virus-educated t cells

Abstract

Compositions and methods for treating general persistent viral infections, particularly long COVID, are described. The compositions comprise T cells, antigen presenting cells (APCs), and optionally NK cells derived from a patient's whole blood. The APCs are genetically engineered to comprise nucleic acids encoding antigenic peptide sequences for MHC-complexed cell surface expression, where further exposure to an IL-15 agonist and patient T cells can produce activation and expansion of virus-educated T cells for readministration to the patient, thereby targeting cells harboring the virus to T cell-mediated cytotoxicity.

Description

[0001] This application claims priority to our co-pending U.S. provisional application filed on November 6, 2023, serial number 63 / 596,583, which is incorporated herein by reference in its entirety. sequence list

[0002] The XML content of the sequence list, named 102690.0067PCT.XML and of size 5,415 bytes, was created on November 5, 2024, and was submitted electronically via EFS-Web together with this application, and is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to compositions and methods for treating viral diseases, and more particularly to treating subjects using ex vivo modified single-harvest products. Background Technology

[0004] The background description includes information that may be used to understand the invention. This is not an admission that any information provided herein is prior art or related to the currently claimed invention, or an admission that any publication specifically or implicitly referenced is prior art.

[0005] 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. Where a definition or use of a term in the incorporated references is inconsistent or contradictory to the definition of that term provided herein, the definition provided herein shall apply, and the definition in the references shall not apply.

[0006] Long Covid (also known as Post-Covid Conditions, PCC) refers to a wide range of symptoms and symptoms that some people experience four weeks or longer after their initial infection with SARS-CoV-2 (the virus that causes Covid-19). These symptoms and symptoms (which may last for weeks, months, or years) can be persistent (meaning they arise during the acute Covid-19 illness and do not disappear), recurrent (meaning they may disappear after the initial illness and then recur), or new (meaning they were not initially present but occur later).

[0007] The severity of prolonged COVID-19 symptoms can range from mild to debilitating. Some people report that prolonged COVID-19 symptoms prevent them from returning to work or school, while others experience difficulty performing daily chores or even walking short distances.

[0008] Long COVID-19 most commonly occurs in people who have had severe Covid-19, but it is not limited to those who are critically ill or hospitalized. People with mild illness and even those who are asymptomatic can also be affected. In fact, most people with long COVID-19 have had mild acute Covid-19. This is because far more people have mild Covid-19 than severe Covid-19, so even if only a small fraction of them develop long COVID-19, they still make up the majority of long COVID-19 cases. Both adults and children can be affected, but long COVID-19 appears to be more common in adults. Researchers do not yet know how common this condition is, but studies estimate it occurs in 5% to 30% of people who have Covid-19.

[0009] Besides PCC, long-term COVID has many other names, including long-haul COVID, acute sequelae of COVID-19 (PASC), long-term effects of COVID-19, and chronic COVID.

[0010] It remains unclear whether the symptoms of long-term COVID-19 are caused by the initial infection or are a manifestation of persistent infection. The fact that viral DNA and RNA can still be found in some patients up to two years after acute infection suggests viral persistence. Furthermore, SARS-CoV-2 can reactivate other dormant viruses, such as endogenous retroviruses and EBV. Currently, there is no treatment for long-term COVID-19, and patients are usually treated only for their symptoms.

[0011] Therefore, there is still a need in the field for new compositions and methods for treating chronic COVID-19. Summary of the Invention

[0012] The subject matter of this invention relates to various compositions and methods for viral vaccine compositions. This document discloses pharmaceutical compositions for use in patients with viral infections, comprising IL-15 or an agonist derivative thereof; and viral-educated T cells. The IL-15 agonist derivative may comprise IL-15:IL:15RaSu and / or stabilizing derivatives such as Fc domain conjugates, HCW-9218,0, or N-803. Methods for preparing and using such compositions are also disclosed herein.

[0013] Virus-educated T cells comprise T cells isolated from a patient's whole blood and exposed in vitro to dendritic cells, wherein these dendritic cells 1) are isolated from the patient's whole blood and 2) are transfected with a nucleic acid vector encoding at least one antigenic viral peptide sequence. In some embodiments, the composition further comprises memory-like cytokine-enhanced natural killer (m-ceNK) cells, wherein these m-ceNK cells are isolated from the patient's whole blood. Autologous T cells and dendritic cells are preferably derived from apheresis products from the patient. In some embodiments, T cells are exposed in vitro to dendritic cells in the presence of IL-15 or an agonist derivative thereof or in the presence of mesenchymal stem cells (MSCs).

[0014] In a preferred embodiment, the nucleic acid vector comprises an adenovirus (Ad) vector encoding an antigenic viral peptide, such as the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins. In one embodiment, the Ad vector comprises a first nucleic acid portion and a second nucleic acid portion; wherein the first nucleic acid portion encodes the CoV2 spike (S) protein having the amino acid sequence of SEQ ID NO: 3; and wherein the second nucleic acid portion encodes a chimeric protein comprising 1) the CoV2 nucleocapsid (N) protein having the amino acid sequence of SEQ ID NO: 1 and 2) an endosome targeting sequence (ETSD) having the amino acid sequence of SEQ ID NO: 2. Alternatively, the nucleic acid vector comprises an RNA vector. In some cases, the viral peptide sequence is derived from SARS-CoV-2, HPV, or HIV.

