Compositions and methods for MAIT cell activation

Abstract

In some embodiments, B cell proliferation and / or memory B cell formation / proliferation may be enhanced by contacting MAIT cells with Noogatointerleukin alpha due to chibercept (N-803) to produce stimulated MAIT cells, which in turn stimulate B cell proliferation and / or memory B cell formation / proliferation. Such stimulation may be performed in the presence of an antigen or antigen presenting cell. In particularly contemplated embodiments, MAIT cell stimulation is performed in vitro to produce an immunostimulatory composition and vaccine, or in vivo to enhance an immune response in airway tissue. Most typically, in vivo immune stimulation is carried out by inhalation or intranasal delivery of a composition comprising N-803 and optionally a vaccine component.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 425,517, filed on Nov. 15, 2022, the content of which is incorporated herein by reference in its entirety. Field of the Invention

[0002] The field of the invention is compositions and methods for immune stimulation (and in particular MAIT cell-mediated immune stimulation with enhanced B cell proliferation and / or memory B cell formation). Background of the Invention

[0003] The background description includes information that may be useful for an understanding of the present invention. It is not admitted that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is it admitted that any publication specifically or implicitly referenced is prior art.

[0004] All publications and patent applications herein are incorporated by reference to the extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If the definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, then the definition of that term provided herein applies and the definition of that term in the incorporated reference does not apply.

[0005] Mucosal-associated invariant T (MAIT) cells are innate-like T cells defined by their semi-invariant αβ T cell receptor (TCR), and are very abundant in human tissues, typically accounting for 1%-4% of all T cells in peripheral blood, up to 10% of airway T cells, and 20%-40% of liver T cells. The semi-invariant αβ T cell receptor recognizes small molecule reaction products of bacterial riboflavin synthesis (which represent unique pathway elements in microbes relative to many eukaryotes), which are presented on major histocompatibility complex (MHC)-related protein 1 (MR1). Thus, the MAIT cell TCR provides an innate ability to respond to a set of specific potential pathogen-derived ligands without the need for antigen-specific expansion otherwise required for conventional T cells.

[0006] Similar to conventional T cell signaling, MR1-TCR signaling alone is not sufficient to fully activate MAIT cells, which also require co-stimulation by CD28, TLR agonists, bacterial products, or cytokines. Such cytokines include interleukin (IL)-7, tumor necrosis factor (TNF), type I interferon (IFN), IL-1β, and / or IL-23. MAIT cells express several cytokine receptors, including IL-7R, IL-12R, IL-15R, IL-18R, and IL-23R. IL-7 is thought to enhance the response of MAIT cells to bacteria and promote cytotoxicity. IL-12 and IL-18 are thought to enhance MR1-dependent bacterial MAIT cell activation. In addition, agonists of various pathogen recognition receptors TLR1, TLR2, and TLR6 in humans and TLR3, TLR4, TLR6 / 2, and TLR9 in mice promote MAIT cell activation in an indirect manner as follows: by activating antigen-presenting cells via enhancing MR1 presentation, stimulating cytotoxic molecules and inflammatory cytokines, or upregulating co-stimulatory ligands.

[0007] Notably, in the absence of TCR-mediated antigen recognition, MAIT cells can also be partially activated by cytokines such as IL-7, IL-12, IL-15, IL-18, and type I IFN, thereby expanding the potential range of pathogens to which MAIT cells can respond, and exemplary pathogens include various viruses. Different from bacterial pathogens, viruses lack the metabolic pathways to activate MAIT cells via MR1, but instead activate them in an IL-18-dependent manner together with IL-12, IL-15, and other pro-inflammatory cytokines such as IFNα / β. TCR-independent activation is typically dominated by the secretion / release of IFN-γ, perforin, Fas ligand, and granzyme B. MAIT cells also secrete selected cytokines such as IL-17A and IL-22.

[0008] Interestingly, in addition to cytotoxic responses against pathogens and pathogen-infected cells, human studies have also shown an association between MAIT cells and pathogen-specific antibody responses. For example, supernatants from bacteria- or cytokine-stimulated MAIT cells showed increased frequencies of plasmablasts and promoted the production of IgA, IgG, and IgM when added to purified autologous B cells. However, these effects were found to be mainly MR1-dependent. In addition, the increase in plasmablasts may be the result of increased differentiation from memory B cells. In addition, supernatants from bacteria-activated MAIT cells contain various cytokines known to stimulate B cells, including IL-6, IL-10, and IL-21. Thus, B cell stimulation via MAIT cells is thought to require microbial infection.

