WO2023081835A1 - Expanding t-cell using mitofusin activators and uses in cancer therapies - Google Patents

Abstract

This disclosure relates to contacting T cells with mitofusin activators and/or PI3K inhibitors to improve efficacy in cell therapies. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of activated T cells using methods as disclosed herein to a subject in need thereof. In certain embodiments, the T cells express a chimeric antigen receptor.

Description

EXPANDING T-CELL USING MITOFUSIN ACTIVATORS AND USES IN CANCER

THERAPIES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/263,525 filed November 4, 2021. The entirety of this application is hereby incorporated by reference for all purposes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 22025PCT.xml. The XML file is 2 KB, was created on November 3, 2022, and is being submitted electronically via the USPTO patent electronic filing system.

BACKGROUND

Chronic lymphocytic leukemia (CLL), a cancer of B-lymphocytes, is the most common leukemia in adults. Frontline therapies for CLL, such as ibrutinib or a combination of venetoclax and obinutuzumab, have significantly improved clinical outcomes. With the advent of chimeric antigen receptor T cells (CART) therapies, complete response rates have increased; however, the percentage of patients that succumb to relapsed and refractory CLL (RR-CLL) disease are considerable. Thus, there is a need to identify improved therapies.

Chen et al. report duvelisib promotes mitochondrial fusion and epigenetic reprogramming to drive therapeutic T cell persistence and function. Blood, 2021, 138, 1714-1715.

Zacharioudakis et al. report a direct activator of mitofusins, MASM7, capable of potently promoting mitochondrial fusion. BioRxiv, 2018, 301713. See also Zacharioudakis et al. Nature Communications, volume 13, Article number: 3775 (2022) and WO2019/079243.

Coma et al. report synergistic anti-tumor efficacy in the dual PI3K-delta/PI3K-gamma inhibitor duvelisib with PD-1 blockade in solid tumor and lymphoma models. Cancer Res, 2020, 80 (16_Supplement): CT045. See also US Published Application No. 2019/0144825.

References cited herein are not an admission of prior art. SUMMARY

This disclosure relates to contacting T cells with mitofusin activators to improve efficacy in cancer cell therapies. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of activated T cells using methods as disclosed herein to a subject in need thereof. In certain embodiments, the T cells express a chimeric antigen receptor.

In certain embodiments, this disclosure relates to methods of expanding T cells comprising contacting isolated and purified T cells with a mitofusin activator. In certain embodiments, the methods are performed in a cell culture as reported herein. In certain embodiments, the mitofusin activator comprises a 2-((4-phenyl-4H-l,2,4-triazol-3-yl)thio)acetamide core structure such as the compound 2-{2-[(5-cyclopropyl-4-phenyl-4H-l,2,4-triazol-3-yl)sulfanyl]propanamido}- 4H, 5H, 6H-cy cl openta[b]thiophene-3 -carboxamide (MASM7).

In certain embodiments, the mitofusin activator is 4-chloro-2-(l-((2,3- dimethylphenyl)amino)ethyl)phenol (MF 18), derivative, prodrug, or salt thereof.

In certain embodiments, this disclosure relates to in vitro cell culture compositions comprising purified and isolated T cells and a mitofusin activator, salt, prodrug, or derivative thereof. In certain embodiments, the cell culture further comprises anti-CD3 antibodies and/or anti-CD28 antibodies optionally immobilized on a bead, in the form of a tetramer, or conjugated to a solid surface.

In certain embodiment more than 15 % of the T cells are negative for CD27 and/or CD28. In certain embodiments, the cell culture further comprises a PI3K inhibitor such as idelali sib and/or dual-PI3Kdelta/gamma inhibitor such as duvelisib.

In certain embodiments, this disclosure relates to methods of expanding T cells comprising contacting isolated and purified T cells with a mitofusin activator. In certain embodiments, the methods are performed in a cell culture as reported herein.

In certain embodiments, the replicated T cells have increased expression of CD8+ T cells compared with levels prior to replication.

In certain embodiments, the replicated T cells have increased or decreased expression of CD4+ T cells compared with levels prior to replication.

In certain embodiments, the replicated T cells have increased expression of CD8+ CD27+ T cells compared with levels prior to replication. In certain embodiments, the replicated T cells have decreased expression of TIM3+, LAG3+ T cells compared with levels prior to replication.

In certain embodiments, the replicated T cells have increased expression of CD8+CD27+CD45RO- T cells compared with levels prior to replication.

In certain embodiments, the replicated T cells have increased expression of

CD8+CD45RO+CCR7+ central memory T cells compared with levels prior to replication.

In certain embodiments, the replicated T cells have decreased expression of

CD8+CD45RO+CCR7- effector T cells compared with levels prior to replication.

In certain embodiments, the replicated T cells have decreased expression of late- activation/exhausted (CD25+PD1+TIM3+) cells compared with levels prior to replication.

In certain embodiments, the replicated T cells have increased expression of CD27 and/or CD28 T cells compared with levels prior to replication. In certain embodiments, the mitofusin activator is used in combination with a PI3K inhibitor such as idelalisib and/or dual- PI3Kdelta/gamma inhibitor such as duvelisib.

In certain embodiments, it is contemplated that prior to, during, or after proliferating the T cells, the T cells are contacted with a recombinant vector having a nucleic acid encoding a chimeric antigen receptor, thereby inserting or transfecting the cell with the recombinant vector, wherein the chimeric antigen receptor comprises a cancer targeting sequence, a transmembrane domain, a T cell costimulatory molecule domain, and a signal -transduction component of a T-cell antigen receptor domain, such that the cells express the chimeric antigen receptor on the surface of the cells providing antigen specific T cells.

In certain embodiments, frequency of antigen specific T cells within expanded T cells is greater than 2%, 5%, or 10% of the T cells.

