Compositions and methods for MAIT cell activation

Abstract

B cell proliferation and / or memory B cell formation / expansion can be enhanced by contacting MAIT cells with nogapendekin alfa invaxcept (N-803) to produce stimulated MAIT cells, which stimulate B cell proliferation and / or memory B cell formation / expansion. Such stimulation can be performed in the presence of antigen or antigen-presenting cells. In particularly contemplated embodiments, MAIT cell stimulation is performed in vitro to produce immunostimulatory compositions and vaccines, or in vivo to enhance immune responses in airway tissue. Most typically, in vivo immune stimulation is performed by inhalation or intranasal delivery of a composition comprising N-803 and, optionally, vaccine components.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 425,517, filed November 15, 2022, the entire contents of which are incorporated by reference.

[0002] The field of the invention is compositions and methods for immune stimulation, particularly MAIT cell-mediated immune stimulation with enhanced B cell proliferation and / or memory B cell formation. [Background technology]

[0003] The background discussion includes information that may be useful in understanding the present invention. No admission is made that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

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

[0005] Mucosal-associated invariant T (MAIT) cells, defined by their semi-invariant αβ T cell receptor (TCR), are innate-like T cells that are remarkably abundant in human tissues, typically constituting 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 the small molecule reaction product of bacterial riboflavin synthesis (representing a pathway element unique in microbes compared to many eukaryotes) displayed on major histocompatibility complex (MHC)-related protein-1 (MR1). Thus, the MAIT cell TCR provides the natural ability to respond to a specific set of potential pathogen-derived ligands without the need for antigen-specific proliferation required for conventional T cells.

[0006] Similar to conventional T cell signaling, MR1-TCR signaling alone is insufficient to fully activate MAIT cells; costimulation by CD28, TLR agonists, bacterial products, or cytokines is also required. Such cytokines include interleukin (IL)-7, tumor necrosis factor (TNF), type I interferon (IFN), IL-1b, 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 MAIT cell responses to bacteria and promote cytotoxicity. IL-12 and IL-18 are thought to enhance MR1-dependent bacterial MAIT cell activation. Furthermore, 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 indirectly through activation of antigen-presenting cells via enhanced MR1 presentation, stimulation of cytotoxic molecules and inflammatory cytokines, or upregulation of costimulatory 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 IFNs, broadening the potential range of pathogens to which MAIT cells can respond. Exemplary pathogens include various viruses. Unlike bacterial pathogens, viruses lack the metabolic pathways that activate MAIT cells via MR1, along with other proinflammatory cytokines such as IL-12, IL-15, and IFNα / β, but instead activate them in an IL-18-dependent manner. TCR-independent activation is typically governed by the secretion / release of IFNγ, perforin, Fas ligand, and granzyme B. MAIT cells also secrete select 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 demonstrated a link between MAIT cells and pathogen-specific antibody responses. For example, supernatants from bacteria- or cytokine-stimulated MAIT cells, when added to purified autologous B cells, have been shown to increase plasmablast frequency and promote IgA, IgG, and IgM production. However, these effects were found to be primarily MR1-dependent. Furthermore, the increase in plasmablasts was likely the result of increased differentiation from memory B cells. Furthermore, supernatants from bacteria-activated MAIT cells contained multiple cytokines known to stimulate B cells, including IL-6, IL-10, and IL-21. Therefore, MAIT cell-mediated B cell stimulation is thought to be required for microbial infection. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, although various compositions and methods for MAIT cell-mediated immunostimulation are known in the art, all or almost all of them suffer from several drawbacks. Thus, there remains a need for improved compositions for MAIT cell-mediated immunostimulation that result in immune stimulation, particularly enhanced B cell proliferation and / or memory B cell formation / proliferation. [Means for solving the problem]

[0010] The present subject matter 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 / expansion. Advantageously, such stimulation and enhancement can be performed in the context of vaccination, and therapeutic or prophylactic treatments targeting one or more antigens.

