. It should be understood that the term “prognosis” does not necessarily mean the ability to predict the course or outcome of a condition with 100% accuracy. Instead, those skilled in the art will understand that the term “prognosis” means a high probability that a particular course or outcome will occur; i.e., that the course or outcome is more likely to occur in patients exhibiting a given condition compared to those individuals not exhibiting the condition.

[0054] The terms “treatment,” “treating,” and “treat” are used herein in general to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in relation to completely or partially preventing a disease or its symptom(s), and / or therapeutic in relation to partially or completely stabilizing or curing a disease and / or adverse effects resulting from the disease. The term “treatment” encompasses any treatment of a disease in mammals, in particular humans, and includes: (a) preventing the occurrence of a disease and / or symptom(s) in a subject who is susceptible to the disease or symptom but has not yet been diagnosed with it; (b) inhibiting a disease and / or symptom(s), i.e., stopping their occurrence; or (c) reducing a disease symptom(s), i.e., causing a regression or reversal of the disease and / or symptom(s). Those requiring treatment include individuals who already have the disease (e.g., those with hyperglycemia or prediabetes) and those for whom prevention is desirable (e.g., those with a high susceptibility to diabetes, those with a genetic predisposition to developing diabetes, etc.). The terms "treatment," "treating," and "medication" may encompass the suppression of the onset of diabetes.

[0055] The term “suppressing the onset of diabetes” is a type of treatment used herein to generally refer to preventing or delaying the onset of diabetes. Delaying the onset of diabetes includes delays of one day or more, one week or more, one month or more, or longer. Preventing the onset of diabetes includes preventing the onset of diabetes over a specific period of time, or preventing the onset of diabetes over an unlimited period of time. The onset of diabetes may be identified by any appropriate measurement, such as measuring blood glucose levels or measuring insulin production.

[0056] Hyperglycemia, as used herein, refers to a condition in which there is an excess of sugar in the bloodstream. Hyperglycemia is also referred to as prediabetes or stage 2 disglycemia. Hyperglycemia can be characterized as mild, moderate, or severe based on blood glucose levels. In people without diabetes, a healthy fasting blood glucose level is approximately 70–100 milligrams (mg / dL) per deciliter of blood. Hyperglycemia is diagnosed when the fasting blood glucose level is approximately 100 mg / dL–125 mg / dL. A fasting blood glucose level higher than 126 mg / dL indicates the onset of clinical diabetes. In the NOD mouse model, mild hyperglycemia is defined as hyperglycemia with a fasting blood glucose level or morning blood glucose level of approximately 140 mg / dL, and severe hyperglycemia is defined as hyperglycemia with a fasting blood glucose level or morning blood glucose level of approximately 180 mg / dL or higher. Individuals with severe hyperglycemia may also be referred to as having "highly hyperglycemic." Moderate hyperglycemia refers to hyperglycemia in the NOD mouse model where fasting or morning blood glucose levels fall between mild and severe hyperglycemia, for example, between approximately 140 mg / dL and approximately 180 mg / dL.

[0057] Therapeutic treatments are those in which the subject is suffering prior to administration, while prophylactic treatments are those in which the subject is not suffering prior to administration. In some embodiments, the subject is suspected to have a high probability of developing or developing suffering prior to treatment. In some embodiments, the subject is suspected to have a high probability of developing suffering. Methods for administering therapeutic treatments are well known in the art and include oral, topical, transdermal or intradermal, inhalation, parenteral, sublingual, buccal, rectal, transvaginal, and intranasal administration. The term “parenteral” as used herein includes subcutaneous injection (e.g., transdermal or intradermal injection), intravenous, intramuscular, intrasternal injection or infusion techniques. In some embodiments, administration includes administration by a route selected from intradermal and mucosal.

[0058] In particular, with respect to a given quantity, the term "approximately" means to include a deviation of plus or minus 5%.

[0059] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject, particularly humans, for which diagnosis, treatment, or therapy is desired. “Mammal” for therapeutic purposes refers to any animal classified as a mammal, e.g., humans, domestic and farm animals, as well as zoo, sports, or pet animals, e.g., dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is human.

[0060] A "therapeutic dose" or "therapeutic dose" is the amount sufficient to produce the desired clinical outcome (i.e., achieve therapeutic efficacy). A therapeutic dose may be administered in one or more doses.

[0061] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms also apply to amino acid polymers, which are artificial chemical mimics of corresponding naturally occurring amino acids, as well as naturally occurring and non-naturally occurring amino acid polymers, in which one or more amino acid residues constitute the amino acid polymer. Both full-length proteins and their fragments are included in these definitions. The terms also include post-expression modifications of polypeptides, such as phosphorylation, glycosylation, acetylation, hydroxylation, and oxidation.

[0062] The terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” and “nucleic acid molecule” are used herein to include polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms refer only to the primary structure of molecules. Therefore, these terms include triple-stranded, double-stranded, and single-stranded DNA, as well as triple-stranded, double-stranded, and single-stranded RNA. This also includes modified and unmodified forms, such as those by methylation and / or capping of polynucleotides. More specifically, the terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” and “nucleic acid molecule” include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other types of polynucleotides that are N- or C-glycosides of purine or pyrimidine bases. There is no intended distinction in length between the terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” and “nucleic acid molecule,” and these terms are used interchangeably.

