Apheresis products transduced by adenovirus vectors

Abstract

An immunotherapy composition is envisioned comprising peripheral blood mononuclear cells (PBMCs) derived from a target and at least one recombinant adenovirus subtype 5 (Ad5) containing 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 in vitro exposed to at least one Ad5 vector. Advantageously, the same PBMC composition may also be used to prepare modified NK cells, and modified NK cells in particular include CIML NK cells, CENK cells, and mCENK cells.

Description

[Technical Field]

[0001] This application claims priority to the inventors' concurrently pending U.S. Provisional Patent Application No. 63 / 500,235, filed on 4 May 2023 and incorporated herein by reference.

[0002] The field of the present invention is, in particular, that of the treatment of a target using an in vitro modified apheresis product in which cells are transfected with recombinant therapeutic viruses and optionally differentiated or transfected to express a chimeric antigen receptor, and therefore the present invention relates to compositions and methods for the treatment of cancer. [Background technology]

[0003] The background information includes information that may be useful in understanding the present invention. This does not constitute prior art, nor does it acknowledge that any information provided herein is prior art, or relates to the claimed invention herein, or that any publication specifically or implicitly referenced is prior art.

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

[0005] Cancer vaccines, and especially viral cancer vaccines, have shown great promise in recent years, but are often limited by various factors, including the immunogenicity of the viral medium and / or the low presentation of recombinant (neo)antigens. In efforts to improve the therapeutic effects of cancer vaccines, the selected neoepitope may follow an in vitro testing protocol in which the immune response of the patient's immune cells is determined before administration of the vaccine containing the selected neoepitope, as described in International Publication No. 2018 / 106699. While conceptually interesting, the selected neoepitope is typically encoded in recombinant viral nucleic acid, and the viral vaccine thus produced is then administered to the patient. Unfortunately, due to the typically systemic delivery of the viral medium, extensive training of various components in the immune system (e.g., dendritic cells, CD8+ T cells, CD4+ helper T cells, B cells) is often not achieved or is simply poorly achieved. Furthermore, even when antigen presentation is achieved to some extent, a therapeutically effective immune response may not be produced, for example, due to the relatively low amount of viral medium. These problems are further exacerbated by significant hepatotoxicity and, in many cases, the rapid attachment of recombinant virus particles to host cells, often leading to a variety of side effects and even death (see, for example, Cancer Gene Therapy (2002) 9, 979-986).

[0006] To overcome some of the problems associated with systemic delivery of tumor antigens, various efforts have been made to induce in vitro stimulation of specific cells in the immune system. For example, one method involves incubating dendritic cells with NK cells to produce mature dendritic cells in the presence of a TLR agonist (see, e.g., Experimental & Molecular Medicine (2010), 42(6), pp. 407-419). Another method involves stimulating antigen-presenting cells with chimeric proteins containing cancer-specific antigens (see, e.g., Nat Rev Cancer; 12(4): 265-277). Here, the antigen was directly delivered in vivo to dendritic cells (DCs) using a chimeric protein containing an antibody specific to the DC receptor fused to the selected antigen. Such specific targeting of the antigen to DCs in vivo induced potent antigen-specific CD4+ and CD8+ T cell-mediated immunization. However, the induction of immunity also required the provision of DC maturation signals. In other cases, such methods induced antigen-specific resistance contrary to the desired outcome. Furthermore, in vitro DC targeting using currently known DC receptors requires the creation of custom molecular entities, which is intensive in terms of both time and resources. Moreover, such methods also require specific maturation signals, further complicating the vaccination process.

[0007] As a further option to reduce systemic exposure, the therapeutic virus may be administered via a subcutaneous route. While this route of administration significantly reduces the risk of serious adverse events, it introduces further problems. Most significantly, subcutaneous administration substantially limits the exposure of the virus to antigen-presenting cells, and especially dendritic cells. Therefore, the expected therapeutic effect is much less than that of systemic administration. [Overview of the project] [Problems that the invention aims to solve]

[0008] Therefore, although various compositions and methods for cancer vaccines are known in the art, all or almost all of them have some drawbacks, particularly when recombinant viral media are used in cancer vaccines to produce neoantigens in infected cells. Thus, there is still a need to improve viral vaccine compositions. [Means for solving the problem]

[0009] The subject of this invention relates to various compositions and methods of viral vaccine compositions for in vitro infection of donor cells, including target peripheral blood mononuclear cells (PBMCs), with a recombinant viral vaccine to a degree sufficient to reduce or even completely deplete the composition of free extracellular viral particles, thereby avoiding particularly likely adverse effects. In further compositions and methods envisioned, at least a portion of the donor cells and especially lymphocytes are further genetically modified, for example, to express CARs and / or treated to differentiate into more mature immune cells. Most preferably, the in vitro infection of PBMCs is preferential or even more selective with respect to dendritic cells, and more preferably carried out in the presence of other immune cells such as macrophages, monocytes, T cells, and B cells.

[0010] In one aspect of the subject matter of the present invention, the inventors intend an immunotherapy composition comprising peripheral blood mononuclear cells (PBMCs) derived from a subject 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 to the Ad5 vector in vitro. Most typically, the Ad5 vector contains nucleic acids for the expression of at least one tumor-associated antigen, neoantigen, cytokine, gene therapy, and immunomodulatory protein.