[0011] In one aspect of the inventive subject matter, the inventors contemplate various methods of administering a composition comprising nogapendekin alfa invaxcept (N-803) to a subject by the intranasal route. Most typically, such methods are directed to stimulating mucosal-associated invariant T (MAIT) cell proliferation, stimulating mucosal-associated invariant T (MAIT) cell-mediated B cell proliferation, and / or stimulating an immune response in respiratory epithelial tissue.

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

[0013] In some embodiments, the composition may further comprise a pathogen antigen or a tumor antigen. Thus, viewed from a different perspective, the composition may also comprise a vaccine component, such as a viral vaccine (e.g., a recombinant adenovirus vaccine). It is further contemplated that the subject may be diagnosed with an infectious disease or cancer, or that the subject may have previously been exposed to or is suspected of being exposed to a pathogen of unknown origin or species.

[0014] In a further aspect of the present subject matter, the inventors also contemplate a method of stimulating MAIT cell-mediated B cell proliferation in a subject, such a method typically comprising the step of intranasal administration of an immunotherapeutic combination comprising a protein antigen or a nucleic acid encoding the antigen and a vaccine comprising N-803.

[0015] For example, the protein antigen may be a protein antigen of a bacterial or viral pathogen, or the protein antigen may be a cancer-associated antigen, a tumor-specific antigen, or a patient- and tumor-specific neoantigen. As will be readily understood, the vaccine may be formulated as a protein vaccine, a viral vaccine, a DNA vaccine, or an RNA vaccine. Furthermore, contemplated vaccines may further include an adjuvant. Thus, the subject may be diagnosed with an infectious disease or cancer. Preferably, although not necessarily, administration delivers 1 to 50 μg / kg of N-803 to the subject.

[0016] In yet another aspect of the inventive subject matter, the inventors contemplate methods of stimulating memory B cell proliferation, such methods typically comprising the steps of isolating a) MAIT cells and b) memory B cells from a peripheral blood mononuclear cell fraction from a patient, and the further step of adding N-803 to the MAIT cells and memory B cells.

[0017] In some embodiments, at least 10 6 MAIT cells and at least 10 6It is further contemplated that individual memory B cells may be isolated and / or the MAIT cells and / or memory B cells may be (further) expanded before adding N-803. If appropriate or desired, the MAIT cells and memory B cells may be combined before adding N-803. Furthermore, such methods are also contemplated to include the step of further stimulating the MAIT cells and / or memory B cells with a stimulatory agent selected from the group consisting of cytokines, anti-CD3 antibodies, anti-CD28 antibodies, PMA (phorbol 12-myristate 13-acetate), ConA (concanavalin A), PHA (phytohemagglutinin), LPS (lipopolysaccharide), PWM (pokeweed mitogen), and α-GalCer (alpha-galactosylceramide). In such methods, the MAIT cells and / or memory B cells are exposed to N-803 for a period of 6 to 24 hours.

[0018] If desired, the MAIT cells and memory B cells can be placed in a pharmaceutically acceptable carrier suitable for infusion or injection. Furthermore, a vaccine or 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. If 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 inventive subject matter, the inventors also contemplate a method for preparing an immunotherapeutic composition for administration to a patient in need thereof, comprising the steps of: isolating a) mucosal-associated invariant T (MAIT) cells and b) memory B cells from a peripheral blood mononuclear cell fraction from a patient; treating the combined MAIT cells and memory B cells with nogapendequin alfa invaxcept (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 6Memory B cells are isolated, and the MAIT cells and / or memory B cells are expanded, if necessary or desired, before adding N-803. It is also contemplated that the MAIT cells and memory B cells can be combined before treating the MAIT cells with N-803, as described above. Furthermore, contemplated methods may include the additional step of stimulating the MAIT cells and / or memory B cells with a stimulatory agent selected from the group consisting of cytokines, anti-CD3 antibodies, anti-CD28 antibodies, PMA (phorbol 12-myristate 13-acetate), ConA (concanavalin A), PHA (phytohemagglutinin), LPS (lipopolysaccharide), PWM (pokeweed mitogen), and α-GalCer (alpha-galactosylceramide).