[0063] When referring to proteins, polypeptides, or peptides, "isolated" means that the molecule in question is a distinct entity separated from the whole organism found in nature, or exists in the substantial absence of other biological macromolecules of the same kind. With respect to polynucleotides, the term "isolated" refers to a nucleic acid molecule that is entirely or partially lacking the sequence that would normally bind to it in nature; or a heterologous sequence that would bind to it but remains naturally present; or a molecule separated from a chromosome.

[0064] Antigen-specific apoptotic DNA immunotherapy. Unique and promising immunotherapies are described herein.

[0065] The immunotherapy may comprise two DNA plasmids, one expressing the intracellular apoptosis-inducing signaling molecule BAX and the other expressing islet antigen-secreting glutamate decarboxylase 65 (sGAD55), and certain embodiments are exemplified herein by being referred to as "ADI-100". DNA vaccines for transplantation; Li, AF; Escher, A. DNA vaccines for transplantation. Expert Opin. Biol. Ther 2010, 10, 903-915, doi:10.1517 / 14712591003796546; Li, A.; Ojogho, O.; Franco, E.; Baron, P.; Iwaki, Y.; Escher, A. Pro-apoptotic DNA vaccination ameliorates new onset of autoimmune diabetes in NOD mice and induces foxp3+ regulatory T cells in vitro. Vaccine 2006, 24, 5036-5046, doi: 10.1016 / j. vaccine.2006.03.041 (``Escher 2006''); Li, A.; Chen, J.; Hattori, M.; Franco, E.; Zuppan, C.; Ojogho, O.; Iwaki, Y.; Escher, A. A therapeutic DNA vaccination strategy for autoimmunity and transplantation. Vaccine 2010, 28, 1897-1904, doi: 10.1016 / j .vaccine.2009.10.090 (``Escher 2010 Vaccination Strategy'').

[0066] The efficacy of ADI-100 in a non-obesity diabetic (NOD) mouse model of T1D was previously shown to be significantly increased when the sGAD55 plasmid was hypermethylated, potentially reducing inflammation caused by an unmethylated CpG motif, a ligand for Toll-like receptor 9 expressed on some APCs. (Escher 2010 Vaccination Strategy. ADI-100 treatment also increases sGAD-specific Treg levels in draining lymph nodes of NOD mice along with total CDllc +DCs; though it is not known whether these DCs have a tolerogenic phenotype. Escher 2006; Escher 2010 Vaccination Strategy; and Li, AF; Hough, J.; Henderson, D.; Escher, A. Co-delivery of pro-apoptotic BAX with a DNA vaccine recruits dendritic cells and promotes efficacy of autoimmune diabetes prevention in mice. Vaccine 2004, 22, 1751-1763, see doi: 10.1016 / j.vaccine.2003.10.049. Furthermore, it was found that ADi-100 treatment increased immune tolerance-inducing DCs (tol-DCs) and increased apoptosis-inducing BAX content, thereby enhancing the efficacy of reversing hyperglycemia when administered to NOD mice during late hyperglycemia, a pre-diabetic stage associated with the corresponding clinical diagnostic stage in human T1D. Alleva DG, Rezaee M, Yip L, Ren G, Rosenberg J, Concepcion W, Escher A, Shabahang S, Thakor AS. Reversal of Hyperglycemia and Suppression of Type 1 Diabetes in the NOD Mouse with Apoptotic DNA Immunotherapy TM (ADi TM ), ADI-100. Biomedicines. 2020 Mar 4;8(3):53. doi: 10.3390 / biomedicines8030053; see, for example, International Patent Cooperation Treaty patent application PCT / US2021 / 020711, published as WO2021178565, with the title "Methods of treating hyperglycemia and suppressing onset of type 1 diabetes".

[0067] ADi-100: Plasmid DNA construct. Two DNA plasmids containing the ADI-100 formulation previously described and illustrated herein are pND2-BAX, which contains a bax cDNA sequence under transcriptional regulation of the CMV promoter, and pSG5-GAD55, which contains a cDNA construct encoding the secreted form of human GAD65 (sGAD55) under transcriptional regulation of the SV-40 promoter in the pSG5 vector (Stratagene, San Diego, CA, USA). Escher 2010 Vaccination Strategy. The pSG5-GAD plasmid was permethylated at the CpG motif in E. coli strain ER1821 via the activity of Sssl methylase (New England BioLabs, Ipswich, MA, USA) (msGAD55). This method has been shown to produce 85%–100% methylation at the CpG motif in the plasmid (see Jimenez-Useche et al., Biophys J. 107(7) 1629–1636). However, enzymatic methylation is intended to be used to achieve various levels of methylation of CpG motifs in plasmids. Plasmid DNA was dissolved in sterile saline immediately before intradermal (id) injection. All plasmids containing BAX sequence inserts showed significant and substantial levels of apoptosis in human HeLa cells (using 1 ug / mL DNA in culture; data not shown), confirming the activity of the BAX-induced apoptosis tolerance delivery system of ADI-100.

