Methods for treating cancer and infectious diseases using cell-based therapies

Abstract

The present disclosure provides compositions and methods for reversing T cell senescence by disrupting vasoactive intestinal peptide (VIP) signaling and / or inhibiting phosphatidylinositol-3-kinase (PI3 kinase) inhibitor signaling, and methods for use in the management of cancer and chronic viral infections. [Solution] An in vitro cell culture composition is provided, characterized in that the composition comprises purified T cells and a phosphatidylinositol-3-kinase inhibitor, as well as anti-CD3 and anti-CD28 antibodies optionally immobilized on beads or a solid surface.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 319,957, filed on April 8, 2016. The entire disclosure of this application is incorporated herein by reference for all purposes.

[0002] Statement Regarding Federally Sponsored Research The present invention was made with government support under grants RO1 HL116737 - 01A and RO1 CA188523 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Incorporation by Reference of Materials Submitted as a Text File via the Patent Office Electronic Filing System (EFS - WEB) The Sequence Listing for this application is provided in text format instead of a paper copy and is incorporated herein by reference. The name of the text file containing the Sequence Listing is 16094PCT_ST25.txt. The text file is 11 KB, was created on April 5, 2017, and was electronically submitted via EFS - Web.

Background Art

[0004] Cytotoxic T cells directly kill cells, and their response is antigen-specific. To destroy tumors, cytotoxic cells must proliferate in sufficient numbers to outnumber dividing cancer cells. However, T cells are considered senescent if their proliferative capacity in response to antigen stimulation is limited. Filaci et al. reported that CD8+CD28-T regulatory lymphocytes, a subset of effector T cells, inhibit the proliferative capacity and cytotoxic function of T cells in human cancers. J Immunol, 2007, 179:4323-4334. Montes et al. showed that tumors induce senescent T cells as a potential form of tumor immune evasion. Cancer Res, 2008, 68:870-879. Senescent T cells are characterized by loss of CD27 and CD28 expression, lack of proliferative capacity, and increased expression of senescence-related molecules. See Ramello et al. Cell Death and Disease, 2014.5, e1507. Therefore, it is necessary to identify improved methods for treating cancer by reversing T-cell aging.

[0005] Regarding T cell proliferation, magnetic beads coated with anti-CD3 and anti-CD28 (anti-CD3 / CD28 beads) have been reported to be experimentally used to enhance T cell immunity in immunosuppressed cancer patients. See Porter et al. Phase 1 trials of donor lymphocyte infusion show that they are proliferated and activated ex vivo by CD3 / CD28 costtimulation. Blood, 2006, 107:1325-1331.

[0006] Li et al. reported the regulation of graft-versus-leukemia in allogeneic transplantation by antagonizing vasoactive intestinal peptide signaling. Cancer Res, 2016, 76(23):6802-681. See also Li et al. PLoS One. 2013, 8(5):e63381; Li et al., Blood. 2013, 121(12):2347-51, Li et al., J Immunol. 2011, 187(2):1057-65; U.S. Patent No. 9,458,217; and U.S. Patent Publication No. 2013 / 0302351.

[0007] The references cited herein do not constitute an endorsement of prior art. [Overview of the project]

[0008] This disclosure relates to compositions and methods for reversing T cell senescence by disrupting vasoactive intestinal peptide (VIP) signaling and / or inhibiting phosphatidylinositol-3-kinase (PI3 kinase) inhibitor signaling, and their use in the management of cancer and chronic viral infections. In certain embodiments, this disclosure envisions a method for reversing T cell senescence by mixing T cells in vitro with an agent that prevents VIP from interacting with the VIP receptor, and / or by adding a PI3 kinase inhibitor. In certain embodiments, this disclosure envisions the proliferation of senescent T cells by mixing them with a PI3 kinase inhibitor, an agent that blocks VIP and VIP receptor signaling, a VIP-degrading enzyme, and combinations thereof.

[0009] In certain embodiments, the Disclosure envisions a method for stimulating isolated T cells or promoting the proliferation of senescent T cells by exposing T cells in vitro to a combination of antibodies that bind CD3 and / or CD28, PI3 kinase inhibitors, idelalisib, agents that prevent interaction between VIP and the VIP receptor (e.g., preventing signaling through the VIP receptor), VIP-degrading enzymes, and combinations thereof. In certain embodiments, the Disclosure envisions the use of anti-CD3 antibodies and anti-CD28 antibodies, or binders (optionally linked to a solid substrate such as magnetic beads).

[0010] In certain embodiments, the present disclosure intends to provide a method for growing CD28 and / or CD27-negative T cells using the in vitro cell culture disclosed herein, thereby providing replicated T cells having increased expression of CD28 and / or CD27 compared to pre-replication levels.

[0011] In certain embodiments, the present disclosure describes a method for proliferating T cells, wherein, before, during, or after T cell proliferation, the T cells are mixed with a vector having a nucleic acid sequence encoding a chimeric antigen receptor, the chimeric antigen receptor comprising a cancer target sequence, a transmembrane domain, a T cell costimulatory molecule domain, and a signaling component of the T cell antigen receptor domain, under conditions in which the cells express the chimeric antigen receptor on their cell surface.

[0012] In certain embodiments, the disclosure relates to an in vitro cell culture composition comprising a minimal essential medium and T cells, as well as a VIP receptor antagonist, a PI3 kinase inhibitor, a VIP-degrading enzyme, and combinations thereof, and optionally further comprising anti-CD3 antibodies and anti-CD28 antibodies optionally immobilized on a solid substrate such as beads. In certain embodiments, the T cells are purified from bone marrow cells or blood cells.

[0013] In certain embodiments, the phosphatidylinositol-3-kinase inhibitor is selected from idelalisib, woltmannin, demethoxypyridine, perifosine, buparlisib, duberisib, copanlisib, and alpelisib. In certain embodiments, the phosphatidylinositol-3-kinase inhibitor is selected from idelalisib at concentrations greater than 0.001, 0.1, 1, 10, 100 nM, or 10 nM to 10 μM, or 10 nM to 500 nM, or 10 nM to 1 μM in the culture.