[0009] Thus, even though various compositions and methods of MAIT cell-mediated immune stimulation are known in the art, all or almost all of these compositions and methods have several drawbacks. Accordingly, there remains a need for improved immune stimulation (and in particular MAIT cell-mediated immune stimulation that elicits enhanced B cell proliferation and / or memory B cell formation / proliferation) compositions. SUMMARY OF THE INVENTION

[0010] The subject matter of the present invention relates to various compositions and methods for stimulating MAIT cells in vitro or in vivo and using the stimulated MAIT cells to enhance B cell proliferation and / or memory B cell formation / proliferation. Advantageously, such stimulation and enhancement can be carried out in the context of vaccine administration and therapeutic or prophylactic treatment targeting one or more antigens.

[0011] In one aspect of the subject matter of the present invention, the inventors contemplated a variety of methods in which a composition comprising nogapendekin alfa inbakicept (N-803) is administered to a subject via the intranasal route. Most typically, such methods are used to stimulate mucosal-associated invariant T (MAIT) cell proliferation, stimulate MAIT cell-mediated B cell proliferation, and / or stimulate the immune response in respiratory epithelial tissue.

[0012] In such methods, it is contemplated that the composition can deliver 1 to 50 μg / kg of N-803 to the subject, typically by using a nasal spray and / or an inhalable composition. Accordingly, it is contemplated that the composition is administered to the nasal epithelium, bronchial epithelium, and / or lungs of the subject.

[0013] In some embodiments, the composition can further comprise an antigen of a pathogen, or an antigen of a tumor. From a different perspective, the composition can thus also comprise vaccine components, such as a viral vaccine (e.g., a recombinant adenovirus vaccine). Further, it is contemplated that the subject can be diagnosed with an infection or cancer, or the subject may have been previously exposed to or suspected of having been exposed to a pathogen of unknown origin or type.

[0014] In a further aspect of the subject matter of the present invention, the inventors also contemplated a method for stimulating MAIT cell-mediated B cell proliferation in a subject. Such a method will typically comprise the step of intranasally administering an immunotherapeutic combination comprising a vaccine, wherein the vaccine comprises a protein antigen or a nucleic acid encoding an antigen, and N-803.

[0015] For example, the protein antigen can be a protein antigen of a bacterial or viral pathogen, or the protein antigen can be a cancer-associated antigen, a tumor-specific antigen, or a patient- and tumor-specific neoantigen. As will be readily appreciated, the vaccine can be formulated as a protein vaccine, a viral vaccine, a DNA vaccine, or an RNA vaccine. Additionally, the vaccine under consideration can further include an adjuvant. Accordingly, the subject can be diagnosed with an infection or cancer. Preferably but not necessarily, 1 to 50 μg / kg of N-803 is administered to the subject.

[0016] In yet another aspect of the subject matter of the present invention, the inventors contemplate a method of stimulating the proliferation of memory B cells, and such a method will typically include the steps of isolating a) MAIT cells and b) memory B cells from a patient-derived peripheral blood mononuclear cell fraction; and the further step of adding N-803 to either the MAIT cells and the memory B cells.

[0017] In some embodiments, at least 10 6 MAIT cells and at least 10 6 memory B cells are isolated, and / or it is further contemplated that the MAIT cells and / or the memory B cells can be (further) expanded prior to adding N-803. Optionally or as needed, the MAIT cells and the memory B cells can be combined prior to adding N-803. Additionally, it is contemplated that such a method can further include the step of further stimulating the MAIT cells and / or the memory B cells with a stimulant selected from the group consisting of: cytokines, anti-CD3 antibody, anti-CD28 antibody, PMA (phorbol 12-myristate 13-acetate), ConA (concanavalin A), PHA (phytohemagglutinin), LPS (lipopolysaccharide), PWM (pokeweed mitogen), and α-GalCer (α-galactosylceramide). In such a method, the MAIT cells and / or the memory B cells are exposed to N-803 for a period of 6 - 24 hours.

[0018] Optionally, the MAIT cells and the memory B cells can be placed in a pharmaceutically acceptable carrier suitable for infusion or injection. Additionally, a vaccine or a protein antigen can be added to the carrier. For example, the protein antigen can be a protein antigen of a bacterial or viral pathogen, a cancer-associated antigen, a tumor-specific antigen, or a patient- and tumor-specific neoantigen. In the case where a vaccine is added, the vaccine can be a recombinant viral vaccine, a DNA vaccine, or an RNA vaccine.

[0019] In a further aspect of the subject matter of the present invention, the inventors also contemplate a method of preparing an immunotherapeutic composition for administration to a patient in need thereof, the method comprising the steps of isolating a) mucosal-associated invariant T (MAIT) cells and b) memory B cells from a patient-derived peripheral blood mononuclear cell fraction; treating the combined MAIT cells and memory B cells with nogales interleukin α inchiop (N-803); and formulating the treated MAIT cells and memory B cells into a composition for administration to the patient.