[0015] This document further discloses the use of the composition in the preparation of a medicament for treating viral infections. In a preferred embodiment, the viral infection comprises SARS-CoV-2, HPV, or HIV. In still preferred embodiments, the viral infection comprises SARS-CoV-2.

[0016] On the other hand, this article discloses a method for treating long-term COVID-19 in patients in need, comprising: transfecting dendritic cells isolated from the patient's whole blood with a nucleic acid vector encoding at least one antigenic peptide derived from SARS-CoV-2; exposing T cells isolated from the patient's whole blood to these dendritic cells, thereby expanding these T cells into virus-educated T cells; and treating long-term COVID-19 by administering IL-15 or an agonist derivative thereof to the patient and these virus-educated T cells.

[0017] In some embodiments, the nucleic acid vector comprises an adenovirus (Ad) vector, and preferably comprises an Ad vector containing deletions of the E1 and E2b gene regions. Preferably, the adenovirus (Ad) vector comprises a nucleic acid portion encoding the SARS-CoV2 S protein and a nucleic acid portion encoding a chimeric protein comprising the SARS-CoV2 N protein and an endosome targeting sequence.

[0018] IL-15 agonist derivatives may comprise IL-15:IL:15RaSu and / or stabilizing derivatives such as Fc domain conjugates, HCW-9218, or N-803. Preferably, T cells and dendritic cells are derived from apheresis products from a patient. After transfecting dendritic cells with a nucleic acid vector, the dendritic cells are exposed to T cells in the presence of IL-15 or its agonist derivative to generate virus-educated T cells. Virus-educated T cells can be administered intravenously to long-term COVID-19 patients to treat the disease. In some embodiments, IL-15 or its agonist derivative may also be administered simultaneously or separately. In this regard, it should be recognized that virus-educated T cells and IL-15 or its agonist derivative may be formulated together or separately. Optionally, natural killer (NK) cells are administered to the patient. NK cells may comprise m-ceNK cells, NK-92 cells, aNK cells, haNK cells, and / or t-haNK cells.

[0019] 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

[0020] Figure 1 This is an exemplary schematic flowchart of the treatment of viral diseases according to the subject matter of the present invention.

[0021] Figure 2 The results showed that DC-driven cytokines reduced the overall background and improved DC specificity.

[0022] Figure 3 The roles of GM-CSF and FLT3L superkine in generating DCs are shown.

[0023] Figure 4 The effect of a mixture of cytokines on antigen-specific T cell expansion is shown.

[0024] Figure 5 The use of AdV and poly-L-lysine to deliver antigens is shown. Detailed Implementation

[0025] The inventors hereby disclose pharmaceutical compositions for treating viral diseases, comprising IL-15 or an agonist derivative thereof (such as N-803) and virus-educated T cells. In a preferred embodiment, the viral disease is COVID-19.

[0026] The inventors have discovered that N-803 is effective in treating viral diseases, particularly latent viral diseases. In patients with chronic HIV infection, N-803 induces the expression of latent HIV antigens. The inventors further observed that N-803 has a similar effect on latent SARS-CoV-2 infection in patients with latent COVID-19. T cells expressing antigens on antigen-presenting cells (APCs) that bind to viral antigens specific to APC MHC were then stimulated.

[0027] In one aspect of this disclosure, T cells and antigen-presenting dendritic cells (abortion) are isolated from a patient's whole blood. The dendritic cells are then transfected with a nucleic acid vector encoding at least one viral antigen. These transfected and modified dendritic cells are exposed to T cells isolated from the patient's whole blood. The number of T cells expands after exposure to the dendritic cells. Throughout this disclosure, this population of T cells exposed to one or more viral antigens is referred to as virus-educated T cells. In one embodiment, the nucleic acid vector is an adenovirus vector, such as human adenovirus serotype 5 (hAd5). The hAd5 vector may be replication-defective, for example by deletion of the early 1 [E1] and early 2b [E2b] genes.

[0028] In a preferred embodiment, the viral antigen includes one or more SARS-CoV-2 antigens, such as the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins. SARS-CoV-2 antigens are further described in U.S. Patent Application No. 16 / 883,263, PCT Publication No. WO / 2021 / 183665, PCT Publication No. PCT / IB2021 / 054887, and PCT Publication No. WO 2023 / 102375, each of which is incorporated herein by reference in its entirety (including figures and sequences).

[0029] The viral antigen is preferably encoded in a replication-defective adenovirus (hAd5-E1, hAd5-E2b). The viral antigen is selected from the group consisting of: coronavirus 2 (CoV2) nucleocapsid protein, CoV2 spike protein, and combinations thereof. The CoV2 nucleocapsid protein is expected to have at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 1. In some cases, the CoV2 nucleocapsid protein may be conjugated to an endosome targeting sequence (ETSD) that has at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence of SEQ ID NO: 2. The CoV2 spike protein is expected to have at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 3.