[0014] In certain embodiments, the culture contains a VIP receptor antagonist such as VIPhyb, which contains (SEQ ID NO: 1)KPRRPYTDNYTRLRKQMAVKKYLNSILN having a C-terminal amide. In certain embodiments, the culture contains a VIP receptor antagonist such as VIPhyb added at a concentration of at least 0.001, 0.01, 0.1, 0.5, 1.0, 2, or 3 μM. In certain embodiments, the culture contains a VIP receptor antagonist such as VIPhyb at a concentration of 0.5–10 μM or 1–8 μM.

[0015] In certain embodiments, the culture contains an enzyme that hydrolyzes VIP. In certain embodiments, the culture contains a VIP-degrading enzyme such as peptidase, serine peptidase, tryptase, chymase, human chymase 1 (CMA1), or chymotrypsin serine protease. In certain embodiments, the culture has at least 0.001, 0.01, 0.1, or 1 μg of a VIP-degrading enzyme such as mast cell chymase per mL. In certain embodiments, the disclosure envisions a T cell culture comprising a minimal essential medium, isolated cells expressing CD3 and / or CD4 and / or CD8 and negative for CD27 and / or CD28, and PI3 kinase inhibitors, agents that block VIP and VIP receptor signaling, and combinations thereof. Cells may be isolated by negative or positive selection using a binder attached to a solid support such as beads, magnetic beads, or fluorescent binder particles.

[0016] In certain embodiments, anti-CD3 antibodies and anti-CD28 antibodies are immobilized on beads, magnetic beads, or a solid surface. In certain embodiments, more than 5.0%, more than 10%, or more than 15% of all cells in the culture express CD3 and / or CD4 and / or CD8. In certain embodiments, more than 20%, more than 25%, or more than 50% of all cells express CD3 and / or CD4 and / or CD8. In certain embodiments, more than 15%, more than 20%, or more than 30% of T cells in the culture are negative for CD28 and / or CD27. In certain embodiments, more than 20%, more than 25%, or more than 50% of T cells are negative for CD28 and / or CD27.

[0017] In certain embodiments, purified T cells are obtained by centrifuging blood under conditions such that plasma and red blood cells are separated, and purified T cells are provided in a mixture of white blood cells between the plasma and red blood cells. In certain embodiments, purified T cells are obtained by bone marrow aspiration or bone marrow biopsy.

[0018] In certain embodiments, purified T cells are obtained by mixing cells with a fluorescent marker that binds to CD3 and purifying the cells by fluorescence-activated cell sorting. In certain embodiments, purified T cells are obtained by mixing cells with a magnetizing marker that binds to CD3 and purifying the cells by magnetic sorting. In certain embodiments, purified T cells are obtained by mixing cells with a fluorescent marker that binds to CD3 and / or CD4 and / or CD8 and purifying the cells by fluorescence-activated cell sorting. In certain embodiments, purified T cells are obtained by mixing cells with a magnetizing marker that binds to CD3 and / or CD4 and / or CD8 and purifying the cells by magnetic sorting.

[0019] In certain embodiments, the disclosure envisions a solid substrate, such as beads, having anti-CD3 and anti-CD28 antibodies and a VIP-degrading enzyme coupled to its surface. In certain embodiments, the beads are placed in a culture medium, and T cells are grown on the medium such that the beads are below the cellular level.

[0020] In a specific embodiment, the VIP-degrading enzyme is human CMA1 accession number GenBank:AAI03975.1: Includes TIFF0007896849000001.tif24170. In certain embodiments, the VIP-degrading enzyme is (SEQ ID NO: 3) This is a human recombinant enkephalinase (neutral endopeptidase, EC3.4.24.11) containing TIFF0007896849000002.tif89170.

[0021] In certain embodiments, the cell cultures and methods described herein further comprise IL-12. In certain embodiments, IL-12 is intended to enhance the effect of a VIP receptor antagonist on T cell proliferation stimulated in vitro with antibodies against CD3 and CD28.

[0022] In certain embodiments, this disclosure relates to the proliferation or extension or reversal of senescence in T cells that have a native reactivity to cancer in which infiltration may be found in the tumor of interest. Tumors may be collected, and these tumor-infiltrating lymphocytes (TILs) may be proliferated using the methods disclosed herein. [Brief explanation of the drawing]

[0023] [Figure 1] Figure 1A shows data demonstrating VIPhyb treatment for increased in vitro T cell proliferation stimulated by alloantigens. This data represents the proliferation of luciferase B6 spleen T cells cultured for 3 days with FVB irradiated in an MLR, to which VIP and / or VIPhyb were added daily to achieve the indicated concentrations (0–10 mmol / L). Figure 1B shows data regarding VIPhyb variant concentrations. [Figure 2] Figure 2 shows data on the relative numbers of CD27+ and CD28+ cells produced in an in vitro culture in a sample in which the patient has senescent T cells. "Id" refers to idelalisib. "VA" refers to VIPhyb. "MC" refers to human mast cell protease (CMA1). [Figure 3A] Figure 3A shows data on the expression of CD27 and CD28 in the total CD3+ population on day 14 of proliferation. T cells from DLBCL patients were grown for 14 days with CD3 / CD28 beads in the presence of 30 U / mL of IL-2 with or without the indicated compounds. [Figure 3B] Figure 3B shows the growth curves of the patients' T cells in the presence or absence of the indicated compounds. Preservation of the CD27+CD28+ compartment and an increase in the total yield in T cell proliferation using idelalisib and VIPhyb in DLBCL patients. [Figure 4] Figure 4 shows data indicating that the expression of Bcl-2 in T cells from lymphoma patients was increased by the addition of VIPhyb, idelalisib, or a combination of both. Protein lysates from the patients' T cell growth cultures were prepared on days 7 and 14. The samples were run on an SDS-PAGE gel before being transferred to a nitrocellulose membrane. The membrane was then probed for the survival-promoting Bcl-2 protein, stripped, and then reprobed for actin as a loading control. [Figure 5]Figure 5A shows data on tumor volume on day 18 after tumor cell injection. Mice were subcutaneously injected with 5×105 E.G7-OVA cells and allowed to grow for 7 days until palpable tumors formed. During this period, OT-1, OT-II, and B6T cells were grown with CD3 / CD28 beads for 3 days in the presence of IL-2 and the indicated compounds. On day 7 of tumor growth, a combination of expanded OT-I, OT-II, and B6T cells (2×106 OT-1, 1×106 OT-II, and 2×106 B6 cells) or 2×106 B6T cells as a control were intravenously injected into the mice. The mice were then monitored for tumor growth calculated as (LxW2) / 2. Figure 5B shows a tumor growth curve indicating the growth rate between groups over time. Proliferation of T cells in mice in the presence of idelalisib, VIPhyb, or a combination of both enhances its therapeutic effect in the OVA-expressing lymphoma model.