In certain embodiments, this disclosure relates to methods of treating cancer or a chronic infection comprising: purifying T cells from a subject providing isolated T cells; contacting the isolated T cells with anti-CD3 antibodies and/or anti-CD28 antibodies optionally immobilized on a bead, in the form of a tetramer, or conjugated to a solid surface in combination with a mitofusin activating small molecule; under conditions such that the T cells replicate, providing replicated T cells having increased or decreased expression of CD8+ T cells, and/or, CD4+, CD27+, TIM3+, LAG3+, CD8+CD27+CD45RO-, CD8+CD45RO+CCR7+ central memory T cells and/or decreased expression of CD8+CD45RO+CCR7- effector T cells and/or decreased expression of late-activation/exhausted CD25+PD1+TIM3+ cells, when compared with levels prior to replication; and administering an effective amount of the replicated T cells to a subject in need thereof.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Figures 1 A-1D show data indicating that MASM7 augments T cell expansion. T cells from CLL donors were activated with soluble aCD3/CD28 activator, cultured with 0.1% DMSO (vehicle), 300nM duvelisib, or various doses of MASM7, and immunolabeled for surface marker expression on day 8 of culture. T cell data was acquired on a 5-laser spectral cytometer prior to analysis. Commercial counting beads were used during flow acquisition to calculate cell number.

Figure 1 A shows plots that depict total number of CD3+ T cells expanded from 200,000 plated cells over the course of 8 days.

Figure IB shows data on relative expansion of CD8+ T cells.

Figure 1C shows data on relative expansion of CD8+CD27+ T cells.

Figure ID shows relative frequency of TIM3+LAG3+ co-expressing cells within the CD 8+ compartment.

Figures 2A-2B show data indicating MASM7 does not affect MFN2 expression. T cells from CLL donors were activated with soluble aCD3/CD28 activator, cultured with 0.1% DMSO (vehicle), 300nM duvelisib, or various doses of MASM7, and immunolabeled for surface marker and intracellular MFN2 expression on day 8 of culture. T cell data was acquired on a 5-laser spectral cytometer prior to analysis. Commercial counting beads were used during flow acquisition to calculate cell number.

Figure 2A shows plots that depict MFN2 expression as mean MFI in CD4+ and CD8+ T cells from vehicle treated cultures.

Figure 2B shows data on MFN2 expression plots as mean MFI in CD8+ T cell subsets, including Tn, Tscm, Tcm, Tern, and Temra.

Figure 3 A and 3B shows data indicating MASM7 augments T cell expansion. T cells from CLL donors were activated with soluble anti-CD3/CD28 activator and cultured with 0.1% DMSO (vehicle), 300nM duvelisib, or various doses of MASM7. On day 8 of culture, T cells were harvested, fixed in 4% paraformaldehyde, and stained for surface CD4, CD8, mitochondrial TOM20, and nuclear DNA. Z-stacks of single-cell, widefield images acquired on a SIM microscope were reconstructed as 3D projections.

Figure 3A shows data on mitochondrial volume per individual/networked mitochondria quantified using software.

Figure 3B shows data on mitochondrial membrane potential to mass ratio evaluated by flow cytometry and plotted.

Figure 4A-4C show data indicating duv-CART conferred a survival advantage in B-CLL bearing mice. OSU-CLL engrafted NOG mice were treated with 1 x 10⁶ control- or duv-CART on day 15. CART efficacy was assessed by flow cytometry.

Figure 4A shows data on the frequency of CD20+CD5+ OSU-CLL in blood.

Figure 4B shows data on CD8+ CART cells in blood following infusion.

Figure 4C shows a Kaplan-Meier survival analysis of treated mice.

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated 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 the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of immunology, medicine, organic chemistry, biochemistry, molecular biology, pharmacology, physiology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

A "mitofusin activator" refers to an agent, compound, or small molecule that promotes mitochondrial fusion. Some mitofusin activators have the ability to induce conformational activation and oligomerization of mitofusins (MFNs) that leads to mitochondrial fusion. Examples include MASM7 and MFI8. See Zacharioudakis et al. Nature Communications, volume 13, Article number: 3775 (2022). Mitochondrial fusion is mediated by the GTPases, MFN1/2 in the OMM and optic atrophy 1 (OPA1) in the inner mitochondrial membrane (IMM). MFN1 and MFN2 proteins tether adjacent mitochondria and execute outer mitochondrial membrane (OMM) fusion. Both homologs contain a N-terminal GTPase domain, a coiled-coiled heptad repeat (HR1) domain, a transmembrane domain, and a C-terminal HR2 domain. Prior to fusion, intramolecular HR1- HR2 interactions position mitofusins in an anti-tethering conformation.

The terms, “cell culture” or “growth medium” or “media” refers to a composition that contains components that facilitate cell maintenance and growth through protein biosynthesis, such as vitamins, amino acids, inorganic salts, a buffer, and a fuel, e.g., acetate, succinate, a saccharide and/or optionally nucleotides. Typical components in a growth medium include amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine and others); vitamins such as retinol, carotene, thiamine, riboflavin, niacin, biotin, folate, and ascorbic acid; carbohydrate such as glucose, galactose, fructose, or maltose; inorganic salts such as sodium, calcium, iron, potassium, magnesium, zinc; serum; and buffering agents. Additionally, a growth media may contain phenol red as a pH indication. Components in the growth medium may be derived from blood serum or the growth medium may be serum-free. The growth medium may optionally be supplemented with albumin, lipids, insulin and/or zinc, transferrin or iron, selenium, ascorbic acid, and an antioxidant such as glutathione, 2-mercaptoethanol or 1 -thioglycerol. Other contemplated components contemplated in a growth medium include ammonium metavanadate, cupric sulfate, manganous chloride, ethanolamine, and sodium pyruvate. Minimal Essential Medium (MEM) is a term of art referring to a growth medium that contains calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium phosphate and sodium bicarbonate), essential amino acids, and vitamins: thiamine (vitamin Bl), riboflavin (vitamin B2), nicotinamide (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), folic acid (vitamin B9), choline, and myo-inositol (originally known as vitamin B8). Various growth mediums are known in the art. Dulbecco's modified Eagle's medium (DMEM) is a growth medium which contains additional components such as glycine, serine, and ferric nitrate with increased amounts of vitamins and amino acids.

"Subject" refers any animal, preferably a human patient, livestock, or domestic pet.

As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression.

As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

As used herein, the term "combination with" when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.

As used herein, the term “small molecule” refers to any variety of covalently bound molecules with a molecular weight of less than 900 or 1000. Typically, the majority of atoms include carbon, hydrogen, oxygen, nitrogen, and to a lesser extent sulfur and/or a halogen. Examples include steroids, short peptides, mono or polycyclic aromatic or non-aromatic, heterocyclic compounds.