[0068] Through experiments, it has been shown that ADI-100, containing two plasmids formulated in a 1:2 ratio—one encoding BAX and the other a permethylated plasmid encoding the secreted form of GAD (sGAD55)—addresses anti-PD1-Ab-promoted autoimmune diabetes in a non-obese diabetic NOD-ShiLtj mouse model of type 1 diabetes.

[0069] Immune checkpoint inhibitors. Immune checkpoints are inhibitory receptors that transmit negative signals to immune cells, preventing autoimmunity. The importance of immune checkpoints in supporting tolerance and preventing the onset of autoimmune diseases is best observed in knockout mouse models. For example, deficiencies in CTLA-4, PD-1, BTLA (B and T lymphocyte atenuators), TIGIT (a T cell immune receptor with immunoglobulin and ITIM domains), and VISTA (a V-domain Ig suppressor for T cell activation) have been shown to cause massive lymphoproliferation, onset of autoimmune disease, or lethal multi-organ tissue destruction (marked with CTLA-4 deficiency). In humans, several polymorphisms of immune checkpoint genes have been identified and reported to be involved in the morbidity of autoimmune diseases. For example, Yu L, Shao M, Zhou T, Xie H, Wang F, Kong J, et al. Association of CTLA-4 (+49 A / G) Polymorphism With Susceptibility to Autoimmune Diseases: A Meta-Analysis With Trial Sequential Analysis. Int Immunopharmacol (2021) 96:107617. doi: See 10.1016 / j.intimp.2021.107617.

[0070] Programmed cell death receptor 1 (PD-1) is an immune checkpoint that is important for self-tolerance and the cessation of immune responses, which are targets of cancer immunotherapy. Upon association with its ligand (PD-L1, programmed cell death ligand 1), PD-1 acts as a brake on the immune system, inducing apoptosis of activated T cells. Francisco LM, Sage PT, Sharpe AH. The PD-1 Pathway in Tolerance and Autoimmunity. Immunol Rev (2010) 236:219-42. doi: 10.1111 / j.1600-065X.2010.00923.x. PD-L1 expression can be detected in the pancreas, vascular endothelial cells, and the placenta, which is a cause of tissue protection from autoimmune responses. Keir ME, Liang SC, Guleria I, Latchman YE, Qipo A, Albacker LA, et al. Tissue Expression of PD-L1 Mediates Peripheral T Cell Tolerance. J Exp Med (2006) 203:883-95. doi: 10.1084 / jem.20051776. For example, in T1D (type 1 diabetes), PD-L1 is upregulated in insulin-producing β cells under autoimmune attack, and CD8 in the pancreas +A correlation with the intensity of T cell infiltration was observed. Colli ML, Hill JLE, Marroqui L, Chaffey J, Dos Santos RS, Leete P, et al. PDL1 is Expressed in the Islets of People With Type 1 Diabetes and is Up-Regulated by Interferons-a and-g via IRF1 Induction. EBioMedicine (2018) 36:367-75. doi: 10.1016 / j.ebiom.2018.09.040; Osum KC, Burrack AL, Martinov T, Sahli NL, Mitchell JS, Tucker CG, et al. Interferon-Gamma Drives Programmed Death-Ligand 1 Expression on Islet b Cells to Limit T Cell Function During Autoimmune Diabetes. Sci Rep (2018) 8:8295. doi: 10.1038 / s41598-018-26471-9. Furthermore, PD-1 / PD-L1 interaction has been reported to be involved in the generation of inducible Tregs (iTregs). Francisco et al. showed that PD-L1-negative APCs (antigen-presenting cells) have a impaired ability to generate Tregs, both in vitro and in vivo. Francisco LM, Salinas VH, Brown KE, Vanguri VK, Freeman GJ, Kuchroo VK, et al. PD-L1 Regulates the Development, Maintenance, and Function of Induced Regulatory T Cells. J Exp Med (2009) 206:3015-29. doi: 10.1084 / jem.20090847. These findings are validated in humans by the fact that APCs isolated from patients with systemic lupus erythematosus (SLE) cannot upregulate PD-L1 expression.Mozaffarian N, Wiedeman AE, Stevens AM. Active Systemic Lupus Erythematosus is Associated With Failure of Antigen-Presenting Cells to Express Programmed Death Ligand-1. Rheumatology (2008) 47:1335-41. doi: 10.1093 / rheumatology / ken256.。

[0071] In experimental models of autoimmunity, blockade of PD-1 or PD-L1 led to disease onset and exacerbation, demonstrating the essential role of these immune checkpoints in tolerance and, specifically, Treg maintenance. Pauken KE, Jenkins MK, Azuma M, Fife BT. PD-1, But Not PD-L1, Expressed by Islet-Reactive CD4+ T Cells Suppresses Infiltration of the Pancreas During Type 1 Diabetes. Diabetes (2013) 62:2859-69. doi: 10.2337 / db12-1475; 9. Ke Y, Sun D, Jiang G, Kaplan HJ, Shao H. PD-L1 Hi Retinal Pigment Epithelium (RPE) Cells Elicited by Inflammatory Cytokines Induce Regulatory Activity in Uveitogenic T Cells. J Leukoc Biol (2010) 88:1241-9. doi: 10.1189 / jlb.0610332. These findings are supported by recent reports on autoimmune-related adverse events in cancer patients treated with PD-1 / PD-L1 axis blockers.Zhao Z, Wang X, Bao Komminoth P, Schwegler G, Boehm S. PD-1 Checkpoint Inhibitor Associated Autoimmune Encephalitis. Case Rep Oncol (2017) 10:473-8. doi: 10.1159 / 000477162.