Mode for Carrying Out the Invention

[0024] Before explaining the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described and can naturally change. It should also be understood that the terms used herein are for the purpose of explaining only specific embodiments and are not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, but the preferred methods and materials are described herein.

[0026] All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials relating to the cited publications. Any citation of a publication is a disclosure prior to the filing date, and this disclosure should not be construed as acknowledging that such publication has no prior rights for prior disclosures. Furthermore, the publication dates provided for publications may differ from the actual publication dates and should be independently verified.

[0027] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has separate constituent elements and features that can be readily separated or combined with any of the features of several other embodiments without departing from the scope or spirit of this disclosure. Any described method may be carried out in the order of the described events or in any other logically possible order.

[0028] The embodiments of this disclosure, unless otherwise specified, utilize techniques from immunology, medicine, organic chemistry, biochemistry, molecular biology, pharmacology, physiology, etc., which are within the scope of the art of those skilled in the art. Such techniques are adequately described in the literature.

[0029] Where applicable, the level of a surface biomarker based on at least one protein is measured in a biological sample derived from an organism. The protein level can be measured using any available measurement technique capable of specifically determining the level of the biomarker in the biological sample. The measurement may be quantitative or qualitative, insofar as it can indicate whether the level of the biomarker in the biological sample is above or below a baseline.

[0030] While some assay formats allow testing of biological samples without sample pretreatment, peripheral blood biological fluid samples may be treated before testing. Treatment generally takes the form of elimination of cells (nucleated and non-nucleated) such as red blood cells, white blood cells, and platelets in the blood sample, and may also include the elimination of specific proteins, such as certain coagulation cascade proteins of blood origin. In some examples, peripheral biological fluid samples are collected in a container containing EDTA.

[0031] The process of comparing measured values ​​with reference values ​​can be carried out in any convenient method suitable for the type of measured and reference values ​​of the biomarker in question. As described above, measurements can be performed using quantitative or qualitative measurement techniques, and the method of comparing measured values ​​with reference values ​​may vary depending on the measurement technique used. For example, when measuring biomarker levels using a quantitative colorimetric assay, levels can be compared by visually comparing the intensity of the colored reaction products, or by comparing data from concentration measurements or spectroscopic measurements of the colored reaction products (e.g., numerical data obtained from the measuring instrument or graphical data such as bar graphs). However, it is assumed that the measured values ​​used in the methods of this disclosure are most commonly quantitative values ​​(e.g., quantitative measurements or absolute amounts of concentration, such as nanograms of biomarker per milliliter of sample). Similar to qualitative measurements, comparisons can be made by examining the data representation (e.g., by examining graphical representations such as bar graphs or line graphs) or by examining the numerical data.

[0032] It should be noted that, as used herein and in the claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless otherwise explicitly indicated by the context. In this specification and the following claims, several terms are defined as having the following meanings unless otherwise clearly intended.

[0033] As used herein, the term “idelalisib” refers to the compound (S)-2-(1-(9H-purine-6-ylamino)propyl)-5-fluoro-3-phenylquinazoline-4(3H)-one or an alternative salt thereof.

[0034] As used herein, “CD28 and / or CD27-negative T cells” means T cells that have lower or absent relative concentrations of the CD3 surface antigen markers compared to normal T cells that express these markers in healthy subjects.

[0035] The term "fluorescence-activated cell sorting" or "FACS" refers to a method of sorting a mixture of cells into two or more regions, typically sorting one cell at a time, based on the fluorescence properties of each cell, the charge applied to each, and separation by movement through an electrostatic field. Typically, an oscillatory mechanism causes a flow of cells to split into individual droplets. Immediately before droplet formation, cells in the fluid pass through a region for measuring the cells' fluorescence. A charging mechanism is configured at the point where the flow splits into droplets. Based on fluorescence intensity measurements, each droplet is assigned a charge as it splits from the flow. The charged droplets then move through an electrostatic deflection system that divides the droplets into regions based on their relative charges. In some systems, the charge is applied directly to the flow, and the separating droplets retain a charge of the same sign as the flow. After the droplets split, the flow returns to neutral. In other systems, the charge is supplied to a conduit, inducing a countercharge on the droplets. Cells typically fluoresce by being mixed with antibodies that specifically bind to markers that fluoresce upon binding to fluorescent molecules. However, other methods for producing cell fluorescence, such as the use of molecular beacons, are also being explored.

[0036] "Minimally Essential Medium" refers to a medium containing calcium, magnesium, potassium, sodium, phosphates, as well as bicarbonates, vitamins, and salts of essential amino acids. The 12 essential amino acids are L-arginine; L-cystine; L-glutamine; L-histidine; L-isoleucine; L-leucine; L-methionine; L-phenylalanine; L-threonine; L-tryptophan; L-tyrosine; and L-valine. Minimally Essential Medium is often supplemented with components such as bicarbonates or glutamine. In certain embodiments, this disclosure intends for a Minimally Essential Medium to be supplemented with non-essential amino acids: L-ala; L-asn; L-asp; L-glu; L-gly; L-pro and L-ser. In certain embodiments, this disclosure intends for a Minimally Essential Medium to be supplemented with nucleosides (ribonucleosides and / or deoxyribonucleosides).

[0037] The term "recombinant," when used in reference to nucleic acid molecules, refers to nucleic acid molecules containing nucleic acid segments linked together by molecular biological techniques. The term "recombinant," when used in reference to proteins or polypeptides, refers to protein molecules expressed using recombinant nucleic acid molecules. The term recombinant nucleic acid is distinguished from naturally occurring recombinants resulting from the mating of homologous chromosomes. As used herein, recombinant nucleic acids are typically non-natural combinations of nucleic acids of non-homologous origin, originating from different organisms.