[0011] As is easily understood, the intended immunotherapy 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 the Ad5 vector and the IL-15:IL-15Rα complex. For example, the intended IL-15:IL-15Rα complex comprises an IL-15N72D:IL-15RαSu / Fc complex (e.g., nogapendekin alpha-invacixept (NAI), also known as N-803 or Alt-803) where the Fc portion may be derived from any immunoglobulin and especially IgG (e.g., IgG1, IgG4, etc.). Alternatively, the IL-15:IL-15Rα complex may also be encoded by an Ad5 vector and subsequently expressed and secreted from infected cells.

[0012] In further embodiments, the subject may or may not be diagnosed with an infectious disease, neoplastic disease, or cancer. Most preferably, the PBMCs are induced by apheresis of the subject and will include T cells, B cells, natural killer (NK) cells, natural killer T (NK-T) cells, dendritic cells, mast cells, bone marrow-derived phagocytic cells, or a combination thereof.

[0013] Furthermore, immunotherapy compositions (post-in vivo) are typically intended to be formulated for intravenous (IV) administration used in the treatment of neoplastic diseases, cancer, or infections in patients. For example, the neoplastic diseases or cancers intended include bladder cancer, glioblastoma, prostate cancer, hematological malignancies, B-cell neoplasms, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, cutaneous T-cell lymphoma, T-cell lymphoma, solid tumors, urothelial / 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 and neck cancer, or urothelial / bladder cancer.

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

[0015] Consequently, the inventors also envision a method for treating cancer or infection comprising the steps of: performing therapeutic apheresis on a subject; purifying a peripheral blood mononuclear cell (PBMC) fraction from the apheresis product; exposing the PBMCs in vitro to at least one recombinant adenovirus subtype 5 (Ad5) vector containing a deletion in the El gene region, a deletion in the E2b gene region, and a nucleic acid sequence encoding a peptide antigen; and administering an effective amount of the Ad5 vector-treated PBMCs to the patient.

[0016] Such methods may also include the step of adding interleukin-15 (IL15) or IL15:IL15 receptor α (IL15:IL15Rα) complexes to PBMCs exposed in vitro. Additionally or alternatively, patients may be pre-treated with IL15 or IL15:IL15Rα complexes (e.g., N-803), and / or IL15 or IL15:IL15Rα complexes may be added to PBMCs treated with the Ad5 vector before administration. Similarly, IL15 or IL15:IL15Rα complexes may be administered to patients after administration of PBMCs treated with the Ad5 vector. As can be easily understood, the administered cells may be obtained from the same individual or from different (e.g., at least haplomatched) individuals.

[0017] Various purposes, features, aspects, and advantages of the subject matter of the present invention will become clearer from the following detailed description of preferred embodiments, along with the accompanying drawings in which similar figures represent similar elements. [Brief explanation of the drawing]

[0018] [Figure 1]An exemplary schematic flowchart of cancer treatment according to the subject matter of the present invention. [Figure 2] Another exemplary schematic flowchart of cancer treatment according to the subject matter of the present invention. [Figure 3] An exemplary schematic flowchart for the selective ex vivo Ad5 infection of PBMCs. [Figure 4] Illustrative results for the Ad5 infection of PBMCs using an MOI of 1000 according to the subject matter of the present invention are presented. [Figure 5] Illustrative results for Ad5 infection at various MOIs according to the subject matter of the present invention are presented.

Mode for Carrying Out the Invention

[0019] Here, the inventors have discovered viral cancer vaccine compositions and methods that reduce or even entirely avoid adverse effects and / or efficiency reduction resulting from the rapid attachment of various vaccine compositions and methods, and particularly therapeutic viruses, to non-target tissues.

[0020] In a particularly preferred embodiment, recombinant therapeutic viruses are administered in vitro to cells of a subject diagnosed with cancer, which are obtained from the subject by apheresis. In connection therewith, it should be particularly understood that apheresis can be used not only to isolate PBMCs but also to isolate erythrocytes and platelets, which can be used in their isolated form or as therapeutic entities after modification (e.g., transfection of recombinant nucleic acids and especially mRNA). In addition, natural killer (NK) cells can be isolated from PBMCs or via apheresis to obtain yet another therapeutic mode in which the NK cells can be further genetically modified (e.g., thereby expressing CAR) or differentiated into cytokine-enhanced NK (CENK) cells or memory-type cytokine-enhanced (m-CENK) cells by stimulation. Thus, apheresis can be used to prepare various cell fractions other than PBMCs in a simultaneous or sequential manner for the treatment of the same patient. Most typically, the treatments envisioned herein will involve at least the administration of dendritic cells infected in vitro (most typically preferentially or selectively infected in the presence of other immune cells).