[0072] In cancer, effector T cells that are continuously exposed to antigen stimulation in the TME express high levels of PD-1 over the long term, leading to T cell exhaustion. This results in T cells being unable to eliminate tumor cells, facilitating cancer progression. Furthermore, cancer cells actively utilize PD-L1 to evade the immune system and hijack immune surveillance mechanisms through PD-L1 expression. In addition, results shown by Chen et al. (2018) revealed that, in addition to cell surface expression, PD-L1 is present in extracellular vesicles (exosomes) produced by melanoma cells, suggesting its systematic immunosuppressive effects. Chen G, Huang AC, Zhang W, Zhang G, Wu M, Xu W, et al. Exosomal PD-L1 Contributes to Immunosuppression and is Associated With Anti-PD-1 Response. Nature (2018) 560:382-6. doi: 10.1038 / s41586-018-0392-8. Consequently, this is due to transcriptome changes and CD4's inability to effectively eliminate cancer cells. + and CD8 +This leads to T cell exhaustion. In many cancers, lymphocyte infiltration is positively correlated with PD-L1 expression, a simple compatibility mechanism by which tumors evade immune responses. Even though tumor PD-L1 expression usually suggests a poor prognosis, higher levels of tumor PD-L1 expression correlate with better efficacy of immunotherapy. Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD-L1 on Tumor Cells in the Escape From Host Immune System and Tumor Immunotherapy by PD-L1 Blockade. Proc Natl Acad Sci USA (2002) 99:12293-7. doi: 10.1073 / pnas.192461099.

[0073] Note that in Examples 1, 2, and 3 described herein, the NOD / ShiLtJ mouse models for autoimmune type 1 diabetes, which included both anti-PD1 antibody and antigen-specific apoptotic DNA immunotherapy, contained an initial dose of 500 μg of anti-PD1 antibody, which can further accelerate the onset of diabetic status, as opposed to, for example, a low dose of 250 μg.

[0074] This disclosure provides the following aspects: Embodiment 1: A therapeutic composition comprising an immune checkpoint inhibitor and apoptotic DNA immunotherapy. Embodiment 2: The therapeutic composition of Embodiment 1, wherein the immune checkpoint inhibitor targets at least one of PD-1 and CTLA-4. Embodiment 3: A therapeutic composition of Embodiment 1 or Embodiment 2, wherein the apoptotic DNA immunotherapy comprises a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX). Embodiment 4: A therapeutic composition in any of Embodiments 1 to 3, wherein the apoptotic DNA immunotherapy comprises a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX) and a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65), and optionally the polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65) is provided as a permethylated expression cassette. Embodiment 5: A method for treating one or more adverse effects caused by the administration of an immune checkpoint inhibitor in a subject who requires treatment of one or more adverse effects caused by the administration of an immune checkpoint inhibitor, including the administration of apoptotic DNA immunotherapy. Embodiment 6: The method of Embodiment 5, further comprising the step of administering at least one of an anti-PD-1 immune checkpoint inhibitor and an anti-CTLA-4 immune checkpoint inhibitor. Embodiment 7: The method of Embodiment 5 or Embodiment 6, further comprising the step of administering an apoptotic DNA immunotherapy comprising a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX). Embodiment 8: A method further comprising the step of administering an apoptotic DNA immunotherapy comprising a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX) and a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65), wherein the polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65) is optionally provided as a permethylated expression cassette, any of embodiments 5 to 7. Embodiment 9: Any method from Embodiments 5 to 8 that prevents the onset of type 1 diabetes. Embodiment 10: A method in which hyperglycemia associated with the onset of type 1 diabetes is prevented or reduced. Embodiment 11: Any method of Embodiments 5 to 10, further comprising the step of administering apoptotic DNA immunotherapy at one or more time points before, during, or after the administration of an immune checkpoint inhibitor. Embodiment 12: Any method of Embodiments 5 to 11, further comprising the step of stopping the administration of apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor when indicating that antigen-specific immune tolerance has been achieved in a subject requiring antigen-specific immune tolerance. Embodiment 13: A method in which the effectiveness of an immune checkpoint inhibitor is maintained, as described in any of Embodiments 5 to 11. Embodiment 14: A method for preventing adverse effects caused by the administration of an immune checkpoint inhibitor to a subject who requires prevention of adverse effects caused by the administration of an immune checkpoint inhibitor, including the administration of apoptotic DNA immunotherapy. Embodiment 15: The method of Embodiment 14, further comprising the step of administering at least one of an anti-PD-1 immune checkpoint inhibitor and an anti-CTLA-4 immune checkpoint inhibitor. Embodiment 16: The method of Embodiment 14 or Embodiment 15, further comprising the step of administering an apoptotic DNA immunotherapy comprising a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX). Embodiment 17: A method further comprising the step of administering an apoptotic DNA immunotherapy comprising a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX) and a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65), wherein the polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65) is optionally provided as a permethylated expression cassette, any of embodiments 14 to 16. Embodiment 18: Any method from Embodiments 14 to 17 that prevents the onset of type 1 diabetes. Embodiment 19: Any method from Embodiments 14 to 17, wherein hyperglycemia associated with the onset of type 1 diabetes is prevented or reduced. Embodiment 20: Any method of Embodiments 14 to 19, further comprising the step of administering apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor. Embodiment 21: Any method of Embodiments 14 to 20, further comprising the step of stopping the administration of apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor when indicating that antigen-specific immune tolerance has been achieved in a subject requiring antigen-specific immune tolerance. Embodiment 22: Any method from Embodiments 14 to 21, wherein the effectiveness of the immune checkpoint inhibitor is maintained. [Examples]