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

[0022] In yet a further aspect of the inventive subject matter, the inventors also contemplate methods of preparing an immunotherapeutic composition for administration to a patient in need thereof. Most typically, such methods include the steps of isolating a peripheral blood mononuclear cell fraction from a patient from which MAIT cells are isolated, treating the MAIT cells with N-803, and formulating the treated MAIT cells into a composition for administration to the patient.

[0023] The same considerations as above apply with regard to the number of cells, possible expansion of the cells, method of treatment and / or stimulation, and formulation.

[0024] Furthermore, in yet another aspect of the present subject matter, the inventors contemplate a method for preparing an immunotherapeutic composition for administration to a patient in need thereof. Such a method would typically include the steps of isolating MAIT cells from a peripheral blood mononuclear cell fraction from the patient, treating the MAIT cells with N-803 or IL-15, IL-12, and IL-18, or with a TxM composed of biologically active portions of IL-15, IL-12, and IL-18, and further formulating the treated MAIT cells for administration to the patient.

[0025] In some embodiments, at least 10 6 MAIT cells may be isolated and / or expanded before adding N-803. Preferably, but not necessarily, MAIT cells are treated with N-803 or TxM. If desired, MAIT cells may be further treated with a stimulatory agent selected from the group consisting of cytokines, anti-CD3 antibodies, anti-CD28 antibodies, PMA (phorbol 12-myristate 13-acetate), ConA (concanavalin A), PHA (phytohemagglutinin), LPS (lipopolysaccharide), PWM (pokeweed mitogen), and α-GalCer (alpha-galactosylceramide). As mentioned above, it is also contemplated that such compositions may be formulated for infusion or injection.

[0026] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present inventors have now discovered that various compositions and methods for exposing MAIT cells to N-803, possibly in the presence of antigen or antigen-presenting cells, can be used to enhance B cell proliferation and / or memory B cell formation / proliferation. In particular, MAIT cells so stimulated can stimulate B cell proliferation and / or memory B cell formation / proliferation. Advantageously, such MAIT cell stimulation can be performed in vitro to produce immunostimulatory compositions and vaccines, but can also be performed in vivo in connection with immune stimulation of tissues within the respiratory tract, for example, using inhaled or sprayed (e.g., intranasal) vaccine compositions.

[0028] For example, in one contemplated embodiment, stimulation of MAIT cell proliferation, MAIT cell-mediated B cell proliferation, and / or immune responses in respiratory epithelial tissue in a subject can be achieved by intranasal administration of a composition comprising N-803. In a particularly preferred embodiment, the composition is administered as a nasal spray from a liquid carrier to one or both nostrils to deliver approximately 10 μg / kg of N-803 to the subject, typically once daily for 3 to 14 days. As will be readily understood, such intranasal administration can be performed with or without inhalation to control the delivery of the composition to the respiratory tract. The subject in such an example is typically an individual diagnosed with or at risk of an infectious disease or cancer, or a subject previously exposed to or suspected of being exposed to a pathogen of unknown origin or species. Notably, the inventors have discovered that activation of MAIT cells in respiratory epithelia can be effective even when cytokine function is provided in an IgG-derived construct. This is particularly unexpected, since the predominant antibody classes in mucosal and respiratory epithelia are IgA and IgM. Without wishing to be 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 otherwise not stable or biologically effective.

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

[0030] Thus, it should be understood that intranasal administration need not necessarily be limited to administration of N-803, but that the administered composition may also include vaccine components in addition to N-803. As will be readily appreciated, the specific nature of the vaccine components is not a limitation of the subject matter of the present invention, but all vaccine compositions are considered suitable for use herein. For example, a suitable vaccine composition may include one or more protein antigens (which may be crude, isolated, purified, or synthetic) against which an immune response is desired. Furthermore, it should be recognized that the types of protein antigens may vary considerably, with particularly preferred protein antigens being derived from bacterial or viral pathogens. Among other suitable examples, bacterial antigens include protein antigens from various strains of Pneumococcus, Vibrio, Salmonella, Meningococcus, Bacillus, Mycobacterium, etc. Similarly, contemplated viral antigens include antigens of various strains of influenza virus, flavivirus, parvovirus, coronavirus, orthopneumovirus, lentivirus, etc. Similarly, it should be understood that protein antigens may also be tumor antigens. Thus, contemplated tumor antigens include cancer-associated antigens, cancer-specific antigens, and patient- and tumor-specific neoantigens.