[0021] In one typical example, as schematically illustrated in Figure 1, subjects diagnosed with cancer, when selectively treated with N-803, stimulate the production of T cells and NK cells before apheresis (during which PBMCs are collected). During the same apheresis procedure, cell fractions enriched with red blood cells and cell fractions enriched with platelets can also be obtained from the same subjects. Most typically, PBMCs are CD4 + T cells, CD8 +It will include various immune cells such as T cells, B cells, macrophages, dendritic cells, Kupffer cells, etc. As can be easily understood, by immediately using a diverse mixture of cells for infection with a therapeutic virus (and particularly recombinant human adenovirus serotype 5 (hAd5)), antigen presentation of recombinant antigens encoded by the recombinant adenovirus on antigen-presenting cells such as macrophages and dendritic cells can be induced. Consequently, antigen-presenting cells presenting recombinant neoantigens on MHC class I complexes and / or class II complexes are non-licensed T cells, immature and mature unstimulated T cells, (antigen-responsive) CD4 + T cells and (antigen-responsive) CD8 + T cells can easily come into contact with, and in this way, the expression (activation and expansion) of antigen-specific cytotoxic T cells will be induced. Alternatively or additionally, the recombinant nucleic acid in the recombinant therapeutic virus need not be limited to recombinant nucleic acids encoding one or more neoantigens or tumor-associated antigens, but the recombinant nucleic acid can also encode a CAR having specificity for cancer-specific antigens of cancer cells. Of course, it should be understood that the infection of lymphocytes with the recombinant therapeutic virus can be carried out in the presence of immunostimulatory cytokines (such as IL-2, IL-15, etc.), costimulatory molecules and / or checkpoint inhibitors.

[0022] In particular the compositions and methods intended, it is generally preferred that PBMCs be exposed to recombinant therapeutic virus for a time sufficient to induce measurable T cell expansion and, in particular, clonal proliferation of antigen-responsive T cells. As understood, such expansion can be stimulated by the addition of IL15, IL15:IL15Rα or its IL15Rβγ agonist derivative, IL15:IL15RαIgG4Fc, or N-803 to the PBMCs and therapeutic virus. In a more preferred embodiment, the PBMCs are exposed to recombinant therapeutic virus for a time sufficient to allow the antigen-presenting cells in the PBMCs to take up substantially all of the recombinant therapeutic virus. From a different perspective, during in vitro infection, it is typically preferred that the viral titer in the PBMC supernatant be low or undetectable. Such time can be empirically confirmed by quantifying the viral infectivity in the cell composition (e.g., using a Hexone assay when the virus is an adenovirus). If T cell activation and / or expansion are observed and the viral titer in the supernatant is low or undetectable, the cell suspension thus prepared can be used for injection. Therefore, it should be understood that by using the intended method and composition, infection of antigen-presenting cells with the therapeutic virus is optimized, while potential adverse effects due to off-target viral binding are minimized or avoided entirely. The inventors also intend that the time of in vitro exposure is determined by the MHC presentation of the peptide encoded by Ad, and that IV administration of the apheresis product treated with Ad is performed via the MHC-presenting antigen on in vitro infected cells, thereby activating antigen-responsive CD4 + T cells and CD8 + This leads to subsequent in vivo stimulation of T cells.

[0023] In addition, NK cells can be isolated from a portion of the apheresis product, and it is intended that in vitro expansion will provide further cytotoxic effector cells. Furthermore, if necessary, the isolated NK cells can be differentiated into cytokine-enhancing NK cells (CENK) and / or memory-type cytokine-enhancing NK cells (m-CENK) by further stimulation. Alternatively or additionally, at least a portion of the isolated NK cells can be transfected with recombinant nucleic acids to express CARs with selected target specificity on the cell surface (e.g., neoantigens or tumor-associated antigens, e.g., MUC1, Brachyury, CEA, PSA, PMSA, PD-L1, HER2, etc.). These modified NK cells can then be administered to the target to further enhance the immunotherapy composition containing the in vitro-infected apheresis product. Furthermore, it is intended that further cell-based therapeutics, such as haNK cells or t-haNK cells, can be administered, as described, for example, in U.S. Patent No. 11,643,452 and U.S. Patent Application Publication No. 2021 / 0198342.

[0024] Furthermore, apheresis can also yield red blood cells and platelets, which are intended to be available as additional therapeutic agents. For example, if a subject has previously received chemotherapy or radiation, especially if the chemotherapy or radiation was administered after apheresis, the red blood cells and platelets can be stored and administered. Notably, red blood cells and / or platelets can be genetically modified to express one or more target proteins (e.g., cytokines, checkpoint inhibitors, etc.).

[0025] With regard to the administration of in vitro-infected apheresis compositions, it is intended that they can be injected into cells as a standalone modality or in combination with one or more immunostimulatory cytokines. Most typically, if the administration also includes the injection of NK cells (e.g., CENK, m-CENK, CAR-expressing NK cells, etc.), the NK cells would preferably be administered after the in vitro-infected apheresis composition, for example, at least 1 day, at least 3 days, at least 7 days, or at least 14 days after the injection of the in vitro-infected apheresis composition.

[0026] Figure 2 provides a typical and schematic illustration of therapeutic options and components using in vitro-infected apheresis compositions. As can be readily interpreted from Figure 2, the therapeutic protocol would include, in addition to the in vitro-infected apheresis composition, autologous or allogeneic CENK grafts and / or m-CENK grafts, autologous or allogeneic blood-matched red blood cell grafts, and autologous or allogeneic platelet grafts. Furthermore, the therapeutic scheme envisioned herein would also include supportive administration of IL-15, IL15:IL15Rα construct or N-803 (subcutaneously or intratumorally) or other cytokines (see, e.g., NHS-IL-12, Immunotargets Ther. 2021;10:155-169) to assist the proliferation of NK cells, T cells, and memory T cells. Furthermore, PD-L1 t-haNK cells can be administered to patients to kill bone marrow-derived suppressor cells (MDSCs) and target tumor cells expressing PD-L1. The proposed treatment may also include chemotherapy and / or radiation therapy, and optionally recombinant therapeutic viruses (e.g., administered intratumorally). Thus, the proposed treatment plan can be used as a neoadjuvant or adjuvant therapy as a first-line treatment for metastatic or recurrent disease.