[0030] Optionally, the amplification step is performed in the presence of an IL-15 agonist (such as N-803), which stimulates the MHC complex expression of latent viral peptides. The virus-educated and amplified T cells are then formulated together with the IL-15 agonist (such as N-803) for re-administration to patients.

[0031] Therefore, in one embodiment, the inventors disclosed a pharmaceutical composition comprising virus-educated T cells and N-803. The inventors envision that co-administering an IL-15 agonist (such as N-803) with virus-educated T cells to long-term COVID-19 patients leads to a reversal of the latent state, thereby enabling the body's immune system to attack the virus. Thus, in other words, the co-administration of virus-educated T cells and N-803 produces a synergistic effect that is not observed when either component is administered alone, and is therefore not merely an additive effect.

[0032] Regarding the cytokine IL-15, all forms of IL-15 (including IL-15 superagonists, IL-15 mutants, the IL-15 receptor α (IL-15Rα) complex (IL-15:IL-15Rα), fusion proteins containing IL-15, or combinations thereof) can be used in the compositions disclosed herein. Stabilized IL-15 includes IL-15 superagonists such as nogapendenkin alpha imbakicept (Alt-803, N-803, Vesanktiva), and stabilized IL-15:IL-15Rα fusion proteins. The IL-15:IL-15Rα fusion protein is an IL-15... N72D The IL-15RαSu / Fc complex (ALT-803, also known as Nogren's interleukin-α inzicept) contains the dimer IL-15RαSu / Fc and two IL-15 molecules. N72D Molecules. U.S. Patent Application Nos. 16 / 985,728, 16 / 505,528, 11,351,196, 63 / 156,269, 8,163,879, 8,507,222 and 10,537,615 discuss IL-15 and its derivatives more extensively and are incorporated herein by reference in their entirety.

[0033] In some embodiments, the composition comprising N-803 and virus-educated T cells further comprises cytokine-enhanced NK cells (ceNK cells). The ceNK cells disclosed herein refer to NK cells in which cytotoxic activity is enhanced by cytokine stimulation. ceNK cells are prepared by inducing NK cells with a composition of corticosteroids and optionally cytokine compositions comprising IL-15, IL-15:IL-15Rα, or agonist derivatives thereof, such as N-803. The cytokine composition may comprise a fusion protein comprising IL-15 or an agonist derivative thereof. Fusion proteins comprising IL-15 are preferred, wherein the fusion protein has increased stability relative to IL-15. While not limiting the subject matter of the invention, it is generally preferred that the corticosteroid be hydrocortisone and the optional cytokine be N-803.

[0034] The memory-like cytokine-enhanced NK cells (m-ceNK) disclosed in this article comprise enriched and expanded NK cells obtained from donor peripheral blood using apheresis technology to generate NK cells with a memory-like phenotype. m-ceNK cells exhibit high cytotoxicity and increased interferon-γ production. These m-ceNK cells can be generated from individual donors for autologous cell therapy or as an allogeneic product derived from umbilical cord blood. In addition to their enhanced efficacy, m-ceNK cells can be readily infused in an outpatient setting.

[0035] As a non-limiting example, m-ceNK cells can be generated by the following steps: obtaining a plurality of monocytes and contacting the plurality of monocytes with corticosteroids and optionally cytokines. In another step, the plurality of monocytes are incubated in the presence of corticosteroids and optionally cytokines to enrich monocytes in NK cells, and then the enriched NK cells are induced with a cytokine composition comprising IL-15, IL-12, and IL-18, or agonist derivatives thereof. The composition may comprise one or more fusion proteins, wherein these fusion proteins comprise at least one of IL-15, IL-12, and IL-18 cytokines, or agonist derivatives thereof. The cytokine composition may comprise a TxM fusion protein to generate m-ceNK cells, wherein the TxM fusion protein comprises a protein moiety having IL-12 activity, a protein moiety having IL-15 activity, and a protein moiety having IL-18 activity.

[0036] Further description of the preparation of m-ceNK cells and their advantageous properties is provided in PCT / US2022 / 018290 (which is incorporated herein by reference in its entirety). U.S. Patent Application 17 / 375,985 and U.S. Patent 11,453,862 provide additional alternative methods for inducing NK cell enrichment and expansion. Each of the above references is incorporated herein by reference in its entirety.

[0037] In some embodiments, the viral antigenic peptide is selected before transfecting dendritic cells with the viral antigenic peptide, and the molecular nature of viral infection is confirmed by PCR (e.g., a handheld nudge device). The virus does indeed believe that peptide selection provides information, which can be accomplished using various strategies known in the art. Peptides are selected based on various properties, including size and MHC binding (depending on the patient's HLA type). The nucleic acid encoding the antigenic peptide is added to the dendritic cells using various means known in the art. An adenoviral vector can be used. RNA (optionally, mRNA) can be complexed with lipid nanospheres or mannan nanogels.

[0038] The preferred embodiment disclosed herein is a method for treating long-term COVID-19, in which latent low-level viral expression may lead to various symptoms associated with long-term COVID-19. IL-15 is used in two aspects of this method. First, it is used to prepare virus-educated T cells, i.e., T cells exposed to dendritic cells in response to IL-15 stimulation, which has been shown to promote T cell proliferation and promote the expression of MHC-presented antigenic peptides on the cell surface of antigen-presenting cells. Second, IL-15 is administered together with the educated T cells to stimulate the T cells and simultaneously stimulate the expression of latent COVID antigens. This is referred to as the "activation" phase of a "kick and kill" strategy to induce latent viral expression, thereby enabling cytotoxic lymphocytes to target and kill it. This method in Figure 1 As shown in the image.