As used herein, the term “derivative” refers to a structurally similar compound that retains sufficient functional attributes of the identified analogue. The derivative may be structurally similar because it is lacking one or more atoms, substituted, a salt, in different hydration/oxidation states, or because one or more atoms within the molecule are switched, such as, but not limited to, replacing a oxygen atom with a sulfur atom, replacing an amino group with a hydroxyl group, replacing a nitrogen with a protonated carbon (CH) in an aromatic ring, replacing a bridging amino group (-NH-) with an oxy group (-O-), or vice versa. The derivative may be a prodrug. Derivatives may be prepared by any variety of synthetic methods or appropriate adaptations presented in synthetic or organic chemistry textbooks, such as those provide in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) Michael B. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F. Tietze hereby incorporated by reference.

The term "substituted" refers to a molecule wherein at least one hydrogen atom is replaced with a substituent. When substituted, one or more of the groups are "substituents." The molecule may be multiply substituted. In the case of an oxo substituent ("=O"), two hydrogen atoms are replaced. Example substituents within this context may include halogen, hydroxy, alkyl, alkoxy, alkanoyl, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarb ocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaNRb, -NRaC(=O)ORb, - NRaSO2Rb, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRb, -OC(=O)NRaRb, -ORa, -SRa, -SORa, - S(=O)2Ra, -OS(=O)2Ra and -S(=O)2ORa. Ra and Rb in this context may be the same or different and independently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, alkanoyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

As used herein, "alkyl" means a noncyclic straight chain or branched, unsaturated or saturated hydrocarbon such as those containing from 1 to 10 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl, n-nonyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an "alkenyl" or "alkynyl", respectively). Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2- butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3 -methyl- 1-butenyl, 2-methyl-2-butenyl, 2,3- dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3- methyl- 1-butynyl, and the like.

Non-aromatic mono or polycyclic alkyls are referred to herein as "carbocycles" or "carbocyclyl" groups. Representative saturated carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated carbocycles include cyclopentenyl and cyclohexenyl, and the like

"Heterocarbocycles" or heterocarbocyclyl" groups are carbocycles which contain from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur which may be saturated or unsaturated (but not aromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Heterocarbocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

"Aryl" means an aromatic carbocyclic monocyclic or polycyclic ring such as phenyl or naphthyl. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic.

As used herein, "heteroaryl" or “heteroaromatic” refers an aromatic heterocarbocycle having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono- and polycyclic ring systems. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic. Representative heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated that the use of the term "heteroaryl" includes N-alkylated derivatives such as a 1-methylimidazol- 5-yl substituent. As used herein, "heterocycle" or "heterocyclyl" refers to mono- and polycyclic ring systems having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom. The mono- and polycyclic ring systems may be aromatic, non-aromatic or mixtures of aromatic and non-aromatic rings. Heterocycle includes heterocarbocycles, heteroaryls, and the like.

"Alkylthio" refers to an alkyl group as defined above with the indicated number of carbon atoms attached through a sulfur bridge. An example of an alkylthio is methylthio, (i.e., -S-CH3).

"Alkoxy" refers to an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n- pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, s-butoxy, t- butoxy.

"Alkylamino" refers an alkyl group as defined above with the indicated number of carbon atoms attached through an amino bridge. An example of an alkylamino is methylamino, (i.e., -NH- CH3).

"Alkanoyl" refers to an alkyl as defined above with the indicated number of carbon atoms attached through a carbonyl bride (i.e., -(C=O)alkyl).

The term "nucleic acid" refers to a polymer of nucleotides, or a polynucleotide, e.g., RNA, DNA, or a combination thereof. The term is used to designate a single molecule, or a collection of molecules. Nucleic acids may be single stranded or double stranded and may include coding regions and regions of various control elements.

The term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p- acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

A "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur the same organism but are joined together in a manner that does not naturally occur in the same organism or any natural state.

The term "recombinant" when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques provided that the entire nucleic acid sequence does not occurring in nature, i.e., there is at least one mutation in the overall sequence such that the entire sequence is not naturally occurring even though separately segments may occur in nature. The segments may be joined in an altered arrangement such that the entire nucleic acid sequence from start to finish does not naturally occur. The term "recombinant" when made in reference to a protein or a peptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

The terms "vector" or " expression vector " refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free expression systems. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. In certain embodiments, this disclosure contemplates a vector encoding a peptide disclosed herein in operable combination with a heterologous promoter.

As used herein, a "chimeric antigen receptor" or "CAR" refers to a protein receptor, which introduces an antigen specificity, via an antigen binding domain, onto cells (immune cells) to which it is expressed (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combination thereof) thus combining the antigen binding properties of the antigen binding domain with the T cell activity (e.g. lytic capacity and self renewal) of T cells. A CAR typically includes an extracellular antigen-binding domain (ectodomain), a transmembrane domain and an intracellular signaling domain. The intracellular signaling domain generally contains at least one immunoreceptor tyrosine-based activation motif (ITAM) signaling domain, e.g., derived from CD3zeta, and optionally at least one costimulatory signaling domain, e.g. derived from CD28 or 4- IBB.

In order to improve the ability of immune cells to kill cancerous cells, T cells can be isolated from the blood of a patient and modified with a recombinant vector to express chimeric antigen receptors (CARs) that specifically target proteins expressed on the surface of cancerous cells and stimulate an immune response. When put back into the patient, the cells attack the cancerous cells. Brentjens et al. report that T cells altered to bind CD19 can induce remissions of cancer in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med, 2013, 5(177): 177ra38.

Whole blood is composed of plasma, red blood cells (RBCs; or erythrocytes), platelets, and nucleated white blood cells, also referred to as leukocytes. The leukocytes can be further categorized into mononuclear cells and polymorphonuclear cells (or granulocytes). There are different techniques to obtain peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells, leukocytes, or specific cell subsets, e.g., isolate specific cells directly by using flow cytometry, depleting red blood cells, centrifugation, and/or apheresis.