[0020] Typically but not necessarily, at least 10 6 MAIT cells and at least 10 6 memory B cells are isolated, and, if desired or required, the MAIT cells and / or memory B cells are expanded prior to the addition of N-803. As mentioned above, it is also contemplated that the MAIT cells and memory B cells can be combined prior to treatment with N-803. In addition, the contemplated method can include a further step of stimulating the MAIT cells and / or memory B cells with a stimulant selected from the group consisting of: cytokines, anti-CD3 antibody, anti-CD28 antibody, PMA (phorbol 12-myristate 13-acetate), ConA (concanavalin A), PHA (phytohemagglutinin), LPS (lipopolysaccharide), PWM (pokeweed mitogen), and α-GalCer (α-galactosylceramide).

[0021] In a further embodiment, the MAIT cells and / or memory B cells are exposed to N-803 for a period of 6 - 24 hours and / or the composition can be formulated for infusion or injection. Additionally, it is contemplated that the patient can be diagnosed with an infection or cancer.

[0022] In a still further aspect of the subject matter of the present invention, the inventors also contemplate a method of preparing an immunotherapeutic composition for administration to a patient in need thereof. Most typically, such a method will include the steps of isolating MAIT cells from a patient-derived peripheral blood mononuclear cell fraction; treating the MAIT cells with N-803; and a further step of formulating the treated MAIT cells into a composition for administration to the patient.

[0023] Regarding the cell numbers, possible expansion of the cells, manner of treatment and / or stimulation, and formulation, the same considerations as mentioned above apply.

[0024] In yet another aspect of the subject matter of the present invention, the inventors contemplate a method of preparing an immunotherapeutic composition for administration to a patient in need thereof. Such a method will typically include the step of isolating MAIT cells from a patient-derived peripheral blood mononuclear cell fraction; the step of treating the MAIT cells with N-803 or IL-15, IL-12, and IL-18, or alternatively with TxM consisting of the bioactive portions of IL-15, IL-12, and IL-18; and a further step of formulating the treated MAIT cells for administration to the patient.

[0025] In some embodiments, at least 10 6 MAIT cells are isolated and / or the MAIT cells can be expanded prior to the addition of N-803. Preferably but not necessarily, the MAIT cells are treated with N-803 or TxM. Optionally, the MAIT cells can be further treated with a stimulant selected from the group consisting of: cytokines, anti-CD3 antibody, anti-CD28 antibody, PMA (phorbol 12-myristate 13-acetate), ConA (concanavalin A), PHA (phytohemagglutinin), LPS (lipopolysaccharide), PWM (pokeweed mitogen), and α-GalCer (α-galactosylceramide). As previously described, it is also contemplated that such compositions can be formulated for infusion or injection.

[0026] The various objects, features, aspects and advantages of the subject matter of the present invention will become more apparent from the following detailed description of the preferred embodiments. Detailed Description of the Preferred Embodiments

[0027] The inventors have now found that various compositions and methods in which MAIT cells are exposed to N-803 (which can be carried out in the presence of an antigen or antigen-presenting cell) can be used to enhance B cell proliferation and / or memory B cell formation / proliferation. Notably, MAIT cells so stimulated are capable of stimulating B cell proliferation and / or memory B cell formation / proliferation. Advantageously, such MAIT cell stimulation can be carried out in vitro to produce immunostimulatory compositions and vaccines, and also in vivo in the context of immunostimulation of respiratory tissues (e.g., using an inhaled or spray (e.g., intranasal) vaccine composition).

[0028] For example, in one contemplated embodiment, MAIT cell proliferation in a subject, MAIT cell-mediated B cell proliferation, and / or stimulation of an immune response in respiratory epithelial tissue can be achieved by intranasal administration of a composition comprising N-803. In a particularly preferred aspect, the composition is administered as a nasal spray via a liquid carrier into one or both nostrils to deliver to the subject an amount of N-803 of about 10 μg / kg, typically once daily for a period of 3 to 14 days. As will be readily appreciated, such intranasal administration can be performed with or without inhalation to control the reach of the composition within the respiratory tract. In such instances, the subject will typically be an individual diagnosed with or at risk of infection or cancer, or a subject who has previously been exposed or is suspected of having been exposed to a pathogen of unknown origin or type. Notably, the inventors have found that activation of MAIT cells in the respiratory epithelium can be effective even where cytokine function is provided by an IgG-derived construct. This is particularly unexpected since the major antibody classes in mucosal and respiratory epithelia are IgA and IgM. Without being bound by any theory or hypothesis, it is contemplated that the long-term stability of IgG-derived constructs such as N-803 or TxM may contribute to activation in environments where IL-15 is unstable or biologically ineffective.

