CXCR3 overexpression in CAR-NK cells stimulates migration / homing to the tumor microenvironment

Abstract

Nucleic acid constructs encoding CXCR3 and CAR, and cells containing the same, for the treatment of cancer are disclosed.

Description

Technical Field

[0001] Government Support This invention was made with government support under grant number R01CA190394 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 340,217, filed on May 10, 2022, under 35 U.S.C. § 119(e), the entire disclosure of which is incorporated herein by reference.

[0003] Sequence Listing This application includes a sequence listing submitted electronically in XML format, the entire disclosure of which is incorporated herein by reference. The XML copy created on March 13, 2023, is named 52095_765001WO_ST.xml and is 78 KB in size.

Background Art

[0004] The activation pathway of stimulator of interferon genes (STING) of the interferon gene promotes antitumor immunity. However, STING agonists have not yet achieved clinical success.

[0005] Activation of innate antitumor immunity involving the STING pathway can overcome barriers to therapeutic responses such as immune exclusion and exhaustion. The clinical development of STING agonists has mainly focused on the myeloid priming of CD8+ T cells that reject transplanted syngeneic tumors in mice (Corrales et al., Cell Rep. 11:1018-30 (2015); Sivick et al., Cell Rep. 25:3074-3085 (2018); Amouzegar et al., Cancers (Basel) 13:2695 (2021)). However, STING activation induces stress, cell cycle arrest, and death in T cells (Cerboni et al., J. Exp. Med. 214:1769-1785 (2017); Larkin et al., J. Immunol. 199:397-402 (2017); Gulen et al., Nat. Commun. 8:427 (2017)), limiting its clinical activity. Human tumors also undergo immune editing over months to years, making it difficult to extrapolate STING-induced T cell death mechanisms across species (O’Donnell et al., Nat. Rev. Clin. Oncol. 16:151-167 (2019)). Despite these limitations, recent mouse studies have investigated the complex interplay of STING signaling in the tumor immune microenvironment (TIME), identified novel effector mechanisms including NK cells, and juxtaposed the importance of STING activity in immune cells versus tumor cells (Sivick et al., Cell Rep. 25:3074-3085 (2018); Marcus et al., Immunity 49:754-763 (2018); Nicolai et al., Sci Immunol 5:eaaz2738 (2020); Chen et al., Nature 533:493-498 (2016); Sen et al., Cancer Discov. 9:646-661 (2019)).

[0006] Chimeric antigen receptor (CAR)-expressing cells have shown significant efficacy and improved patient outcomes and have received FDA approval for treating liquid tumors. In contrast, the efficacy of CAR T cell therapy and CAR NK cell therapy has been less effective in solid tumors due to a number of factors including the presence of an immunosuppressive tumor microenvironment (TME), vascular barriers, lack of chemokine gradients, and dysregulation of immune cell trafficking. Therefore, there is a great need for effective cell therapies for treating solid tumors.

Summary of the Invention

Means for Solving the Problems

[0007] In one aspect, the present disclosure provides a nucleic acid construct containing a first nucleic acid having a promoter operably linked to a nucleic acid encoding C-X-C motif chemokine receptor 3 (CXCR3) and a second nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR contains a ligand-binding domain containing a single-chain antibody fragment that binds to an antigen on a tumor cell, a transmembrane domain, and an intracellular domain containing a signaling domain.

[0008] In another aspect, the present disclosure provides a vector, for example, containing the nucleic acid construct, which is incorporated therein or cloned therein.

[0009] In another aspect, the present disclosure provides a genetically modified immune cell containing one or more vectors containing a nucleic acid encoding CXCR3 and a nucleic acid encoding CAR. In some embodiments, the genetically modified immune cell is a NK cell.

[0010] In another aspect, the present disclosure provides a pharmaceutical composition containing an effective number of genetically modified immune cells that express the vector and a pharmaceutically acceptable carrier.

[0011] In yet another aspect, the present disclosure provides a method of treating cancer. The method involves administering to a subject in need thereof an effective amount of a pharmaceutical composition. In some embodiments, the method further involves administering to the subject an effective amount of a STING agonist before, substantially simultaneously with, or after administration of the pharmaceutical composition.

[0012] The examples disclosed herein demonstrate that NK cells are resistant to STING-mediated cytotoxicity and that co-contact with a STING agonist enhances NK cell migration and death and improves their therapeutic activity. This effect is further enhanced in genetically modified NK cells that overexpress CXCR3 and / or contain an anti-methotrexate CAR. The examples further show that malignant pleural mesothelioma cells reliably express STING and that MPM cells responded to STING agonist treatment ex vivo in adoptive cell therapy.

Brief Description of the Drawings

[0013]

Figure 1

[0014]

Figure 2

[0015]

Figure 3

[0016]

Figure 4

[0017]

Figure 5

[0018]

Figure 6

[0019]

Figure 7

[0020]

Figure 8

[0021]

Figure 9

[0022]

Figure 10

[0023]

Figure 11

[0024]

Figure 12

[0025]

Figure 13

[0026]

Figure 14

[0027]

Figure 15

[0028]

Figure 16

[0029]

Figure 17

[0030]

Figure 18

[0031]

Figure 19

[0032]

Figure 20

[0033]

Figure 21

[0034]

Figure 22

[0035]

Figure 23

[0036]

Figure 24

[0037]

Figure 25

[0038]

Figure 26

[0039]

Figure 27

[0040]

Figure 28

[0041]

Figure 29

[0042]

Figure 30

DETAILED DESCRIPTION OF THE INVENTION

[0043] 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 the subject matter of this specification belongs. As used in this specification and the appended claims, unless the contrary is clearly stated, the following terms have the meanings set forth below for the purpose of facilitating an understanding of the present disclosure.

[0044] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a composition" includes mixtures of two or more such compositions, a reference to "an inhibitor" includes mixtures of two or more such inhibitors, a reference to "a construct" includes instances of having two or more constructs, and the like.

[0045] Unless otherwise specified, the term "about" is understood within the normal tolerance range in the relevant technical field, for example, within 2 standard deviations of the average. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided in this specification are modified by the term "about".

[0046] As used herein, the term "about", unless otherwise stated or otherwise clear from the context (except when such a number exceeds 100% of the possible value), refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) from the stated reference value.

[0047] The transitional phrase "comprising", which is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended and does not exclude additional unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or component not specified in the claims. The transitional phrase "consisting essentially of" limits the claims to those that do not materially affect the "specific materials or steps" of the claimed disclosure and the basic and novel features.

[0048] The terms "overexpression", "overexpressing", and "overexpressed" are used interchangeably herein when referring to CXCR3 expression as an increase of 30% or more of the protein or messenger RNA compared to an appropriate control.

[0049] Nucleic acid construct In one aspect, the present disclosure provides a nucleic acid construct comprising a first nucleic acid containing a first promoter operably linked to a nucleic acid encoding C-X-C motif chemokine receptor 3 (CXCR3), and a second nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a ligand-binding domain containing a single-chain antibody fragment that binds to an antigen on a tumor cell, a transmembrane domain, and an intracellular domain containing a signaling domain. In some embodiments, the second nucleic acid is operably linked to a second promoter that may be the same as or different from the first promoter. In some embodiments, a third nucleic acid encoding a self-cleaving peptide is disposed between the first nucleic acid and the second nucleic acid, and the first promoter drives the expression of the CXCR3 nucleic acid, the self-cleaving peptide, and the CAR nucleic acid.

[0050] As used herein, the term "nucleic acid" refers to a polymer of nucleotides, each of which is an organic molecule consisting of a nucleoside (nucleic acid base and pentose sugar) and a phosphate. The term nucleotide includes a nucleoside having a ribose sugar (i.e., ribonucleotide forming ribonucleic acid, RNA) or 2'-deoxyribose sugar (i.e., deoxyribonucleotide forming deoxyribonucleic acid, DNA), unless otherwise specified or apparent from the context. Nucleotides serve as monomeric units of nucleic acid polymers or polynucleotides. The four nucleic acid bases in DNA are guanine (G), adenine (A), cytosine (C), and thymine (T). The four nucleic acid bases in RNA are guanine (G), adenine (A), cytosine (C), and uracil (U). A nucleic acid is a linear chain of nucleotides (e.g., at least three nucleotides) chemically linked by a series of ester bonds between the phosphoryl group of one nucleotide and the hydroxyl group of a sugar (i.e., ribose or 2'-deoxyribose) in an adjacent nucleotide.