In a typical procedure, T cells and other immune cells are purified and isolated from blood or bone marrow. For example, T cells are collected via apheresis, a process that withdraws blood from the body and removes one or more blood components (such as plasma, platelets, or other white blood cells). The cells are exposed to a recombinant vector such that a chimeric antigen receptor (CAR) protein is produced and presented in the cell membrane. Before and/or after infecting the isolated cells with the recombinant vector, the cells may be induced to replicate. The genetically modified T cells may be expanded by growing cells in the laboratory until there are sufficient number. Optionally, these CAR T cells are frozen. The modified cells are then administered back to the patient. Various T cell subsets, as well as T cell progenitors can be targeted with a CAR. In certain embodiments, the targeting sequence in a chimeric antigen receptor refers to any variety of polypeptide sequences capable of selectively binding to a targeted associated molecule. The targeting sequences may be derived from variable binding regions of antibodies, single chain antibodies, and antibody mimetics. In certain embodiments, targeting sequence is a single-chain variable fragment (scFv) derived from an antibody. The targeting sequence is typically connected to intracellular domains by a hinge/transmembrane region, commonly derived from CD8 or IgG4. The intracellular domains may contain co-stimulatory domains such as CD80, CD86, 4-1BBL, IL- 2Rbeta, OX40L and CD70 and/or CD28 linked to the cytoplasmic signaling domain of CD3zeta.

Peripheral blood mononuclear cells (PBMCs) may be isolated by leukapheresis. T cells can be enriched by mononuclear cells counter-flow elutriation and expanded by addition of anti- CD3/CD28 antibody coated paramagnetic beads for activation of T cells. Cells may be expanded, harvested, and cryopreserved in infusible medium sometime after the subject has had an allogeneic stem-cell transplantation.

Cells may be obtained by isolation from peripheral blood and optionally purified by fluorescent activated cells sorting e.g., mixing cells with fluorescent antibodies or other fluorescent agents (molecular beacons) and separating the cells by flow cytometry based fluorescent sorting. CD3 is expressed on T cells as it is associated with the T cells receptor (TCR). The majority of TCR are made up of alpha beta chains (alpha beta T-cells). Alpha beta T-cells typically become double-positive intermediates (CD4+CD8+) which mature into single-positive (CD4+CD8-) T helper cells or (CD4-CD8+) cytotoxic T cells. T helper cells interact with antigen presenting dendritic cells and B cells. Upon activation with cognate antigen by dendritic cells, antigen specific CD4 T cells can differentiate to become various types of effector CD4 T cells with specific roles in promoting immune responses.

T cells may be isolated and separated from a human sample (blood or PBMCs or bone marrow) based on the expression of alpha beta T cells receptor (TCR), gamma delta T cells receptor, CD2, CD3, CD4, CD8, CD4 and CD8, NK1.1, CD4 and CD25 and other combinations based on positive or negative selection. In certain embodiments, the immune cells are CD8+, CD4+, alpha beta T cells, delta gamma T cells, natural killer cells and/or double-negative alpha beta T cells, macrophages, NK T cells, and cells derived from umbilical cord blood, bone marrow, or peripheral blood from human samples. Cell culturing, chimeric antigen receptor T cells (CART), and uses cancer therapies

The lower response rate for relapsed and refractory CLL (RR-CLL) patients is believed to be in part due to the inherently immunosuppressive nature of CLL, such that CLL patients are significantly deficient in CD8 co-receptor expressing (CD8+) T cells, including stem cell-like memory T (Tscm) and central memory T (Tcm) cells. As the prevalence of Tscm and Tcm cell populations is directly correlated to the in vivo persistence and efficacy of CART, elucidating translatable methods to selectively expand Tscm and Tcm from CLL patients is desirable to improving the efficacy CART cell therapy for CLL patients.

Mitochondria undergo fusion and fission processes. Fusion of the outer mitochondrial membrane (OMM) is facilitated by mitofusins (MFNs), e.g., MFN1 and MFN2. The compound MASM7 is a direct activator of MFN1/2 and mitochondrial fusion. Experiments were developed to determine whether MASM7 and other activators are useful in therapeutic T cell expansion.

Data indicates that a threshold frequency of more than 29% CD8+ central memory T cells prior to CART manufacturing predicted complete responses to CART therapy. Experiments using MASM7 demonstrates that dosing T cell cultures stimulated with anti-CD3/CD28-bound beads with MASM7 daily over the course of an 8-day expansion alters T cell populations phenotypically, favoring CD8+CD45RO+CCR7+ central memory T cells with coincident decreases in numbers of CD8+CD45RO+CCR7- effector T cells. Importantly, this subset of MASM7-expanded T cells contains fewer late-activation/exhausted (CD25+PD1+TIM3+) cells. While the total number of CD8 T cells did not appear increase with MASM7 treatment, a concentration of 5 uM MASM7 seems to optimally expand central memory T cells to exceed the number and fraction of central memory T cells present in the vehicle control group.

As such, in certain embodiments, this disclosure relates to in vitro cell culture compositions comprising purified and isolated T cells and a mitofusin activator. In certain embodiments, the mitofusin activator is a small molecule, antibody peptide, or nucleic acid encoding the peptide.

In certain embodiments, the small molecule is MASM7 (2-{2-[(5-cyclopropyl-4-phenyl- 4H-l,2,4-triazol-3-yl)sulfanyl]propanamido}-4H,5H,6H-cyclopenta[b]thiophene-3- carboxamide), CAS Number: 920868-45-7, derivative, prodrug, or salt thereof or any mitofusin activating compound derivative, prodrug, or salt thereof such as: MASM19, (N-carbamoyl-2-((5-cyclopropyl-4-phenyl-4H-l,2,4-triazol-3-yl)thio)propan amide), derivative, prodrug, or salt thereof;

MASM20, (2-(2-((4,5-diphenyl-4H-l,2,4-triazol-3-yl)thio)propanamido)-5,6-dihydro-

4H-cyclopenta[b]thiophene-3-carboxamide) derivative, prodrug, or salt thereof;

MASM21, (2-(2-((5-cyclopropyl-4-phenyl-4H-l,2,4-triazol-3-yl)thio)acetamido)-5,6- dihydro-4H-cyclopenta[b]thiophene-3-carboxamide) derivative, prodrug, or salt thereof;

MASM22, (2-(2-((5-(furan-2-yl)-4-phenyl-4H-l,2,4-triazol-3-yl)thio)propanamido)-5,6- dihydro-4H-cyclopenta[b]thiophene-3-carboxamide) derivative, prodrug, or salt thereof;

MASM23, (2-(2-((5-(5-methylfuran-2-yl)-4-phenyl-4H-l,2,4-triazol-3-yl)thio)propan amido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide) derivative, prodrug, or salt thereof; or

MASM24, (2-(2-((5-cyclopropyl-4-phenyl-4H-l,2,4-triazol-3-yl)thio)propanamido)-6- methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide) derivative, prodrug, or salt thereof.