[0029] However, it should be understood that the compositions and methods presented herein can be extended to many other applications and uses that would benefit from the stimulation of MAIT cells in vitro or in vivo, particularly where such MAIT cells will further enhance B cell proliferation and / or memory B cell formation / proliferation. For example, in the context of vaccination and particularly vaccination with delivery of vaccine components to the respiratory tract, enhanced B cell proliferation and / or memory B cell formation / proliferation may be advantageous.

[0030] Accordingly, it should be understood that intranasal administration need not be limited to the administration of N-803, but rather the administered composition may include vaccine components in addition to N-803. As will be readily appreciated, the specific nature of the vaccine components does not limit the subject matter of the present invention, but rather all vaccine compositions are considered suitable for use herein. For example, suitable vaccine compositions may contain one or more protein antigens (which may be crude, isolated, purified, or synthetic), against which an immune response is desired. In addition, it should be recognized that the types of protein antigens can vary widely, and protein antigens that are particularly preferred will be (derived) from bacterial or viral pathogens. In other suitable examples, bacterial antigens include protein antigens from various strains of pneumococcus, vibrio, salmonella, meningococcus, bacillus, mycobacterium, etc. Similarly, viral antigens contemplated include antigens from various strains of influenza virus, flavivirus, parvovirus, coronavirus, orthopneumovirus, lentivirus, etc. Similarly, it should be understood that protein antigens may also be tumor antigens. Accordingly, tumor antigens contemplated include cancer-associated antigens, cancer-specific antigens, and patient- and tumor-specific neoantigens.

[0031] While in some embodiments the antigen is a protein antigen or a peptide antigen, it is also contemplated that the antigen may be encoded in a recombinant nucleic acid such that, upon administration of the recombinant nucleic acid, the cells of the subject will express the antigen from the recombinant nucleic acid. Accordingly, nucleic acids contemplated will include fragments encoding one or more antigens, wherein the fragment is under the control of regulatory sequences such that expression of the antigen protein is permitted. As will be readily understood, such recombinant nucleic acids can be configured in a variety of ways, and particularly suitable recombinant nucleic acids include recombinant RNA sequences and recombinant DNA sequences, which may be part of an expression vector, a linear sequence containing regulatory elements and the fragment, or may be part of a viral vector (such as an adenovirus vector).

[0032] Accordingly, in the case where an antigen or vaccine is delivered together with N-803 and / or stimulated MAIT cells, it should be understood that, due to the presence of the stimulated MAIT cells (and N-803, if also present), the antigen or vaccine will produce an enhanced immunogenic effect, and particularly a B cell response and memory B cell formation. Accordingly, it should be recognized that the methods contemplated may also provide stimulation of MAIT cell-mediated B cell proliferation and / or memory B cell proliferation in the presence of a vaccine or protein antigen. In such methods, N-803 is administered intranasally to the subject (as already mentioned above).

[0033] For the administration of N-803 to a subject, typically a dosage range of N-803 administration is preferably between about 1-50 μg / kg, and more typically between 5 and 25 μg / kg. In addition, it should be noted that while N-803 administration is typically preferred, various alternative or additional stimulants can also be administered. Stimulants specifically contemplated include IL-2, IL-12, IL-15, IL-18, and / or IL-21, and / or TxM consisting of the bioactive portions of IL-15, IL-12, IL-18 (most preferably at their clinically known and accepted concentrations, routes of administration, and schedules).

[0034] A preferred formulation of N-803 will typically be a liquid suitable for intranasal administration using a nebulizer or atomizer, but other formulations suitable for generating a mist or vapor are also considered applicable herein. Similarly, N-803 can be adsorbed onto inhalable solid phase particles to allow the compound to be administered to the lower respiratory tract. Thus, as will be appreciated, intranasal administration of N-803 can also be suitable for prophylactically stimulating an immune response, or therapeutically stimulating an immune response against an unknown pathogen to which the subject is believed or suspected to have been exposed. Thus, from a different perspective, MAIT cell stimulation can be performed in vivo by administering N-803 (or other or additional stimulants) to MAIT cells in situ. Since a large number of MAIT cells are also found in the liver, administration of the contemplated compounds and compositions to the liver by injection or targeting the hepatic circulation is also specifically contemplated.

[0035] In a further contemplated aspect of the subject matter of the present invention, MAIT cells can also be activated in vitro in the presence or absence of a vaccine or protein antigen, and / or in the presence or absence of B cells and / or memory B cells. For example, immune stimulation can also be achieved in a variety of ways, wherein the activation of stimulated MAIT cells on MAIT cells and / or B cells is performed in vitro. In such a method, MAIT cells and memory B cells can be isolated from a patient-derived peripheral blood mononuclear cell fraction, and then N-803 (Nogueira interleukin α inchiop) can be added to the MAIT cells and memory B cells in an amount and for a time sufficient to stimulate the proliferation of memory B cells. Thus, such a method will be suitable for preparing an immunotherapeutic composition for administration to a patient in need. As will be readily understood, the contemplated immunotherapeutic composition does not necessarily need to include B cells (autologous or from the same patient or subject). Thus, MAIT cells can be isolated only from a patient-derived peripheral blood mononuclear cell fraction, and then N-803 can be added to the MAIT cells in an amount and for a time sufficient to stimulate the MAIT cells. Then, the cells so treated can be formulated into a composition for administration to a subject.