[0051] As used herein, the term "promoter" refers to a nucleic acid that directly or indirectly regulates the transcription of a corresponding nucleic acid coding sequence to which it is operably linked. A promoter may function alone to regulate transcription or may act in concert with one or more other regulatory sequences (e.g., enhancers or silencers, or regulatory elements that may be present in a vector). A promoter is located upstream of the DNA (towards the 5' region of the sense strand) on the same strand, near the transcription start site of the open reading frame. A promoter typically ranges from about 100 to 1000 base pairs in length.

[0052] As used herein, the term "operably linked" should be understood to mean that a nucleic acid is spatially located or arranged in a nucleic acid construct relative to a promoter to drive the expression of a protein encoded by the nucleic acid (e.g., CXCR3).

[0053] CXCR3 CXCR3 is a chemokine receptor that induces cell responses involved in immune cell trafficking. As demonstrated in the following examples, genetically modified immune cells containing a nucleic acid encoding CXCR3 increased their migration to the TME.

[0054] CXCR3 is a G protein-coupled receptor that binds to three chemokines known as monokines induced by interferon-γ (Mig / CXCL9), interferon-γ-inducible 10 kDa protein (IP10 / CXCL10), and interferon-inducible T cell α-chemoattractant (I-TAC / CXCL11). Binding of chemokines to CXCR3 induces cell responses including integrin activation, cytoskeletal changes, and chemotactic migration.

[0055] A representative amino acid sequence of CXCR3 is provided by NCBI accession number NP_001495, version NP_001495.1, which is incorporated herein by reference and shown in the Sequence Listing as SEQ ID NO: 1. The nucleic acid sequence (SEQ ID NO: 1) encoding the CXCR3 protein is provided by NCBI accession number NC_000023, version NC_000023.11, which is incorporated herein by reference and shown in the Sequence Listing as SEQ ID NO: 2. Another representative nucleic acid sequence encoding CXCR3 is shown in the Sequence Listing as SEQ ID NO: 3.

[0056] CAR CAR binds to an antigen on the surface of a cancer cell. CAR contains a ligand-binding domain containing a single-chain antibody fragment that binds to an antigen on the surface of a cancer (e.g., tumor cell), a transmembrane domain, and an intracellular domain containing a signaling domain. In some embodiments, the ligand-binding domain is an antibody fragment (e.g., scFv).

[0057] In some embodiments, the CAR is specific for and binds to a malignant pleural mesothelioma (MPM) antigen. In some of these embodiments, the MPM antigen is mesothelin. In some embodiments, the CAR ligand binding domain is derived from an anti-mesothelin antibody, antibody fragment, or derivative thereof. In some embodiments, the CAR ligand binding domain is derived from YP218, amatuximab, RC88, 19C3, 3C10, or 7B1. The nucleic acid sequences of YP218 VH (SEQ ID NO: 4) and VL (SEQ ID NO: 5), amatuximab VH (SEQ ID NO: 6) and VL (SEQ ID NO: 7), RC88 VH (SEQ ID NO: 8) and VL (SEQ ID NO: 9), 19C3 VH (SEQ ID NO: 10) and VL (SEQ ID NO: 11), 3C10 VH (SEQ ID NO: 12) and VL (SEQ ID NO: 13), and 7B1 VH (SEQ ID NO: 14) and VL (SEQ ID NO: 15) are shown in the Sequence Listing. The amino acid sequences of YP218 VH (SEQ ID NO: 16), and VL (SEQ ID NO: 17), amatuximab VH (SEQ ID NO: 18) and VL (SEQ ID NO: 19), RC88 VH (SEQ ID NO: 20) and VL (SEQ ID NO: 21), 19C3 VH (SEQ ID NO: 22) and VL (SEQ ID NO: 23), 3C10 VH (SEQ ID NO: 24) and VL (SEQ ID NO: 25), and 7B1 VH (SEQ ID NO: 26) and VL (SEQ ID NO: 27) are shown in the Sequence Listing.

[0058] In some embodiments, the CAR ligand binding domain contains a VH having the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CAR ligand binding domain contains a VL having the amino acid sequence of SEQ ID NO: 5.

[0059] Additional anti-methotrexate antibodies and their methotrexate-binding fragments are known in the art. See, for example, U.S. Pat. Nos. 7,081,518; 7,943,133; 8,460,660; 8,911,732; 9,272,002; 9,719,996; 10,022,452; 10,183,993; 10,793,641; and 10,919,975, as well as U.S. Patent Application Publication Nos. 2009 / 0047211; 2015 / 0252118; and 2022 / 0056147.

[0060] The transmembrane domain of the CAR connects the CAR ligand-binding domain to the intracellular domain. In some embodiments, the transmembrane domain is directly connected to the CAR ligand-binding domain. In some embodiments, the transmembrane domain is derived from CD3α, CD3β, CD3γ, CD3ζ, CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 (4-1BB or tumor necrosis factor receptor superfamily member 9 (TNFRSF9)), CD154, FcεRIα, FcεRIβ, FcεRIγ, ICOS, KIR2DS2, MHC class I, MHC class II, or NKG2D. Representative amino acid sequences of transmembrane domains are shown in the Sequence Listing as SEQ ID NOs: 14-18.

[0061] The amino acid sequence of a naturally occurring transmembrane domain can be modified by amino acid substitution to minimize its interaction with other members of the receptor complex and to avoid the binding of such regions to the transmembrane domains of the same or different surface membrane proteins. See, for example, U.S. Patent Application Publication No. 2021 / 0101954; Soudais et al., Nat. Genet. 3:77-81 (1993); Muller et al., Front. Immunol. 12:639818-13 (2021); and Elazar et al., elife 11:e75660-29 (2022).

[0062] In some embodiments, the CAR further includes a hinge domain disposed between the ligand-binding domain and the transmembrane domain. The hinge domain provides flexibility with respect to enabling the ligand-binding domain to obtain an optimal orientation for antigen binding, thereby enhancing the anti-tumor activity of the genetically modified immune cells expressing the CAR. In some embodiments, the hinge domain is derived from IgA, IgD, IgE, IgG, or IgM. In some embodiments, the hinge domain is derived from CD3ζ, CD4, CD8α, CD28, IgG1, IgG2, or IgG4, and its representative amino acid sequences are shown in the Sequence Listing as SEQ ID NOs: 19-25, respectively.

[0063] The intracellular domain of the CAR contains a signaling domain that enables intracellular signaling and immune cell function. The signaling domain can include a primary signaling domain and / or a co-stimulatory signaling domain. In some embodiments, the intracellular domain can deliver a signal similar to the signal of the natural ligation of a receptor complex such as an ITAM-containing molecule or a TCR receptor complex.

[0064] The intracellular domain contains a signaling domain that enables intracellular signaling and immune cell function. The signaling domain can include a primary signaling domain and / or a co-stimulatory signaling domain. In some embodiments, the intracellular domain includes one or more phosphorylatable intracellular motifs (ITAMs) that can deliver an immune activation signal. In some embodiments, the intracellular domain can deliver a signal similar to the signal of the natural ligation of a receptor complex such as an ITAM-containing molecule or a TCR receptor complex.

[0065] In some embodiments, the signaling domain comprises a plurality of, for example, 2 or 3 co-stimulatory signaling domains selected from, for example, 4-1BB, CD3ζ, CD28, CD27, ICOS, and OX40. In some embodiments, the signaling domain can comprise the CD3ζ domain as the primary signaling domain and any of the following pairs of co-stimulatory signaling domains from extracellular to intracellular direction: 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB; OX40-CD28; CD28-OX40; 4-1BB-CD3ζ; CD3ζ-4-1BB; CD28-CD3ζ; and CD3ζ-CD28. In some embodiments, the primary signaling domain is derived from CD3ζ, CD27, CD28, CD40, KIR2DS2, MyD88, or OX40. In some embodiments, the co-stimulatory signaling domain is derived from one or more of 4-1BB (CD137; TNFRSF9), CD3γ, CD3δ, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD68, CD72, CD80, CD86, CD154, CLEC-1, DAP10 (hematopoietic cell signal transducer (HCST)), DAP12 (TYROBP), dectin-1, FcαRI, FcγRI, FcγRII, FcγRIII, IL-2RB, ICOS, KIR2DS2, MyD88, OX40, and ZAP70. The amino acid sequences of representative signaling domains are shown in the sequence listing as SEQ ID NOs: 26 to 43, respectively.

[0066] In some embodiments, the signaling domain is derived from CD3ζ and the co-stimulatory domain is derived from 4-1BB. In some embodiments, the signaling domain is derived from CD3ζ and the co-stimulatory domain is derived from CD28. In some embodiments, the signaling domain is derived from CD3ζ and the co-stimulatory domains are derived from 4-1BB and CD28. The amino acid sequences of representative 4-1BB and CD28 are shown as SEQ ID NO: 26 and SEQ ID NO: 32, respectively, and further isoforms of CD28 are shown in the sequence listing as SEQ ID NOs: 44 to 46.