[0038] The term "vector" or "expression vector" refers to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the coding sequence operably linked in a particular host organism or expression system (e.g., a cell system or a cell-free system). Nucleic acid sequences required for expression in prokaryotes typically include a promoter, an operator (optionally selected), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0039] Unless otherwise indicated in the context, the terms “vasoactive intestinal peptide” and “VIP” refer to (SEQ ID NO: 12)HSDAVFTDNYTRLRKQMAVKKYLNSILN. VIP is a multifunctional endogenous polypeptide that modulates both innate and adaptive immunity at multiple levels of immune cell differentiation and activation.

[0040] VIP is typically secreted by various cells, including neurons (in both the central and peripheral nervous systems), B cells, T cells, and accessory cells. VIP and the closely related neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP) bind to three known receptors: VPAC1, VPAC2, and PAC1. T cells and dendritic cells (DCs) are thought to express VPAC1 and VPAC2, but not PAC1. PAC1 is primarily expressed on neurons and endocrine cells in the brain and pituitary and adrenal glands, and selectively binds to PACAP in most forms.

[0041] The term “VIP antagonist” or “VIP receptor antagonist” refers to any molecule that inhibits or reduces the ability of VIP to alter the immune response. Known VIP receptor antagonists include VIP analogs, VIP fragments, growth hormone-releasing factor analogs, and hybrid peptides. Numerous VIP receptor antagonists are disclosed in U.S. Patent No. 5,565,424; U.S. Patent No. 7,094,755; and U.S. Patent No. 6,828,304, all of which are incorporated herein by reference.Some examples of VIP receptor antagonists include [Ac-Tyr1,D-Phe2]GRF1-29, an amide, i.e., (SEQ ID NO: 4)YFDAIFTNSYRKVLGQLSARKLLQDIMSR (modified: Tyr-1=N-terminal Ac, Phe-2=D-Phe, Arg-29=C-terminal amide); VIP(6-28), i.e., (SEQ ID NO: 5)FTDNYTRLRKQMAVKKYLNSILN (modified: Asn-23=C-terminal amide); Mido); [Dp-Cl-Phe6,Leu17]-VIP, i.e. (SEQ ID NO: 6)HSDAVFTDNYTRLRKQLAVKKYLNSILN (modified: Phe-6=p-Cl-D-Phe, Asn=C-terminal amide); VIP-hyb, also known as VIPhybrid (SEQ ID NO: 1)KPRRPYTDNYTRLRKQMAVKKYLNSILN, i.e., neurotensin (6-11) N-terminal (SEQ ID NO: 6-11) 7) A hybrid peptide of VIP and neurotensin consisting of KPRRPY, followed by the C-terminal 22 amino acids of VIP, namely (SEQ ID NO: 8)TDNYTRLRKQMAVKKYLNSILN, also called VIP(7-28); the N-terminal stearyl, norleucine 17VIPhyb, namely (SEQ ID NO: 9)KPRRPYTDNYTRLRKQXAVKKYLNSILN (wherein X is norleucine); AcHis1[D-Phe(2),Lys(15),Arg(16),Leu(27)]-VIP(1-7) / GRF(8-27), namely (SEQ ID NO: 10)HFDAVFTNSYRKVLKRLSARKLLQDIL, a C-terminal amide; and the pituitary adenylyl cyclase-activating polypeptide, PACAP(6-38) C-terminal amide, namely (SEQ ID NO: 11)TDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK. To provide improved properties such as solubility, bioavailability, and / or biodegradability, it is intended that any of these molecules may be modified with hydrocarbon or polyethylene glycol groups.

[0042] As used herein, the terms “to treat” and “treatment” are not limited to cases where the subject (e.g., a patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also intend treatments that merely alleviate symptoms and / or slow the progression of the disease.

[0043] Immunophenotypes of patients with relapsed lymphoma In individuals undergoing physiological aging and those exposed to repeated rounds of chemotherapy or chronic inflammatory states, T cells develop senescence, either becoming anerious or exhibiting limited proliferative capacity in response to antigenic stimulation. The phenotype of senescent T cells has been described as lacking the costimulatory receptors CD27 and CD28, or possessing high levels of the CD57 marker. Furthermore, the expression of PD1, a receptor for programmed cell death ligand, has been associated with anerious states. The presence of anerious T cells is a crucial cofactor in a patient's ability to respond to vaccines or to confer a protective and durable immune response against chronic viral infections or cancer. Novel approaches that reverse T cell senescence and anergy are needed to offer the potential for effective cell-based immunotherapy in patients with chronic infections and cancer.

[0044] The immunophenotype of patients with relapsed lymphoma is characterized by overexpression of T cells with an anergy phenotype: CD3-positive, CD27-negative, and CD28-negative. We investigated whether signaling via vasoactive intestinal polypeptide receptors contributed to T cell senescence and antigen-specific energy. Addition of a peptide antagonist to the VIP receptor called VIPhyb increased T cell proliferation in vitro and reduced the inhibitory effect of the native VIP peptide on T cell proliferation.

[0045] The addition of a VIPhyb antagonist was tested to see if it could reverse the proliferative deficiency of anergic T cells from patients with long-term recurrent cancer. The immunophenotype of T cells in patients with recurrent lymphoma was evaluated. There was relative overexpression of cells lacking the costimulatory receptors CD27 and CD28. Furthermore, T cells from this observation site exhibited high levels of CD57 expression, PD1, and Tim-3.

[0046] Using rapid cell sorting, T cell subsets were defined and isolated from patients with relapsed lymphoma and healthy controls based on their CD27 and CD28 expression levels. CD3-positive T cells with each of the four phenotypes defined by CD27 and CD28 expression were sorted and cultured in vitro in the presence of anti-CD3 / anti-CD28 beads. All T cells from healthy donors expressed 100% of the Ki67 proliferation marker after 4 days of culture, whereas the corresponding all T cell population from patients with anergistic T cells after lymphoma treatment showed only 90% Ki67 marker expression in the T cells.