[0036] MAIT cell isolation can be performed in a variety of ways, but is preferably isolated from the PBMC fraction of a subject's whole blood. For example, following the manufacturer's protocol, PBMCs are isolated by gradient centrifugation using Ficoll-Paque Plus reagent (e.g., GE, 17-1440-02, Chicago, USA). In the desired case, an additional platelet removal step (e.g., using a low-speed centrifugation / washing step) can be added. Surface marker antigens are stained with the following monoclonal fluorescent dye-conjugated antibodies for 15 - 20 minutes at 4 °C in the dark: Percep5.5-TCRγδ (B1), PE-CD161 (HP-3G10), APC-Cy7-CD3 (HIT3a), PE-Cy7 or FITC-TCRVα7.2 (3C10). All antibodies are commercially available from BioLegend (San Diego, USA). Freshly isolated PBMCs are analyzed and sorted / isolated by flow cytometry. MAIT cells will be gated as 7-AAD-TCRγδ - , CD3 + , TCRVα7.2 hi或+ , and CD161 hi或+ . Additional suitable protocols for isolating and characterizing peripheral blood and tissue-infiltrating MAIT cells by using multicolor flow cytometry are described elsewhere (e.g., Methods Mol Biol [Methods in Molecular Biology] 2020; 2098:23 - 38; or Methods Mol Biol [Methods in Molecular Biology] 2020; 2111:285 - 293). Then, the MAIT cells so isolated are cultured in, for example, RPMI-1640 medium (SH30809.1, USA) supplemented with 10% heat-inactivated FBS and placed in a cell culture incubator at 37 °C and 5% CO2 until the desired cell density and number are reached.

[0037] Then, the thus isolated MAIT cells can be stimulated by contacting the cells with N-803 at a concentration between about 0.1 nM and 10 nM. In addition, there are also IL-12 / 15 / 18Txm and other tricytokine Txm constructs (e.g., IL-7 / 15 / 21, or IL-15 / 21, or IL-15 / 18) (as described in WO2020 / 023713). In a less preferred aspect, individual cytokines can be used to stimulate MAIT cells, and particularly contemplated cytokines include IL-2, IL-7, IL-15, IL-18, and IL-21. Alternatively or additionally, stimulation can also be carried out using α-CD3 (anti-CD3 antibody), α-CD28 (anti-CD28 antibody) stimulation, PMA (phorbol 12-myristate 13-acetate), ConA (concanavalin A), PHA (phytohemagglutinin P), LPS (lipopolysaccharide), PWM (pokeweed mitogen), α-GalCer (α-galactosylceramide), etc. The stimulation of MAIT cells can follow with their enhanced ability to secrete cytokines and / or chemokines that are associated with helping B cell activation, migration, and regulation.

[0038] Similarly, B cell isolation typically follows well-known protocols starting from whole blood or the PBMC fraction of whole blood. For example, following the manufacturer's protocol, PBMCs are separated by gradient centrifugation using Ficoll-Paque Plus reagent (e.g., General Electric (GE), 17-1440-02, Chicago, USA). In the desired case, an additional platelet removal step (e.g., using a low-speed centrifugation / washing step) can be added.

[0039] After isolating PBMCs, B cells can be separated by two main methods: negative selection, where B cells remain "untouched" in their native state. Negative selection techniques are advantageous because B cells are likely to remain functionally intact during this process. Positive selection, where B cells are labeled and actively removed from the sample. There are various commercially available kits that allow simple and effective negative or positive separation, such as the Miltenyi B cell isolation kit II, Miltenyi naive B cell isolation kit, or Miltenyi CD43 microbead kit for negative selection, and the Miltenyi CD19 positive isolation kit for positive separation. To isolate memory B cells, positive selection can be carried out from total B cells or PBMCs using CD27 microbeads with compositions and methods well-known in the art (e.g., using the Miltenyi memory B cell isolation kit).

[0040] Once isolated, B cells can be cultured as is well-known. For example, B cells are seeded at 1×10 5Cells were seeded at TM a density of cells / well in a 24-well tissue culture plate and cultured using serum-free medium and supplements (such as ImmunoCult TM -XF B cell basal medium and ImmunoCult

[0041] -ACF human B cell expansion supplement). Cells were passaged every 3 - 4 days for up to 14 days or until the desired cell density or number was reached. Alternatively, B cells can also be cultured using feeder cells following a known protocol as described elsewhere (J Immunol. November 15, 2016; 197(10):4163–4176).