[0031] In some embodiments, the antigen is a protein antigen or a peptide antigen, but it is also contemplated that the antigen can be encoded by a recombinant nucleic acid such that upon administration of the recombinant nucleic acid, the subject's cells express the antigen from the recombinant nucleic acid. Thus, contemplated nucleic acids include segments encoding one or more antigens, which are under the control of regulatory sequences to allow expression of the antigenic protein. As will be readily understood, such recombinant nucleic acids can be configured in a variety of ways, with particularly suitable recombinant nucleic acids including recombinant RNA and DNA sequences that can be part of an expression vector, a linear sequence containing regulatory elements and segments, or that can be part of a viral vector, such as an adenoviral vector.

[0032] As a result, it should be understood that when an antigen or vaccine is delivered together with N-803 and / or stimulated MAIT cells, the antigen or vaccine will have an enhanced immunogenic effect, particularly B cell responses and memory B cell formation, due to the presence of stimulated MAIT cells (and N-803, if present). Accordingly, it should be recognized that contemplated methods may also provide for 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 a subject as already described above.

[0033] Regarding administration of N-803 to a subject, N-803 is typically administered in a dosage range of about 1-50 μg / kg, more typically 5-25 μg / kg. It is further noted that while administration of N-803 is typically preferred, various alternative or additional stimulating agents can be administered as well. Particularly contemplated stimulating agents include TxMs consisting of IL-2, IL-12, IL-15, IL-18, and / or IL-21, and / or biologically active portions of IL-15, IL-12, and IL-18, most preferably at their clinically known and accepted concentrations, routes of administration, and schedules.

[0034] Preferred formulations of N-803 are typically liquids suitable for intranasal administration using a nebulizer or spray, although other formulations suitable for generating mist or vapor are also contemplated for use herein. Similarly, N-803 may be adsorbed onto inhalable solid particles, allowing administration of the compound to the lower respiratory tract. It will be appreciated, therefore, that intranasal administration of N-803 may also be suitable for prophylactically stimulating an immune response or therapeutically stimulating an immune response against an unknown pathogen to which a 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 stimulatory agents) to MAIT cells in situ. Because MAIT cells are also found in significant quantities in the liver, administration of contemplated compounds and compositions to the liver by injection or targeting the hepatic circulation is also expressly contemplated.

[0035] In further contemplated aspects of the present subject matter, MAIT cells can also be activated in vitro in the 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 various ways in which MAIT cell and / or B cell activation by stimulated MAIT cells is performed in vitro. In such an approach, MAIT cells and memory B cells can be isolated from a peripheral blood mononuclear cell fraction from a patient, and then nogapendequin alfa invaxept (N-803) is added to the MAIT cells and memory B cells in an amount and for a time sufficient to stimulate memory B cell proliferation. Thus, such methods are suitable for preparing immunotherapeutic compositions for administration to patients in need thereof. As will be readily understood, contemplated immunotherapeutic compositions do not necessarily need to include B cells (autologous or derived from the same patient or subject). As a result, MAIT cells can be isolated solely from the peripheral blood mononuclear cell fraction from a patient, and then N-803 is added to the MAIT cells in an amount and for a time sufficient to stimulate the MAIT cells. The cells so treated can then be formulated into a composition for administration to a subject.

