Adenoviral vector-transduced apheresis products

Abstract

An immunotherapeutic composition comprising a subject-derived peripheral blood mononuclear cell (PBMC) and at least one recombinant adenoviral subtype 5 (Ad5) comprising a deletion in the E1 gene region, a deletion in the E2b gene region and a nucleic acid sequence encoding a peptide antigen wherein the PBMC is exposed ex vivo to the at least one Ad5 vector is contemplated. Advantageously, the same PBMC composition can also be used to prepare modified NK cells, and in particular the modified NKs include CIML NK cells, CENK cells and mCENK cells.

Description

[0001] This application claims priority to our co-pending U.S. provisional application, filed May 4, 2023, serial number 63 / 500,235, which is incorporated herein by reference. Technical Field

[0002] The field of the present invention is compositions and methods for cancer treatment, particularly when it involves treating subjects with ex vivo modified single-harvest products, wherein cells are transfected with recombinant therapeutic viruses, and optionally wherein the cells are differentiated or transfected to express chimeric antigen receptors. Background Technology

[0003] The background description includes information that may be used to understand the invention. No information provided herein is acknowledged to be prior art or related to the currently claimed invention, nor is any publication specifically or implicitly referenced considered prior art.

[0004] All publications and patent applications herein are incorporated by reference to the same extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference. If a definition or use of a term in an incorporated reference is inconsistent with or contradicts the definition of that term provided herein, the definition provided herein shall apply, and the definition in the incorporated reference shall not apply.

[0005] Cancer vaccines, particularly viral cancer vaccines, have shown great promise in recent years, but are often limited by various factors, including the immunogenicity of viral vectors and / or poor presentation of recombinant (novel) antigens. To improve the therapeutic efficacy of cancer vaccines, selected novel epitopes can be subjected to in vitro testing protocols, where the immune response of the patient's immune cells is determined prior to administration of a vaccine containing the selected novel epitope, as described in WO 2018 / 106699. While conceptually attractive, the selected novel epitopes are typically encoded in recombinant viral nucleic acids, and the resulting viral vaccine is then administered to the patient. Unfortunately, due to the typically systemic delivery of viral vectors, the general training of various components of the immune system (e.g., dendritic cells, CD8+ T cells, CD4+ helper T cells, B cells) is often not achieved or only poorly achieved. Furthermore, even when antigen presentation is achieved to at least some extent, a therapeutically effective immune response may not be generated, for example, due to the relatively low amount of viral vector. These difficulties are often further exacerbated by significant hepatotoxicity and the often rapid attachment of recombinant viral particles to host cells, leading to various side effects and even death (see, for example, Cancer Gene Therapy [Cancer Gene Therapy] (2002) 9, 979 – 986.

[0006] To overcome some of the difficulties associated with the systemic delivery of tumor antigens, various efforts have been made to trigger in vitro stimulation of certain cells of the immune system. For example, in one approach, dendritic cells are incubated with NK cells in the presence of a TLR agonist to generate mature dendritic cells (see, for example, Experimental & Molecular Medicine [Experimental and Molecular Medicine] (2010), 42(6), pp. 407-419. In another approach, antigen-presenting cells are stimulated with chimeric proteins containing cancer-specific antigens (see, for example, Nat Rev Cancer [Nature Review - Cancer]; 12(4): 265-277). Here, chimeric proteins are used to deliver antigens directly to dendritic cells (DCs) in vivo, these chimeric proteins containing antibodies specific to DC receptors fused with selected antigens. Such antigen-specific targeting of DCs in vivo elicits effective antigen-specific CD4+ and CD8+ T cell-mediated immunity. However, inducing immunity also requires the provision of DC maturation signals. Otherwise, such methods induce antigen-specific tolerance, which is contrary to the desired outcome. Furthermore, currently known in vitro DC targeting using DC receptors requires the generation of customized molecular entities, which is time- and resource-intensive. In addition, such methods require specific maturation signals, which further complicates vaccination strategies.

[0007] Among the alternatives to reduce systemic exposure, therapeutic viruses can be administered subcutaneously. While this route of administration significantly reduces the risk of serious adverse events, additional problems arise. Most notably, subcutaneous administration substantially limits viral exposure to antigen-presenting cells, particularly dendritic cells. Consequently, the expected therapeutic effect is far lower than that of systemic administration.

[0008] Therefore, although various compositions and methods for cancer vaccines are known in the art, all or almost all of them have several drawbacks, especially in cancer vaccines that use recombinant viral vectors to generate neoantigens in infected cells. Therefore, there remains a need for improved viral vaccine compositions. Summary of the Invention

[0009] The subject matter of this invention relates to various compositions and methods for viral vaccine compositions, wherein donor cells (including peripheral blood mononuclear cells (PBMCs)) of a subject are in vitro infected with a recombinant viral vaccine to a degree sufficient to reduce or even completely deplete the free extracellular viral particles of the composition, thereby avoiding adverse reactions that might otherwise occur. In further contemplated compositions and methods, at least some of the donor cells, and particularly lymphocytes, are further genetically modified, for example, to express CAR, and / or treated to differentiate into more mature immune cells. Most preferably, in vitro infection of PBMCs is preferential or even selective towards dendritic cells, and further preferably is carried out in the presence of other immune cells, such as macrophages, monocytes, T cells, B cells, etc.

[0010] In one aspect of the subject matter of this invention, the inventors have conceived of an immunotherapeutic composition comprising subject-derived peripheral blood mononuclear cells (PBMCs) and at least one recombinant adenovirus subtype 5 (Ad5) vector having a deletion in the E1 gene region, a deletion in the E2b gene region, and a nucleic acid sequence encoding a peptide antigen, wherein the PBMCs are exposed in vitro to the Ad5 vector. Most typically, the Ad5 vector comprises nucleic acid for expressing at least one of tumor-associated antigens, neoantigens, cytokines, gene therapies, and immunomodulatory proteins.