[0042] Then, the isolated and stimulated MAIT cells can be combined with the isolated B cells and cultured for a time sufficient to stimulate memory B cell proliferation and / or memory B cell formation / proliferation. Alternatively, it should be noted that the culture supernatant from the MAIT cell culture can also be used in the B cell culture after contact with N-803, thereby activating the B cells via secreted factors from the MAIT cell supernatant. 5 In the presence of N803 or other stimulators, the co-culture of MAIT cells with B cells is preferably carried out at a cell ratio (MAIT cells to B cells) between 100:1 and 1:100, more typically between 50:1 and 1:50, or between 10:1 and 1:10, or between 5:1 and 1:5, or between 3:1 and 1:3. The total cell density during stimulation is typically at least 100 cells / mL, or at least 1,000 cells / mL, or at least 10 5 cells / mL, or at least 10

[0043] cells / mL, etc. In addition, the appropriate length of stimulation will depend at least in part on the specific manner of MAIT cell activation and the cell ratio, but will generally be between 10 - 120 minutes, or between 120 and 480 minutes, or between 480 minutes and 12 hours, or between 12 hours and 24 hours, or between 24 - 36 hours, or between 36 and 48 hours, or between 2 and 4 days, and even longer.

[0044] Once co-cultured or otherwise activated, the MAIT cells and / or B cells can then be used directly for infusion or washed with a suitable medium and suspended in a pharmaceutically acceptable liquid carrier for infusion into a subject. Accordingly, the inventors also contemplate various formulations that can be infused or injected into a subject. Most typically, the cells are prepared as a cell suspension in a pharmaceutical carrier suitable for intravenous administration, which can further include one or more immunostimulants such as cytokines, checkpoint inhibitors, and the like.

[0045] The formulations contemplated for infusion or injection can further include various functional reagents, and particularly preferred reagents include antigens against which immunity is desired (such as bacterial antigens, viral antigens, antigens from prokaryotic or eukaryotic pathogens), as well as cancer-associated antigens (such as cancer or tumor-specific antigens and patient- and tumor-specific neoantigens). As will be readily appreciated, such antigens can be provided as polypeptides or encoded in nucleic acids (e.g., recombinant RNA or DNA sequences containing fragments encoding one or more antigens, and wherein the encoded antigen sequences are under the control of one or more regulatory elements permitting expression of the antigen-encoding fragments). In a further contemplated aspect, the nucleic acid can be naked nucleic acid or encapsulated in a lipid fraction or otherwise associated with a lipid fraction to form a nucleic acid-based vaccine. Similarly, the recombinant nucleic acid can be part of a viral vector (such as an adenovirus vector) commonly used in vaccine compositions.

[0046] In addition, the inventors also contemplate another therapeutic composition, wherein MAIT cells are activated in vitro as described herein, B cells are exposed to the activated cells or the cell culture supernatant from such activated MAIT cells, and wherein the antibodies produced by the B cells are then isolated and used as a therapeutic product. Most preferably, both the MAIT cells and the B cells are freshly isolated from a subject using the methods known and described above. After appropriate stimulation of the B cells, the antibodies can then be harvested from the medium using one or more affinity reagents such as protein A or protein G immobilized on a solid phase.

[0047] In some embodiments, the numerical values that describe and claim certain embodiments of the present invention, such as the amounts of the components, properties (such as concentrations), reaction conditions, etc., should be understood to be modified in some instances by the term "about". Accordingly, in some embodiments, the numerical parameters set forth in the written description and the appended claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. The description of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

[0048] As used herein, the term "administering" a pharmaceutical composition or a medicament refers to both direct and indirect administration of the pharmaceutical composition or the medicament, where direct administration of the pharmaceutical composition or the medicament is typically performed by a healthcare professional (e.g., a physician, a nurse, etc.), and where indirect administration includes the steps of providing the pharmaceutical composition or the medicament to a healthcare professional or making the pharmaceutical composition or the medicament available to a healthcare professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). It should also be noted that the terms "prognosis" or "predicting" the susceptibility of a disorder, disease progression, or response to a contemplated treatment are intended to encompass the act of making a prediction or prediction of a disorder, susceptibility, and / or response (including the rate of progression, improvement, and / or duration of a disorder in a subject), but not the act of treatment or diagnosis.

[0049] All of the methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., "such as") provided with respect to certain embodiments herein is intended merely to better illustrate the invention and does not limit the scope of the invention as otherwise claimed. The language in this specification should not be construed as indicating any non-claimed element as essential to the practice of the invention.