[0067] The expression of the first nucleic acid encoding CXCR3 and the expression of the second nucleic acid encoding the CAR are controlled by one or more promoters, which can be natural or synthetic. In some embodiments, a third nucleic acid encoding a self-cleaving peptide or an internal ribosome entry site (IRES) is disposed between the first nucleic acid and the second nucleic acid. In these embodiments, the first nucleic acid and the second nucleic acid are controlled by the same promoter. In some embodiments, the second nucleic acid is controlled by a second promoter different from the first promoter.

[0068] In some embodiments, the first promoter is a strong promoter that overexpresses the nucleic acid to which it is operably linked. Overexpression can be achieved by providing a vector encoding a protein controlled by a constitutive promoter, or by removing a repressor, adding multiple copies of a gene to a cell, or upregulating an endogenous gene. In some embodiments, one or both of the promoters are derived from the elongation factor 1α (EF-1α), cytomegalovirus (CMV), β-actin, simian virus 40 (SV40) early promoter, human phosphoglycerate kinase (PGK), RPBSA (synthetic, made by Sleeping Beauty), or CAG (synthetic, CMV early enhancer element, chicken β-actin, and rabbit β-globin splice acceptor) promoter. As used herein when referring to a protein or nucleic acid, the term "derived from" refers to a protein or nucleic acid derived from another parent protein or nucleic acid. The derived protein or nucleic acid can have a sequence identical to the parent sequence, can be a part of the parent sequence, or can have at least one variant from the parent sequence. Variants can include amino acid and nucleotide substitutions, insertions, or deletions. Thus, for example, an amino acid sequence derived from a parent sequence can be identical for a particular range of the parent amino acids but not include the amino acids outside of that particular region.

[0069] In some embodiments, the promoter may have a core region located near the starting point of the nucleic acid coding sequence. In some embodiments, the promoter is modified with respect to the native promoter. One modification involves the removal of methylation-sensitive sites, for example, where a guanine nucleotide follows a cytosine nucleotide, i.e., "CpG". Another modification involves the addition of regulatory sequences that bind to DNA methylation-inhibiting transcription factors. In some embodiments, the expression vector includes an A / T-rich nuclear matrix interaction sequence known as a scaffold / matrix attachment region (S / MAR) that can enhance transformation efficiency and improve the stability of transgene expression.

[0070] In some embodiments, the first and second promoters are derived from EF-1α. In some embodiments, the first promoter is derived from CMV and the second promoter is derived from EF-1α. The sequence of the EF-1α promoter is provided by NCBI accession number J04617.1. The sequences of CMV promoters from different CMV isolates are provided by NCBI accession numbers AY218848, AF477200, M64754, and AF286076. The sequence of the PGK promoter is provided by NCBI accession number NC_000023.11, range 78104248 - 78129295. The sequence of the RPBSA promoter is provided by NCBI accession number MN811119.1. The sequence of the CAG promoter is provided by NCBI accession number MG763233.1.

[0071] In some embodiments, the nucleic acid construct contains a nucleic acid encoding a self-cleaving polypeptide disposed between a nucleic acid encoding CXCR3 and a nucleic acid encoding a CAR. The nucleic acid sequences of representative self-cleaving polypeptides are set forth in the Sequence Listing as SEQ ID NOs: 476 - 49.

[0072] In some embodiments, the nucleic acid construct contains a selectable marker to assist in isolation, capture, or detection. The selectable marker typically involves the addition of an in-frame nucleic acid that is translated into an amino acid along with the protein to which it is attached. Representative examples of selectable markers include enhanced green fluorescent protein (EGFP) (SEQ ID NO: 50), AU1 epitope (SEQ ID NO: 51), AU5 epitope (SEQ ID NO: 52), polyhistidine (SEQ ID NO: 53), FLAG epitope (SEQ ID NO: 54), FLAG His tag (SEQ ID NO: 55), histidine affinity tag (HAT) (SEQ ID NO: 56), herpes simplex virus (HSV) epitope (SEQ ID NO: 57), human influenza hemagglutinin (HA), glutathione S-transferase (GST), KT3 epitope, maltose binding protein (MBP), bacteriophage T7 epitope, myc tag. The amino acid sequences of representative selectable markers are shown in the Sequence Listing as SEQ ID NOs: 50-57.

[0073] Vector The nucleic acids encoding CXCR3 and CAR can be introduced into immune cells by the same or separate vectors. The nucleic acid construct is introduced into immune cells by an appropriate vector. The vector is configured to contain additional regulatory elements necessary to achieve transport into immune cells and expression of the nucleic acid after transformation. Such elements include an origin of replication or promoter, polyA tail sequence, selectable marker, one or more sites suitable for insertion of the nucleic acid sequence, such as a multiple cloning site (MCS), and a selectable marker, as well as any additional optional regulatory elements.

[0074] In some embodiments, the vector is a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, a herpes viral vector, an adenovirus, or an adeno-associated virus (AAV) vector. As used herein, the term "lentiviral vector" is intended to mean an infectious lentiviral particle. Lentiviruses (lentivirinae or lentivirus) are a subfamily of enveloped retroviruses (retrovirinae or retroviruses) that can be distinguished from other viruses by virion structure, host range, and pathological effects. Infectious lentiviral particles can enter target host cells, including infecting and transducing non-dividing cells and immune cells. Characteristics of lentiviruses include, for example, the ability to infect or transduce non-dividing host cells, including immune cells.

[0075] In some embodiments, the vector is a recombinant lentivirus comprising a recombinant genome that includes a lentiviral packaging psi sequence, an RNA nuclear export element, a transgene, a promoter and / or a sequence that favors nuclear import of the RNA, and a mutant integrase that prevents integration of its genome into the genome of the host cell, between the LTR 5' and 3' lentiviral sequences. The construction of lentiviral vectors is described, for example, in U.S. Pat. Nos. 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119,119, and 10,954,530.

[0076] In some embodiments, the vector is a non-integrating and non-replicating recombinant lentiviral vector. The construction of lentiviral vectors is described, for example, in U.S. Patent Nos. 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119,119, and 10,954,530. Lentiviral vectors include those in which at least one of the defective lentiviral genome, i.e., the lentiviral genes gag, pol, and env, is inactivated or deleted.

[0077] Lentiviral vectors can also exhibit additional functions compared to naturally occurring lentiviruses or functions different from those of naturally occurring lentiviruses. For example, lentiviral vectors can be modified to change or reduce the characteristics of lentiviruses. Lentiviral vectors can also be modified to exhibit the characteristics of one or more other retroviruses, retroviral vectors, host cells, or heterologous cells. Modifications can include, for example, pseudotyping, modification of the binding and / or fusion functions of envelope polypeptides, incorporation of heterologous, chimeric, or multifunctional polypeptides into the vector, incorporation of non-lentiviral genomes, or incorporation of heterologous genes into the lentiviral vector genome.

[0078] The terms "pseudotyping," "pseudotyped," "pseudotyped vector," and "pseudotyped vector particle" are used herein to refer to vectors having components (e.g., envelope or capsid) from more than one source. The source can be from a heterologous virus or a non-viral protein. Non-viral proteins can include antibodies and antigen-binding fragments thereof. A representative pseudotyped vector is a vector having a non-glycoprotein component derived from a first virus and an envelope glycoprotein derived from a second virus. Thus, the host range of a pseudotyped vector can be expanded or altered depending on the type of cell surface receptor bound by the glycoprotein derived from the second virus.

[0079] In some embodiments, the lentiviral vector is pseudotyped with baboon endogenous virus (BaEV) glycoprotein (BaEV-gp). The amino acid sequence of a representative BaEV-gp is shown in the Sequence Listing as SEQ ID NO: 58. The nucleic acid sequence encoding BaEV-gp (SEQ ID NO: 58) is shown as SEQ ID NO: 59. Additional BaEv pseudotyped lentiviral vectors are known in the art. See, e.g., Levy et al., J. Thromb. Haemost. 14:2478-2492 (2016), Costa et al., Leukemia 31:977-980 (2017), and Bari et al., Front. Immunol. 10:2001 (2019). The nucleic acid sequence of a representative BaEV vector is shown in the Sequence Listing as SEQ ID NO: 60.

[0080] In some embodiments, the vector comprises a plx307-based nucleic acid construct. In some embodiments, the vector contains a pHIV-based nucleic acid construct. The nucleic acid sequence of a representative vector (pHIV-aMesoCAR-GFP) containing a pHIV-based nucleic acid construct encoding a CAR that binds to mesothelin is shown in the Sequence Listing as SEQ ID NO: 61. The nucleic acid sequence of a representative vector (pHIV-aMesoCAR-CXCR3; illustrated in FIG. 30B) containing a pHIV-based nucleic acid construct encoding a CAR that binds to CXCR3 and mesothelin is shown in the Sequence Listing as SEQ ID NO: 62.