[0039] While COVID-19 is an exemplary use of this disclosure, the compositions and methods disclosed herein can be used for any other viral disease, such as HIV, influenza, HPV, EBV, etc.

[0040] Other therapeutic agents contemplated for inclusion in the compositions and methods disclosed herein include m-CENK, NK-92 cells (hank, t-haNK), antiviral drugs, bNAb, and HDAC inhibitors.

[0041] m-ceNK cells comprise natural killer cells enhanced with memory-like cytokines (PCT / 2021 / 006876). They can be obtained from patient whole blood using methods known in the art and administered autologously. m-ceNK cells are characterized by enhanced cytotoxicity against tumor or infected cells. In embodiments, m-ceNK cells can be expanded with a composition comprising at least one of the following: a corticosteroid (such as dexamethasone), IL-15, IL-18, IL-12, anti-CD16 Ab, and optionally anti-CD3 Ab.

[0042] In a particularly preferred aspect, single-agent harvesting can be used to prepare the various cell fractions disclosed herein for treating the same patient in a parallel or sequential manner.

[0043] In a typical example, such as Figure 1 The illustration schematically shows that, prior to apheresis collection of PBMCs, subjects diagnosed with a viral disease (e.g., COVID-19) are optionally treated with N-803 to stimulate the production of T and NK cells. Most typically, PBMCs will include various immune cells, such as CD4+. + T cells, CD8 + T cells, B cells, macrophages, dendritic cells, Kupffer cells, etc. NK cells, T cells, and dendritic cells can be isolated from each other. Alternatively, intact PBMC formulations can be transfected with hAd5 encoding one of a variety of viral antigens, wherein DCs specifically take up the Ad5 vector. Dendritic cells and / or macrophages are used for viral antigen infection, thereby achieving antigen presentation of recombinant antigens on antigen-presenting cells (such as macrophages and dendritic cells). In a preferred embodiment, the viral antigen is encoded by a recombinant adenovirus (such as hAd5). The virally loaded dendritic cells, which present recombinant neoantigens on MHC class I and / or II complexes, are then subjected to T cells (including unpermitted T cells, immature and mature unprimed T cells, CD4+, etc.). + T cells and CD8 + Contact with T cells triggers the development (activation and expansion) of antigen-specific cytotoxic T cells and helper T cells. It should be recognized that infection of antigen-presenting cells with viral antigens can occur in the presence of immunostimulatory cytokines (IL-2, IL-15, etc.), co-stimulatory molecules, and / or checkpoint inhibitors.

[0044] In particularly envisioned compositions and methods, it is generally preferred to expose T cells to antigen-loaded dendritic cells 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 the addition of IL15, IL15:IL15Rα or an IL15Rβγ agonist derivative thereof, IL15:IL15RαIgG4Fc, HCW-9218, or N-803. HCW-9218 is further described in U.S. Patent No. 11,518,792, which is incorporated herein by reference in its entirety.

[0045] In a further preferred aspect, dendritic cells are exposed to viral antigens for a duration sufficient for the dendritic cells to take up substantially all of the viral antigens. From a different perspective, following in vitro infection, it is typically preferred that the viral titer in the antigen-presenting cell fraction of the PBMCs is so low as to be undetectable, meaning that substantially all of the virus has been taken up by the antigen-presenting cells (APCs). Such a time can be empirically determined by quantifying the viral infectivity in the cell composition (e.g., using a hexagon 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. Thus, by using the envisioned method and composition, it should be understood that infection of antigen-presenting cells with therapeutic viral antigens is optimized while minimizing or completely avoiding potential adverse effects due to off-target viral binding. The inventors also envision that the duration of in vitro exposure is determined by MHC presentation of an Ad-encoded peptide, wherein intravenous (IV) administration of the Ad-treated apheresis product results in the subsequent in vivo stimulation of antigen-responsive CD4 cells via MHC presentation of the antigen on in vitro infected cells. + and CD8 + T cells.

[0046] Furthermore, it is envisioned that some of the apheresis products could be subjected to NK cell isolation, and these NK cells could be expanded in vitro to provide additional cytotoxic effector cells. Moreover, 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). 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. Further still, 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.

[0047] Furthermore, it is envisioned that the apheresis product also contains red blood cells and platelets, which could be used as additional therapeutic agents. 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.).

[0048] 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.

[0049] Several different treatment options using the ex vivo infected apheresis compositions disclosed in this invention are envisioned. In addition to the ex vivo infected apheresis compositions, 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, treatment regimens envisioned herein will also include supportive administration of IL-15, IL15:IL15Rα constructs, HCW-9218 or N-803 (subcutaneously or intratumorally), or other cytokines (e.g., NHS-IL-12, see Immunotargets Ther. [Immune Targets and Therapies] 2021; 10: 155–169), for example, to aid the proliferation of NK cells, T cells, and memory T cells. Further methods disclosing treatment options can be found in PCT application PCT / US24 / 26806, which is incorporated herein by reference in its entirety.