In certain embodiments, the cell culture further comprises anti-CD3 antibodies and/or anti- CD28 antibodies optionally immobilized on a bead, in the form of a tetramer, or conjugated to a solid surface.

In certain embodiments, this disclosure relates to methods of expanding T cells comprising contacting isolated and purified T cells with a mitofusin activator.

In certain embodiments, the method is performed in a cell culture as provided for in any of the embodiments disclosed herein.

In certain embodiments, the mitofusin activator is at a concentration between 100 nM and 250 nM, or between 100 nM and 500 nM.

In certain embodiments, the mitofusin activating small molecule is MASM7, derivative, prodrug or salt thereof which is at a concentration between 100 nM and 250 nM, or between 100 nM and 500 nM.

In certain embodiments, the mitofusin activator, e.g., small molecule, MASM7, derivative, prodrug or salt thereof, is at a concentration of 5, 0.5, or 0.05, or 0.005 micromolar or less.

In certain embodiments, the mitofusin activating small molecule, e.g., MASM7, or derivative, prodrug or salt thereof is in a concentration between 0.5 micromolar and 50 micromolar, or between 2 micromolar and 20 micromolar. In certain embodiments, the mitofusin activating small molecule, e.g., MASM7, or derivative, prodrug or salt thereof is in a concentration of 5 micromolar or less.

In certain embodiments, the replicated T cells have increased expression of CD8+ T cells compared with levels prior to replication.

In certain embodiments, the replicated T cells have increased expression of

CD8+CD27+CD45RO- T cells compared with levels prior to replication.

In certain embodiments, the replicated T cells have increased expression of

CD8+CD45RO+CCR7+ central memory T cells compared with levels prior to replication.

In certain embodiments, the prior to, during, or after proliferating the T cells, the T cells are mixed with a vector having a nucleic acid encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a cancer targeting sequence, a transmembrane domain, a T cell costimulatory molecule domain, and a signal -transduction component of a T-cell antigen receptor domain, under conditions such that the cells express the chimeric antigen receptor on the surface of the cells.

In certain embodiments, the frequency of antigen specific T cells within expanded T cells is greater than 2%, 5%, or 10% of the T cells.

In certain embodiments, this disclosure relates to the methods of treating cancer or a chronic infection comprising: purifying T cells from a subject providing isolated T cells; contacting the isolated T cells with anti-CD3 antibodies and/or anti-CD28 antibodies optionally immobilized on a bead, in the form of a tetramer, or conjugated to a solid surface in combination with a mitofusin activating small molecule; under conditions such that the T cells replicate, providing replicated T cells; and administering an effective amount of the replicated T cells to a subject in need thereof.

In certain embodiments, the T cells have increased expression of CD8+CD45RO+CCR7+ central memory T cells compared with levels prior to replication.

In certain embodiments, the replicated T cells are antigen specific or express a chimeric antigen receptor on the surface of the cells such as those that specifically bind CD 138, CD 19, immunoglobulin kappa (Ig-Kappa) and B-cell maturation antigen (BCMA).

In certain embodiments, the chimeric antigen receptor specifically binds (EGFR) epidermal growth factor receptor, (HER2) human epidermal growth factor receptor 2, (MUC1) mucin 1, (MUC16) mucin 16, (EpCAM) epithelial cell adhesion molecule, (AFP) alpha-fetoprotein, (FAP) familial adenomatous polyposis, (CEA) carcinoembryonic antigen, (PSCA) prostate stem cell antigen, (PSMA) prostate-specific membrane antigen, (PSA) prostate-specific antigen, (AXL) AXL receptor tyrosine kinase, (DLL3) delta-like 3, (EPHA2) EPH receptor A2, (FRa) folate receptor alpha, (LMP1) Epstein-Barr virus latent membrane protein 1, (MAGE) melanoma antigen gene protein, MAGE-A1, MAGE- A3, MAGE-A4, (DR5) death receptor 5, (NKG2D) natural killer group 2 member D receptor, (CAIX) carbonic anhydrase IX, (TAG-72) tumor-associated glycoprotein 72, (GUCY2C) guanylate cyclase 2C, (ANTXR1) anthrax toxin receptor 1, (GSPG4) general secretion pathway protein G, (ROR) RAR-related orphan receptors, IL13RA2 (Interleukin 13 Receptor Subunit Alpha 2), Wilms' tumor 1 (WT1), Survivin, Tn (aGalNAc-O-Ser/Thr), sialyl- Tn (aNeuAc2,6-aGalNAc-O-Ser/Thr), TF (bGall,3-aGalNAc-O-Ser/Thr), CA 19-9 (Neu5Aca2- 3Gaipi-3[Fucal-4]GlcNAcP), Telomerase reverse transcriptase (TERT), Beta-hCG (Human chorionic gonadotropin), p53, Ras, bladder tumor antigen (BTA), antibody specific antigen Om5, GD2 (Ganglioside GD2), integrin alpha-v/beta-6, or mesothelin antigen. In certain embodiments, the chimeric antigen receptor is an antibody single-chain variable fragment (scFv).

In certain embodiments, the cancer is a hematological malignancy such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronic myelogenous leukemia, acute monocytic leukemia (AMOL), chronic myeloid leukemia (CML), myeloproliferative neoplasms (MPNs), and lymphomas, Hodgkin's lymphomas, and non-Hodgkin's lymphomas such as Burkitt lymphoma, B-cell lymphoma.

In certain embodiments, the cancer is a solid tumor, cellular malignancy, or hematological malignancy. In certain embodiments, the cancer is ependymoma, lung cancer, non-small cell lung cancer, small cell lung cancer, bronchus cancer, mesothelioma, malignant pleural mesothelioma, lung adenocarcinoma, breast cancer, prostate cancer, colon cancer, rectum cancer, colorectal cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, cervical cancer, melanoma, kidney cancer, pancreatic cancer, pancreatic ductal adenocarcinoma (PDA), thyroid cancer, brain cancer, glioblastoma (GBM), medulloblastoma, glioma, neuroblastoma, liver cancer, bladder cancer, uterine cancer, bone cancer, osteosarcoma, sarcoma, rhabdomyosarcoma, Ewing's sarcoma, retinoblastoma, nasopharyngeal carcinoma.