[0081] In some embodiments, the vector contains a pCMV-based nucleic acid construct. The nucleic acid sequence of a representative vector (pCMV-dR8.91) containing a pCMV-based nucleic acid construct is described in the Sequence Listing as SEQ ID NO: 63. In some embodiments, the vector contains a pAdv-based nucleic acid construct. The nucleic acid sequence of a representative vector (pAdvAntage) containing a pAdv-based nucleic acid construct is shown in the Sequence Listing as SEQ ID NO: 64.

[0082] Cell One aspect of the present disclosure is a genetically modified (or transformed) immune cell containing a vector containing nucleic acid constructs encoding CXCR3 and CAR. As used herein, "immune cell" refers to a hematopoietic-derived cell that is functionally involved in initiating and / or executing innate and / or adaptive immune responses. Representative examples of immune cells include natural killer (NK) cells, T cells, macrophages, and dendritic cells. Combinations of different genetically modified immune cells may be used. In some embodiments, the genetically modified immune cell is an NK cell. In some embodiments, the genetically modified immune cell is derived from an NK cell line, primary NK cell, stem cell-derived NK cell, umbilical cord blood-derived NK cell, peripheral blood mononuclear cell (PBMC)-derived NK cell, memory-like NK cell, or induced memory-like NK cell. Suitable NK cell lines suitable for this method include NK-92, NKG, NKL, KHYG-1, YT, NK-YS, SNK-6, IMC-1, YTS, NKL cells, and high-affinity NK (haNK, NK / T cell lymphoma cell line).

[0083] In some embodiments, the genetically modified immune cell is a memory-like NK cell. Memory-like NK cells can be generated by harvesting NK cells from a subject, for example, purified from a peripheral blood sample, stimulated with cytokines (e.g., IL-12, IL-15, and IL-18) for an appropriate period (e.g., between about 12 hours and less than 7 days), the cytokines are removed, and they can be transduced to express CXCR3 and CAR. In some embodiments, the genetically modified immune cell is a cytokine-induced memory-like (CIML) NK cell. CIML NK cells can be produced by stimulating NK cells with one or more of IL-12, IL-15, and IL-18, typically in combination. See, for example, Cooper et al., Proc. Natl Acad. Sci. USA 106:1915-9 (2009); Ni et al., J. Exp. Med. 209:2351-65 (2012); Keppel et al., J. Immunol. 190:4754-62 (2013).

[0084] In some embodiments, the cell is a T cell. In some embodiments, the T cell is a naive T cell, a memory stem cell T cell, a central memory T cell, an effector memory T cell, a helper T cell, a CD4+ T cell, a CD8+ T cell, a CD8 / CD4+ T cell, an αβ T cell, a γδ T cell, and a natural killer T (NKT) cell, and a Th17 T cell. Isolation of T cells and fractionation into T cell subsets are known in the art. See, for example, U.S. Pat. Nos. 10,507,219, 11,135,245, and 11,242,376, and U.S. Patent Application Publication Nos. 2013 / 0060011, 2019 / 0276540, 2020 / 0347350, and 2021 / 0106622.

[0085] Methods for introducing vectors containing nucleic acid constructs into immune cells are known in the art. See, for example, U.S. Pat. Nos. 7,399,633, 7,575,925, 10,072,062, 10,370,452, and 10,829,735, and U.S. Patent Application Publication Nos. 2019 / 0000880 and 2021 / 0407639.

[0086] In some embodiments, the lentiviral vector is transduced into immune cells. In other embodiments, the method involves the use of gammaretroviral vectors. See, for example, U.S. Pat. Nos. 9,669,049, 11,065,311, and 11,230,719. In some embodiments, the method involves the use of adenovirus, adeno-associated virus (AAV), dsRNA, ssDNA, or dsRNA to deliver the nucleic acid construct. See, for example, U.S. Pat. No. 10,563,226, and U.S. Patent Application Publication Nos. 2019 / 0225991, 2020 / 0080108, and 2022 / 0186263.

[0087] Pharmaceutical composition The pharmaceutical composition of the present disclosure comprises an effective number of genetically modified immune cells and a pharmaceutically acceptable carrier. As used herein, the term "effective number of genetically modified immune cells" (indirectly including corresponding amounts of CXCR3 and CAR) refers to genetically modified immune cells containing a sufficient number of nucleic acids encoding CXCR3 and CAR to bring about the desired effect.

[0088] The composition can be provided as a sterile liquid preparation that can be buffered to a selected pH, for example, an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition. The liquid carrier similarly includes aqueous or non-aqueous carriers. Representative examples of liquid carriers include physiological saline, phosphate-buffered saline, soluble proteins, dimethyl sulfoxide (DMSO), polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. In some embodiments, the liquid carrier contains proteins dissolved or dispersed therein, and representative examples include serum albumin (such as human serum albumin, recombinant human albumin), gelatin, and casein. The composition is typically isotonic, i.e., having the same osmotic pressure as blood. Sodium chloride and isotonic electrolyte solutions (such as Plasma-Lyte®) can be used to achieve the desired isotonicity. Depending on the carrier and the genetically modified immune cells, other excipients known in the art, such as wetting agents, dispersing agents, or emulsifying agents, gelling agents and thickening agents, preservatives, etc., can be added.

[0089] cancer In some aspects, the present disclosure relates to treating cancer in a subject. The method involves administering to a subject in need thereof an effective number of genetically modified immune cells (also referred to herein as "genetically modified immune cells") containing a nucleic acid construct containing a first nucleic acid encoding CXCR3 and a second nucleic acid encoding a CAR. As used herein, the term "cancer" refers to a disease characterized by uncontrolled cell growth, reduced cell apoptosis, and the spread of abnormal cells that invade and destroy non-cancerous tissue. Cancer cells may be in the form of a tumor (i.e., a solid tumor) or may exist alone within the subject, also referred to as a liquid tumor. The term cancer includes pre-malignant as well as malignant cancers.

[0090] In some embodiments, the cancer is a solid tumor. Solid tumors are highly heterogeneous because the type of tissue in which the solid tumor arises differs in the characteristics of tumor growth. In some embodiments, the solid tumor is a sarcoma or a carcinoma. In some embodiments, the cancer is MPM. Some embodiments relate to a method of treating MPM by administering to a subject in need thereof an effective amount of NK cells or a pharmaceutical composition thereof containing a nucleic acid construct having CXCR3 and a CAR. In some embodiments, the method further involves administering to the subject an effective amount of a STING agonist before, substantially simultaneously with, or after administration of the NK cells or the pharmaceutical composition thereof.

[0091] In some embodiments, the cancer comprises hypermethylation of the cyclic GMP-AMP synthase (cGAS) or STING gene promoter. In some of these embodiments, the cancer is bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), cervical pre-cancerous lesions (CPL), colon adenocarcinoma (COAD), glioma (e.g., glioblastoma), head and neck squamous cell carcinoma (HNSC), kidney clear cell carcinoma (KIRC), kidney papillary cell carcinoma (KIRP), hepatocellular liver carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), melanoma, ovarian cancer, pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), sarcoma (SARC), skin melanoma (SKCM), stomach adenocarcinoma (STAD), thyroid cancer (THCA), and uterine corpus cancer (UCEC). See Konno et al., Oncogene 37:2037-2051(2018), de Queiroz et al., Mol. Cancer Res. 17:974-986(2019), Huang et al., Front. Genet. 10:1-11(2019), Falahat et al., Proc. Natl. Acad. Sci. U.S.A. 118:1-9(2021), Low et al., Cancer Cell 40:439-440(2022).

[0092] In some embodiments, the cancer has high basal STING expression, also referred to herein as STING+. As used herein, the term "high basal" expression of a gene refers to an elevated expression of the gene in a disease state as compared to a reference non-disease state. In some embodiments, STING+ cancers are melanoma (e.g., malignant melanoma), gastric cancer, liver cancer (e.g., hepatocellular carcinoma (HCC)), lung cancer (e.g., non-small cell lung cancer (NSCLC)), bladder cancer, colorectal cancer, or breast cancer. Additional cancers in which STING has been shown to play a role are known in the art and include leukemia (e.g., acute myeloid leukemia), lymphoma (e.g., malignant lymphoma), breast cancer, colorectal cancer, glioma, head and neck squamous cell carcinoma, lung cancer, melanoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, and tongue squamous cell carcinoma. See Zhu et al., Mol. Cancer 18(1):152(2019).