[0011] As will be readily understood, the envisioned immunotherapeutic composition may further comprise an IL-15 or interleukin-15:interleukin-15 receptor α (IL-15:IL-15Rα) complex. Most typically, immune effector cells are simultaneously exposed to an Ad5 carrier and the IL-15:IL-15Rα complex. For example, the envisioned IL-15:IL-15Rα complex comprises an IL-15N72D:IL-15RαSu / Fc complex (e.g., Nogapendekin alfa inbakicept (NAI), also known as N-803 or Alt-803), wherein the Fc portion may be derived from any immunoglobulin, and particularly from IgG (e.g., IgG1, IgG4, etc.). Alternatively, the IL-15:IL-15Rα complex may also be encoded in an Ad5 carrier and then expressed and secreted from infected cells.

[0012] In another embodiment, the subject may have been diagnosed with or may not have been diagnosed with an infectious disease, adenoid disease, or cancer. Most preferably, the PBMC is derived from the subject through apheresis and will include T cells, B cells, natural killer (NK) cells, natural killer T (NK-T) cells, dendritic cells, mast cells, myeloid-derived phagocytes, or combinations thereof.

[0013] Furthermore, it is envisioned that immunotherapeutic compositions (after ex vivo infection) will be formulated for intravenous (IV) administration, typically for use in treating aplastic diseases, cancers, or infectious diseases in patients. Examples of envisioned aplastic diseases or cancers include bladder cancer, glioblastoma, prostate cancer, hematologic malignancies, B-cell tumors, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, cutaneous T-cell lymphoma, T-cell lymphoma, solid tumors, urothelial carcinoma / bladder cancer, melanoma, lung cancer, renal cell carcinoma, breast cancer, gastric and esophageal cancer, head and neck cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, B-cell non-Hodgkin lymphoma, squamous cell head or neck cancer, or urothelial carcinoma / bladder cancer.

[0014] Additionally, treatment may further include radiotherapy, chemotherapy, surgery, and / or administration of therapeutic antibodies, immunomodulators, proteasome inhibitors, pan-DAC inhibitors, H-DAC inhibitors, and / or checkpoint inhibitors, and / or administration of adoptive cell therapy and / or immunotherapeutic agents, including CAR T and / or NK cell therapy.

[0015] Therefore, the inventors have also conceived of a method for treating cancer or infectious diseases, comprising the steps of therapeutic apheresis on a subject, purification of peripheral blood mononuclear cell (PBMC) fractions from the apheresis product, ex vivo exposure of PBMCs to at least one recombinant adenovirus subtype 5 (Ad5) vector (which contains a deletion in the E1 gene region, a deletion in the E2b gene region, and a nucleic acid sequence encoding a peptide antigen), and administration of an effective amount of Ad5 vector-treated PBMCs to a patient.

[0016] Such a method may also include the step of adding interleukin-15 (IL15) or an IL15:IL15 receptor α (IL15:IL15Rα) complex to ex vivo exposed PBMCs. Alternatively or alternatively, patients may be pretreated with IL15 or an IL15:IL15Rα complex (e.g., N-803), and / or the IL15 or IL15:IL15Rα complex may be added to Ad5-carrier-treated PBMCs prior to administration. Similarly, the IL15 or IL15:IL15Rα complex may be administered to patients after administration of Ad5-carrier-treated PBMCs. As will be readily understood, the cells administered may be obtained from the same individual or from different (e.g., at least haploidentical) individuals.

[0017] Various objects, features, aspects, and advantages of the subject matter of the invention will become clearer from the following detailed description of preferred embodiments and the accompanying drawings (where the same numerals represent the same components). Attached Figure Description

[0018] Figure 1 This is an exemplary schematic flowchart of cancer treatment according to the subject matter of the present invention.

[0019] Figure 2 This is an exemplary schematic flowchart of another cancer treatment according to the subject matter of the present invention.

[0020] Figure 3 This is an exemplary schematic flowchart of selective in vitro PBMC Ad5 infection.

[0021] Figure 4 Exemplary results of Ad5 infection of PBMCs using 1000 MOIs according to the subject matter of the present invention are depicted.

[0022] Figure 5 Exemplary results of Ad5 infection under different MOIs according to the subject matter of the present invention are depicted. Detailed Implementation

[0023] The inventors have now discovered various vaccine compositions and methods, and in particular viral cancer vaccine compositions and methods, which reduce or even completely avoid adverse reactions and / or decreased efficacy due to the rapid attachment of therapeutic viruses to non-target tissues.

[0024] In a particularly preferred aspect, recombinant therapeutic viruses are administered ex vivo to cells of a subject diagnosed with cancer, wherein these cells are obtained from the subject via apheresis. In this context, it should be understood in particular that apheresis can be used not only to isolate PBMCs but also to isolate erythrocytes and platelets, which can be used as therapeutic entities either in isolated form or after modification (e.g., transfection with recombinant nucleic acids, and especially mRNA). Additionally, natural killer (NK) cells can be isolated from PBMCs or via apheresis to produce yet another therapeutic modality, wherein the NK cells can be further genetically modified (e.g., to express CAR) or stimulated to differentiate into cytokine-enhanced NK (CENK) or memory cytokine-enhanced (m-CENK) cells. Thus, apheresis can be used to prepare various cell fractions beyond PBMCs for treating the same patient in parallel or sequentially. Most typically, the treatments envisioned herein will at least involve the administration of ex vivo infected dendritic cells (most typically dendritic cells preferentially or selectively infected in the presence of other immune cells).