In certain embodiments, the cells are administered in combination with another anticancer agent. In certain embodiments, the anticancer agent is abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfilzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, filgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa- 2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof such as cyclophosphamide, methotrexate, 5 -fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5 -fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin, cisplatin (MV AC). In certain embodiments, the anticancer agent is an anti-PD-1, anti-PD-Ll anti-CTLA4 antibody or combinations thereof, such as an anti-CTLA4 (e.g., ipilimumab, tremelimumab) and anti-PDl (e.g., nivolumab, pembrolizumab, cemiplimab) and anti-PD-Ll (e.g., atezolizumab, avelumab, durvalumab).

In certain embodiments, the cells are administered to a subject with a lymphodepleted environment due to prior or concurrent administration of lymphodepleting agents such as cyclophosphamide and fludarabine).

In certain embodiments, this disclosure relates to in vitro cell culture compositions comprising purified and isolated T cells and a PI3K inhibitor optionally in combination with a mitofusin activator, salt, prodrug, or derivative thereof. In certain embodiments, the PI3K inhibitor is duvelisib and/or idelalisib.

In certain embodiment, the cells culture further comprises an agent that blocks VIP and VIP receptor signaling or VIP receptor antagonist such as VIPhyb comprising (SEQ ID NO: 1) KPRRPYTDNYTRLRKQMAVKKYLNSILN having a C-terminal amide. In certain embodiment, the cells culture does not contain an agent that blocks VIP and VIP receptor signaling or VIP receptor antagonist such as VIPhyb comprising (SEQ ID NO: 1) KPRRPYTDNYTRLRKQMAVKKYLNSILN having a C-terminal amide.

In certain embodiments, the cell culture further comprises anti-CD3 antibodies and/or anti- CD28 antibodies optionally immobilized on a bead, in the form of a tetramer, or conjugated to a solid surface.

In certain embodiments, more than 15 % of the T cells are negative for CD27 or CD28.

In certain embodiments, this disclosure relates to methods of expanding T cells comprising contacting isolated and purified T cells with a PI3K inhibitor optionally in combination with a mitofusin activator as reported herein.

In certain embodiments, the method is performed in a cell culture as provided for in any of the embodiments disclosed herein.

In certain embodiments, the replicated T cells have increased expression of CD8+CD45RO+CCR7+ central memory T cells compared with levels prior to replication. In certain embodiments, this disclosure relates to the methods of treating cancer or a chronic infection comprising: purifying T cells from a subject providing isolated T cells; contacting the isolated T cells with anti-CD3 antibodies and/or anti-CD28 antibodies optionally immobilized on a bead, in the form of a tetramer, or conjugated to a solid surface in combination with a PI3K inhibitor optionally in combination with a mitofusin activating small molecule; under conditions such that the T cells replicate, providing replicated T cells; and administering an effective amount of the replicated T cells to a subject in need thereof.

In certain embodiments, the prior to, during, or after proliferating the T cells, the T cells are contacted with a recombinant vector having a nucleic acid encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a cancer targeting sequence, a transmembrane domain, a T cell costimulatory molecule domain, and a signal-transduction component of a T-cell antigen receptor domain, under conditions such that the cells express the chimeric antigen receptor on the surface of the cells providing antigen specific T cells.

In certain embodiments, the antigen specific T cells are autologous, allogeneic or syngeneic.

In certain embodiments, the effective amount is between 10⁷ and 10⁹ cells or 10⁵ to 10¹⁰ of T cells or antigen specific T cells.

In certain embodiments, the vector is a recombinant viral vector.

In certain embodiments, the frequency of antigen specific T cells within transduced T cells is greater than 2%, 5%, or 10% of the T cells.

In certain embodiments, the transduced cells are culture-expanded, formulated and/or cryopreserved.

T-cells from untreated, early-stage patients with CLL have a senescent phenotype

T cells lacking expression of CD27/CD28 are senescent and do not proliferate after T-cell receptor (TCR)-mediated stimulation. Because CD27+CD8+ CART-cell subsets positively correlate with remissions in CLL, the differentiation status and expression of costimulatory molecules on T cells in treatment-naive patients were characterized with Rai stage 0 or 1 CLL. CD8+ T cells comprised a mean of 28% of the total CD3+ population with significant reductions in frequencies of naive CD8+ (P = 9.3 x 10-5) and naive CD4+ (P = 1.3 x 10-5) T cells in patients with CLL, relative to healthy donors. Notably, patients with CLL have fewer CD27+CD28+CD8+ and CD27+CD28+CD4+ T cells, with 51% of CD8+ T cells lacking either CD27 or CD28. A threshold frequency of > 29% of the peripheral CD8+ T cells with the phenotype CD27+CD45RO- before manufacture of CART cells predicted positive outcomes. T cells from 100% of healthy volunteers exceeded the >29% threshold of (CD27+CD45RO-) T cells expressing CD8+, whereas only 45% of patients with CLL of Rai stages 0 and 1 exceeded this threshold. Thus, efficacy of CART therapy in CLL is limited by T-cell senescence.

PI3Kdelta/gamma inhibition increased yields of T cells from healthy donors

Experiments were performed to test the effects of PI3Kdelta- selective and dual- PI3Kdelta/gamma inhibition on T cells from healthy donors by adding graded amounts of idelalisib (PI3Kdelta selective) or duvelisib (PI3Kdelta, gamma dual inhibitor) during culturing. Dosedependent fourfold increases in the mean number of viable T cells were observed for idelalisib and duvelisib. The maximal stimulatory effect of either PI3Ki was seen at doses predicted to inhibit both PI3Kdelta and PI3Kgamma. Addition of PI3Ki led to a dose-dependent increase in the expression of Fas and Fas-L, leading us to hypothesize that PI3Ki prevents PI3K-mediated apoptosis through Fas signaling. In contrast, adding ibrutinib, a Bruton’s tyrosine kinase- and IL- 2-inducible kinase inhibitor, did not increase the number of T cells and significantly decreased CD8+ T-cell frequencies. Combining duvelisib and ibrutinib in T-cell cultures from patients with CLL led to a decreased number of total and CD8+ T cells.