[0050] As used in the specification and throughout the following claims, unless the context clearly indicates otherwise, the meaning of "a," "an," and "the" includes plural referents. Similarly, as used in the specification herein, unless the context clearly indicates otherwise, the meaning of "in" includes "in" and "on." Also as used herein, and unless the context otherwise indicates, 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). Thus, the terms "coupled to" and "coupled with" are used synonymously.

[0051] It will be apparent to those skilled in the art that, without departing from the inventive concept herein, further modifications are possible in addition to those already described. Accordingly, the subject matter of the present invention should not be limited except within the scope of the appended claims. Further, 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 elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present or utilized, or combined with other elements, components, or steps not expressly recited. When the specification or claims refer to at least one selected from the group consisting of A, B, C... and N, the text should be interpreted as requiring only one element from the group, rather than A plus N, or B plus N, etc.

Claims

1. A method for stimulating the proliferation of mucosal-associated invariant T (MAIT) cells, the method comprising: Administering to a subject, by an intranasal route, a composition comprising nogales interleukin alpha inchipt (N-803).

2. A method for stimulating the proliferation of B cells mediated by mucosal-associated invariant T (MAIT) cells, the method comprising: Administering to a subject, by an intranasal route, a composition comprising nogales interleukin alpha inchipt (N-803).

3. A method for stimulating an immune response in respiratory epithelial tissue, the method comprising: Administering to a subject, by an intranasal route, a composition comprising nogales interleukin alpha inchipt (N-803).

4. The method according to any one of claims 1-3, wherein the composition delivers 1 to 50 μg / kg of N-803 to the subject.

5. The method according to any one of claims 1-3, wherein the composition is administered by a nasal spray or an inhalable composition.

6. The method according to any one of claims 1-3, wherein the composition is administered to the nasal epithelium, bronchial epithelium, and / or lungs of the subject.

7. The method according to any one of claims 1-3, wherein the composition further comprises an antigen of a pathogen.

8. The method according to any one of claims 1-3, wherein the composition further comprises an antigen of a tumor.

9. The method according to any one of claims 1-3, wherein the composition further comprises a vaccine component.

10. The method according to claim 9, wherein the vaccine component is a viral vaccine and optionally a recombinant adenovirus vaccine.

11. The method according to any one of claims 1-3, wherein the subject is diagnosed with an infection or cancer.

12. The method according to any one of claims 1-3, wherein the subject has been exposed to or is suspected of having been exposed to a pathogen of unknown origin or type.

13. A method for stimulating the proliferation of B cells mediated by mucosal-associated invariant T (MAIT) cells in a subject, the method comprising: Intranasally administering an immunotherapeutic combination comprising a vaccine; Wherein the vaccine comprises a protein antigen or a nucleic acid encoding an antigen, and nogales interleukin alpha inchipt (N-803).

14. The method according to claim 13, wherein the protein antigen is a protein antigen of a bacterial or viral pathogen.

15. The method according to claim 13, wherein the protein antigen is a cancer-associated antigen, a tumor-specific antigen, or a patient- and tumor-specific neoantigen.

16. The method according to any one of claims 13-15, wherein the vaccine is formulated as a viral vaccine, a DNA vaccine, or an RNA vaccine.

17. The method according to any one of claims 13-16, wherein the vaccine further comprises an adjuvant.

18. The method according to any one of claims 13-17, wherein the subject is diagnosed with an infection or cancer.

19. The method according to any one of claims 13-17, wherein the administration delivers 1 to 50 μg / kg of N-803 to the subject.

20. A method for stimulating the proliferation of memory B cells, the method comprising: Isolate a) mucosal-associated invariant T (MAIT) cells and b) memory B cells from a patient-derived peripheral blood mononuclear cell fraction; and Add nogral interleukin α inchiop (N-803) to these MAIT cells and memory B cells.

21. The method according to claim 20, wherein at least 10 6 MAIT cells and at least 10 6 memory B cells are isolated.

22. The method according to claim 20, wherein these MAIT cells and / or these memory B cells are expanded before adding the N-803.

23. The method according to any one of claims 20-22, wherein these MAIT cells and these memory B cells are combined before adding N-803.

24. The method according to any one of claims 20-23, the method further comprising the step of further stimulating these MAIT cells and / or these memory B cells with a stimulant selected from the group consisting of: cytokines, anti-CD3 antibody, anti-CD28 antibody, PMA (phorbol 12-myristate 13-acetate), ConA (concanavalin A), PHA (phytohemagglutinin), LPS (lipopolysaccharide), PWM (pokeweed mitogen), and α-GalCer (α-galactosylceramide).

25. The method according to any one of claims 20-24, wherein these MAIT cells and / or these memory B cells are exposed to the N-803 for a period of 6-24 hours.

26. The method according to any one of claims 20-25, the method further comprising the step of placing these MAIT cells and these memory B cells in a pharmaceutically acceptable carrier suitable for infusion or injection.