[0027] Regarding in vitro infection of PBMCs, Figure 3 shows an exemplary workflow using previously frozen PMBCs from LeucoPak (LP186). More specifically, human peripheral blood mononuclear cells (PBMCs) were isolated from LeucoPak using a FicolPak gradient and then frozen under liquid nitrogen. To test AdV's ability to transduce dendritic cells into a mixed cell population, PBMCs were thawed and cultured for 24 hours at 37°C in XVIVO15 medium containing GM-CSF and FLT3L. The cells were then transduced for 24 hours with AdV-GFP (a recombinant adenovirus encoding green fluorescent protein as a marker) at the multiplicity of infection (MOI) specified below, and then analyzed by flow cytometry. While Figure 3 shows peptide antigen stimulation, we also performed stimulation with AdV-GFP, as shown in the data below. Antibodies against cell type-specific surface markers were used to identify granulocytes (CD56), antigen-presenting cells (HLA-DR), monocytes (CD14), and T cells (CD3). Each cell type was gated and tested for GFP expression using Flowjo software. In particular, the results in Figure 4 show that only HLA-DR-positive antigen-presenting cells exhibited GFP expression.

[0028] To test the effect of adjuvants on APC transduction by AdV-GFP, PBMCs were transduced and analyzed as described above, with the simultaneous addition of the TLR7 / 8 ligand rexiquimod (R848), the TLR4 ligand lipopolysaccharide (LPS), or both. Surprisingly, the results in Figure 5 show that the simultaneous addition of TLR ligands (TLR7 / 8 ligand and TLR4 ligand) and AdV-GFP transduction resulted in a decrease in transduction efficiency across almost the entire range of MOI tested.

[0029] Based on the results obtained in the above experiments, the inventors then envisioned that fresh or frozen PBMCs obtained from the subject could be treated with GM-CSF and FLT3L for a sufficient time (e.g., 8–16 hours, 12–24 hours, 18–36 hours, or 24–48 hours) typically in the presence of interleukin-4 (IL-4) to allow differentiation of antigen-presenting cells. Subsequently, the cells thus treated were activated by stimulating them with, optionally, one or more TLR ligands, for example, 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 to activate the antigen-presenting cells. The thus differentiated and stimulated cells were then nourished, and the T cell subpopulation was expanded in the presence of activated antigen-presenting cells using a fresh medium containing suitable cytokines (e.g., IL-2, IL-7, IL-15). Most preferably, the cell population thus obtained is then stimulated again with a peptide antigen, inactivated virus, recombinant virus, or other pathogen (preferably in the presence of anti-CD28 antibody and / or anti-CD49d antibody) for at least 6–8 hours. If necessary, the T cells may then be tested for function (e.g., IFN-γ secretion in a conventional erythropoiesis assay) before injection into the target.

[0030] As should be readily understood, the initial stimulation by peptide antigens, inactivated viruses, recombinant viruses or other pathogens can be carried out at substantially higher doses or relative concentrations of viral particles to antigen-presenting cells than would be achieved particularly with in vivo stimulation. Furthermore, for restimulation, an amount significantly higher than that administered in vivo can be equally used. In fact, peptide antigens, inactivated viruses, recombinant viruses or other pathogens can be easily removed simply by exchanging the medium after (re)stimulation and prior to administration to the subject. Thus, while high transfection efficiency can be achieved using the subject's antigen-presenting cells in an in vitro protocol, it should be understood that systemic side effects or non-specific infection or adhesion of viral particles to non-target tissues from reduced recombinant viruses can be entirely avoided. Advantageously and particularly when a recombinant Ad5 virus is used, the transfection is selective for antigen-presenting cells (and particularly dendritic cells) and will not adversely affect other cell types in the PBMC preparation (and particularly T cells) that will be reactive to the antigen presented by the transfected antigen-presenting cells.

[0031] For example, in some embodiments, the PBMCs are exposed to the recombinant therapeutic virus for a time sufficient for the antigen-presenting cells in the PBMCs to take up substantially all of the recombinant therapeutic virus (during stimulation and / or restimulation). Among other options, suitable times are typically at a MOI of 1:1 to 100,000 (and even more typically at a MOI of 100 to 50,000) for 60 to 180 minutes, or 180 to 600 minutes, or 8 to 12 hours, or 12 to 24 hours and even longer. Instead and as already mentioned above, residual virus can be removed and the numbers reduced by exchanging the medium in which the cells are maintained or grown. Thus, during in vitro infection, the virus titer in the PBMC supernatant is low to undetectable (e.g., 10 4 virus particles / mL or less, 10 3 virus particles / mL or less, 10 2 virus particles / mL or less or even lower).

[0032] When recombinant viruses are used for in vitro transfection, it is generally preferable that in vitro infection of PBMCs involves antigen-presenting cells / dendritic cells in the PBMC preparation with preferential or even more selective involvement compared to non-dendritic cells (for example, antigen-presenting cells / dendritic cells have the highest transfection rate, or antigen-presenting cells / dendritic cells have a transfection rate at least 5 times, at least 10 times, at least 50 times, or at least 100 times faster than the transfection rate of non-dendritic cell subpopulations (e.g., T cells, NK cells, monocytes, etc.)). Therefore, recombinant adenoviruses are particularly preferred.