[0050] In the embodiments, dendritic cells in the immunotherapeutic composition disclosed herein were transfected with a human adenovirus serotype 5 (hAd5) vector containing deletions of the early 1 [E1] and early 2b [E2b] genes and a nucleic acid encoding at least one immunogenic viral peptide. In this context, it is worth noting that the term "human adenovirus serotype 5 (hAd5) vector" encompasses both recombinant hAd5 nucleic acid and recombinant hAd5 viral particles containing recombinant hAd5 nucleic acid.

[0051] For example, in some embodiments, dendritic cells are exposed to a recombinant therapeutic virus (a recombinant hAd5 vector encoding at least one immunogenic viral peptide) for a duration sufficient for the dendritic cells to take up substantially all of the recombinant therapeutic virus. 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 or additionally, residual virus and its quantity can be removed by replacing the culture medium in which the cells are maintained or grown. Therefore, it should be understood that, following 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).

[0052] Antigen-loaded dendritic cells are exposed to T cells (especially CD4+ and CD8+ T cells) to obtain a cell population that can be infused into a subject to elicit an effective immune response against a viral antigen encoded by a recombinant adenovirus and expressed in the transfected cells. This cell population is referred to herein as virus-educated T cells. Alternatively, in vitro transfection of antigen-presenting / dendritic cells can be performed in the presence of other immune cells, such as macrophages, monocytes, T cells, B cells, etc.

[0053] In this embodiment, the composition comprises virus-educated 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 virus-educated T cells.

[0054] In the examples, the composition is intended for use in the treatment of infectious diseases, particularly COVID-19. The Ad5-encoded immunogenic peptide encodes a viral antigen. Identification of the antigen sequence can be derived from sequencing, mRNA expression, and peptide expression analysis.

[0055] In a preferred embodiment, fresh or frozen PBMCs obtained from the subject may be treated with GM-CSF and FLT3L for a duration sufficient to allow antigen-presenting cell differentiation (e.g., 8-16 hours, 12-24 hours, 18-36 hours, or 24-48 hours), typically in the presence of interleukin-4 (IL-4). Subsequently, the treated cells are optionally stimulated in the presence of one or more SARS-CoV2 peptides (e.g., N and / or S peptides) or recombinant viruses (e.g., Ad5 virus having recombinant nucleic acids encoding one or more SARS-CoV2 peptides) or other viral pathogens to activate antigen-presenting cells. The differentiated and stimulated cells are then fed, and T cell subsets are expanded in the presence of activated antigen-presenting cells using fresh medium containing suitable 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, anti-CD3, and / or anti-CD-49d antibodies) for at least 6-8 hours. If necessary, T cell function can then be tested before infusion to the subject (e.g., testing IFN-γ secretion in a routine ELISpot assay).

[0056] Figure 2 The results show that DC-driven cytokines reduce overall background and appear to improve specificity. In this example, PBMCs from CMV pp65-responsive donors were thawed on day 0 and treated with the listed DC-generating cytokine mixture. On day 1, cells were treated with LPS, R848 (resiquimod; TLR 7 / 8 agonist), and the CMV pp65 pepmix. On day 2, T-cell expansion cytokines (IL-2, IL-7, IL-15) were added, followed by medium replacement on days 4 and 7, respectively. Cells were harvested on day 9, and their responsiveness to the CMV pepmix was tested using the standard ELISPOT protocol. The data indicate that DC-driven cytokines are required to reduce overall background and improve specificity.

[0057] Figure 3 This study demonstrates the use of superfactors GM-CSF and FLT3L in the culture of dendritic cells (DCs) derived from peripheral blood mononuclear cells (PBMCs). This combination was found to effectively support DC expansion and development.

[0058] Figure 4The effect of a mixture of cytokines on the expansion of immediate antigen-specific T cells is illustrated. 5. Cytokines IL-2, IL-7, and IL-15 are known to be used in T cell growth, but the optimal cytokines or combinations for maximizing this effect remain unclear. Existing literature generally indicates that IL-2 and IL-15 are equally effective in promoting T cell growth. However, in the compositions and methods disclosed in this invention, the inventors observed that when expansion was performed using a combination of IL-2, IL-7, and IL-15, NLV-specific T cells unexpectedly increased by 813-fold. Removal of IL-2 resulted in a sharp decrease in this increase to only 32-fold, while exclusion of IL-15 or IL-7 resulted in even more pronounced decreases.

[0059] Figure 5 This study demonstrates the use of adenovirus and poly-L-lysine for antigen delivery. Based on the disclosed GM-CSF / Flt-3 superfactor, AdV expressing pp65 (containing a poly-L-lysine enhancer), without the use of TLR agonists, and IL-2 / IL-7 / IL-15 T cell-expanding cytokines, low CMV pp65 NLV responders (LP381) and high responders (LP186) were compared. The results showed that a response to NLV could be generated efficiently and effectively.