[0075] Examples The following examples are provided to those skilled in the art to provide a complete disclosure and description of how the present invention may be made and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to indicate that the following experiments are all or only experiments performed.

[0076] Example 1 Example 1 provides preliminary data and results from an ongoing study, with two objectives: The first objective was to establish an enhanced type 1 diabetes (T1D) mouse model in female non-obese diabetic NOD-ShiLtj mice in which the disease is enhanced by anti-PD-1 antibody treatment. The NOD-ShiLtj, or NOD mouse line, is a polygene autoimmune T1D model that develops apparent hyperglycemia due to immune-mediated islet loss in females at approximately 12 weeks of age. In this study, the effects of anti-PD-1 antibody treatment on disease onset and disease progression were evaluated. The second objective was to determine the efficacy of ADI-100 BAX+msGAD treatment at two dose levels on the following parameters: blood glucose concentration (blood glucose), whole-pancreatic morphology and islet histopathology, and islet insulin content by immunohistochemistry.

[0077] In this study, 8-week-old female NOD-ShiLtj mice (JAX strain 001976, Jackson Laboratories, Bar Harbor, ME) were administered either anti-PD-1 antibody (BioXCell; catalog number BE0033-2) or an IgG isotype (BioXCell; catalog number BE0091) as a control, starting on day 0 and ending on day 10, every two days. Findings from these preliminary studies include tolerance induction using DNA plasmids performed on days 0, 7, 14, and 21.

[0078] Figure 1 and Table 1 below provide details of the test designs and treatment groups from which these preliminary results were obtained.

[0079] Figure 1 shows the study design and time course. Eight-week-old mice were obtained and acclimatized for one week. After random assignment to one of four groups, each animal received an initial dose of 500 μg of anti-PD1 antibody (day 0), followed by doses of 250 μg (days 2, 4, 6, 8, and 10), or IgG as a negative control. As shown in Figure 1, treatment using vehicle control or ADI-100 was initiated on day 0 and administered once weekly (QW) for eight weeks. Blood glucose levels were monitored during treatment administration and for several weeks after discontinuation of treatment.

[0080] Table 1 below shows the treatment groups and planned dosing regimens for the ongoing trial, from which these preliminary results were obtained during the first 25 days of the trial. Forty female NOD mice were randomly assigned to four groups (N=10). Mice started with 500 μg on day 0 and received six doses of 250 μg of IgG (group 1) or anti-PD1 antibody (groups 2-4) on days 2, 4, 6, 8, and 10. For these preliminary results, mice in groups 2-4 also received the first four of eight planned weekly doses of the test substance on days 0, 7, 14, and 21, as shown in the preliminary trial data provided in Figures 2A, 2B, 2C, and 2D. Animals in group 2 received a vehicle control, animals in group 3 received 50 μL of ADI-100 at a low dose of 1 μg / μL in a 1:2 ratio of BAX / msGAD, and animals in group 4 received 50 μL of ADI-100 at a high dose of 2 μg / μL in a 1:2 ratio of DNA BAX / msGAD. The 1 μg / μL (low dose) of ADI-100 in a 1:2 ratio of BAX / msGAD (BAX 17 μg + msGAD 33 μg) was supplied by Aditxt, Inc. in Tris-EDTA (TE) buffer. The 2 μg / μL (high dose) of ADI-100 in a 1:2 ratio of BAX / msGAD (BAX 34 μg + msGAD 66 μg) was supplied by Aditxt, Inc. in Tris-EDTA (TE) buffer. Intraperitoneal (IP) and intradermal (ID) dose administrations are shown in Table 1 below. [Table 1]

[0081] The preliminary results of the first 25 days of the experiment are shown in Figures 2A, 2B, 2C, and 2D (absolute glucose reads). Mice in the control group (Group 1) showed relatively stable blood glucose levels during the 25-day observation period, while most (9 / 10) of the mice in Group 2, treated with anti-PD1 antibody and vehicle, showed obvious hyperglycemia within 10 days, indicating an earlier and more synchronized onset of the disease. Most (7 / 10) of the mice in Group 3, treated with a low-dose ADI-100, also showed hyperglycemia within the first 10 days. In contrast, 7 out of 10 mice (7 / 10) in Group 4, treated with a high-dose ADI-100, showed normal blood glucose levels during the observation period.