PI3K inhibitors promote dose-dependent decreases in exhaustion markers for T cells of patients. T cells from untreated CLL donors expanded less than those of healthy donors. T cells from patients with CLL have been reported to exhibit decreased signaling of AKT, a downstream target of PI3Kdelta and PI3Kgamma, suggesting decreased TCR and costimulatory signaling in CLL T cells. Accordingly, western blots of healthy and CLL donor T-cells confirmed that CLL T cells had reduced phosphorylation of AKT after anti-CD3/CD28 bead activation. Addition of duvelisib or idelalisib to ex vivo CLL T-cell cultures led to a mean 150% increase in the number of T cells. The addition of duvelisib and idelalisib at concentrations sufficient for dual- PI3Kdelta/gamma antagonism led to a dose-dependent increased frequency of CD8+ T cells. Modulating Mitofusins to Enhance Therapeutic T Cell Expansion and Persistence

Chronic lymphocytic leukemia (CLL), a cancer of B-lymphocytes, is the most common leukemia in adults. T cells from CLL patients are significantly deficient in CD8 co-receptor expressing (CD8+) T cells, including naive T (Tn), stem cell-like memory T (Tscm), and central memory T (Tcm) cells. Thus, elucidating translatable mechanisms for selective expansion of Tn, Tscm and Tcm from CLL patients is needed to improve the efficacy of CART cell therapy for CLL patients.

Dual inhibition of Phosphoinositide 3-Kinase (PI3K) isoforms with IPI-145 (duvelisib) preferentially expands CD8+ T cells, including Tn, Tscm and Tcm, as well as improves the in vivo persistence and cytotoxicity of CD19-targeted CART (CD19-CART). Furthermore, immunoblot analysis of T cells cultured with duvelisib for 15 days reveals increases in the expression of epigenetic and transcriptional regulators of memory T cell programs, including sirtuins 1/3/5, FOXO1/3, TCF1/7, and ID3. In addition, ex vivo duvelisib treatment of CLL patient T cells increased expression of essential mitochondrial fusion proteins, mitofusins 1 and 2 (MFN1/2), and decreased serine 637 phosphorylation, and thereby inactivation, of mitochondrial fission protein, DRP1, both consistent with an increase in mitochondrial fusion. Interestingly, duvelisib increased the spare respiratory capacity of CD8+ T cells but did not alter the average mitochondrial cross- sectional area of bulk T cells, assessed by extracellular flux analysis and transmission electron microscopy (TEM), respectively. These data, taken together, demonstrate intersections between PI3K inhibition, mitochondrial fusion, and T cell memory.

Given the potential role of mitochondrial fusion in enhancing Tn/Tscm and Tcm persistence and function, one approach is to induce mitochondrial fusion pharmacologically. Using mitofusin activating small molecule, MASM7, one can enhance MFN1/2 activity in CLL patient T cells. T cell cultures treated with MASM7 doses between 100 nM - 250 nM daily for nine days have >50% more CD8+ and CD8+CD27+CD45RO- T cells, compared to duvelisib and vehicle control groups. In addition, MASM7 induces significant increases in mitochondrial volume per mitochondrion and mitochondrial membrane potential to mass ratios, indicative of enhanced mitochondrial fusion.

Either dual inhibition of Phosphoinositide 3-Kinase (PI3K) with IPI-145 (duvelisib) or

MFN1/2 agonism with MASM7 improved the ex vivo expansion of CLL patient T cells. It is contemplated that there is synergy between duvelisib and MASM7 during ex vivo T cell expansion and CART generation to provide improved outcomes among CLL patients.

Duvelisib increases CART-cell mitochondrial fusion

Mitochondrial content was measured by staining with nonyl-acridine orange (NAO), which binds cardiolipin in mitochondrial membranes. The addition of either idelalisib or duvelisib to T- cell cultures doubled the median fluorescence intensity of NAO emission on CD4+ and CD8+ T- cell subsets at culture day 9. To directly visualize mitochondrial content, transmission electron microscopic images of nontransduced, control CART, and Duv-CART cells generated from patients with CLL were obtained after 3, 9, and 14 to 15 days of culture. Duvelisib treatment led to decreased T-cell size at all time points with significant decreases in total T-cell cross-sectional area by day 14. A 1.45-fold increase in mitochondrial cross-sectional area relative to total T-cell cross-sectional area was observed in Duv-CART cells, which imputes a 175% increase in mitochondrial volume for Duv-CART cells relative to control CART cells, but no difference in the number of mitochondria. Protein levels of mitochondrial fusion proteins MFN1 and MFN2 were increased, while activation of mitochondrial fission, p-DRPl, was decreased.

MASM7 promotes CD8+ T cell expansion and mitochondrial fusion

To evaluate MASM7 as a suitable T cell expansion reagent, CLL patient T cells were activated with soluble anti-CD3/CD28 antibodies and expanded in the presence of 30 lU/mL IL-2 and 62.5 nM-500 nM MASM7, 300nM duvelisib, or vehicle. T cells were cultured to 8 days. On day 8, T cells were harvested and evaluated for T cell phenotype and three-dimensional (3D) mitochondrial morphology by flow cytometry and widefield microscopy. Soluble anti-CD3/CD28 antibodies were used in place of beads for these experiments to facilitate downstream imaging experiments. For flow cytometry experiments, T cell populations were gated on viable cells using an analysis platform such that T cells with compromised permeabilized membranes were excluded from analysis.

Paired comparisons of T cell cultures from individual donors indicate that MASM7 consistently improves T cell expansion. Due to stark differences in the number of viable T cells between cultures from different CLL donors, differences in numbers of expanded T cells do not appear significant when analyzed across multiple donors. To account for the fold differences in viable cell number between donor T cell samples, the fold-expansion of each T cell culture was calculated (dividend of day 8 and day 0 numbers of viable cells). The fold-expansion and frequencies of T cell subsets on day 8 were then normalized to that of the respective vehicle group.

CLL patient-derived T cells treated with 125 nM of MASM7 expanded greater than 50% more than vehicle and greater than 100% more than duvelisib counterparts. Treatment with 125 nM MASM7 significantly enhanced the expansion of CD8 and CD27 co-expressing T cells (CD8+CD27+) and decreased the frequency of TIM-3 and LAG-3 co-expressing CD8+ T cells, suggesting that T cells expanded from MASM7-treated cultures have clinically significant phenotypes. Higher or lower doses of MASM7 (62.5 nM, 250 nM, and 500 nM) resulted in nonsignificant trends towards increased T cell expansion, consistent with an optimal effect at 125 nM MASM7.