[0060] 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, IL-15:IL-15Rα or a derivative thereof, HCW-9218, or N-803. NK cells can be activated and expanded from PBMCs as described previously in US 11,453,862, US 2021 / 0009954; and / or activated and expanded as described in US 20210008107, US 2021 / 0008112; US 20210361711 to generate CIML-NK cells. Alternatively or additionally, the NK cells can be differentiated into m-ceNK 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 disclosed compositions are intended for use in the treatment of infectious diseases. A treatment method is also disclosed in which the contemplated composition is administered IV to a patient in need. The method further comprises subcutaneously administering IL-15, IL-15:IL-15Rα or a derivative thereof, or N-803 (an IL-15 superagonist) to the patient.

[0061] In this embodiment, sorted NK cells are cultured in IL-15 and optionally glucocorticoids, followed by an expansion phase in which NK cells are cultured in IL-15 / IL-18 / IL-12, 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 infectious disease antigens such as SARS-CoV-2 antigens. m-ceNK cells can be combined with virus-educated T cells of the present invention for autologous administration. m-ceNK cells can be combined with the virus-educated T cells of the present invention and N-803 for IV administration in autologous or allogeneic haploidentical co-administration.

[0062] According to the methods provided herein, an effective amount of one or more of the compositions and agents provided herein is administered to a patient for the treatment of chronic COVID-19. The terms effective amount and effective dose are used interchangeably. The term effective amount is defined as any amount necessary to produce the desired physiological response (e.g., reduction of inflammation). Those skilled in the art can empirically determine the effective amount and schedule for administering the agent. The range of doses administered is those sufficiently large to produce the desired effect, in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dose should not be so large as to cause serious adverse side effects, such as unwanted cross-reactions, allergic reactions, etc. Generally, the dose will vary with age, condition, sex, type of disease, severity of disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by those skilled in the art. If any contraindications occur, the dose may be adjusted by an individual physician. The dose may be varied and may be administered once or more daily for one or several days. For a given class of pharmaceutical products, guidelines for appropriate dosages can be found in the literature. For example, for a given parameter, the effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as an increase or decrease in "multiples". For example, the therapeutically effective amount can have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more relative to the control. The exact dosage and formulation will depend on the purpose of treatment and will be determined by a person skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Gennaro (2012); and Pickar, Dosage Calculations (1999)).

[0063] In a preferred embodiment, the virus-educated T cells and / or ceNK cells described herein are used at 10 4 Up to 10 9 Cells / kg body weight, preferably 10 5 Up to 10 6The dosage is administered in units of cells per kg body weight, including all integer values ​​within these ranges. In preferred embodiments, the dosage of N-803 is approximately 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 mg / kg body weight.

[0064] Pharmaceutically acceptable compositions may include a variety of carriers and excipients. A variety of aqueous carriers, such as buffered saline, can be used. These solutions are sterile and generally do not contain undesirable substances. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 22nd ed., edited by Loyd V. Allen et al., Pharmaceutical Press (2012). A pharmaceutically acceptable carrier means a material that is not biologically or otherwise undesirable; that is, administration of the material to a subject will not cause undesirable biological effects or interact with other components contained in the pharmaceutical composition in a harmful manner. If administered to a subject, the carrier is optionally selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject. As used herein, the term pharmaceutically acceptable is used synonymously with physiologically acceptable and pharmacologically acceptable. Pharmaceutical compositions typically contain agents for buffering and preservation during storage and may include buffers and carriers for appropriate delivery, depending on the route of administration.

[0065] These compositions may contain acceptable adjuvants close to those required for physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The cell concentration in these formulations and / or other agents may vary and will be selected based on the specific administration method chosen and the needs of the subject, primarily according to fluid volume, viscosity, body weight, etc.

[0066] In some embodiments, the figures representing the amount or characteristics of components (such as concentration, reaction conditions, etc.) used to describe and claim certain embodiments of the invention should be understood to be modified in some cases by the term “about.” As used herein, the terms “about” and “approximately” when referring to a specified measurable value (such as a parameter, quantity, duration of time, etc.) are intended to cover the specified value and variations of that specified value and variations relative to that specified value, such as + / - 10% or less, alternatively + / - 5% or less, alternatively + / - 1% or less, alternatively + / - 0.1% or less, and such variations relative to that specified value, provided that such variations are suitable for implementation in 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 for individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were described separately herein.

[0067] Unless otherwise specified herein or otherwise clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such”) provided herein with respect to certain embodiments is intended only to better illustrate the invention and does not constitute a limitation on the scope of the originally claimed invention. The language in the specification should not be construed as indicating that any unclaimed element is necessary for the practice of the invention.

[0068] 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”. As also used herein, and unless the context clearly indicates otherwise, the term “coupled to” is intended to include both direct coupling (where two elements coupled to each other 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.