[0025] In a typical example, such as Figure 1As illustrated, prior to apheresis collection of PBMCs, subjects diagnosed with cancer are optionally treated with N-803 to stimulate the production of T and NK cells. During the same apheresis procedure, erythrocyte-rich and platelet-rich fractions can also be obtained from the same subject. Most typically, PBMCs will include various immune cells, such as CD4+. + T cells, CD8 + T cells, B cells, macrophages, dendritic cells, Kupffer cells, etc. As will be readily understood, different mixtures of cells can be immediately used for infection with therapeutic viruses (and especially recombinant human adenovirus serotype 5 (hAd5)), resulting in antigen presentation of recombinant antigens encoded by the recombinant adenovirus on antigen-presenting cells (such as macrophages and dendritic cells). Therefore, antigen-presenting cells that present recombinant neoantigens on MHC class I and / or II complexes will readily interact with unpermitted T cells, immature and mature unsensitized T cells, and (antigen-responsive) CD4+ cells. + T cells and (antigen-responsive) CD8 + T cell contact will thus trigger the development (activation and expansion) of antigen-specific cytotoxic T cells. Alternatively or additionally, the recombinant nucleic acid in the recombinant therapeutic virus is not limited to a recombinant nucleic acid encoding one or more neoantigens or tumor-associated antigens, but may also encode a CAR specific to cancer-specific antigens of cancer cells. Of course, it should be recognized that lymphocyte infection with recombinant therapeutic viruses can be performed in the presence of immunostimulatory cytokines (IL-2, IL-15, etc.), co-stimulatory molecules, and / or checkpoint inhibitors.

[0026] In particularly envisioned compositions and methods, it is generally preferred to expose PBMCs to the recombinant therapeutic virus for a duration sufficient to induce measurable T cell expansion, and particularly clonal expansion of antigen-responsive T cells. As will be appreciated, such expansion can be stimulated by adding IL15, IL15:IL15Rα or its IL15Rβγ agonist derivative, IL15:IL15RαIgG4Fc, or N-803 to the PBMCs and the therapeutic virus. In a further preferred aspect, the exposure of PBMCs to the recombinant therapeutic virus is sufficient to allow antigen-presenting cells in the PBMCs to take up substantially all of the recombinant therapeutic virus. From a different perspective, after in vitro infection, it is typically preferred that the viral titer in the PBMC supernatant is so low as to be undetectable. Such a time can be empirically determined by quantifying the viral infectivity in the cell composition (e.g., using a hexagonal assay where the virus is adenovirus). Once T cell activation and / or expansion are observed and the viral titer in the supernatant is so low as to be undetectable, the cell suspension thus prepared can be used for infusion. Therefore, by using the envisioned methods and compositions, it should be understood that the infection of antigen-presenting cells with a therapeutic virus is optimized while minimizing or completely avoiding potential adverse reactions due to off-target viral binding. The inventors have also envisioned that the duration of in vitro exposure is determined by MHC presentation of an Ad-encoded peptide, wherein IV administration of the Ad-treated single-absorption product results in subsequent in vivo stimulation of antigen-responsive CD4+ via MHC presentation on in vitro infected cells. + and CD8 + T cells.

[0027] Furthermore, it is envisioned that some of the apheresis products could be subjected to the isolation of NK cells, which could be expanded in vitro to provide additional cytotoxic effector cells. Additionally, and when needed, the isolated NK cells could be further stimulated to differentiate into cytokine-enhanced NK cells (CENK) and / or memory cytokine-enhanced NK cells (m-CENK). Alternatively or additionally, at least some of the isolated NK cells could also be transfected with recombinant nucleic acids to express CARs with selected target specificity (e.g., neoantigens or tumor-associated antigens such as MUC1, Brachyury, CEA, PSA, PMSA, PD-L1, HER2, etc.) on the cell surface. These modified NK cells could then be administered to a subject, thereby further enhancing the immunotherapeutic composition containing the apheresis products from ex vivo infection. Still further, it is envisioned that additional cell-based therapeutic agents, such as haNK cells or t-haNK cells, could be administered, as described, for example, in US 11,643,452 and US 2021 / 0198342.

[0028] Furthermore, it is envisioned that apheresis can also produce red blood cells and platelets, which can be used as additional therapeutic agents. For example, red blood cells and platelets can be preserved and administered in cases where the subject has previously undergone chemotherapy or radiation, especially in cases where chemotherapy or radiation is administered after apheresis. It should also be noted that red blood cells and / or platelets can be genetically modified to express one or more target proteins (e.g., cytokines, checkpoint inhibitors, etc.).

[0029] Regarding the administration of the ex vivo infection apheresis composition, it is envisioned that cells can be infused into the subject either as a standalone form or in combination with one or more immunostimulatory cytokines. Most typically, in cases where administration also includes the infusion of NK cells (e.g., CENK, m-CENK, CAR-expressing NK cells, etc.), the NK cells will preferably be administered at least 1 day, at least 3 days, at least 7 days, or at least 14 days after the infusion of the ex vivo infection apheresis composition.

[0030] Figure 2 Therapeutic options and components for using ex vivo infection single-agent compositions are illustrated exemplary and schematically. From Figure 2 It is readily apparent that, in addition to apheresis compositions for ex vivo infection, treatment regimens will also include autologous or allogeneic CENK and / or m-CENK transplantation, autologous or allogeneic blood-matched erythrocyte transplantation, and autologous or allogeneic platelet transplantation. Furthermore, the treatment regimens envisioned herein will also include supportive administration of IL-15, IL15:IL15Rα constructs, or N-803 (subcutaneously or intratumorally) or other cytokines (e.g., NHS-IL-12, see [link to article]). Immunotargets Ther [Immune Targets and Therapies] 2021; 10: 155-169), for example, to help NK cells, T cells, and memory T cells proliferate. Furthermore, PD-L1 t-haNK cells can be administered to patients to kill myeloid-derived suppressor cells (MDSCs) and target tumor cells expressing PD-L1. In addition, the envisioned treatment may also include chemotherapy and / or radiation, and in some cases, treatment with recombinant therapeutic viruses (e.g., intratumoral administration). Therefore, the envisioned treatment regimens can be used as neoadjuvant therapy or as adjuvant therapy for first-line treatment of metastatic or recurrent disease.