Direct comparison of MFN2 expression between CD4+ and CD8+ T cells reveals that MFN2 expression varies significantly between patient samples following expansion. While Tscm and Tcm subsets express the highest levels of MFN2, MASM7 treatment at the effective doses of 125nM and 250nM do not affect MFN2 expression level compared to control. Furthermore, only addition of MASM7 to T cell cultures significantly augmented the mitochondrial volume of individual mitochondrion (p<0.05, n>212 mitochondria from >10 CD8+ T cells from one donor), suggesting that MASM7, but not duvelisib, promotes mitochondrial fusion in CLL patient T cells. Coincidentally, the mitochondrial membrane potential to mass ratio is higher in CD8+ T cells treated with MASM7 compared to duvelisib and vehicle, further supporting the specific activity of MASM7 on MFN1/2 activity and mitochondrial fusion. Future studies utilizing a MFN1/2 genetic knockout T cell model will improve our understanding of the MFNl/2-specific effects of MASM7. In addition, doses between 125 nM and 250 nM of MASM7 should be interrogated to identify the optimal dose of MASM7 conducive to mitochondrial fusion and therapeutic T cell expansion.

B-ALL B-cell Acute Lymphoblastic Leukemia

CART Chimeric Antigen Receptor T cell

CD19-CART CD 19 antigen-targeted Chimeric Antigen Receptor T cells

CD8+ CD8 co-receptor expressing

CLL Chronic Lymphocytic Leukemia duv-T/CART Duvelisib-treated T cells or duvelisib-treated CD19-CART

DRP1 Dynamin-related protein 1 ; GTPase that promotes mitochondrial fission

ECAR Extracellular Acidification Rate

ETC Electron Transport Chain

FAO Fatty Acid Oxidation

IMM Inner mitochondrial membrane

Ki-67 Antigen KI-67; nuclear protein that is associated with cellular proliferation

LAG-3 Lymphocyte Activation Gene 3; CD223

MASM7 Mitofusin Activating Small Molecule 7

MFN1/2 Mitofusins 1 and 2; GTPases that promotes mitochondrial outer membrane fusion

NOG NOD/Shi-scid/IL-2Rynull mouse; severely immunodeficient mouse

OCR Oxygen Consumption Rate

OMM Outer mitochondrial membrane

OPA1 Optic Atrophy 1; GTPase that promotes mitochondrial inner membrane fusion

OSU-CLL Ohio State University-Chronic Lymphocytic Leukemia; cell line

OXPHOS Oxidative Phosphorylation

PD-1 Programmed cell Death protein 1; CD279

PGCla Protein encoded by the PPARGC1 A gene; promotes mitochondrial biogenesis

PI3K d/g Phosphoinositide 3 -kinase delta and gamma isoforms

RR- Relapsed and Refractory -

SIRT Sirtuin; NAD+-dependent deacetylases (i.e. SIRT1/3) and deacylases (i.e.

SIRT5)

TCF1 T cell factor 1; T-cell-specific transcription factor

Tcm Central Memory T cell

Tern Effector Memory T cell

TEM Transmission Electron Microscope/Microscopy

Temra/Tte Effector Memory CD45RA-expressing T cell/Terminal Effector T cell

TIM-3 T cell Immunoglobulin and Mucin domain-containing protein 3; CD366 TMRM Tetramethylrhodamine, methyl ester; readily sequestered by active mitochondria

Tn Naive T cell

Tscm Stem Cell-like Memory T cell

Claims

1. An in vitro cell culture composition comprising isolated T cells and a mitofusin activator.

2. The composition of claim 1 wherein the mitofusin activator is a small molecule.

3. The composition of claim 2 wherein the small molecule is 2-{2-[(5-cyclopropyl-4-phenyl- 4H-l,2,4-triazol-3-yl)sulfanyl]propanamido}-4H,5H,6H-cyclopenta [b]thiophene-3 -carboxamide (MASM7), prodrug, or salt thereof.

4. The composition of claim 1, wherein the cell culture further comprises anti-CD3 antibodies and/or anti-CD28 antibodies optionally immobilized on a bead, in the form of a tetramer, or conjugated to a solid surface.

5. The composition of claim 1, wherein more than 15 % of the T cells are negative for CD27 or CD28 and replicated T cells in the culture have increased expression of CD27 or CD28 compared with levels prior to replication.

6. A method of expanding T cells comprising contacting purified T cells with a mitofusin activator providing replicated T cells.

7. The method of claim 6 wherein the method is performed in a cell culture.

8. The method of claim 6, wherein the mitofusin activator is a small molecule.

9. The method of claim 8, wherein the small molecule is 2-{2-[(5-cyclopropyl-4-phenyl-4H- l,2,4-triazol-3-yl)sulfanyl]propanamido}-4H,5H,6H-cyclopenta [b]thiophene-3 -carboxamide

(MASM7), prodrug, or salt thereof.

10. The method of claim 6, wherein the replicated T cells have increased expression of CD8+ T cells compared with levels prior to replication.

27

11. The method of claim 6, wherein prior to, during, or after expanding the T cells, T cells are mixed with a vector having a nucleic acid encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a cancer targeting sequence, a transmembrane domain, a T cell costimulatory molecule domain, and a signal -transduction component of a T-cell antigen receptor domain, under conditions such that the T cells express the chimeric antigen receptor on the surface of the cells providing antigen specific T cells.

12. A method of treating cancer comprising: purifying T cells from a subject providing isolated T cells; contacting the isolated T cells with anti-CD3 antibodies and/or anti-CD28 antibodies optionally immobilized on a bead, in the form of a tetramer, or conjugated to a solid surface in combination with a mitofusin activating small molecule providing replicated T cells having increased expression of CD8+ T cells compared with levels prior to replication; and administering an effective amount of the replicated T cells to a subject in need thereof.

13. The method of claim 12, wherein the replicated T cells are antigen specific or express a chimeric antigen receptor on the surface.

14. The method of claim 12, wherein the cancer is a hematological malignancy.

15. The method of claim 12, wherein the cells are administered in combination with another anticancer agent.