[0036] MAIT cell isolation can be performed in a variety of ways, but preferably, isolation is performed from the PBMC fraction of the subject's whole blood. For example, PBMCs are isolated by gradient centrifugation using Ficoll-Paque Plus reagent (e.g., GE, 17-1440-02, Chicago, USA) according to the manufacturer's protocol. If desired, an additional step of platelet removal can be added (e.g., using a low-speed centrifugation / washing step). Staining of surface marker antigens will be performed in the dark at 4°C for 15–20 minutes using the following monoclonal fluorochrome-conjugated antibodies: 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 express 7-AAD-TCRγδ - , CD3 + , TCRVα7.2 hi又は+ , and CD161 hi又は+ The MAIT cells will be gated as . Further suitable protocols for isolating and characterizing peripheral blood and tissue-infiltrating MAIT cells by using multicolor flow cytometry have been described elsewhere (e.g., Methods Mol Biol 2020;2098:23-38; or Methods Mol Biol 2020;2111:285-293). The MAIT cells thus isolated are then cultured, for example, in RPMI-1640 medium (SH30809.1, America) supplemented with 10% heat-inactivated FBS and placed in a cell incubator at 37°C and 5% CO2 to achieve the desired cell density and quantity.

[0037] Thus, isolated MAIT cells can be stimulated by contacting the cells with N-803 at a concentration of about 0.1 nM to 10 nM. This also applies to IL-12 / 15 / 18 Txm and other avian cytokine Txm constructs described in WO 2020 / 023713 (e.g., IL-7 / 15 / 21, or IL-15 / 21, or IL-15 / 18). In less preferred embodiments, individual cytokines can be used to stimulate MAIT cells; particularly contemplated cytokines include IL-2, IL-7, IL-15, IL-18, and IL-21. Alternatively or additionally, stimulation can also be performed 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 (alpha-galactosylceramide), etc. Stimulation of MAIT cells can be followed by their enhanced ability to secrete cytokines and / or chemokines associated with B cell activation, migration, and regulatory support.

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

[0039] After isolation of PBMCs, B cells can be isolated by two main approaches: negative selection, in which B cells remain "untouched" in their natural state. Negative selection techniques are advantageous because B cells are likely to remain functionally untouched by this process. Positive selection, in which B cells are labeled and actively removed from the sample. There are various commercially available kits that allow for simple and effective negative or positive isolation, such as the Miltenyi B Cell Isolation Kit II, Miltenyi Naive B Cell Isolation Kit, or the Miltenyi CD43 Microbead Kit for negative selection and the Miltenyi CD19 Positive Isolation Kit for positive isolation. For isolation of memory B cells, positive selection from total B cells or PBMCs using CD27 microbeads can be performed using compositions and methods well known in the art (e.g., the Miltenyi Memory B Cell Isolation Kit).

[0040] After isolation, B cells can be cultured as known in the art. For example, B cells can be cultured at 1 x 10 in a 24-well tissue culture plate. 5 Cells are seeded per well and cultured using serum-free media and supplements such as ImmunoCult™-XF B Cell Base Medium and ImmunoCult™-ACF Human B Cell Expansion Supplement. Cells are passaged every 3-4 days for up to 14 days or until the desired cell density or number is reached. Alternatively, B cells can also be cultured using feeder cells according to known protocols, as described elsewhere (J Immunol. 2016 Nov 15;197(10):4163-4176).

[0041] The isolated and stimulated MAIT cells can then be combined with isolated B cells and cultured for a time sufficient to stimulate memory B cell proliferation and / or memory B cell formation / expansion. Alternatively, it should be noted that the culture supernatant from MAIT cell cultures after contact with N-803 can be used in B cell cultures to activate B cells via factors secreted from the MAIT cell supernatant.

[0042] Co-culture of MAIT cells with B cells in the presence of N803 or other stimulatory agents is preferably carried out at a cell ratio (MAIT cells with B cells) of 100:1 to 1:100, more typically 50:1 to 1:50, or 10:1 to 1:10, or 5:1 to 1:5, or 3:1 to 1:3. The total cell density during stimulation is generally at least 100 cells / mL, or at least 1,000 cells / mL, or at least 10 5 cells / mL, or at least 10 5 Furthermore, the appropriate length of stimulation will depend, at least in part, on the particular mode of MAIT cell activation and cell ratio, but will generally be 10-120 minutes, or 120-480 minutes, or 480 minutes to 12 hours, or 12 hours to 24 hours, or 24-36 hours, or 36-48 hours, or 2-4 days, or even longer.