[0093] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention, process, or administration of an agent for a therapeutic purpose ("therapeutic effect") of reversing, alleviating, improving, inhibiting, reducing, decelerating, halting, stabilizing, or preventing the onset, progression, occurrence, severity, or recurrence of a symptom, complication, or condition associated with cancer, or a biochemical hallmark, to a subject in need thereof.

[0094] As used herein, the term "subject" (or "patient") includes all members of the animal kingdom that are susceptible to or have been diagnosed with the indicated cancer. Thus, a "cancer having" or "in need of treatment" subject according to the present disclosure includes a subject having an active disease that may have been treated previously with one or more treatments, and a subject not currently being treated (e.g., in remission) but still at risk of recurrence, and a subject not diagnosed as positive but having a predisposition to cancer (e.g., based on a previous medical history and / or family history, or otherwise, exhibiting one or more risk factors such that a medical professional can reasonably suspect that the subject has a predisposition to cancer), broadly encompassing subjects diagnosed as positive.

[0095] Administration The number of genetically modified immune cells administered to a subject varies within a wide range depending on the location, type, and severity of the cancer, the age, weight, and condition of the individual being treated, etc. The physician ultimately determines the appropriate number and dosage of cells to be used. Typically, the genetically modified immune cells are administered as a single dose. In some embodiments, the effective number of genetically modified immune cells is about 1×10 5 ~ about 1×10 10 cells per subject. In some embodiments, the effective number of genetically modified immune cells is about 1×10 5 ~ about 6×10 8 cells per kg of the subject's body weight.

[0096] Compositions containing a therapeutically effective number of genetically modified immune cells can be administered to a subject for the treatment of cancer by any medically acceptable route. The genetically modified immune cells are typically delivered intravenously, but can also be introduced into other convenient sites, such as the affected organ or tissue, intratumorally or in a manner determined by the attending physician. Proliferation and differentiation agents can be provided before, during, or after the administration of the cells to increase the differentiation, proliferation, or persistence of the genetically modified immune cells (e.g., NK cells).

[0097] In some embodiments, the genetically modified immune cells are administered as a single intravenous infusion over a period of time. Representative infusion times are 30 minutes, 60 minutes, and 90 minutes. In some embodiments, the infusion time is 30 - 60 minutes. In some embodiments, the first administration is infused into the patient for 90 minutes, and subsequent administrations are infused into the patient for 30 minutes.

[0098] Combination therapy In some embodiments, the method includes co - administration of a STING agonist. The term "co - administered" includes administration by the same or separate dosage forms, or as part of the same treatment regimen, or by a continuous treatment regimen, e.g., sequential, substantially simultaneous administration. The order and time intervals can be determined such that the co - administered therapies can act together, e.g., synergistically, to provide an increased benefit compared to when they are administered in another manner. For example, the therapeutic agents can be administered simultaneously, or sequentially in any order at different time points, but if not administered simultaneously, they can be administered at sufficiently close times to provide the desired therapeutic effect in a synergistic manner.

[0099] In some embodiments, the genetically modified immune cells of the present disclosure are used in combination with a STING agonist. In some embodiments, the STING agonist is ADU - S100, TAK - 676, BI - STING, BMS - 986301, GSK532, DMXAA (ASA - 404), GSK3745417, JNJ - 4412, MK - 1454, SB11285, 3’3’ - scylic AIMP, ALG - 031048, E7766, JNJ - ’6196, MK - 2118, MSA - 1, MSA - 2, SNX281m SR - 717, KAT676, TTI - 10001, XMT - 2056, CRD - 5500, c - di - AMP, synthetic cyclic dinucleotide (DCN) molecules, analogs thereof, or combinations thereof. In some embodiments, the STING agonist is ADU - S100 or TAK - 676. In some embodiments, the STING agonist is delivered by intratumoral injection or systemically (i.e., intravenously). See Woodward et al., Science 328:1703 - 5(2010), Motedayen Aval et al., J. Clin. Med. 9:3323(2020), as well as U.S. Patent Nos. 11,285,131 and 11,312,772, and U.S. Patent Application Publication Nos. 2018 / 0028553, 2019 / 0328762, 2020 / 0330556, and 2021 / 0170043.

[0100] In some embodiments, the method includes co - administering genetically modified immune cells, with or without a STING agonist, and another anti - cancer agent. Representative examples of additional anti - cancer agents are shown below.

[0101] Anti - cancer agents that can be used in combination with the cells of the present invention are known in the art. See, for example, U.S. Patent No. 9,101,622, Section 5.2. An “anti - cancer” agent can, for example, kill cancer cells, induce apoptosis in cancer cells, reduce the growth rate of cancer cells, reduce the incidence or number of metastases, reduce tumor size, inhibit tumor growth, reduce the blood supply to a tumor or cancer cells, promote an immune response against cancer cells or tumors, prevent or inhibit the progression of cancer, or extend the lifespan of a subject having cancer, thereby having a negative impact on cancer in the subject.

[0102] In some embodiments, the genetically modified immune cells of the present disclosure, or genetically modified immune cells in combination with a STING agonist, are used in combination with a type I IFN agonist. In some embodiments, the type I INF agonist is a recombinant synthetic type I INF protein, such as interferon alpha con - 1 (Infergen®), recombinant interferon α - 2b (Intron A®, Roferon® - A), interferon β - 1b (Betaseron®, Extavia®, Rebif®, Avonex®), interferon α - 2c (Berofor Alpha®), interferon α - n4 (Alferon N®), or a pegylated IFN such as pegylated interferon β - 1a (Plegridgy®).

[0103] In some embodiments, the genetically modified immune cells of the present disclosure are used in combination with a DNA methylation inhibitor. In some embodiments, the DNA methylation inhibitor is a DNA methyltransferase (DNMT) enzyme inhibitor. Representative DNMT inhibitors include azacitidine (Vidaza®) and decitabine (5-azacytidine) (Dacogen®). In some embodiments, the additional anti-cancer agent includes epigenetic therapy. In some embodiments, the epigenetic therapy includes azacitidine (Vidaza®, Onureg®), decitabine (5-azacytidine) (Dacogen®), zebularine (pyrimidin-2-one β-D-ribofuranoside), guadecitabine, 5-fluoro-2′-deoxycytidine, (−)-epigallocatechin gallate, curcumin, hydralazine, procainamide, RG-108, and SG-1027. See Nepali et al., J. Biomed. Sci. 28:27 (2021); Giri et al., Front. Pharmacol. 10:1-11 (2019).

[0104] Immunotherapy In some embodiments, the additional anti-cancer agent includes immunotherapy, such as immune checkpoint inhibitors. Representative examples of immune checkpoint molecules that can be targeted by the additional therapy include PD-1, PDL1, CTLA4, KIR, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD47, and NKG2A. Clinically available examples of immune checkpoint inhibitors include durvalumab (Imfinzi®), atezolizumab (Tecentriq®), and avelumab (Bavencio®). Clinically available examples of PD-1 inhibitors include nivolumab (Opdivo®), pembrolizumab (Keytruda®), and cemiplimab (Libtayo®).

[0105] Chemotherapy Anticancer therapies also include various combination therapies with both chemical and radiation-based treatments. Combinatorial chemotherapy includes, for example, Abraxane®, altretamine, docetaxel, Herceptin®, methotrexate, Novantrone®, Zoladex®, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, emcitabine, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binders, Taxol®, gemcitabine, Navelbine®, farnesylation enzyme inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, or any analogs or derivative variants of the foregoing, and combinations thereof.

[0106] Radiation therapy Anticancer therapies also include radiation-based DNA damage treatments. Combinatorial radiation therapy includes gamma rays, those commonly known as X-rays, and / or the targeted delivery of radioisotopes to cancer cells that cause extensive damage to DNA, DNA replication and repair, and the assembly and maintenance of chromosomes. The dose range of the radioisotope varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by neoplastic cells, and is determined by the attending physician.

[0107] These and other aspects of the present application are intended to illustrate certain embodiments of the present application, but are not intended to limit the scope of the present application as defined by the claims. It will be further understood by considering the following examples.

Example

[0108] Example 1: Materials and Methods

[0109] Patient samples. Formalin-fixed paraffin-embedded (FFPE) tissue microarray slides from patients with SCLC, NSCLC, and thymoma were purchased from Biomax (LC245, LC817, LC2081, THY761). Additionally, FFPE slides were collected from DFCI / BWH patients with SCLC (n = 58), MPM (n = 68), and benign pleura (n = 9) under Dana-Farber / Harvard Cancer Center protocols 02-180 and 98-063. Tumors from MPM patients treated at DFCI / BWH between July 2018 and October 2021 were collected postoperatively under protocol 98-063. Patient samples analyzed by flow cytometry in Figures 1A–1D and 7A–7E were different from those treated ex vivo in the remaining figures. NK cells in blood collected from patients with head and neck squamous cell carcinoma or oral proliferative verrucous leukoplakia were tested under protocols 17-255 and 18-387 (Figure 1D). Mann–Whitney test: ***p < 0.001. Details of flow cytometry antibodies are shown in Table 1. TIM-3 = T cell immunoglobulin and mucin domain-containing protein 3; PD-1 = programmed cell death protein 1; LAG3 = lymphocyte activation gene 3; EMRA = effector memory re-expressing CD45 RA; EM = effector memory; CM = central memory. These studies were conducted in accordance with the Helsinki Declaration and approved by the DFCI and BWH Institutional Review Boards. Written informed consent was obtained from all patients whose tumors were studied.