[0031] Regarding in vitro infection of PBMCs, Figure 3An exemplary workflow using previously frozen PMBCs from leukocyte apheresis (LP186) is shown. More specifically, human peripheral blood mononuclear cells (PBMCs) were isolated from the leukocyte apheresis using a Ficoll Paque gradient and then frozen in liquid nitrogen. To test the ability of AdV to transduce dendritic cells in a mixed cell population, the PBMCs were thawed and cultured at 37°C for 24 hours in XVIVO15 medium containing GM-CSF and FLT3L. Next, the cells were transduced for 24 hours with AdV-GFP (a recombinant adenovirus encoding green fluorescent protein as a marker) at the indicated multiple of infection (MOI) and then analyzed by flow cytometry. While Figure 3 Peptide antigen stimulation is shown, but the inventors also performed AdV-GFP stimulation as shown in the following data. Antibodies against cell type-specific surface markers were used to identify granulocytes (CD56), antigen-presenting cells (HLA-DR), monocytes (CD14), and T cells (CD3). Gating was performed on individual cell types, and GFP expression was examined using Flowjo software. Notably, Figure 4 The results showed that only HLA-DR positive antigen-presenting cells exhibited GFP expression.

[0032] To test the effect of adjuvants on APC transduction by AdV-GFP, PBMCs were transduced and analyzed as described above, with the addition of TLR7 / 8 ligand remiquimod (R848), TLR4 ligand lipopolysaccharide (LPS), or both. Unexpectedly, Figure 5 The results showed that adding TLR ligands (TLR7 / 8 and TLR4 ligands) during AdV-GFP transduction resulted in reduced transduction efficiency across almost the entire tested MOI range.

[0033] Based on the results obtained in the above experiments, the inventors then conceived of treating fresh or frozen PBMCs obtained from subjects with GM-CSF and FLT3L for a duration sufficient to allow antigen-presenting cell differentiation (e.g., 8–16 hours, or 12–24 hours, or 18–36 hours, or 24–48 hours), typically in the presence of interleukin-4 (IL-4). Subsequently, the treated cells are then optionally stimulated to activate antigen-presenting cells in the presence of one or more TLR ligands, for example, with one or more peptide antigens, inactivated viruses, recombinant viruses (e.g., Ad5 viruses having recombinant nucleic acids encoding one or more cancer antigens, cancer-associated antigens, and / or patient- and tumor-specific neoantigens), or other pathogens. The differentiated and stimulated cells are then fed, and T cell subsets are expanded in the presence of activated antigen-presenting cells using fresh culture medium containing appropriate cytokines (e.g., IL-2, IL-7, IL-15). Most preferably, the resulting cell population is then restimulated with one or more peptide antigens, inactivated viruses, recombinant viruses, or other pathogens (preferably in the presence of anti-CD28 and / or anti-CD-49d antibodies) for at least 6-8 hours. If necessary, T cell function can then be tested (e.g., IFN-γ secretion can be tested in a routine ELISpot assay) before infusion to the subject.

[0034] As should be readily understood, initial stimulation with one or more peptide antigens, inactivated viruses, recombinant viruses, or other pathogens can be performed at doses or relative concentrations of viral particles to antigen-presenting cells that are substantially higher than those otherwise achieved in vivo stimulation. Furthermore, restimulation can also be performed at doses significantly higher than those administered in vivo. In practice, one or more peptide antigens, inactivated viruses, recombinant viruses, or other pathogens can be readily removed after (re)stimulation by simply exchanging the culture medium before administration to the subject. Therefore, it should be recognized that high transfection efficiency can be achieved in an ex vivo protocol using the subject's antigen-presenting cells, while completely avoiding systemic side effects or loss of recombinant virus due to nonspecific infection or viral particle adhesion to non-target tissues. Advantageously, and particularly in the case of recombinant Ad5 virus, transfection is selective for antigen-presenting cells (and especially dendritic cells) and does not adversely affect other cell types (and especially T cells) in the PBMC formulation that will be responsive to the antigens presented by the transfected antigen-presenting cells.

[0035] For example, in some embodiments, PBMCs are exposed to recombinant therapeutic viruses for a duration sufficient for antigen-presenting cells in the PBMCs to take up substantially all of the recombinant therapeutic virus (during stimulation and / or restimulation). In other options, suitable times will be between 60 and 180 minutes, or between 180 and 600 minutes, or between 8 and 12 hours, or between 12 and 24 hours, and even longer, typically at an MOI between 1:1 and 100,000 (and even more typically between 100 and 50,000 MOIs). Alternatively, and as already mentioned above, residual virus and its quantity can be removed and reduced by replacing the culture medium in which the cells are maintained or grown. Therefore, it should be understood that after in vitro infection, the viral titer in the PBMC supernatant is so low as to be undetectable (e.g., equal to or less than 10). 4 10 virus particles / mL, equal to or less than 10 3 10 virus particles / mL, equal to or less than 10 2 (1 virus particle / mL, or even less).

[0036] In cases where recombinant viruses are used for in vitro transfection, it is generally preferred that, in the PBMC formulation, in vitro infection of PBMCs exhibits preferential or even selective transmission of antigen-presenting / dendritic cells compared to non-dendritic cells (e.g., antigen-presenting / dendritic cells have the highest transfection rate, or the transfection rate of antigen-presenting / dendritic cells is at least 5, 10, 50, or 100 times that of non-dendritic cell subsets (e.g., T cells, NK cells, monocytes, etc.). Therefore, recombinant adenoviruses are particularly preferred.