[0033] It is even more preferable that the in vitro transfection of antigen-presenting cells / dendritic cells be carried out in the presence of other immune cells such as macrophages, monocytes, T cells, and B cells. As can be easily understood, such a mixed cell population is suitable for directing the exposure of T cells (and especially CD4+ and CD8+ T cells) to the processed and presented antigen, and injecting it into a target, resulting in a cell population capable of inducing an effective immune response to the antigen encoded by the recombinant virus and expressed within the transfected cells.

[0034] Accordingly, in one embodiment, the inventors envision an immunotherapy composition comprising a patient-derived apheresis product containing peripheral blood mononuclear cells (PBMCs) and a human adenovirus serotype 5 (hAd5) vector containing deletions of the initial 1[E1] and initial 2b[E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence. In this regard, it should be noted that the term “human adenovirus serotype 5 (hAd5) vector” encompasses recombinant hAd5 nucleic acids and recombinant hAd5 virus particles containing recombinant hAd5 nucleic acids. The composition is intended for use in 1) a cell medium for administering the Ad5 vector to a patient in need, 2) an in vitro composition comprising antigen-presenting cells (APCs) expressing MHC-presented immunogenic peptides encoded by the Ad5 nucleic acid, and / or 3) the generation of activated T cells stimulated to proliferate by T cell receptor (TCR)-mediated contact with APC cells.

[0035] In one embodiment, the immunotherapy composition is determined by the exposure time, and the composition comprises PBMCs and an Ad5 vector, and the duration of in vitro exposure of PBMCs to Ad5 is determined by the results of the desired composition (1-3 above).

[0036] In one embodiment, the composition comprises at least one antigenic peptide, activated T cells derived from PBMCs, and an Ad5 vector encoding IL-15 or its IL15Rβγ agonist derivative. In another embodiment, the composition comprises activated T cells and N-803. IL-15 may act to promote binding between IL15Rα expressed in APCs and IL15Rβγ expressed in T cells. N-803 will stimulate the expansion of activated T cells. In another embodiment, the composition further comprises 3M-052 (a Toll-like receptor (TLR) 7 / 8 agonist).

[0037] In some embodiments, the composition is for use in the treatment of cancer or infection. The immunogenic peptide encoded by Ad5 may encode a tumor-associated antigen, a tumor-specific antigen, or a neoantigen. The immunogenic peptide encoded by Ad5 may encode a viral, yeast, or bacterial antigen. Antigen sequence identification can be obtained from normal sequencing of the tumor, mRNA expression, and peptide expression analysis of normal and affected tissues. For example, normal sequencing of a tumor may be performed using algorithms such as those described in U.S. Patent No. 1,0971,248, U.S. Patent No. 1,0991,451, and U.S. Patent No. 1,113,3085.

[0038] In one embodiment, the composition comprises an Ad5 vector in which nucleic acid encodes alpha-fetoprotein. In another embodiment, the composition comprises 1) an Ad5 vector in which 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.

[0039] In one embodiment, the inventors have conceived a method of treatment in which the composition is administered to a patient in need. The method comprises intravenous (IV) administration of the composition to the patient. The method further comprises subcutaneous administration to the patient of IL-15, IL15:IL15Rα or a derivative thereof, or N-803 (an IL-15 superagonist). The method further comprises administration of one or more agents, including, for example, a Nant cancer vaccine (as described in U.S. Patent Publication No. 2023 / 0034802, U.S. Patent No. 11,207,392, U.S. Patent No. 11,071,774, and U.S. Patent No. 11,439,697). The method may further comprise surgery and / or radiation.

[0040] In one embodiment, the composition comprises NK cells purified from an apheresis product. The NK cells are sorted and removed by flow sorting or other methods. The NK cells are identified as CD56+ / CD3-. The 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 U.S. Patent No. 11,453,862 and U.S. Patent Publication No. 2021 / 0009954, and / or activated and expanded to produce CIML-NK cells as described in U.S. Patent Publication No. 20210008107, U.S. Patent Publication No. 2021 / 0008112 and U.S. Patent Publication No. 20210361711. Alternatively or additionally, NK cells can be differentiated into mCENK cells as described in International Publication No. 2022 / 187207. When NK cells are transfected with recombinant nucleic acid to express CAR, compositions and methods particularly intended are described in U.S. Patent Publication No. 2022 / 0282216. The compositions intended are for use in the treatment of cancer or infectious diseases. Further envisioned is a method of treatment in which the envisioned composition is administered intravenously to a patient in need. The method further comprises subcutaneous administration to a patient of IL-15, IL15:IL15Rα or a derivative thereof, or N-803 (an IL-15 superagonist). The method further comprises administration of one or more agents, including the Nant cancer vaccine, which are known from U.S. Patent Publication No. 2023 / 0034802, U.S. Patent No. 11,207,392, U.S. Patent No. 11,071,774, and U.S. Patent No. 11,439,697.