[0069] 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 the invention is not 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 explicitly referenced. When the specification or claims refer to at least one item 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 (as amended under Article 19 of the Treaty) 1. A pharmaceutical composition for use in a patient suffering from a viral infection, the composition comprising: a.IL-15:IL-15RαSu; and b. Virus-educated T cells comprising T cells isolated from the patient’s whole blood and exposed in vitro to dendritic cells, wherein these dendritic cells 1) are isolated from the patient’s whole blood and 2) are transfected with a nucleic acid vector encoding at least one antigenic viral peptide sequence. 2. The composition of claim 1, further comprising memory-like cytokine-enhanced natural killer (m-ceNK) cells, wherein these m-ceNK cells are isolated from the patient's whole blood. 3. Delete. 4. The composition according to any one of claims 1-2, wherein the IL-15:IL-15RαSu comprises a stabilizing derivative. 5. The composition of claim 4, wherein the stabilized IL-15:IL-15RαSu comprises an Fc domain. 6. The composition of claim 5, wherein the stabilized IL-15:IL-15RαSu comprises N-803. 7. The composition as described in any of the preceding claims, wherein the autologous T cells and dendritic cells are derived from apheresis products. 8. The composition as claimed in any of the preceding claims, wherein the nucleic acid vector comprises an adenovirus (Ad) vector. 9. The composition of claim 8, wherein the Ad vector comprises SARS-CoV-2 spike (S) and nucleocapsid (N) proteins. 10. The composition of claim 8, wherein the Ad vector comprises a first nucleic acid portion and a second nucleic acid portion; wherein the first nucleic acid portion encodes a CoV2 spike (S) protein having the amino acid sequence of SEQ ID NO: 3; and wherein the second nucleic acid portion encodes a chimeric protein comprising 1) a CoV2 nucleocapsid (N) protein having the amino acid sequence of SEQ ID NO: 1 and 2) an endosome targeting sequence (ETSD) having the amino acid sequence of SEQ ID NO: 2. 11. The composition as claimed in any of the preceding claims, wherein the nucleic acid vector comprises an RNA vector. 12. The composition as claimed in any of the preceding claims, wherein the viral peptide sequence is derived from SARS-CoV-2, HPV, or HIV. 13. The composition of any one of the preceding claims, wherein the T cells are exposed to dendritic cells in vitro in the presence of IL-15 or an agonist derivative thereof. 14. The composition of any one of the preceding claims, wherein the T cells are exposed to dendritic cells in vitro in the presence of mesenchymal stem cells (MSCs). 15. Use of the composition as described in any of the preceding claims in the preparation of a medicament for treating viral infections. 16. The use as described in claim 15, wherein the viral infection comprises SARS-CoV-2. 17. The use as described in claim 16, wherein the viral infection comprises long-term COVID-19. 18. Use of IL-15 or an agonist derivative thereof and virus-educated T cells in the manufacture of a medicament for treating patients in need of long-term COVID-19, wherein the medicament is prepared by a method comprising: a. Transfect dendritic cells isolated from the patient’s whole blood with an adenovirus (Ad) vector containing deletions of the E1 and E2b gene regions and encoding at least one antigenic peptide derived from SARS-CoV-2. b. T cells isolated from the patient's whole blood are exposed to these dendritic cells, thereby expanding these T cells into virus-educated T cells; and c. Treating long-term COVID-19 patients by administering IL-15 or its agonist derivatives and these virus-educated T cells. 19. Delete. 20. Delete. 21. The use as claimed in claim 18, wherein the adenovirus (Ad) vector comprises a nucleic acid portion encoding a SARS-CoV-2 S protein and a nucleic acid portion encoding a chimeric protein comprising a SARS-CoV-2 N protein and an endosome targeting sequence. 22. The use as described in claim 21, wherein the T cells are exposed to the dendritic cells in the presence of IL-15 or an agonist derivative thereof. 23. The use as claimed in claim 21, wherein the IL-15 agonist derivative comprises IL-15:IL-15RαSu. 24. The use as described in claim 21, wherein the IL-15 agonist derivative comprises a stabilizing derivative. 25. The use as claimed in claim 21, wherein the stabilized IL-15 agonist derivative comprises an Fc domain. 26. The use as claimed in claim 21, wherein the stabilized IL-15 agonist derivative comprises N-803. 27. The use as described in any one of claims 18 and 21-26, wherein these T cells and dendritic cells are derived from single-harvest products. 28. The use as described in any one of claims 18 and 21-27, wherein the T cells are administered intravenously. 29. The use as described in any one of claims 18 and 21-28, wherein the T cells and the IL-15 or an agonist derivative thereof are administered simultaneously. 30. The use as described in claim 29, wherein the T cells are formulated together with the IL-15 or an agonist derivative thereof. 31. The use as described in claim 29, wherein the T cells and the IL-15 or its agonist derivative are formulated separately. 32. The use as described in any one of claims 18 and 21-31, wherein natural killer (NK) cells are administered to the patient. 33. The use as described in claim 32, wherein the NK cells comprise m-ceNK cells isolated from the patient's whole blood. 34. The use as described in claim 32, wherein these NK cells are NK-92 cells. 35. The use as described in claim 34, wherein the NK-92 cells comprise aNK, haNK, or t-haNK cells. 36. The use as described in any one of claims 18 and 21-35, wherein the antigenic peptide is a SARS-CoV-2 variant.

Claims

1. A pharmaceutical composition for use in a patient suffering from a viral infection, the composition comprising: a. IL-15 or its agonist derivatives; and b. Virus-educated T cells comprising T cells isolated from the patient’s whole blood and exposed in vitro to dendritic cells, wherein these dendritic cells 1) are isolated from the patient’s whole blood and 2) are transfected with a nucleic acid vector encoding at least one antigenic viral peptide sequence.