[0082] Any mouse with a BG measurement of >400 mg / dL was euthanized according to the test protocol. Figure 2A shows that all control mice in Group 1 survived for the first 25 days of the study. Figure 2B shows that 9 out of 10 mice in Group 2 experienced a BG measurement of >400 mg / dL within the first 11 days. Figure 2C shows that 7 out of 10 mice in the low-dose test group 3 experienced a BG measurement of >400 mg / dL within the first 8 days. Figure 2D shows that 3 out of 10 mice in the high-dose test group (Group 4) experienced a BG measurement of >400 mg / dL within the first 7 days (1 week).

[0083] As shown in Figure 2D, mouse subject 225 in group 4 (high dose) had multiple fluctuating blood glucose (BG) readings from above and below 200 mg / dL on test days 7, 9, and 11 to above 250 mg / dL on test days 8, 10, and 13; note that mouse subject 225 showed normal blood glucose levels from test day 14 to test day 25. These data support the efficacy of ADI-100 in controlling the effect of anti-PD1 antibodies in promoting autoimmune diabetes in NOD mice. These data also demonstrate that the efficacy of ADI-100 is dose-dependent.

[0084] Example 2 Example 2 provides further data obtained from the continuation of Example 1 described above.

[0085] Figures 3A, 3B, 3C, and 3D (logarithmic glucose readings) and Figures 4A, 4B, 4C, and 4D (absolute glucose readings) show an observation period of 45 days, and the findings follow those of Example 1, Figures 2A, 2B, 2C, and 2D. Note that the three-digit mouse subject identification names in Figures 2A, 2B, 2C, and 2D begin with the number "2", while these same mice are identified again in Figures 3A, 3B, 3C, and 3D, as well as in Figures 4A, 4B, 4C, and 4D, and have three-digit mouse subject identification names that begin with the number "0" instead of "2".

[0086] Figures 3A, 3B, 3C, and 3D, as well as Figures 4A, 4B, 4C, and 4D, show that mice exhibiting normal blood glucose levels on day 25 of the study continue to exhibit normal blood glucose levels for at least 45 days or at least approximately 6 weeks. That is, mice exhibiting normal blood glucose levels on day 25 do not subsequently experience a relapse indicated by a BG measurement higher than 200 mg / dL within 45 days of the study. These findings add to the information learned from Example 1, where mice in either Example 1 or Example 2 exhibiting normal blood glucose levels on day 14 did not subsequently experience a relapse indicated by a BG measurement higher than 200 mg / dL within 45 days of the study.

[0087] Note that administration of the test substance was stopped on D29 after the fifth dose of the test substance, and blood glucose (BG) was continuously monitored. The study design provided for the administration of additional test substance doses three times per week (3) in the event of recurrence (i.e., mice showing normal blood glucose levels on day 25, followed by BG measurements higher than 250 mg / dL), but no such recurrences occurred within this 45-day study period. These data indicate that the test substance was effective for at least two weeks after the last dose on day 28 of the study, demonstrating longer-term ADI-100 efficacy for normalizing blood glucose levels without further administration of test substance doses.

[0088] Animals from vehicle group 2 and low-dose BAX+msGAD treatment group 3 were collected on day 84 (D84) for final measurements and tissue sampling.

[0089] Example 3 The studies described in Examples 1 and 2 were extended beyond the original study end date of D84 for animals in control group 1 and high-dose ADI-100 BAX+msGAD treatment group 4. This extension was to determine the endurance of the treatment, including prophylactic measures beyond the time when ADI-100 was administered, and whether animals from group 4, whose administration was discontinued on D29 after the fifth dose of the test substance, would relapse and develop hyperglycemia and T1D disease at a later stage. In the event of relapse, administration is resumed to determine whether high concentrations of the test substance can reverse disease progression at this later stage or after relapse has occurred. Further resumption of ADI-100 administration acts as a T1D treatment after the onset of the disease, as seen by the appearance of hyperglycemia suggesting a breakdown of tolerance.

[0090] Figure 5 shows the study design and time course. The remaining mice from groups 1 and 4 continued the study, and body weight (BW), body condition score (BCS), clinical observation (CO), and blood glucose (BG) were continued to be measured. If a blood glucose (BG) increase of >250 mg / dL was observed in mice from group 4, administration was resumed for those animals (one or more). Once resumed, the dose was administered once a week (QW) until a BG measurement of <250 mg / dL was observed, followed by three further QW doses; any recurrent mice with a BG measurement of >400 mg / dL were euthanized.

[0091] Table 2 below shows the treatment groups and administration methods. [Table 2]

[0092] Figure 6A shows absolute glucose readings for control mice in Group 1. Note that only 3 out of the original 10 mice in control Group 1 survived from the previous 112 days to 301 days. In contrast, Figure 6B shows that 6 out of the original 10 mice in High-Dose Group 4 survived to 301 days. Furthermore, Figure 6B shows that 6 out of the original 10 mice experienced prolonged normalization of blood glucose levels throughout 301 days after the last dose of the high-dose test substance on day 28. These results demonstrate the treatment of persistent hyperglycemia and prevention of T1D onset in mice in ADI-100 High-Dose Group 4.