[0043] As will be readily appreciated, B cell activation may also be performed in the presence of additional stimuli, contemplated stimuli including N-803, TxM stimuli, one or more cytokines, and / or 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 (alpha-galactosylceramide), and the like.

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

[0045] Contemplated formulations for transfusion or injection may further include various functional agents, particularly preferred agents including antigens against which immunity is desired, such as bacterial antigens, viral antigens, antigens forming pro- or eukaryotic pathogens, and cancer-associated antigens, such as cancer- or tumor-specific antigens and patient- and tumor-specific neoantigens. As will be readily understood, such antigens can be provided as polypeptides or encoded by nucleic acids (e.g., recombinant RNA or DNA sequences containing an antigen-encoding segment, the encoded antigen sequence being under the control of one or more regulatory elements that enable expression of the antigen-encoding segment). In yet further contemplated embodiments, the nucleic acid may be naked, or may be encapsulated in or otherwise associated with a lipid fraction to form a nucleic acid-based vaccine. Similarly, the recombinant nucleic acid may be part of a viral vector, such as an adenoviral vector, commonly used in vaccine compositions.

[0046] Furthermore, the inventors also contemplate additional therapeutic compositions in which MAIT cells are activated in vitro as described herein, B cells are exposed to activated cells or cell culture supernatant from such activated MAIT cells, and antibodies produced by the B cells are then isolated and used as a therapeutic product. Most preferably, both MAIT cells and B cells are freshly isolated from a subject using known methods, such as those described above. Once the B cells have been appropriately stimulated, antibodies can then be recovered from the culture medium using one or more affinity reagents, such as solid-phase-immobilized protein A or protein G.

[0047] In some embodiments, numbers expressing properties such as amounts and concentrations of ingredients, reaction conditions, and the like, used to describe and claim particular embodiments of the present invention should be understood to be modified in some cases by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by a particular embodiment. The recitation of ranges 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 herein as if it were individually recited herein.

[0048] As used herein, the term "administering" a pharmaceutical composition or drug refers to both direct and indirect administration of the pharmaceutical composition or drug, where direct administration of a pharmaceutical composition or drug is typically performed by a medical professional (e.g., a doctor, a nurse, etc.), and indirect administration includes providing or making available the pharmaceutical composition or drug to a medical professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). It should be further noted that the terms "prognosing" or "predicting" a condition, susceptibility to developing a disease, or response to an intended treatment is meant to encompass the act of predicting or forecasting (but not treating or diagnosing) the condition, susceptibility, and / or response, including the rate of progression, improvement, and / or duration of the condition in a subject.

[0049] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided with respect to specific embodiments herein is intended merely to better clarify the invention and does not otherwise limit the scope of the invention as claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0050] As used throughout this description and the claims that follow, the meanings of "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Also, as used in this description, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise. Also, as used herein, unless the context dictates otherwise, the term "coupled" is intended to include both direct coupling (two elements coupled to each other are in contact with each other) and indirect coupling (at least one additional element is disposed between the two elements). Thus, the terms "coupled" and "coupled with" are used interchangeably.

[0051] It will be apparent to those skilled in the art that many more modifications beyond those already described are possible without departing from the inventive concepts herein. Accordingly, the inventive subject matter should not be limited except as by the appended claims. Moreover, 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 element, component, or step may be present, utilized, or combined with other elements, components, or steps not expressly referenced. When the specification or claims refer to at least one of something selected from the group consisting of A, B, C... and N, the text should be interpreted as requiring only one element from the group, not A plus N or B plus N, etc.