[0041] In one embodiment, selected NK cells are cultured with IL15 and optionally glucocorticoids, followed by an expansion phase in which the NK cells are cultured with IL15 / IL18 / IL12, thereby inducing memory cytokine-enriched natural killer (m-CENK) cells. See, for example, International Publication No. 2022 / 187207. m-CENK cells can be further genetically modified to express CARs specific to cancer or infectious disease antigens. m-CENK cells may be used for autologous administration in combination with the stimulated T cells of the present invention. m-CENK cells may be used for autologous or allohaplomatched administration in combination with IV administration of the Ad5-treated apheresis product of the present invention. m-CENK cells may be administered in combination with tumor-specific antibodies or checkpoint inhibitor antibodies. Alternatively, IL15-enlarged NK cells may be administered as an adjuvant to lymphocyte-depleting chemotherapy agents administered in combination with IV administration of the Ad5-treated apheresis product of the present invention.

[0042] In one embodiment, the inventors have conceived a method for inducing differential in vitro expansion of antigen-specific T cells, comprising exposing a patient-derived apheresis product, including peripheral blood mononuclear cells (PBMCs), to a human adenovirus serotype 5 (hAd5) vector containing deletions of the initial 1[E1] and initial 2b[E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence for a period of time sufficient to induce measurable T cell expansion. The method may further include enhancing T cell stimulation by adding IL15, IL15:IL15Rα or its IL15Rβγ agonist derivative, IL15:IL15RαIgG4Fc, or N-803 to the PBMCs and Ad5 vector. In one embodiment, the differentially stimulated T cells are specific to a tumor antigen or neoantigen. In another embodiment, the differentially stimulated T cells are specific to an infectious disease antigen.

[0043] In one embodiment, the inventors have conceived a method for intravenous administration of an hAd5 vector comprising deletions of the initial 1[E1] and initial 2b[E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence, comprising exposing a patient-derived apheresis product, including peripheral blood mononuclear cells (PBMCs), to a human adenovirus serotype 5 (hAd5) vector comprising deletions of the initial 1[E1] and initial 2b[E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence for a period of time sufficient for the APC of the PBMCs of the apheresis product to take up the Ad5 vector, wherein the toxicity of the IV or subcutaneous administration of the Ad5 vector is mitigated by cellular uptake, and thus by cell-mediated delivery via IV administration of the Ad5 vector to the patient, and the viral titer of the PBMC supernatant is virtually zero.

[0044] In one embodiment, the inventors have conceived a method for inducing antigen expression in apheresis product (PBMC-derived) antigen-presenting cells, comprising exposing a patient-derived apheresis product, including peripheral blood mononuclear cells (PBMCs), to a human adenovirus serotype 5 (hAd5) vector containing deletions of the initial 1[E1] and initial 2b[E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence, for a period of time sufficient to induce immunogenic peptide expression on the surface of the PBMC-derived APC.

[0045] In one embodiment, the inventors have conceived of adding IL15:IL15RαIgG4Fc to an in vitro composition containing PBMCs and an Ad5 vector. The IgG4 moiety provides stability to the IL15:IL15Rα construct but inhibits the ability of endogenous NK cells to be bound to and directed to stimulated T cells by IL15:IL15RαIgG4Fc. This is because IgG4Fc does not bind to the Fc receptor (CD16) on NK cells. Therefore, antibody-dependent cell-mediated cytotoxicity of NK cells interacting with IgG1 of N-803 is blocked.

[0046] In one embodiment, the patient is administered (subcutaneously) IL15, IL15:IL15Rα or its IL15Rβγ agonist derivative, IL15:IL15RαIgG4Fc, or N-803 before apheresis.

[0047] In one embodiment, red blood cells are purified from the apheresis product. The RBCs can be modified to express one or more therapeutic proteins. The RBCs can be enlarged, differentiated into enucleated mature RBCs, and formulated for IV administration with the apheresis product treated with Ad5.

[0048] In one embodiment, platelets are purified from the apheresis product. The platelets may be modified to express one or more therapeutic proteins (typically from RNA). The platelets may be formulated for intravenous administration with the apheresis product treated with Ad5.

[0049] In embodiments, the hAd5 E2b deletion vector includes transgenes for tumor-associated antigens (e.g., MUC1, Brachyury1, CEA, PSA, PMSA, PD-L1, HER2, etc.) and / or neoantigens. In embodiments, the hAd5 E2b deletion vector further includes transgenes encoding cytokines, gene therapies, immunomodulatory proteins, alpha-fetoproteins, checkpoint inhibitors, and costimulatory proteins.

[0050] In one embodiment, PBMCs derived from apheresis products are exposed to an hAd5 vector containing deletions of the initial 1[E1] and initial 2b[E2b] genes, as well as nucleic acids encoding at least one immunogenic or therapeutic peptide sequence. The exposure time may be at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 4 days, at least 6 days, or at least 8 days.

[0051] The inventors have conceived a method for optimizing hAd5 uptake in an in vitro composition comprising a patient-derived apheresis product containing peripheral blood mononuclear cells (PBMCs) and a human adenovirus serotype 5 (hAd5) vector containing deletions of the initial 1[E1] and initial 2b[E2b] genes and nucleic acids encoding at least one immunogenic peptide sequence, wherein the optimal exposure time is empirically determined by the reduction in Ad5 infectivity in the supernatant of the apheresis product. Infectivity can be determined indirectly, for example, by a hexone assay of the supernatant.