[0037] Further preferred is that the in vitro transfection of antigen-presenting / dendritic cells is performed in the presence of other immune cells (such as macrophages, monocytes, T cells, B cells, etc.). As will be readily understood, such a mixed cell population will be suitable for directly exposing processed and presented antigens to T cells (and especially CD4+ and CD8+ T cells), thereby generating a cell population that can be infused into the subject to elicit an effective immune response against antigens encoded in the recombinant virus and expressed in the transfected cells.

[0038] Therefore, in the embodiments, the inventors have conceived of an immunotherapeutic composition comprising a patient-derived apheresis product and a human adenovirus serotype 5 (hAd5) vector, the patient-derived apheresis product comprising peripheral blood mononuclear cells (PBMCs), and the human adenovirus serotype 5 (hAd5) vector comprising deletions of the early 1 [E1] and early 2b [E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence. In this context, it is noteworthy that the term "human adenovirus serotype 5 (hAd5) vector" encompasses recombinant hAd5 nucleic acid and recombinant hAd5 viral particles containing recombinant hAd5 nucleic acid. This composition is intended for use in: generating 1) a cell-mediated delivery of an Ad5 vector to a patient in need; 2) an ex vivo composition comprising antigen-presenting cells (APCs) expressing an MHC-presented immunogenic peptide encoded by the Ad5 nucleic acid; and / or 3) activated T cells, wherein the T cells have been stimulated to proliferate through T cell receptor (TCR)-mediated contact with APC cells.

[0039] In the examples, the immunotherapeutic composition is determined by exposure time, wherein the composition comprises PBMC and an Ad5 carrier, and the duration of ex vivo exposure of PBMC to Ad5 is determined by the desired composition outcome (1-3 above).

[0040] In some embodiments, the composition comprises an Ad5 carrier encoding at least one antigenic peptide, PBMC-derived activated T cells, and IL-15 or an IL15Rβγ agonist derivative thereof. In some embodiments, the composition comprises activated T cells and N-803. IL-15 can promote the binding between IL15Rα expressed by APCs and IL15Rβγ expressed by T cells. N-803 will stimulate the expansion of activated T cells. In some embodiments, the composition further comprises 3M-052 (a Toll-like receptor (TLR) 7 / 8 stimulator).

[0041] In the embodiments, the composition is intended for use in the treatment of cancer or infectious diseases. The Ad5-encoded immunogenic peptide may encode a tumor-associated antigen, a tumor-specific antigen, or a neoantigen. The Ad5-encoded immunogenic peptide may encode a viral, yeast, or bacterial antigen. Identification of the antigen sequence may be derived from normal tumor sequencing, mRNA expression, and peptide expression analysis of normal and diseased tissues. For example, sequencing of tumors relative to normal tissues can be performed using algorithms as described in US 10971248, US 10991451, and US 11133085.

[0042] In one embodiment, the composition comprises an Ad5 carrier wherein the nucleic acid encodes alpha-fetoprotein. In another embodiment, the composition comprises 1) an Ad5 carrier wherein the nucleic acid encodes alpha-fetoprotein, 2) granulocyte-macrophage colony-stimulating factor (GMCSF), and 3) interleukin-4 (IL-4). In this embodiment, the composition is intended for use in the treatment of ulcerative colitis.

[0043] In embodiments, the inventors envision a treatment method in which the composition is administered to a patient in need. The method includes intravenous (IV) administration of the composition to the patient. The method further includes subcutaneous administration of IL-15, IL15:IL15Rα or a derivative thereof, or N-803 (an IL-15 superagonist) to the patient. The method further includes administration of one or more agents comprising Nant cancer vaccines, such as those described in US 2023 / 0034802, US 11,207,392, US 11,071,774, and US 11,439,697. The method may further include surgery and / or radiation.

[0044] In the embodiments, the composition comprises NK cells, wherein the NK cells are purified from apheresis products. NK cells are screened by flow cytometry or other methods. The NK cells are identified as CD56+ / CD3-. These NK cells are in vitro combined with an Ad5 vector containing a nucleic acid encoding at least one chimeric antigen receptor. Optionally, the composition further comprises IL-15, IL15:IL15Rα or a derivative thereof, or N-803. NK cells can be activated and expanded from PBMCs as previously described in US 11,453,862, US2021 / 0009954; and / or activated and expanded to generate CIML-NK cells as described in US 20210008107, US 2021 / 0008112, US 20210361711; alternatively or additionally, the NK cells can be differentiated into mCENK cells as described in WO / 2022 / 187207. In the case of transfecting NK cells with recombinant nucleic acids to express CAR, particularly contemplated compositions and methods are described in US 2022 / 0282216. The contemplated compositions are intended for use in the treatment of cancer or infectious diseases. A treatment method is also contemplated in which the contemplated composition is administered IV to a patient in need. The method further comprises subcutaneous administration of IL-15, IL15:IL15Rα or a derivative thereof, or N-803 (an IL-15 superagonist) to the patient. The method further comprises administration of one or more agents comprising a Nant cancer vaccine, as known from US 2023 / 0034802, US11,207,392, US 11,071,774, and US 11,439,697.

[0045] In this embodiment, sorted NK cells are cultured in IL15 and optionally glucocorticoids, followed by an expansion phase in which NK cells are cultured in IL15 / IL18 / IL12, thereby inducing natural killer (m-CENK) cells enriched with memory cytokines. Referring, for example, WO / 2022 / 187207, m-ceNK cells can be further genetically engineered to express CARs specific to cancer or infectious disease antigens. m-ceNK cells can be combined with stimulated T cells of the present invention for autologous administration. m-CENK cells can be used for autologous or allogeneic haploidentical co-administration in combination with IV administration of the Ad5-treated apheresis product of the present invention. m-CENK cells can be administered in combination with tumor-specific antibodies or checkpoint inhibitor antibodies. Alternatively, IL15-expanded NK cells can be administered as adjunctive therapy to lymphocyte-depleting chemotherapy agents in combination with IV administration of the Ad5-treated apheresis product of the present invention.