[0110] Immunohistochemistry. STING and phosphoro-IRF3 immunohistochemistry (IHC) were performed on a Leica Bond III automated staining platform. Antibodies against STING (Cell Signaling Technology #13647, clone D2P2F) were run at a 1:50 dilution using the Leica Biosystems Refine Detection Kit with citrate antigen retrieval. Phosphoro-IRF3 (Cell Signaling Technology #29047, clone D6O1M) was run at a 1:100 dilution using the Leica Biosystems Refine Detection Kit with EDTA antibody retrieval. This was optimized from a comparison of dilution ranges and citrate vs. EDTA antigen retrieval in MPM cell lines treated in vitro with 50 μM ADU-S100 for 24 hours prior to paraformaldehyde fixation and paraffin embedding (Figure 7C). STING IHC staining was quantified using QuPath software (version 0.2.3) (Bankhead et al., Sci. Rep. 7:16878 (2017)). Positive pixel detection analysis was used with default settings for DAB staining to detect and quantify positive pixels in each of three randomly selected individual fields per tumor, which were then averaged.

[0111] Flow cytometry immune profiling. Fresh tumors were mechanically and enzymatically dissociated in dissociation buffer consisting of RPMI (Life Technologies) + 10% fetal bovine serum (FBS; HyClone), 100 U / ml type IV collagenase (Life Technologies), and 50 μg / ml DNase I (Roche). The suspension was incubated at 37 °C for 45 minutes and then further dissociated mechanically. Red blood cells were removed from the sample using red blood cell lysis buffer (Biolegend). The sample was pelleted and then resuspended in fresh RPMI + 10% FBS and filtered through a 40 μm filter. Cells were incubated with Live / Dead Zombie NIR (Biolegend) for 5 minutes at room temperature in the dark. Fc receptors were blocked using Human TruStain FcX blocking reagent (Biolegend) prior to surface antibody staining. Cells were stained in the dark on ice for 15 minutes and washed twice with PBS + 2% FBS. Cells were analyzed on a BD LSRFortessa using FACSDiva software (BD Biosciences). Data were analyzed using FlowJo software version 10.5.3. Antibodies are described in parentheses by clone, manufacturer, catalog number for the protein target, CD69 (FN50, BioLegend, 310904), CD16 (3G8, BioLegend, 302006), CD8 (RPA-T8, Thermo Fisher, BDB560662), CCR2 (K036C2, Biolegend, 357203), CD38 (HIT2, BioLegend, 303506), CD11c (3.9, BioLegend, 301605), CCR7 (150503, Thermo Fisher, BDB62381), CD56 (GDC56, BioLegend, 318348), LAG-3 (11C3C65, BioLegend, 369309), CD103 (B-Ly7, Thermo Fisher, 25-1038-41), TIM-3 (F38-2E2, BioLegend, 345012), PD-L1 (29E.2A3, BioLegend, 329708), CD3 (UCHT1, BioLegend, 300424), PD-1 (EH12.They are listed as follows: 2H7 (BioLegend, 329920), HLA-DR (G46-6, Thermo Fisher, BDB562804), CD45RA (HL100, BioLegend, 304142), CD15 (SSEA-1, BioLegend, 323028), CTLA-4 (BNI3, BioLegend, 369609), CD19 (HIB19, BioLegend, 302243), CD45 (H130, BioLegend, 304050), CD4 (PRA-T4, BioLegend, 300554), CD14 (M5E2, BioLegend, 301840), Mesothelin (REA1057, Miltenyi, 130-118-168), STING (D2P2F, Cell Signaling, 13647), pTBK1 (D52C2, Cell Signaling, 5483), TBK1 (Polyclonal, Cell Signaling, 3013), pIRF3 (4D4G, Cell Signaling, 4947), IRF3 (D6I4C, Cell Signaling, 11904), pSTAT1 (58D6, Cell Signaling, 9167), STAT1 (Polyclonal, Cell Signaling, 9172), IFNAR-1 (Polyclonal, Thermal Fischer, PA5-79441), and β-actin (C4, Santa Cruz, sc-47778).

[0112] Patient-derived organotypic tumor spheroids (PDOTS). PDOTS were generated as described above (Jenkins et al., Cancer Discov. 8:196-215 (2018); Aref et al., Lab Chip 18:3129-3143 (2018). Briefly, fresh tumor specimens were minced in a 15 mL Falcon tube in complete medium preheated to 37°C (DMEM + 10% FBS from Thermo Fisher Scientific) + 100 U / mL type IV collagenase (Life Technologies) and 50 μg / mL DNase I (Roche) using sterile scissors and pipetting for approximately 20 minutes. The dissociated material was filtered through 100-μm and 40-μm filters to generate S1 (>100 μm), S2 (40-100 μm), and S3 (<40 μm) spheroid fractions, which were subsequently maintained in ultra-low attachment (ULA) tissue culture plates (Corning). The S1 fraction was treated with 50 μM ADU-S100 (Chemietek) for cytokine analysis and single-cell RNA sequencing. The S2 fraction was resuspended in type I rat tail collagen (Corning) at a concentration of 2.8 mg / mL and incubated at 37°C for 40 minutes in a humidified chamber to polymerize prior to loading into the central gel region of a 3-D microfluidic culture device (AIM Biotech) for ex vivo culture. Collagen hydrogels containing PDOTS were hydrated with medium, with or without the indicated treatments. TAK-676 was provided by Takeda and diluted in dH20. Recombinant human interferon beta (100 ng / mL; R&D Systems) was used as a positive control downstream of STING for STAT1 pathway activation. CD8a was neutralized with 50 μg / mL InVivoMAb antibody against an IgG control (BE0092). CXCR3 was neutralized with 5 μg / mL human CXCR3 antibody (R&D MAB160).

[0113] PDOTS immunofluorescence and live / dead quantification. Double labeling was performed by loading the microfluidic device with Nexcelom ViaStain acridine orange / propidium iodide (AO / PI) staining solution (Nexcelom, CS2-0106) or a 10 μg / mL solution of Hoechst 33342 (Thermo Fisher Scientific) and a 1 μg / mL solution of PI (Thermo Fisher Scientific). After incubation with the dyes (20 minutes at room temperature in the dark for AO / PI or 45 minutes for Hoechst 33342 / PI), images were captured using a 4x objective lens of a Nikon Eclipse 80i fluorescence microscope equipped with an automated motorized stage (Proscan), a Z-stack (Prior), and a Zyla 5.5 sCMOS camera (Andor). Image capture and analysis were performed using the NIS-Elements AR software package. Quantification of live and dead cells was performed by measuring the total cell area of each dye. For further immunofluorescence studies, PDOTS were washed with PBS and blocked with an FcR blocking reagent (Miltenyi) for 30 minutes at room temperature. Direct conjugate antibodies CD326 EpCAM-AlexaFluor647 (clone 9C4), CD45-AlexaFluor647 (HI30) (BioLegend), and mesothelin-PE (clone REA1057, Miltenyi) were diluted 1:50 in a 10 μg / mL solution of Hoechst 33342 (Thermo Fisher Scientific) in PBS and loaded onto the microfluidic device for 1 hour incubation in the dark at room temperature. The spheroids were washed twice with PBS containing 0.1% Tween20 and then with PBS. For viability assessment, a 1:1,000 solution of calcein AM (Thermo Fisher Scientific) in PBS was loaded onto the microfluidic device. For IRF3 IF, PDOTS were treated with dH20 control or 50 μM ADU-S100 for 3 hours, washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton-X for 10 minutes.Cell Signaling Antibody #11904 (clone D6I4C) was diluted 1:50 in PBS, incubated for 45 minutes, washed, and then incubated for 30 minutes in FITC-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific) diluted 1:100. PDOTS were washed twice with PBS containing 0.1% Tween 20 and counterstained with 1 μg / mL Hoechst 33342 solution. Images were captured for live / dead dual staining as described above using a 20× objective lens.