[0052] Furthermore, various exemplary methods are intended in the intended embodiments. For example, these include the following: Experiment 1: Testing of N-803 versus IL15:IL15RαSuIgG4 to determine the effect of the IgG4Fc moiety on T cell activation and / or sibling killing via antibody-dependent cytotoxicity. Here, various test agents with IL-15 activity, such as IL-15, N-803, and IL15:IL15RαSuIgG4, are intended to be added to the PBMC fraction derived from apheresis in the presence of Ad5 / tumor antigen. Alternatively, PBMCs can be pulsed using CEFT, and subsequent T cell activation / proliferation can be evaluated in the presence of IL15, IL15:IL15RαSu, IL15:IL15RαSuIgG4, or N803.

[0053] Experiment 2: Optimization of in vitro uptake, antigen presentation, and / or T cell stimulation. Here, viral uptake can be measured using recombinant Ad5 virus encoding GFP or other reporter genes, which can then be used to identify the optimized MOI. Using the same system, Ad5 uptake (and conversely, viral clearance from the supernatant) can also be monitored using either recombinant Ad5 virus or Ad5 null virus. In such a system, residual virus in the cell supernatant can be quantified using the Hexone assay. Furthermore, using an in vitro system, MHC expression and MHC-mediated antigen presentation of antigen-presenting cells can also be quantified (e.g., via flow cytometry or the Erispot assay). As is readily apparent, antigen presentation can be further stimulated by co-incubation with various ligands such as GMCSF, IL4, and / or Flt3 ligands. It should also be noted that in such an in vitro system, T cell stimulation and proliferation can be measured using the Erispot or other assays. If necessary, the effects of adjuvants, co-stimulatory molecules (e.g., CD40 ligand, GITR ligand, Ox40 ligand, etc., which may be encoded in recombinant Ad5) or checkpoint inhibitors may be evaluated in vitro.

[0054] Experiment 3: Stimulation of memory versus novel immunization. Here, the system intended for memory immunization may use a universal antigen sequence (e.g., E1 vector, E2B vector, SARS-CoV-2 spike, or wt Ad5 peptide without nucleocapsid protein) or an antigen sequence from which the individual from which PMBC was obtained was immunized (e.g., from known prior exposure or vaccination). Alternatively, a standard vaccine (e.g., an adenovirus vaccine formulation encoding or expressing Brachyury, CEA, MUC1, PSA, etc.) may be used to induce novel immunization.

[0055] Experiment 4: Optimization of MHC Antigen Peptide Presentation: Various vaccine formulations can be tested in vitro to confirm the effects on antigen presentation, such as intracellular transport and encapsulation of recombinant costimulatory molecules and / or cytokines. Such test systems preferably include quantification of MHC-presenting antigens and / or antigen-responsive T cell proliferation. Antigen presentation can be optimized using genetically modified intracellular localization tags at the N or C terminus of the antigen peptide.

[0056] Experiment 5: Comparison of SubQ versus in vitro administration. Here, using animal systems, quantitative differences in immune response can be determined between SubQ-administered formulations and treated apheresis products administered by infusion into a medium suitable for cell injection (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 the Erispot assay). For example, the C57BL / 6 inbred mouse model can be used for both 1) SubQ administration of the Ad5 vaccine and 2) in vitro IV administration of apheresis products exposed to the Ad5 vaccine.

[0057] It should be understood that in some embodiments, numbers representing properties such as the amount or concentration of components, reaction conditions, etc., used to describe and claim specific embodiments of the present invention are, in some cases, modified by the term “about.” As used herein, the terms “about” and “approximately” are intended to encompass, when referring to a specific measurable value (e.g., a parameter, quantity, length of time, etc.), the specific value and its variation and variation from that value, e.g., variation of ±10%, ±5%, ±1%, or ±0.1% or less of the specific value, and such variation from the specific value, to the extent that such variation is appropriate for carrying out embodiments of the present disclosure. Thus, the values ​​themselves referred to by the modifiers “about” or “approximately” are also specifically disclosed. The listing of value ranges herein is intended solely as a way of referring individually to each separate value within the range. Unless otherwise indicated herein, each individual value is incorporated herein as it is individually listed herein.

[0058] All methods described herein may be carried out in any preferred order unless otherwise indicated herein or unless it is clearly inconsistent in the context. The use of any exemplary terms (e.g., "etc.") provided herein in reference to any example or particular embodiment is intended solely to illustrate the invention in more detail and does not limit the scope of the claimed invention in particular. There is no term herein that should be construed to indicate any unclaimed element that is essential to the carrying out of the invention.

[0059] In this description and throughout the following claims, “one (a),” “one (an),” and “it” include multiple references unless explicitly indicated otherwise by the context. In this description, “inside” also includes “inside” and “on top,” unless explicitly indicated otherwise by the context. In this description and unless otherwise specified by the context, the term “joined to ~” is also intended to include both direct joining (where two joined elements are in contact with each other) and indirect joining (where at least one additional element is located between the two elements). Thus, the terms “joined to ~” and “joined with ~” are used synonymously.