[0046] In embodiments, the inventors have envisioned a method for inducing differential in vitro expansion of antigen-specific T cells, comprising exposing a patient-derived apheresis product comprising peripheral blood mononuclear cells (PBMCs) to a human adenovirus serotype 5 (hAd5) vector for a duration sufficient to induce measurable T cell expansion, the hAd5 vector comprising deletions of the early 1 [E1] and early 2b [E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence. The method may further comprise the addition of IL15, IL15:IL15Rα or an IL15Rβγ agonist derivative thereof, IL15:IL15RαIgG4Fc, or N-803 to the PBMCs and the Ad5 vector to enhance T cell stimulation. In embodiments, the differentially stimulated T cells are specific to tumor antigens or neoantigens. In embodiments, the differentially stimulated T cells are specific to infectious disease antigens.

[0047] In an embodiment, the inventors envision an IV administration method of hAd5 vectors comprising deletions of the early 1 [E1] and early 2b [E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence, wherein the toxicity of IV or subcutaneous administration of the Ad5 vector is mitigated by cellular uptake, and thus cell-mediated delivery of the Ad5 vector to a patient is achieved via IV administration. The method comprises exposing a patient-derived apheresis product comprising peripheral blood mononuclear cells (PBMCs) to a human adenovirus serotype 5 (hAd5) vector for a duration sufficient to allow APCs of the apheresis product PBMCs to absorb the Ad5 vector, the human adenovirus serotype 5 (hAd5) vector comprising deletions of the early 1 [E1] and early 2b [E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence, wherein the viral titer of the PBMC supernatant is zero within an effective range.

[0048] In the embodiments, the inventors have envisioned a method for inducing antigen expression in PBMC-derived antigen-presenting cells, the method comprising exposing a patient-derived PBMC containing peripheral blood mononuclear cells (PBMCs) to a human adenovirus serotype 5 (hAd5) vector for a duration sufficient to induce expression of an immunogenic peptide on the surface of PBMC-derived APCs, the hAd5 vector comprising deletions of the early 1 [E1] and early 2b [E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence.

[0049] In the embodiments, the inventors envision adding IL15:IL15RαIgG4Fc to an ex vivo composition comprising PBMCs and an Ad5 vector. The IgG4 portion provides stability to the IL15:IL15Rα construct but eliminates the ability to recruit endogenous NK cells to T cells bound and stimulated by IL15:IL15RαIgG4Fc, because IgG4Fc does not bind to the Fc receptor (CD16) on NK cells. Therefore, antibody-dependent cytotoxicity of NK cells interacting with IgG1 of N-803 is blocked.

[0050] In the embodiments, prior to apheresis, IL15, IL15:IL15Rα or its IL15Rβγ agonist derivative, IL15:IL15RαIgG4Fc or N-803 are administered to the patient (subcutaneously).

[0051] In this embodiment, red blood cells are purified from the apheresis product. The RBCs can be engineered to express one or more therapeutic proteins. The RBCs can be amplified and differentiated into enucleated mature RBCs and formulated with Ad5-treated apheresis products for intravenous administration.

[0052] In this embodiment, platelets are purified from the apheresis product. Platelets can be engineered to express one or more therapeutic proteins (typically derived from RNA). Platelets can be formulated with Ad5-treated apheresis products for intravenous administration.

[0053] In the embodiments, the hAd5 E2b-deficient vector contains transgenes encoding tumor-associated antigens (e.g., MUC1, Brachyury1, CEA, PSA, PMSA, PD-L1, HER2, etc.) and / or neoantigens. In the embodiments, the hAd5 E2b-deficient vector further contains transgenes encoding cytokines, gene therapies, immunomodulatory proteins, alpha-fetoprotein, checkpoint inhibitors, and co-stimulatory proteins.

[0054] In this embodiment, PBMCs derived from single-harvest products are exposed to hAd5 vectors containing deletions of the early 1 [E1] and early 2b [E2b] genes and nucleic acids encoding at least one immunogenic or therapeutic peptide sequence. The exposure time can be at least one hour, at least two hours, at least four hours, at least eight hours, at least sixteen hours, at least twenty-four hours, at least forty-eight hours, at least seventy-two hours, at least four days, at least six days, or at least eight days.

[0055] The inventors have also envisioned a method for optimizing hAd5 uptake in an ex vivo composition comprising a patient-derived apheresis product and a human adenovirus serotype 5 (hAd5) vector. The patient-derived apheresis product comprises peripheral blood mononuclear cells (PBMCs), and the human adenovirus serotype 5 (hAd5) vector comprises deletions of the early 1 [E1] and early 2b [E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence. The optimal exposure time is empirically determined by the loss of Ad5 infectivity in the apheresis product supernatant. Infectivity can be determined indirectly, for example, by hexazometry of the supernatant.

[0056] In aspects that are still being further envisioned, various exemplary methods are envisioned. For example: Experiment 1: An assessment of N-803 versus IL15:IL15RαSuIgG4 to determine the effect of the IgG4 Fc moiety on T cell activation and / or self-destruction via antibody-dependent cytotoxicity. Here, it is envisioned that various assays with IL-15 activity (such as IL-15, N-803, IL15:IL15RαSuIgG4, etc.) be added to apheresis-derived PBMC fractions in the presence of Ad5 / tumor antigen. Alternatively, CEFT can be used for pulsed PBMCs, and T cell activation / proliferation can subsequently be assessed in the presence of IL15, IL15:IL15RαSu, IL15:IL15RαSuIgG4, or N803.