27. The method according to claim 26, the method further comprising the step of adding a vaccine or protein antigen to the carrier.

28. The method according to claim 27, wherein the protein antigen is a protein antigen of a bacterial or viral pathogen, a cancer-associated antigen, a tumor-specific antigen, or a patient- and tumor-specific neoantigen.

29. The method according to claim 27, wherein the vaccine is a recombinant viral vaccine, a DNA vaccine, or an RNA vaccine.

30. A method of preparing an immunotherapeutic composition for administration to a patient in need thereof, the method comprising: Isolate a) mucosal-associated invariant T (MAIT) cells and b) memory B cells from a patient-derived peripheral blood mononuclear cell fraction; and Treat the combined MAIT cells and memory B cells with nogral interleukin α inchiop (N-803); and Formulate these treated MAIT cells and memory B cells into a composition for administration to the patient.

31. The method according to claim 30, wherein at least 10 6 MAIT cells and at least 10 6 memory B cells are isolated.

32. The method according to claim 30, wherein these MAIT cells and / or these memory B cells are expanded before adding the N-803.

33. The method according to any one of claims 30-32, wherein these MAIT cells and these memory B cells are combined before treating these MAIT cells with N-803.

34. The method according to any one of claims 30 - 33, the method further comprising the step of further stimulating these MAIT cells and / or these memory B cells with a stimulant selected from the group consisting of: cytokines, anti-CD3 antibody, anti-CD28 antibody, PMA (phorbol 12-myristate 13-acetate), ConA (concanavalin A), PHA (phytohemagglutinin), LPS (lipopolysaccharide), PWM (pokeweed mitogen), and α-GalCer (α-galactosylceramide).

35. The method according to any one of claims 30 - 34, wherein these MAIT cells and / or these memory B cells are exposed to the N-803 for a period of 6 - 24 hours.

36. The method according to any one of claims 30 - 35, wherein the composition is formulated for infusion or injection.

37. The method according to any one of claims 30 - 36, wherein the patient is diagnosed with an infection or cancer.

38. A method of preparing an immunotherapeutic composition for administration to a patient in need thereof, the method comprising: isolating mucosal-associated invariant T (MAIT) cells from a patient-derived peripheral blood mononuclear cell fraction; treating these MAIT cells with nogal interleukin α inchiopsis (N-803); and formulating these treated MAIT cells into a composition for administration to the patient.

39. The method according to claim 38, wherein at least 10 6 MAIT cells and at least 10 6 memory B cells are isolated.

40. The method according to claim 38, wherein these MAIT cells and / or memory B cells are expanded before adding the N-803.

41. The method according to any one of claims 38 - 40, wherein these MAIT cells and these memory B cells are combined before treating these MAIT cells with N-803.

42. The method according to any one of claims 38 - 41, the method further comprising the step of further stimulating these MAIT cells and / or these memory B cells with a stimulant selected from the group consisting of: cytokines, anti-CD3 antibody, anti-CD28 antibody, PMA (phorbol 12-myristate 13-acetate), ConA (concanavalin A), PHA (phytohemagglutinin), LPS (lipopolysaccharide), PWM (pokeweed mitogen), and α-GalCer (α-galactosylceramide).

43. The method according to any one of claims 38 - 42, wherein these MAIT cells and / or these memory B cells are exposed to the N-803 for a period of 6 - 24 hours.

44. The method according to any one of claims 38 - 43, wherein the composition is formulated for infusion or injection.

45. A method of preparing an immunotherapeutic composition for administration to a patient in need thereof, the method comprising: isolating mucosal-associated invariant T (MAIT) cells from a patient-derived peripheral blood mononuclear cell fraction; Treat these MAIT cells with N-803 (Nogueira interleukin-α inchi peptide), IL-12, and IL-18, or TxM consisting of the bioactive portions of IL-15, IL-12, and IL-18; and Formulate these treated MAIT cells for administration to the patient.

46. The method according to claim 45, wherein at least 10 6 MAIT cells are isolated.

47. The method according to claim 45, wherein these MAIT cells are expanded before adding the N-803.

48. The method according to any one of claims 45-47, wherein these MAIT cells are treated with N-803 or the TxM.

49. The method according to any one of claims 45-48, the method further comprising the step of further stimulating these MAIT cells with a stimulant selected from the group consisting of: cytokines, anti-CD3 antibody, anti-CD28 antibody, PMA (phorbol 12-myristate 13-acetate), ConA (concanavalin A), PHA (phytohemagglutinin), LPS (lipopolysaccharide), PWM (pokeweed mitogen), and α-GalCer (α-galactosylceramide).

50. The method according to any one of claims 45-49, wherein these MAIT cells are treated for a period of 6-24 hours.

51. The method according to any one of claims 45-50, wherein the composition is formulated for infusion or injection.