[0093] As shown in Figure 6B, two mice, mouse 214 and mouse 247, had relapses in which their measured blood glucose (BG) increased to >250 mg / dL. The arrows in Figure 6B indicate the respective treatments. Mouse 214 relapsed on test day 124 or test day 96, after the last dose of the test substance on test day 28. Mouse 214 responded to treatment and was treated 4(4) times, becoming normal without further relapses. Mouse 247 relapsed on test day 160 or test day 132, after the last dose of the test substance on test day 28. Mouse 247 was treated (see arrow), but did not respond to treatment, could not be saved, and died due to the natural course of the disease. Therefore, administration of the high-dose test substance was resumed as described above. The relapse in mouse 214 was successfully reversed after the resumption of high-dose test substance administration, which indicates the treatment of hyperglycemia after disease onset. Mouse 247, which relapsed on test day 160, was successfully treated within a certain timeframe. However, mouse 247 relapsed again 36 days later, on test day 196, and was euthanized according to the test protocol.

[0094] This study was conducted in NOD mice, which are known to develop diabetes mellitus. Here, the rate and timing of disease progression were enhanced by the application of checkpoint inhibitors. The development of disease induced by checkpoint inhibitors was inhibited in most animals receiving the ADI-100 test, with the strongest effect observed in Group 4 high-dose test mice. However, a second underlying pathology, distinct from the enhanced diabetes mellitus process induced by checkpoint inhibitors, which is the natural course that drives NOD mice to disease, did not appear to develop during the subsequent observation period. Thus, there was no further disease development in the remaining healthy animals, except for one of the two relapsed animals that developed hyperglycemic levels later in the study after successful temporary reversal and suppression of elevated blood glucose levels. Therefore, the data indicate prevention of disease progression, firstly by treatment with checkpoint inhibitors, and secondly by suppressing the natural course of disease development.

[0095] Example 4 The objective of this study was to test the antitumor efficacy of anti-PD-1 (BAX 34 μg + msGAD 66 μg) in combination with the vehicle alone, anti-PD-1 alone, and the ADI-100 test compound. Anti-PD-1 has been reported to be highly effective in the Hepa1-6 mouse tumor model.

[0096] ADI-100 was supplied by Aditxt, Inc. in a Tris-EDTA (TE) buffer vehicle in a frozen vial. One ADI-100 vial per administration day was thawed overnight at 4°C the day before administration. Anti-PD-1 in phosphate-buffered saline (PBS) buffer vehicle was supplied by Bio X Cell, Inc. (catalog number BP0146).

[0097] This study was conducted in a Hepa1-6 liver model using C57BL / 6 mice. Thirty mice were enrolled in the study. All animals were randomly assigned to one of three different study groups. Randomization was performed on day 1 using Study Log software. Table 3 below shows the treatment groups and administration regimens. [Table 3]

[0098] Mean tumor volume (mm) for each group in randomization 3 The values ​​of ) + SD are shown in Table 4, and were as follows: [Table 4]

[0099] The animals were administered the drug on test days 1, 4, 8, and 11. Table 5 below shows the test reference dates. However, due to the rapid treatment results with anti-PD-1, the study was terminated after the fourth dose (day 11). [Table 5]

[0100] The results of this test are shown in Figures 7A, 7B, and 7C.

[0101] As shown in Figure 7A, up to day 11 after the start of medication, both group 2 (anti-PD-1 monotherapy) and group 3 (anti-PD-1 + ADI-100) showed >80% tumor inhibition compared to control group 1 (vehicle monotherapy). Furthermore, ADI-100 in group 3 (anti-PD-1 + ADI-100) did not negatively interfere with the efficacy shown by anti-PD-1 compared to group 2 (anti-PD-1 monotherapy).

[0102] Figures 7B and 7C show the mean absolute body weight ± SEM (standard error of the mean) and the mean percentage change in body weight ± SEM (standard error of the mean), respectively, for groups 1, 2, and 3. ADI-100 showed no signs of toxicity.

[0103] Example 5 In this study, a syngeneic mouse model will be used to evaluate the performance of the therapies disclosed herein in an animal model with a complete and functional immune system. Immunoprofiling of Tregs, including antigen-specific Tregs, will be performed.

[0104] The various embodiments described above may be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature referenced herein and / or enumerated in the application datasheet, including, but not limited to, U.S. Provisional Patent Application No. 63 / 495,984 filed on April 13, 2023, are incorporated herein by reference in their entirety. If it is still necessary to provide further embodiments using the concepts of various patents, applications, and publications, aspects of the embodiments may be modified.

[0105] In light of the detailed description above, these and other modifications may be made to the embodiments. In general, the terms used in the following claims should not be interpreted to limit the claims to specific embodiments disclosed in the specification and claims, but rather to include all possible embodiments, along with the entire scope of equivalents to which such claims are granted. Thus, the claims are not limited by such disclosure.

Claims

1. A therapeutic composition comprising an immune checkpoint inhibitor and an apoptotic DNA immunotherapy.

2. The therapeutic composition according to claim 1, wherein the immune checkpoint inhibitor targets at least one of PD-1 and CTLA-4.