[0047] After daily addition of 3 μM VIPhyb to these cultures, the percentage of Ki67-positive T cells derived from anerergic lymphoma patients increased to 100%. Subsets of T cells sorted from these patients based on CD27 and CD28 expression showed that the CD27-positive, CD28-positive (double-positive) population proliferated normally in response to anti-CD3 / CD28 beads. In contrast, when incubated with anti-CD3 / CD28 beads, T cell subsets lacking expression of the CD27 or CD28 markers or both impaired the proliferation response.

[0048] The addition of the antagonist VIPhyb increased T cell proliferation in subsets lacking either CD27 or CD28. Addition of IL12 to these cultures significantly increased the proliferation fraction of CD28-negative but not CD27-negative T cells. Flow cytometry analysis of T cells cultured with anti-CD3 / anti-CD28 beads showed that the addition of 3 μM VIPhyb reduced PD-1 expression levels compared to control cultures without the VIP antagonist. These data suggest that VIP is induced during T cell antigen-specific activation, and that strategies to block VIP signaling may increase T cell proliferation and reverse anergy among senescent T cells from individuals with chronic inflammatory conditions or those exposed to multiple chemotherapy cycles.

[0049] Because VIP peptides have a very short half-life of less than 2 minutes and are cleaved by endopeptidases, overexpression of VIP-specific peptidases near T cells activated via T cell receptors is thought to increase T cell activation and proliferation, potentially reversing immunosenescence.

[0050] Method of therapeutic use In certain embodiments, this disclosure envisions an in vivo method for reversing T-cell senescence by injecting, transplanting, or administering effective amounts of anti-CD3 antibodies and anti-tumor antibodies as well as recombinant mast cell chymase. In certain embodiments, the recombinant mast cell chymase is administered or intravenously infused into a subject / patient who is receiving anti-CD3 antibodies and antibodies against tumor-associated antigens.

[0051] In certain embodiments, the Disclosure envisions a method for treating cancer or a chronic infection, comprising: purifying T cells from a subject providing isolated T cells; mixing the isolated T cells with an anti-CD3 antibody and an anti-CD28 antibody optionally immobilized on beads or a solid surface, in combination with a PI3 kinase inhibitor, a VIP receptor antagonist, a VIP-degrading enzyme, or a combination thereof; providing replicated T cells having increased CD28 expression compared to pre-replication levels under conditions that induce T cell replication; and administering an effective amount of the replicated T cells to a subject in need.

[0052] In certain embodiments, the Disclosure envisions a method for treating cancer comprising: purifying T cells from a subject providing isolated T cells; mixing the isolated T cells with an anti-CD3 antibody and an anti-CD28 antibody optionally immobilized on beads or a solid surface, in combination with a PI3 kinase inhibitor, a VIP receptor antagonist, a VIP-degrading enzyme, or a combination thereof; providing replicated T cells having increased CD28 expression compared to pre-replication levels under conditions that induce T cell replication; and administering an effective amount of the replicated T cells to a subject requiring it. In certain embodiments, the replicated T cells express a chimeric antigen receptor on the cell surface. In certain embodiments, the Method further comprises administering a PI3 kinase inhibitor, a VIP receptor antagonist, a VIP-degrading enzyme, or a combination thereof before, during, or after administration of the replicated T cells.

[0053] In certain embodiments, the Disclosure envisions a method for treating cancer comprising: purifying T cells from a subject providing isolated T cells; culturing the isolated T cells in vitro by exposing the T cells to a combination of an antibody binding to CD3 and / or CD28, a PI3 kinase inhibitor, idelalisib, a drug that prevents interaction between VIP and the VIP receptor (e.g., preventing signaling through the VIP receptor), a VIP-degrading enzyme, and a combination thereof, thereby providing proliferating T cells having increased CD28 expression; and administering an effective amount of the proliferating T cells to the subject.

[0054] In certain embodiments, the present disclosure envisions a method for treating cancer, comprising administering an effective amount of a bispecific antibody in combination with a VIP receptor antagonist and / or a phosphatidylinositol-3-kinase inhibitor to a subject requiring such treatment, wherein the bispecific antibody comprises a cancer target binding sequence and a CD3 binding sequence. In certain embodiments, the bispecific antibody is catumakisomab or blinatumomab.

[0055] In certain embodiments, the disclosure envisions a method of treating cancer by administration or parenteral administration of a bispecific antibody or a combination of an anti-cancer antibody or an antibody against a tumor-associated antigen, and a VIP receptor antagonist such as VIPhyb. In certain embodiments, the anti-tumor-specific antibody is directed to CD19. In certain embodiments, the anti-cancer antibody is directed to CD123. In certain embodiments, the anti-cancer antibody is directed to HER2 / neu. In certain embodiments, the anti-cancer antibody is directed to BMCA, a myeloma-associated antigen. In certain embodiments, the anti-cancer antibody is directed to EGFR. In certain embodiments, the anti-cancer antibody is directed to PD-L1 or PD1.

[0056] In certain embodiments, the present disclosure envisions a method for treating cancer, comprising administering an effective amount of cells having a chimeric antigen receptor in combination with a VIP receptor antagonist or a phosphatidylinositol-3-kinase inhibitor to a subject requiring such treatment, wherein the chimeric antigen receptor comprises a cancer target sequence, a transmembrane domain, a T cell costimulatory molecule domain, and a signaling component of the T cell antigen receptor domain.

[0057] In certain embodiments, the disclosure envisions an in vivo method for reversing T cell senescence, comprising genetically modifying T cells to express a VIP-degrading enzyme on their surface. In certain embodiments, the VIP-degrading enzyme is recombinant human CMA1 mast cell chymase. In certain embodiments, the genetically modified T cells also express a chimeric antigen receptor that targets cancer cells. In certain embodiments, the genetically modified T cells are administered or injected into subjects with cancer.

[0058] In certain embodiments, the Disclosure relates to a method for treating cancer comprising: purifying T cells from a subject expressing a T cell receptor, wherein the T cells express CD3 and optionally CD4 and / or CD8; providing isolated T cells; mixing the isolated T cells with a cell culture disclosed herein under conditions that promote cell proliferation; and transplanting or administering an effective amount of the proliferated cells to a subject.