[0114] Cytokine analysis. CXCL10 ELISA (R&D Systems DIP100) and Granzyme B ELISA (R&D Systems DY008) were performed according to the manufacturer's instructions on conditioned media collected from cell cultures. Cytokine analysis of conditioned media 3 days after explant (S1) culture (Figure 9B) utilized the MSD U-PLEX Viral Combo 1 assay (Hu:K15343K-2), which was performed according to the manufacturer's instructions.

[0115] 2’3’cGAMP ELISA. The Cayman Chemical 2’3’cGAMP ELISA kit was performed according to the manufacturer's instructions to detect the levels of 2’3’cGAMP in the supernatants of MPM cell lines. For these experiments, 3 - 5×10 5 cells were plated in 6-well plates and transfected with X-tremeGENE HP DNA transfection reagent combined with Opti-MEM Reduced-Serum medium and 1 μg of poly(dG:dC) (Invivogen) in a 30-minute incubation. 2’3’cGAMP (Invivogen) was used as a positive control.

[0116] Cell culture. The MPM cell lines were cultured in RPMI-1640 (Thermo Fisher Scientific) supplemented with 10% FBS (Gemini Bio-products). H226, H28, MSTO-211H, H2452, and H2052 were purchased from ATCC. MS428 was provided by the Richards Lab. H2461 and H2591 were provided to Dr. Janne by the NIH (Pass et al. Thorac. Surg. 59:835-44 (1995)). JMN1B (Demetri et al., Blood 74:940-6 (1989)) and MS589 (Gordon et al., Am. J. Pathol. 166:1827-40 (2005)) were derived at BWH / DFCI and shared internally under license. All experiments were performed before reaching passage 10. Mycoplasma infection was regularly checked by PCR using the conditioned media from each cell line with previously described primers (Kitajima et al., Cancer Discov. 9:34-45 (2019)).

[0117] Immunoblotting. Cells were lysed in RIPA buffer containing 1× protease inhibitor (Roche 11-836-145-001) and phosphatase inhibitors (50 mmol / L NaF and 100 mmol / L Na3VO4). Immunoblotting was performed according to the method previously described (Kitajima et al., Cancer Discov. 9:34-45 (2019)) using the antibodies shown in Table 1. The secondary antibodies were from LI-COR Biosciences: IRDye 680LT goat anti-mouse IgG (#926-68020) and IRDye 800CW goat anti-rabbit IgG (#926-32211). Blot imaging was performed using the LI-COR Odyssey system.

[0118] Dynamic single-cell RNA sequencing and data analysis. A previous protocol (Sehgal et al., J. Clin. Invest. 131:e135038 (2021)) was adapted to test S1 explants from MPM PDOTS. The sample tested (#26) showed a 63% baseline viability and 18-hour cytokine release in response to treatment (Figure 10B). After 24-hour treatment in a ULA dish, tumor spheroids were digested with trypsin for 5 minutes in a 37°C incubator to obtain a single-cell suspension. Cells were loaded onto a 10× Chromium instrument (10× Genomics) according to the manufacturer's instructions. The ScRNA library was generated using the Single Cell 3' Reagent Kit (10× Genomics) according to the user guide. Quality control of the completed library was performed using the Bioanalyzer High Sensitivity DNA Kit (Agilent) and then sequenced using the Illumina NextSeq 500 platform.

[0119] Raw sequencing reads were processed using the 10× Genomics CellRanger bioinformatics pipeline v6.0.1. The assembled matrix was then fed into the standard workflow of the R package, Seurat v4.0.4. Only genes expressed in at least 3 cells and cells expressing at least 2 genes were retained for downstream processing. Additionally, cells expressing more than 7000 genes and cells with more than 10% of UMIs mapped to mitochondrial genes were excluded from the analysis. All samples were prepared and sequenced together on the same platform. The filtered matrix was log-normalized using global scaling in Seurat. UMI and mitochondrial transcript content were used as regression parameters. The normalized matrix was scaled and centered gene-wise and then dimensionality reduction was performed using principal component analysis (PCA) for highly variable genes. After visual inspection of the PCA elbow plot, the top 10 PCs were selected for further analysis. Clustering was performed on the selected PCs using Seurat's shared nearest neighbor algorithm with default parameters.

[0120] Using Seurat's DimPlot module, Uniform Manifold Approximation and Projection (UMAP) maps were calculated and plotted. Cluster differential expression analysis was performed in Seurat using the FindMarkers command with a Wilcoxon rank sum test without a threshold. Contour plots overlaid on the UMAP were created using the R package ggplot2 (Wickham, Springer-Verlag New York (2016)).

[0121] Cell types were identified based on a comparative analysis of previously published signatures (Han et al., Cell 172:1091-1107(2018); Muhl et al., Nat. Commun. 11:3953(2020); Correia et al., Proc. Natl. Acad. Sci. U.S.A. 115:E5980-E5989(2018); Gueugnon et al., Am. J. Pathol. 178:1033-42(2011)) and marker genes identified in this study, and were used to remove genes that are ubiquitously expressed across cell subpopulations. Genes encoding collagen were added to the fibroblast signature. A list of gene signatures used for enrichment analysis is shown in Table 1.

[0122]

Table 1

[0123] Isolation of tumor-infiltrating lymphocytes. TILs were isolated from patient specimens under IRB protocol 02-180 and filtered as described above for PDOTS. The S3 fraction was expanded in 24-well plates using RPMI-1640 containing L-glutamine, 1% penicillin-streptomycin solution, 1 mM sodium pyruvate, 0.0375% sodium bicarbonate, 50 nM mercaptoethanol, 10% human AB serum and 6000 U / mL IL-2, and split 1:2 every other day over an 8- to 10-day period. After expansion, they were frozen / stored in liquid nitrogen.

[0124] Proliferation and transduction of primary T cells. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors after informed consent and isolated by using Ficoll density centrifugation. The isolated PBMCs were activated with TransAct (1:100, Miltenyi) in complete medium (RPMI-1640 supplemented with 10% FBS in the presence of 10 ng / ml of IL-2). Two days after activation, the T cells were transduced lentivirally by spinoculation with BCMA CAR virus (1% virus volume) in the presence of Lentiboost (1:100, Sirion Biotech). The BCMA CAR sequence has been described previously (Works et al., Mol. Cancer Ther. 18:2246-2257 (2019)), cloned into the pHAGE lentiviral vector (Addgene plasmid #24526), and the generated plasmid was subjected to sequencing verification. For packaging and production of lentiviral particles, 293 Lenti-X packaging cells (Takara) were seeded in 15-cm plates at 8×10 6 cells / plate) and left for 24 hours, after which the plasmid encoding the CAR (pHIV-aMeso-CAR), the plasmid encoding the VSV-G envelope pMD.2G, and the packaging vector psPAX2 were transfected using PEI transfection reagent (Polysciences). The viral supernatant was collected 24 and 48 hours after transfection, filtered through a 0.45-μm membrane, concentrated by ultracentrifugation, and stored at -80°C before transduction. After transduction, the T cells were grown in RPMI-1640 supplemented with 10% FBS using the cytokines IL-2 (10 ng / ml), IL-7 (3 ng / ml), and IL-15 (10 ng / ml), and their transduction efficiency was determined by FACS 3 days after transduction.

[0125] Proliferation and transduction of primary NK cells. For experiments using non-manipulated primary NK cells, CD56+CD3− NK cells were expanded from human PBMCs (Lonza) using the CellXVivo Human NK Cell Expansion Kit (R&D Systems). After 14 days of proliferation, the cells were transferred to culture in CTS OpTmizer T cell expansion medium supplemented with 5% human AB serum (Sigma Aldrich), 1% GlutaMAX, 1% HEPES, and 1% penicillin-streptomycin in the presence of IL-2 (PeproTech or Miltenyi; 200 U / mL for flow cytometry experiments, 500 U / mL for killing experiments including PDOTS). All NK cell culture reagents were purchased from Life Technologies unless otherwise specified.

[0126] For experiments using transduced and control-treated primary NK cells, they were isolated from leukapheresis under an approved Crimson Study protocol T0197 using RosetteSep (StemCell technologies) and Ficoll-Paque density gradient centrifugation. The isolated NK cells were tested for purity and cultured for 2 days in RPMI (Gibco) supplemented with 10% heat-inactivated (HI)-FBS (Gibco), 1% penicillin-streptomycin, 2 mM L-glutamine and HEPES in the presence of IL-15 (1 ng / mL; Miltenyi). The isolated NK cells were then either transduced as follows or cultured in NK MAC medium (Miltenyi) supplemented with 5% human serum (Sigma) and 1% v / v penicillin-streptomycin (Gemini Bio-products) in the presence of IL-2 (500 U / mL; Miltenyi).