2. The composition of claim 1, further comprising memory-like cytokine-enhanced natural killer (m-ceNK) cells, wherein these m-ceNK cells are isolated from the patient's whole blood.

3. The composition of any one of claims 1-2, wherein the IL-15 agonist derivative comprises IL-15:IL-15RαSu.

4. The composition of any one of claims 1-2, wherein the IL-15 agonist derivative comprises a stabilizing derivative.

5. The composition of claim 4, wherein the stabilized IL-15 agonist derivative comprises an Fc domain.

6. The composition of claim 5, wherein the stabilized IL-15 agonist derivative comprises N-803.

7. The composition as described in any of the preceding claims, wherein the autologous T cells and dendritic cells are derived from apheresis products.

8. The composition as claimed in any of the preceding claims, wherein the nucleic acid vector comprises an adenovirus (Ad) vector.

9. The composition of claim 8, wherein the Ad vector comprises SARS-CoV-2 spike (S) and nucleocapsid (N) proteins.

10. The composition of claim 8, wherein the Ad vector comprises a first nucleic acid portion and a second nucleic acid portion; wherein the first nucleic acid portion encodes a CoV2 spike (S) protein having the amino acid sequence of SEQ ID NO: 3; and wherein the second nucleic acid portion encodes a chimeric protein comprising 1) a CoV2 nucleocapsid (N) protein having the amino acid sequence of SEQ ID NO: 1 and 2) an endosome targeting sequence (ETSD) having the amino acid sequence of SEQ ID NO:

2.

11. The composition as claimed in any of the preceding claims, wherein the nucleic acid vector comprises an RNA vector.

12. The composition as claimed in any of the preceding claims, wherein the viral peptide sequence is derived from SARS-CoV-2, HPV, or HIV.

13. The composition of any one of the preceding claims, wherein the T cells are exposed to dendritic cells in vitro in the presence of IL-15 or an agonist derivative thereof.

14. The composition of any one of the preceding claims, wherein the T cells are exposed to dendritic cells in vitro in the presence of mesenchymal stem cells (MSCs).

15. Use of the composition as described in any of the preceding claims in the preparation of a medicament for treating viral infections.

16. The use as described in claim 15, wherein the viral infection comprises SARS-CoV-2.

17. The use as described in claim 16, wherein the viral infection comprises long-term COVID-19.

18. Use of IL-15 or an agonist derivative thereof and virus-educated T cells in the manufacture of a medicament for treating patients in need of long-term COVID-19, wherein the medicament is prepared by a method comprising: a. Transfect dendritic cells isolated from the patient's whole blood with a nucleic acid vector encoding at least one antigenic peptide derived from SARS-CoV-2; b. T cells isolated from the patient's whole blood are exposed to these dendritic cells, thereby expanding these T cells into virus-educated T cells; as well as c. Treating long-term COVID-19 patients by administering IL-15 or its agonist derivatives and these virus-educated T cells.

19. The use as described in claim 18, wherein the nucleic acid vector comprises an adenovirus (Ad) vector.

20. The use as described in claim 19, wherein the adenovirus (Ad) vector comprises an E1 gene region deletion and an E2b gene region deletion.

21. The use as claimed in claim 19, wherein the adenovirus (Ad) vector comprises a nucleic acid portion encoding a SARS-CoV-2 S protein and a nucleic acid portion encoding a chimeric protein comprising a SARS-CoV-2 N protein and an endosome targeting sequence.

22. The use as described in any one of claims 18-21, wherein the T cells are exposed to the dendritic cells in the presence of IL-15 or an agonist derivative thereof.

23. The use as claimed in any one of claims 18-21, wherein the IL-15 agonist derivative comprises IL-15:IL-15RαSu.

24. The use as claimed in any one of claims 18-21, wherein the IL-15 agonist derivative comprises a stabilizing derivative.

25. The use as described in any one of claims 18-21, wherein the stabilized IL-15 agonist derivative comprises an Fc domain.

26. The use as described in any one of claims 18-21, wherein the stabilized IL-15 agonist derivative comprises N-803.

27. The use as described in any one of claims 18-26, wherein these T cells and dendritic cells are derived from single-harvest products.

28. The use as described in any one of claims 18-27, wherein the T cells are administered intravenously.

29. The use as described in any one of claims 18-28, wherein the T cells and the IL-15 or an agonist derivative thereof are administered simultaneously.

30. The use as described in claim 29, wherein the T cells are formulated together with the IL-15 or an agonist derivative thereof.

31. The use as described in claim 29, wherein the T cells and the IL-15 or its agonist derivative are formulated separately.

32. The use as described in any one of claims 18-31, wherein natural killer (NK) cells are administered to the patient.

33. The use as described in claim 32, wherein the NK cells comprise m-ceNK cells isolated from the patient's whole blood.

34. The use as described in claim 32, wherein these NK cells are NK-92 cells.

35. The use as described in claim 34, wherein the NK-92 cells comprise aNK, haNK, or t-haNK cells.

36. The use as described in any one of claims 18-35, wherein the antigenic peptide is a SARS-CoV-2 variant.

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