3. The therapeutic composition according to claim 1 or 2, wherein the apoptotic DNA immunotherapy comprises a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX).

4. The therapeutic composition according to any one of claims 1 to 3, wherein the apoptotic DNA immunotherapy comprises a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX) and a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65), and optionally the polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65) is provided as a permethylated expression cassette.

5. A method for treating one or more adverse effects caused by the administration of an immune checkpoint inhibitor in a subject who requires treatment of one or more adverse effects caused by the administration of an immune checkpoint inhibitor, including the administration of apoptotic DNA immunotherapy.

6. The method according to claim 5, further comprising the step of administering at least one of an anti-PD-1 immune checkpoint inhibitor and an anti-CTLA-4 immune checkpoint inhibitor.

7. The method according to claim 5 or 6, further comprising the step of administering an apoptotic DNA immunotherapy comprising a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX).

8. A method further comprising the step of administering an apoptotic DNA immunotherapy comprising a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX) and a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65), wherein the polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65) is optionally provided as a permethylated expression cassette, according to any one of claims 5 to 7.

9. A method according to any one of claims 5 to 8, which prevents the onset of type 1 diabetes.

10. The method according to any one of claims 5 to 8, wherein hyperglycemia associated with the development of type 1 diabetes is prevented or reduced.

11. The method according to any one of claims 5 to 10, further comprising the step of administering an apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor.

12. The method according to any one of claims 5 to 11, further comprising the step of stopping the administration of apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor, when indicating that antigen-specific immune tolerance has been achieved in a subject requiring antigen-specific immune tolerance.

13. The method according to any one of claims 5 to 11, wherein the effectiveness of the immune checkpoint inhibitor is maintained.

14. The method according to any one of claims 5 to 11, further comprising the step of restarting the administration of apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor when antigen-specific immune tolerance is lost in a subject requiring antigen-specific immune tolerance.

15. A method for preventing adverse effects caused by the administration of immune checkpoint inhibitors, including the administration of apoptotic DNA immunotherapy, in subjects who require prevention of adverse effects caused by the administration of immune checkpoint inhibitors.

16. The method according to claim 15, further comprising the step of administering at least one of an anti-PD-1 immune checkpoint inhibitor and an anti-CTLA-4 immune checkpoint inhibitor.

17. The method according to claim 15 or 16, further comprising the step of administering an apoptotic DNA immunotherapy comprising a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX).

18. A method further comprising the step of administering an apoptotic DNA immunotherapy comprising a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX) and a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65), wherein the polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65) is optionally provided as a permethylated expression cassette, according to any one of claims 15 to 17.

19. The method according to any one of claims 15 to 18, which prevents the onset of type 1 diabetes.

20. The method according to any one of claims 15 to 18, wherein hyperglycemia associated with the development of type 1 diabetes is prevented or reduced.

21. The method according to any one of claims 15 to 20, further comprising the step of administering an apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor.

22. The method according to any one of claims 15 to 21, further comprising the step of stopping the administration of apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor, when indicating that antigen-specific immune tolerance has been achieved in a subject requiring antigen-specific immune tolerance.

23. The method according to any one of claims 14 to 21, wherein the effectiveness of the immune checkpoint inhibitor is maintained.

24. The method according to any one of claims 15 to 23, further comprising the step of restarting the administration of apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor when antigen-specific immune tolerance is lost in a subject requiring antigen-specific immune tolerance.

25. A method for treating one or more adverse effects resulting from the administration of immune checkpoint inhibitors to a subject with cancer, including the administration of apoptotic DNA immunotherapy.

26. The method according to claim 25, further comprising the step of administering at least one of an anti-PD-1 immune checkpoint inhibitor and an anti-CTLA-4 immune checkpoint inhibitor.

27. The method according to claim 25 or 26, further comprising the step of administering an apoptotic DNA immunotherapy comprising a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX).

28. A method according to any one of claims 25 to 27, further comprising the step of administering an apoptotic DNA immunotherapy comprising a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX) and a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65), wherein the polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65) is optionally provided as a permethylated expression cassette.

29. The method according to any one of claims 25 to 28, wherein tumor growth is inhibited.

30. The method according to any one of claims 25 to 28, wherein weight loss in a subject being treated is prevented or reduced.

31. The method according to any one of claims 25 to 28, which prevents the onset of type 1 diabetes.

32. The method according to any one of claims 25 to 31, wherein hyperglycemia associated with the development of type 1 diabetes is prevented or reduced.

33. The method according to any one of claims 25 to 32, further comprising the step of administering an apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor.

34. The method according to any one of claims 25 to 33, further comprising the step of stopping the administration of apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor, when indicating that antigen-specific immune tolerance has been achieved in a subject requiring antigen-specific immune tolerance.

35. The method according to any one of claims 25 to 34, further comprising the step of restarting the administration of apoptotic DNA immunotherapy at any one or more time points before, during, or after the administration of an immune checkpoint inhibitor when antigen-specific immune tolerance is lost in a subject requiring antigen-specific immune tolerance.

36. The method according to any one of claims 25 to 35, wherein the effectiveness of the immune checkpoint inhibitor is maintained.

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