Claims

1. 1. A method for stimulating mucosal-associated invariant T (MAIT) cell proliferation, comprising: administering a composition comprising nogapendekin alfa invaxept (N-803) to a subject by intranasal route; 10. A method for stimulating mucosal-associated invariant T (MAIT) cell proliferation, comprising:

2. 1. A method for stimulating mucosal-associated invariant T (MAIT) cell-mediated B cell proliferation, comprising: administering a composition comprising nogapendekin alfa invaxept (N-803) to a subject by intranasal route; 1. A method for stimulating mucosal-associated invariant T (MAIT) cell-mediated B cell proliferation, comprising:

3. 1. A method for stimulating an immune response in respiratory epithelial tissue, comprising: administering a composition comprising nogapendekin alfa invaxcept (N-803) to a subject by an intranasal route; 10. A method for stimulating an immune response in respiratory epithelial tissue, comprising:

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

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

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

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

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

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

10. 10. The method of claim 9, wherein the vaccine component is a viral vaccine, and optionally a recombinant adenoviral vaccine.

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

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

13. 1. A method of stimulating mucosal-associated invariant T (MAIT) cell-mediated B cell proliferation in a subject, comprising: Intranasal administration of an immunotherapeutic combination comprising a vaccine, The vaccine comprises a protein antigen or a nucleic acid encoding the antigen, and nogapendekin alfa invaxcept (N-803). A method for stimulating mucosal-associated invariant T (MAIT) cell-mediated B cell proliferation in a subject.

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

15. 14. The method of claim 13, wherein the protein antigen is a cancer-associated antigen, a tumor-specific antigen, or a patient- and tumor-specific neo-antigen.

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

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

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

19. 18. The method of any one of claims 13 to 17, wherein said administering delivers 1 to 50 μg / kg of N-803 to said subject.

20. 1. A method for stimulating memory B cell proliferation, comprising: Isolating a) mucosal-associated invariant T (MAIT) cells and b) memory B cells from a peripheral blood mononuclear cell fraction from a patient; adding nogapendekin alfa invaxcept (N-803) to the MAIT cells and the memory B cells; 20. A method for stimulating memory B cell proliferation, comprising:

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

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

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

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

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

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

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

28. 28. The method of 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. 28. The method of claim 27, wherein the vaccine is a recombinant viral vaccine, a DNA vaccine, or an RNA vaccine.

30. 1. A method for preparing an immunotherapeutic composition for administration to a patient in need thereof, comprising: Isolating a) mucosal-associated invariant T (MAIT) cells and b) memory B cells from a peripheral blood mononuclear cell fraction from a patient; treating the combined MAIT cells and the memory B cells with nogapendequin alfa invaxcept (N-803); formulating the treated MAIT cells and memory B cells into a composition for administration to the patient; 10. A method for preparing an immunotherapeutic composition for administration to a patient in need thereof, comprising:

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

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

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

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

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

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

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

38. 1. A method for preparing an immunotherapeutic composition for administration to a patient in need thereof, comprising: Isolating mucosal-associated invariant T (MAIT) cells from a peripheral blood mononuclear cell fraction from a patient; Treating the MAIT cells with nogapendequin alfa invaxcept (N-803); formulating the treated MAIT cells into a composition for administration to the patient; 10. A method for preparing an immunotherapeutic composition for administration to a patient in need thereof, comprising:

39. At least 10 ６ MAIT cells and at least 10 ６ 39. The method of claim 38, wherein the memory B cells are isolated.

40. 39. The method of claim 38, wherein the MAIT cells and / or the memory B cells are expanded prior to adding the N-803.

41. The method of any one of claims 38 to 40, wherein the MAIT cells and the memory B cells are combined before N-803 is used to treat the MAIT cells.

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

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

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

45. 1. A method for preparing an immunotherapeutic composition for administration to a patient in need thereof, comprising: Isolating a peripheral blood mononuclear cell fraction from mucosal-associated invariant T (MAIT) cells from a patient; treating the MAIT cells with a TxM consisting of nogapendequin alfa invaxcept (N-803), IL-12 and IL-18, or biologically active portions of IL-15, IL-12, and IL-18; formulating the treated MAIT cells for administration to the patient; 10. A method for preparing an immunotherapeutic composition for administration to a patient in need thereof, comprising:

46. At least 10 ６ 46. ​​The method of claim 45, wherein MAIT cells are isolated.

47. The method of claim 45, wherein the MAIT cells are expanded prior to adding the N-803.

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

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

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

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

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