[0057] Experiment 2: Optimization of in vitro uptake, antigen presentation, and / or T cell stimulation. Viral uptake can be measured using a recombinant Ad5 virus encoding GFP or other reporter genes, which can then be used to identify the optimized multiplicity of infection (MOI). The same assay system can also be used to monitor Ad5 uptake using recombinant Ad5 virus or Ad5 null virus (and conversely, the virus is removed from the supernatant). In such a system, hexagonal assays can be used to quantify residual virus in the cell supernatant. Furthermore, the in vitro system can be used to quantify MHC expression in antigen-presenting cells and MHC-mediated antigen presentation (e.g., via flow cytometry or ELISPOT assays). As will be readily understood, antigen presentation can be further stimulated by co-incubation with various ligands, such as GMCSF, IL4, and / or Flt3 ligands. Further, it should be noted that T cell stimulation and proliferation can be determined in such an in vitro system using ELISPOT or other assays. When needed, the effects of adjuvants, co-stimulatory molecules (e.g., CD40 ligands, GITR ligands, Ox40 ligands, etc., which can be encoded in recombinant Ad5) or checkpoint inhibitors can be evaluated in vitro.

[0058] Experiment 3: Memory Stimulation vs. De novo Immunization. Here, the envisioned memory immune system could employ a test utilizing a universal antigen sequence (e.g., the wt Ad5 peptide absent in E1, E2B-deficient vectors, the SARS-CoV-2 spike protein, or the nucleocapsid protein) or an antigen sequence already immunized for individuals with PMBC (e.g., due to known prior exposure or vaccination). Alternatively, standard vaccines (e.g., adenovirus vaccine formulations encoding or expressing Brachyury, CEA, MUC1, PSA, etc.) could be used to induce de novo immunization.

[0059] Experiment 4: Optimization of MHC antigen peptide presentation: Various vaccine formulations can be tested in vitro to determine the effects of intracellular transport, the addition of recombinant co-stimulatory molecules, and / or cytokines on antigen presentation. Such a testing system will preferably include quantification of MHC-presented antigens and / or cell proliferation of antigen-responsive T cells. Subcellular localization tags genetically engineered to the N- or C-terminus of the antigen peptide can be used to optimize antigen presentation.

[0060] Experiment 5: Comparison of SubQ with in vitro administration. Animal systems can be used to, for example, determine the quantitative difference in immune response between the SubQ-administered formulation and the treated apheresis product administered via infusion in a culture medium suitable for cell infusion (e.g., 5% human serum albumin in 0.9% sodium chloride). Efficacy can be measured in vitro by antigen-specific T cells (e.g., using ELISPOT assays). For example, the C57BL / 6 inbred mouse model can be used for 1) SubQ-administered Ad5 vaccine and 2) IV-administered in vitro apheresis product exposed to Ad5 vaccine.

[0061] In some embodiments, the numbers used to describe and claim certain embodiments of the invention regarding the quantities of expressed components, properties (such as concentrations), reaction conditions, etc., should be understood to be modified in some cases by the term "about". As used herein, when referring to a specified measurable value (such as a parameter, quantity, duration, etc.), the terms "about" and "approximately" are intended to cover the specified value and variations thereof, such as + / - 10% or less, alternatively + / - 5% or less, alternatively + / - 1% or less, alternatively + / - 0.1% or less, provided that such variations are suitable for the disclosed embodiments. Thus, the values ​​referred to by the modifiers "about" or "approximately" are also specifically disclosed themselves. The description of value ranges herein is intended only as a shorthand method of individually referring to each individual value falling within that range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were described separately herein.

[0062] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise obviously contradicted by the context. The use of any and all instances or exemplary language (e.g., "such") provided with respect to certain embodiments herein is intended only to better illustrate the invention and does not constitute a limitation on the originally claimed scope of the invention. The language in this specification should not be construed as indicating that any unclaimed element is necessary for practicing the invention.

[0063] As used herein and throughout the claims, unless the context clearly indicates otherwise, the meanings of “a” and “the” include plural pronouns. Similarly, as used herein, unless the context clearly indicates otherwise, the meaning of “in” includes both “in” and “on”. Likewise, as used herein, and unless the context clearly indicates otherwise, the term “coupled to” is intended to include both direct coupling (where two coupled elements are in contact with each other) and indirect coupling (where at least one additional element is located between the two elements). Therefore, the terms “coupled to” and “coupled with” are used synonymously.

[0064] It will be apparent to those skilled in the art that further modifications are possible beyond those already described without departing from the inventive concept described herein. Therefore, the subject matter of this invention should not be limited except within the scope of the appended claims. Furthermore, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to an element, component, or step in a non-exclusive manner, indicating that the referenced element, component, or step may be present or utilized, or combined with other elements, components, or steps not expressly referenced. When the specification or claims refer to at least one selected from the group consisting of A, B, C… and N, the text should be interpreted as requiring only one element from that group, rather than A plus N, or B plus N, etc.

Claims

1. An immunotherapeutic composition comprising: Subject-derived peripheral blood mononuclear cells (PBMCs); and At least one recombinant adenovirus subtype 5 (Ad5) vector comprising a deletion in the E1 gene region, a deletion in the E2b gene region, and a nucleic acid sequence encoding a peptide antigen, wherein the PBMC is exposed in vitro to the at least one Ad5 vector.

2. The immunotherapeutic composition of claim 1, further comprising IL15 or an interleukin-15:interleukin-15 receptor α (IL15:IL15Rα) complex, wherein immune effector cells are simultaneously exposed to the Ad5 carrier and IL15 or the IL-15:IL-15Rα complex.