[0059] In certain embodiments, the present disclosure envisions a method for treating cancer, comprising administering T cells containing a vector configured to express a chimeric antigen receptor, for example, cells infected with a recombinant virus having nucleic acid encoding a chimeric antigen receptor, in combination with the administration of a VIP receptor antagonist such as VIPhyb, VIP-degrading enzyme, PI3 inhibitor, or a combination thereof.

[0060] In certain embodiments, the Disclosure envisions a method for treating cancer comprising: purifying cells derived from a subject expressing CD3 and / or CD4 and / or CD8 to provide isolated T cells; measuring the expression of CD27 and / or CD28 on the isolated T cells to provide measured values ​​of CD27 and / or CD28; comparing the measured values ​​of CD27 and / or CD28 to reference levels; and, if the measured CD27 level is lower than normal and / or the measured CD28 level is lower than normal, then administering an effective amount of a VIP receptor antagonist, VIP-degrading enzyme, PI3 inhibitor, or a combination thereof to the subject.

[0061] In certain embodiments, the Disclosure envisions a method for treating cancer comprising: purifying cells derived from a subject expressing CD3 and / or CD4 and / or CD8 to provide isolated T cells; measuring the expression of CD27 and / or CD28 on the isolated T cells to provide a measurement of CD27 and / or CD28; if the measurement of CD27 is lower than a normal value that provides replicated T cells and / or the measurement of CD28 is lower than a normal value that provides replicated T cells, then mixing the isolated T cells with a VIP receptor antagonist, VIP-degrading enzyme, PI3 inhibitor, or a combination thereof under conditions that cause the isolated T cells to replicate; and transplanting or administering an effective amount of replicated T cells to a subject in combination with, optionally, the administration of a VIP receptor antagonist, VIP-degrading enzyme, PI3 inhibitor, or a combination thereof to the subject.

[0062] In certain embodiments, the disclosure relates to the proliferation of T cells, such as gamma delta T cells (γδ T cells), which are CD3-positive and CD4 and CD8-negative. These gamma delta T cells have a T cell receptor (TCR) specific to their surface. Most T cells are alpha-beta T cells, which have a TCR composed of two glycoprotein chains called alpha- and beta-TCR chains. In contrast, gamma delta (γδ) T cells have a TCR composed of one gamma chain and one delta chain.

[0063] In certain embodiments, the disclosure envisions T cells genetically modified to express a VIP-degrading enzyme on their surface. In certain embodiments, the VIP-degrading enzyme is a recombinant human CMA1 mast cell chymase. In certain embodiments, the genetically modified T cells also express a chimeric antigen receptor that targets cancer cells. In certain embodiments, the genetically modified T cells are administered or injected into human subjects with cancer.

[0064] In certain embodiments, the disclosure relates to a method for treating cancer, comprising administering an effective amount of a bispecific antibody in combination with a VIP receptor antagonist, a PI3 kinase inhibitor, a VIP-degrading enzyme, and a combination thereof. In certain embodiments, the bispecific antibody is catumaxomab. In certain embodiments, the subject is diagnosed with malignant ascites. In certain embodiments, the bispecific antibody is blinatumomab. In certain embodiments, the cancer is leukemia.

[0065] In certain embodiments, the Disclosure envisions a method for treating cancer comprising: purifying and growing T cells using a method provided herein to provide isolated T cells; mixing the isolated T cells with a bispecific antibody under conditions such that the bispecific antibody binds to the CD3-T cell receptor complex; and administering an effective amount of bispecific antibody-bound T cells to a subject in need, in combination with administering a VIP receptor antagonist, a PI3 kinase inhibitor, a VIP-degrading enzyme, and combinations thereof to the subject.

[0066] A bispecific antibody contains two target sequences: the first sequence targets a tumor-associated antigen, and the second sequence targets the CD3 T cell receptor complex, so that T cells can bind to cancer cells. The bispecific antibody links T cells to cancer cells. See Zhukovsky et al. Bispecific antibodies and CARs: generalized immunotherapeutics harnessing T cell redirection, Current Opinion in Immunology, 2016, 40:24-35. In certain embodiments, this disclosure intends for the bispecific antibody to be directed to a tumor-associated antigen, CD19 epitope, CD123, HER2 / neu, or BMCA, which is a myeloma-associated antigen.

[0067] To enhance the ability of immune cells to kill cancer cells, T cells can be isolated from a patient's blood and genetically modified to express chimeric antigen receptors that specifically target proteins expressed on the surface of cancer cells, thereby stimulating an immune response. When returned to the patient, the cells attack the cancer cells. In certain embodiments, this disclosure intends to use CART cells that target CD22 and / or CD19 antigens. CD19 is a protein expressed on cancerous B cells. Brentjens et al. reported that T cells modified to bind to CD19 may induce cancer remission in adults with chemotherapy-resistant acute lymphoblastic leukemia. Sci Transl Med, 2013, 5(177):177ra38.

[0068] In a typical procedure, T cells are purified and isolated from blood or bone marrow. For example, T cells are collected by apheresis, a process in which blood is collected from the body and one or more blood components (such as plasma, platelets, or other white blood cells) are removed. The remaining blood is then returned to the body. The cells are exposed to a recombinant vector, such as a lentiviral vector, which infects the cells in such a way that the CAR protein is produced to be present on the cell membrane. The T cells may be sent to a laboratory or pharmaceutical facility to be genetically engineered to produce a chimeric antigen receptor (CAR) on its surface. Cell replication can be induced before and / or after infection with the recombinant vector using the methods disclosed herein. Genetically modified T cells can be grown by growing the cells in the laboratory until a sufficient number are obtained. By choice, these CART cells are frozen. The modified cells are then administered to the patient and returned. In certain embodiments, the disclosure envisions administering a VIP receptor antagonist, a VIP-degrading enzyme, and / or an IP3 kinase inhibitor to a subject in combination with one or more chemotherapeutic agents, in an optional manner, prior to the subject receiving CART cell injection.

[0069] In certain embodiments, the disclosure relates to cells produced by a process disclosed herein, comprising a recombinant vector containing a nucleic acid encoding a chimeric polypeptide that includes signaling elements of a target sequence, a transmembrane domain, a T cell costimulatory molecule domain, and a T cell antigen receptor domain.