[0060] It should be obvious to those skilled in the art that many further modifying forms other than those already described are possible without departing from the concept of the invention as set forth herein. Therefore, the subject matter of the invention should not be limited beyond the scope of the appended claims. Furthermore, in the interpretation of both this specification and the claims, all terms should be interpreted in the broadest possible form that is consistent with the context. In particular, the terms “includes” and “contains” should be interpreted to refer to an element, component, or step in a non-exclusive manner indicating that the referenced element, component, or step exists with, is utilized in, or can be combined with, other elements, components, or steps not explicitly referenced. Where this specification or the claims refer to at least one selected from the group consisting of A, B, C, ..., and N, the text should be interpreted to require only one element from the group, such as not A+N or B+N, etc.

Claims

1. Peripheral blood mononuclear cells (PBMCs) derived from the target, A 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, and An immunotherapy composition comprising the PBMC being exposed in vitro to the at least one Ad5 vector.

2. The immunotherapy composition according to claim 1, further comprising IL-15 or interleukin-15:interleukin-15 receptor α (IL-15:IL-15Rα) complex, wherein the immune effector cells are simultaneously exposed to the Ad5 vector and either IL-15 or the IL-15:IL-15Rα complex.

3. The immunotherapy composition according to claim 2, wherein the IL-15:IL-15Rα complex comprises an IL-15N72D:IL-15RαSu / Fc complex.

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

5. The immunotherapy composition according to claim 1, wherein the subject is diagnosed with an infectious disease, a neoplastic disease, or cancer.

6. The subject is an immunotherapy composition according to claim 1, which has not been diagnosed with an infectious disease, neoplastic disease, or cancer.

7. The immunotherapy composition according to claim 1, wherein the PBMC is derived from apheresis.

8. The immunotherapy composition according to claim 7, wherein the PBMC comprises T cells, natural killer (NK) cells, natural killer T (NK-T) cells, dendritic cells, mast cells, bone marrow-derived phagocytic cells, or a combination thereof.

9. The immunotherapy composition according to claim 1, formulated for intravenous (IV) administration.

10. The immunotherapy composition according to claim 9, for use in the treatment of neoplastic diseases, cancer, or infections in a patient, and to be administered IV to the patient.

11. The immunotherapy composition according to claim 10, wherein the neoplastic disease or cancer includes bladder cancer, glioblastoma, prostate cancer, hematological cancer, B-cell neoplasm, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, cutaneous T-cell lymphoma, T-cell lymphoma, solid tumor, urothelial / 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 and neck cancer, or urothelial / bladder cancer.

12. The immunotherapy composition according to claim 10, wherein the treatment further comprises radiation, chemotherapy, surgery, therapeutic antibodies, immunomodulators, proteasome inhibitors, pan-DAC inhibitors, H-DAC inhibitors, checkpoint inhibitors, adoptive cell therapy and / or immunotherapy including CAR T and / or NK cell therapy.

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

14. The immunotherapy composition according to claim 13, wherein the Ad5 vector further comprises nucleic acids for the expression of at least one cytokine, gene therapy, and immunomodulatory protein.

15. A method for treating cancer or infectious diseases, Therapeutic apheresis is performed on the target population, Purification of peripheral blood mononuclear cell (PBMC) fraction from apheresis products, The PBMC is exposed in vitro to at least one recombinant adenovirus subtype 5 (Ad5) vector containing a deletion in the El gene region, a deletion in the E2b gene region, and a nucleic acid sequence encoding a peptide antigen of the pathogen causing the cancer or the infection. Administer an effective amount of PBMC treated with the Ad5 vector to the patient. A method that includes this.

16. The method according to claim 15, further comprising adding interleukin 15 (IL15) or an IL15:IL15 receptor α (IL15:IL15Rα) complex to the PBMC exposed in vitro.

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

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

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

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

21. The method according to claim 15, wherein IL15 or IL15:IL15Rα complex is added to PBMC treated with the Ad5 vector before administration.

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

23. A method for transfecting multiple peripheral blood mononuclear cells (PBMCs), (1) Providing a PBMC fraction prepared in vitro, which contains, in addition to antigen-presenting cells / dendritic cells, cells selected from the group consisting of macrophages, monocytes, T cells and B cells, (2) Exposing the PBMC fraction to GM-CSF and Flt3L to induce differentiation of the antigen-presenting cells / dendritic cells, (3) Exposing the PBMC fraction in vitro to recombinant adenovirus subtype 5 (Ad5) virus particles comprising a deletion in the El gene region, a deletion in the E2b gene region, and a nucleic acid sequence encoding a peptide antigen of a pathogen that causes cancer or infection, at a MOI of at least 100; (4) The step of expanding the T cells in the PBMC fraction using IL-2, IL-7 and IL-15, (5) The step of re-exposing the PBMC fraction in vitro to the recombinant adenovirus subtype 5 (Ad5) virus particles, (6) The step of placing the re-exposed PBMC fraction in a culture medium suitable for injection. A method that includes them in order.

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

25. The method according to 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 according to claim 23, wherein the step of exposing the PBMC fraction to the recombinant Ad5 virus particles in vitro is carried out at an MOI of 10,000 to 100,000.

27. The method according to claim 23, wherein the step of expanding the T cells in the PBMC fraction is carried out over a period of at least four days.

28. The method according to claim 23, wherein the step of placing the re-exposed PBMC fraction into the culture medium suitable for injection reduces the number of Ad5 virus particles in the PBMC fraction by at least 100 times.

29. The method according to claim 23, wherein the step of placing the re-exposed PBMC fraction into the culture medium suitable for injection reduces the number of Ad5 virus particles to 1,000 or fewer virus particles per mL.

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