3. The immunotherapeutic composition of claim 2, wherein the IL-15:IL-15Rα complex comprises the IL-15N72D:IL-15RαSu / Fc complex.

4. The immunotherapeutic composition of claim 3, wherein the IL-15N72D:IL-15RαSu / Fc complex is N-803.

5. The immunotherapeutic composition of claim 1, wherein the subject has been diagnosed with an infectious disease, a neoplastic disease, or cancer.

6. The immunotherapeutic composition of claim 1, wherein the subject has not been diagnosed with an infectious disease, adenoid disease, or cancer.

7. The immunotherapeutic composition of claim 1, wherein the PBMC is derived from apheresis.

8. The immunotherapeutic composition of claim 7, wherein the PBMC comprises T cells, natural killer (NK) cells, natural killer T (NK-T) cells, dendritic cells, mast cells, myeloid-derived phagocytes, or combinations thereof.

9. The immunotherapeutic composition of claim 1, wherein the immunotherapeutic composition is formulated for intravenous (IV) administration.

10. The immunotherapeutic composition of claim 9, for use in treating a patient with a neoplastic disease, cancer, or infectious disease, wherein the composition is administered IV to the patient.

11. The immunotherapeutic composition of claim 10, wherein the proliferative disease or cancer includes bladder cancer, glioblastoma, prostate cancer, hematologic malignancy, B-cell tumor, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, cutaneous T-cell lymphoma, T-cell lymphoma, solid tumor, urothelial carcinoma / bladder cancer, melanoma, lung cancer, renal cell carcinoma, breast cancer, gastric and esophageal cancer, head and neck cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, B-cell non-Hodgkin lymphoma, squamous cell head or neck cancer, or urothelial carcinoma / bladder cancer.

12. The immunotherapeutic composition of claim 10, wherein the treatment further comprises radiation, chemotherapy, surgery, therapeutic antibodies, immunomodulators, proteasome inhibitors, panDAC inhibitors, H-DAC inhibitors, checkpoint inhibitors, adoptive cell therapies including CAR T and / or NK cell therapies, and / or immunotherapeutic agents.

13. The immunotherapeutic composition of claim 1, wherein the peptide antigen is a tumor-associated antigen and / or a neoantigen.

14. The immunotherapeutic composition of claim 13, wherein the Ad5 vector further comprises a nucleic acid for expressing at least one of cytokines, gene therapies, and immunomodulatory proteins.

15. A method for treating cancer or an infectious disease, the method comprising: Therapeutic apheresis was performed on the subjects; Purification of peripheral blood mononuclear cell (PBMC) fractions from apheresis products; The PBMC was exposed in vitro to at least one recombinant adenovirus subtype 5 (Ad5) vector containing deletions in the E1 gene region, deletions in the E2b gene region, and nucleic acid sequences encoding peptide antigens of the cancer or pathogens causing the infectious disease; and Administer an effective amount of PBMCs treated with Ad5 vector to the patient.

16. The method of claim 15, further comprising adding interleukin-15 (IL15) or an IL15:IL15 receptor α (IL15:IL15Rα) complex to the ex vivo exposed PBMCs.

17. The method of claim 15, further comprising pre-treating the patient with IL15 or an IL15:IL15Rα complex.

18. The method of claim 17, wherein the IL15:IL15Rα complex is N-803.

19. The method of claim 15, wherein the subject and the patient are the same individual.

20. The method of claim 15, wherein the subject and the patient are different individuals.

21. The method of claim 15, wherein IL15 or the IL15:IL15Rα complex is added to the PBMC treated with the Ad5 carrier prior to application.

22. The method of claim 15, wherein after administration of the Ad5-carrier-treated PBMC, IL15 or an IL15:IL15Rα complex is administered to the patient.

23. A method for transfecting multiple peripheral blood mononuclear cells (PBMCs), the method comprising the following steps in sequence: (1) Provides PBMC fractions prepared in vitro, wherein the PBMC fractions contain, in addition to antigen-presenting / dendritic cells, cells selected from a group consisting of macrophages, monocytes, T cells and B cells; (2) Expose the PBMC fraction to GM-CSF and Flt3L to trigger the differentiation of these antigen-presenting / dendritic cells; (3) Expose the PBMC-grade isolate to recombinant adenovirus subtype 5 (Ad5) viral particles at a multiplicity of infection (MOI) of at least 100, wherein the recombinant Ad5 particles contain deletions in the E1 gene region, deletions in the E2b gene region, and nucleic acid sequences encoding peptide antigens for cancer or pathogens causing infectious diseases. (4) Use IL-2, IL-7 and IL-15 to expand T cells in this PBMC fraction; (5) Re-expose the PBMC-grade isolate to these recombinant adenovirus subtype 5 (Ad5) virus particles; as well as (6) Place the re-exposed PBMC fraction in a culture medium suitable for infusion.

24. The method of claim 23, wherein the PBMC is a previously frozen cell.

25. The method of claim 23, wherein the step of exposing the PBMC fraction to GM-CSF and Flt3L further comprises exposing the PBMC fraction to IL-4.

26. The method of claim 23, wherein the step of exposing the PBMC-grade isolate to these recombinant Ad5 virus particles is performed at an MOI between 10,000 and 100,000.

27. The method of claim 23, wherein the step of amplifying these T cells in the PBMC fraction is performed for at least four days.

28. The method of claim 23, wherein the step of placing the re-exposed PBMC fraction in the culture medium suitable for infusion reduces the Ad5 virus particles in the PBMC fraction by at least 100-fold.

29. The method of claim 23, wherein the step of distributing the re-exposed PBMC grade in the culture medium suitable for infusion reduces Ad5 virus particles to equal or less than 1,000 virus particles / mL.

Images