[0070] In certain embodiments, the target sequence in a chimeric antigen receptor refers to any variety of polypeptide sequences that can selectively bind to surface proteins on target cells (e.g., cancer cells). Other target sequences may be variable binding regions of antibodies, single-chain antibodies, and antibody mimetic compounds. In certain embodiments, targeting is achieved via a single-chain variable fragment (scFv) derived from a monoclonal antibody. The target sequence is typically linked to an intracellular domain by a hinge / transmembrane region generally derived from CD8 or IgG4. The intracellular domain may contain a co-stimulatory domain, such as a 4-1BB zeta and / or CD28 zeta linked to the cytoplasmic signaling domain of a CD3 zeta. [Examples]

[0071] VIP signaling regulates T cell proliferation induced by alloantigens. To investigate the role of VIP signaling in alloimmune responses, we performed unidirectional mixed lymphocyte reactions (MLRs) containing VIP and / or the antagonist VIPhyb peptide. VIP addition dose-dependently reduced luciferase T cell proliferation, while VIPhyb addition increased T cell proliferation. VIPhyb reversed the suppressive effect of VIP in MLRs, restoring T cell proliferation to higher levels than the control culture.

[0072] Pharmacological blockade during ex vivo T cell proliferation for CART production increases yield and preserves naive and central memory compartments. Diffuse large B-cell lymphoma (DLBCL) is a high-grade B-cell malignancy that primarily affects the elderly patient population. Despite the availability of aggressive treatment regimens, a subset of DLBCL patients exhibit highly resistant lymphoma. Given that these patients' disease is resistant to virtually all current treatment regimens, CART therapy is a very promising option for effective salvage; however, many patients have been unable to receive treatment in clinical trials due to failure of T-cell proliferation ex vivo. This failure is largely attributable to the patient's age, as well as the damage inflicted by multiple rounds of treatment. Furthermore, data indicate that patients with NHL have a skewed ratio of memory cells to naive T cells, impairing cellular immunotherapy.

[0073] Two surface proteins that indicate the potential for successful T cell proliferation in patients are CD27 and CD28. T cells expressing both CD27 and CD28 have the highest proliferative capacity, while T cells lacking expression of both often do not proliferate and die during anti-CD3 / CD28 activation and proliferation. Healthy individuals have abundant CD27+CD28+ (double-positive) T cells, while patients with DLBCL have an excess of CD27-CD28- (double-negative) T cells. This overexpression in the double-negative population in lymphoma patients helps further explain the failure of T cells to proliferate properly during CART production. In addition to the lack of CD27 and CD28 expression, T cells in DLBCL patients also show signs of fatigue and aging, both of which result in dysfunction and inability to proliferate.

[0074] To address the T cell proliferation problem in DLBCL patients during CART cell production, various techniques were utilized to promote the proliferation of specific desired T cell populations. During T cell activation, the inclusion of the PI3Kδ inhibitor idelalisib alone, or in combination with a mast cell chymase or vasoactive intestinal peptide (VIPhyb) antagonist, increased the number of viable T cells and the proportion of naive and central memory cells. Naive and central memory cells are the most effective subsets for adoptive cell immunotherapy. Furthermore, the inclusion of either idelalisib or VIPhyb increased the frequency of CD27+CD28+ cells and decreased the frequency of CD27-CD28- cells. Further studies using mouse and human T cells showed that while PI3Kδ blockade inhibits proliferation, preventing terminal differentiation and preserving the naive compartment enhances T cell viability and yield. This data suggests that the culture conditions used not only increase the overall number of T cells from the patient but also preserve the most effective compartments in adoptive T cell immunotherapy. Thus, by utilizing our method during CART manufacturing, it may be possible to develop a lasting treatment option that many DLBCL patients have not been able to receive before.

[0075] Proliferation of senescent T cells by P13 kinase inhibitors and VIP signaling blockade Idelalisib is a PI3 kinase inhibitor. PI3 kinase is a key signaling pathway for lymphocyte activation and differentiation, and modulates AKT and mTOR1 activity. Idelalisib is FDA approved for the treatment of patients with chronic lymphocytic leukemia and low-grade B-cell malignancies. Patients treated with idelalisib have been observed to continue their anti-cancer response after discontinuation of drug treatment and to develop autoimmune-like signs and symptoms, including colitis and rash, which leads to the hypothesis that idelalisib modulates the immune system and activates or preserves Th1-polarized T cells.

[0076] Idelalisib treatment results in increased T cell activation in patients with CLL. Ex vivo exposure of activated T cells to idelalisib enhances their activation and in vitro proliferation. T cell proliferation and differentiation were measured by adding a range of idelalisib concentrations to T cells cultured in vitro with anti-CD3 / CD28 beads.

[0077] T cell samples from lymphoma patients with senescent T cells that did not proliferate in vitro when attempting to produce CART cells were used. Monocytes were depleted from the T cell samples because they are known to inhibit T cell proliferation in cultures containing anti-CD3 / CD28 beads. The effects of a range of idelalisib concentrations were compared alone and in combination with 3 μM VIP peptide antagonist (VIPhyb) or 1 μg / ml mast cell chymase (an enzyme that degrades endogenous VIP in T cell activation, differentiation, and proliferation). The number of viable T cells and T cell differentiation and activation profiles were measured during 10 days of in vitro culture, focusing on the relative number of T cells with a senescent phenotype lacking both CD27 and CD28 (CD27-CD28-) compared to an activated phenotype with either CD27, CD28, or both costimulatory receptors (CD27+CD28+).

[0078] Aliquots of frozen patient apheresis products or phytochemically ficolated whole blood-derived PBMCs were rapidly thawed in complete RPMI1640 (complete medium) supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 50 μM 2-mercaptoethanol, and left overnight. The following day, erythrocytes were removed from the apheresis products by a ficol gradient. Next, leukocytes were enriched for T cells using the EasySep Human T Cell Concentration Kit according to the manufacturer's instructions. Cells were seeded in 200 μL of complete medium in each well of a 96-well flat-bottom plate. Compounds were added at the indicated concentrations, and the DMSO concentration was standardized to 0.1% for all wells. Anti-CD3 / CD28 beads were added in a 1:1 bead:cell ratio. On day 7 post-stimulation, cells from each treatment were transferred to fresh medium. Fresh compounds and beads were then added, and the cells were cultured for a further 3 days. The beads were removed 10 days after initial stimulation, and the cells were stained for flow cytometry analysis.

[0079] The cells were washed twice with PBS. The cells were also stained with a fixable viability dye. Surface markers were then stained by adding fluorescent dye-conjugated antibodies to CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, PD-1, and CCR7. To assess proliferation, half of the cells were evaluated for Ki67 expression. Ki67 staining was performed using the FoxP3 intracellular staining kit. Accucheck counting beads were added to each tube immediately before running the samples. Samples were acquired using BD FACS Aria and analyzed using FlowJo software. Viable cells were used for analysis. The absolute number of cells was calculated for each population according to the instructions provided by the manufacturer of Accucheck counting beads.

[0080] Addition of idelalisib at concentrations of 1 μM or 100 nM to T cell cultures significantly increased the proportion of T cells co-expressing CD27 and CD28 compared to control cultures without the drug. Notably, the fraction of senescent T cells lacking both CD27 and CD28 costimulatory receptors decreased from 55.2% in control cultures without the drug to 16.1% in cultures with 10 nM idelalisib, 35.7% in cultures with 3 μM VIPhyb, and 37.4% in cultures with 1 μg / ml mast cell chymase.

[0081] Ideralisib at a concentration of 100 nM was synergistic in enhancing the proportion of costimulatory receptor-expressing T cells when combined with VIPhyb or chymase. Compared to 3.5% in control cultures without drug addition, the combination of 100 nM ideralisib with VIPhyb resulted in a 21.5% CD27+CD28+ cell fraction, while the combination of 100 nM ideralisib with chymase resulted in 14.2%.

[0082] Exposure to idelalisib, VIPhyb, and combinations of idelalisib with chymase and VIPhyb also increased the number of effector memory T cells (Tems) in 10-day cultures. The total number of viable T cells in the culture was significantly increased with idelalisib and chymase or idelalisib and VIPhyb combinations compared to a control culture without drug addition. Notably, the total number of CD27+CD28+ T cell subsets was more than 10-fold enriched in cultures containing 100 nM idelalisib, 1 μM idelalisib + 1 μg / ml chymase, or 100 nM idelalisib + 3 μM VIPhyb compared to a control culture without drug addition, and the total number of CD3+ Tems in 10-day cultures was 10-fold increased in cultures containing 1 μM idelalisib + chymase compared to a control culture without drug addition. Cultures containing idelalisib alone, VIPhyb alone, chymase alone, or a combination of idelalisib and VIPhyb or chymase showed a 5- to 10-fold increase in CD3+ central memory T cells (Tcm) compared to control cultures without the drug.

[0083] These data demonstrate that senescent T cells that could not proliferate in culture with anti-CD3 / CD28 beads and did not produce enough CART cells for clinical use could be significantly proliferated in vitro by adding idelalisib, VIPhyb, or mast cell chymase as a single agent or in combination. These results support the addition of these agents during CART production and support their in vitro use to proliferate senescent T cells to enhance cancer immunotherapy and antiviral immunity.

Claims

1. An in vitro cell culture composition for T cell proliferation, The composition comprises purified T cells, a VIP receptor antagonist, a phosphatidylinositol-3-kinase inhibitor, and anti-CD3 and anti-CD28 antibodies immobilized on beads or a solid surface. The VIP receptor antagonist includes KPRRPYTDNYTRLRKQMAVKKYLNSILN) (SEQ ID NO: 1) An in vitro cell culture composition characterized by the following features.

2. The composition according to claim 1, characterized in that more than 15% of the T cells are negative for CD28.

3. The composition according to claim 1, wherein the composition further comprises a VIP-degrading enzyme containing the sequence mllklkekekasltlavgtlpfpsqfnfvppgrmcrvagwgrtgvlkpgsdtlqevklrlmdpqacshfrdfdhnlqlcvgnprktksafkgdsggpllcagvaqgivsygrsdakppavftrishyrpwinqilqan (SEQ ID NO: 2).

4. The composition according to claim 1, wherein the phosphatidylinositol-3-kinase inhibitor is selected from idelalisib, woltmannin, demethoxypyridine, perifosine, buparlisib, duberisib, copanlisib, and alpelisib.

5. A method for proliferating T cells using an in vitro cell culture composition according to any one of claims 1 to 4, characterized by providing replicated T cells.

6. The method according to claim 5, characterized in that the T cells are negative for CD28.

7. The method according to claim 6, characterized in that the replicated T cells have increased CD28 expression compared to the pre-replication level.

8. The method according to claim 7, wherein, before, during, or after proliferation of the T cells, the T cells are mixed with a vector having nucleic acid encoding a chimeric antigen receptor under conditions in which the cells express a chimeric antigen receptor on the surface of the cells, and the chimeric antigen receptor comprises a cancer target sequence, a transmembrane domain, a T cell costimulatory molecule domain, and a T cell antigen receptor domain signaling components.

9. The use of isolated T cells in the manufacture of pharmaceuticals for treating cancer or chronic infection in a subject, Under conditions such that the isolated T cells replicate and provide replicated T cells having increased CD28 expression compared to the pre-replication level, the isolated T cells are mixed with 1) anti-CD3 antibody and anti-CD28 antibody immobilized on beads or a solid surface, 2) PI3 kinase inhibitor, and 3) VIP receptor antagonist. The VIP receptor antagonist includes KPRRPYTDNYTRLRKQMAVKKYLNSILN) (SEQ ID NO: 1) A characteristic use.

10. The use according to claim 9, characterized in that the replicated T cells express a chimeric antigen receptor on the surface of the cell.

11. The use according to claim 9, characterized in that the isolated T cells are further mixed with a VIP-degrading enzyme containing the sequence mllklkekasekasltlavgtlpfpsqfnfvppgrmcrvagwgrtgvlkpgsdtlqevklrlmdpqacshfrdfdhnlqlcvgnprktksafkgdsggpllcagvaqgivsygrsdakppavftrishyrpwinqilqan (SEQ ID NO: 2).

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