[0127] CAR construct. The CAR construct was designed using an extracellular ScFv domain and a transmembrane segment derived from the CD8 protein. This was followed by the conventional 4-1BB and CD3 co-stimulatory domains. The CAR gene was designed to incorporate an HA tag for analysis using flow cytometry. For the gene construct having CXCR3, a P2A self-cleaving peptide nucleic acid and CXCR3 followed the CAR gene. To generate CAR or CAR-CXCR3 NK cells, primary NK cells were purified from peripheral blood and activated using IL-12, IL-15, and IL-18, which resulted in the activation and differentiation of NK cells and the generation of cytokine-induced memory-like (CIML). Conventional NK cells (cNK) maintained with low-dose IL-15 (1 ng / mL) were used as a control. The CAR gene was transduced into cNK or CIML NK cells via the inventors' optimized baboon endogenous retrovirus system to achieve a high transduction efficiency. Anti-mesothelin CAR (αMSLN) was constructed in the pHIV backbone using a mesothelin-specific ScFv derived from the YP218 antibody, followed by a transmembrane domain and co-stimulatory domains (4-1BB and CD3ζ), as shown in Figure 30A. The construct also contained an EGFP fragment separated from the CAR fragment by self-cleaving P2A (Figure 16A). The CAR gene construct was packaged into the BaEV pseudotyped lentivirus system by transfecting HEK-293 cells with pCMV-BaEV, pCMV-Δ8.9, and pAdv plasmids. Virus particles were titrated using Jurkat cells. Assuming a multiplicity of infection (MOI) of 1 for Jurkat cells, the virus titer was calculated and NK cells were transduced at an MOI of 10. NK cells were transduced using RetroNectin and Vectofusin, followed by spinfection with + / - active lentivirus (virus-free cNK control) 2 days after extraction, and then cultured in NK MAC medium (Miltenyi) supplemented with 5% human serum and 1% penicillin-streptomycin (Gemini Bio-products) in the presence of IL-2 (500 U / mL; Miltenyi).The percentage of NK cells expressing CAR was determined by flow cytometry analysis of surface expression of GFP and ScFv using APC human lectin (HA).

[0128] Immunocytotoxicity assay. For the flow cytometry immunocytotoxicity assay, primary NK cells and TIL were seeded into 96-well plates alone or in co-cultures at 200,000 cells per well (NK or TIL alone, or 1:1 of 100,000 cells of each type) and treated for 72 hours with 10 μM or 50 μM ADU-S100 (Chemietek) or dH2O control with or without the indicated concentration of IL-2 (Miltenyi or PeproTech). After treatment, samples were stained with anti-CD45, anti-CD3, anti-CD4, anti-CD8, and anti-CD56 antibodies (Table 1), and Zombie Green live / dead (Biolegend 423111) and analyzed by flow cytometry as described above. Data were analyzed using FlowJo software version 10.5.3. As an orthogonal measure of viability, CellTiter-Glo was performed on primary T cells and NK cells.

[0129] CellTiter-Glo Luminescent Cell Viability Assay. Cell viability was evaluated using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571) according to the manufacturer's instructions. For non-transduced primary T cells and BCMA CAR T cells, 25,000 cells per well were seeded in 96-well plates and treated with ADU-S100 or dH2O as a control at the indicated concentrations for 24 hours. For NK cells, 25,000 cells per well were seeded and treated with ADU-S100 or dH2O as a control at the indicated concentrations for 24 hours. For MPM cell lines, 10,000 cells / well (MS428) or 12,500 cells / well (H2461, H2591) were seeded in 96-well plates and treated with 50 μM ADU-S100 or medium as a control for the indicated times. All conditions were tested in triplicate and the plates were read on a Tecan Infinite Mplex Microplate Reader.

[0130] Autophagy staining. Autophagy was evaluated by vacuole staining to identify autophagolysosomes using the CYTO-ID Autophagy Detection Kit 2.0 (Enzo ENZ-51031-0050) according to the manufacturer's instructions. Briefly, 5×10 5 isolated primary NK cells or TILs were incubated in T cell growth medium (TCGM) containing 500 U / mL of IL-2, which was refreshed every time the medium was changed to ensure proper growth and selection. CLQ from the kit (Enzo 51005-CLQ) was used starting at the recommended initial dose of 10 μM compared to a DMSO control. After 24 hours, the medium was changed and the cells were treated with CLQ + 10 μM ADU-S100 for an additional 24 hours. The medium was collected, stained according to the manufacturer's instructions using CYTO-ID Green Detection Reagent 2, and then flow cytometry was performed.

[0131] NK cell killing assay. Target cells (MPM cell lines) were detached by trypsin treatment, labeled with CellTrace Violet (CTV, Life Technologies), and then seeded in 96-well plates at a cell density of 25,000 cells / well. The target cells were allowed to adhere for 12 - 16 hours, and then NK cells or αMSLN-CAR-NK cells were added at different effector-to-target (E:T) ratios (1:1, 2:1, 5:1, and 10:1) with or without ADU-S100 (50 μM). After 6 hours of co-culture, the cells were harvested and incubated with an antibody against the apoptosis marker annexin V (PE) and the live / dead stain 7-AAD (Biolegend). The cells were analyzed on a BD LSRFortessa using FACSDiva software (BD Biosciences). The data were analyzed using FlowJo software version 10.5.3. Apoptotic cells were evaluated by gating on the CTV+ population and represented as the percentage of viable or dead (late apoptotic) cells. The apoptotic cell analysis was performed using NK cells extracted from four different healthy donors per target MPM cell line to account for baseline donor variability.

[0132] NK cell infiltration assay. Immune cell infiltration was evaluated as described above (Kitajima et al., Cancer Discov. 9:34 - 45 (2019); Mahadevan et al., Cancer Discov. 11:1952 - 1969 (2021)). Briefly, mesothelioma cancer cell spheroids (H2591, H2461, H226) were suspended in ULA dishes at 5 × 10 5Generated by seeding individual cells for 24 hours. H226 cells were treated with 50 μM ADU-S100 during the last 6 hours of spheroid formation to establish a cytokine gradient. The samples were then pelleted, 10× PBS containing phenol red was added, the pH was adjusted using NaOH, and then resuspended in type I rat tail collagen (Corning) at a concentration of 2.5 mg / mL. The pH was confirmed to be 7.0 - 7.5 using PANPEHA Whatman paper (Sigma-Aldrich). The cells and collagen were maintained on ice to prevent polymerization. The spheroid-collagen suspension was then injected into the central gel region of a 3D DAX-1 microfluidic cell culture chip (AIM Biotech). The microfluidic device was utilized as described above (Aref et al., Lab Chip 18:3129-3143 (2018)), and the central region contained the cell-collagen mixture in a 3D microenvironment (3×10 4 cells H2591 and H2461, 2×10 4 cells H226), and two media channels were adjacent. After injection, the collagen hydrogel containing the cells was incubated at 37 °C for 40 minutes in a humidified chamber, then hydrated with culture medium, and labeled primary NK cells (E:T ratio 2:1) were added to one of the side channels. The primary NK cells were labeled with Cell Tracker Red (Thermo Fisher Scientific) according to the manufacturer's instructions. After 96 hours of incubation, viability staining of cancer cell spheroids and infiltrating immune cells was performed (incubated with a 1 μg / mL propidium iodide solution for 20 minutes; Thermo Fisher Scientific). For experiments using the CXCR3 neutralizing antibody (R&D MAB160), the NK cells were pretreated for 30 minutes before loading.

[0133] For quantification, images were captured with a Nikon Eclipse 80i fluorescence microscope equipped with a Z-stack (Prior) and a CoolSNAP CCD camera (Roper Scientific). Image capture and analysis were performed using the NIS-Elements AR software package. Images of the entire device were obtained by stitching multiple captures. Quantification of immune cell infiltration into the 3D tumor microenvironment was performed by measuring the total cell area of the cell-tracking dye throughout the gel region. In experiments using the CXCR3 neutralizing antibody (R&D MAB160; Figure 6B), staining was quantified in a square region at the center of the channel to focus on the effect of the CXCR3 ligand released by tumor cells. Quantification of the proportion of dead cells was performed in the same manner as PDOTS described above.

[0134] 3D vascular model. To generate the tumor-vascular model, H226 spheroids were mixed with collagen rat tail hydrogel (2.5 mg / ml) and injected into the central gel region of the 3D microfluidic chamber (10 - 15 μL per microfluidic chamber). After incubating at 37°C for 30 minutes in a sterile humidified chamber, the side walls of the channel located on one side (media channel) were coated with a 150 μg / ml collagen solution in PBS to enable better adhesion of ECs to the channel. After 15 minutes, the channel was washed once with media. To create the 3D vessel, 25 μL of a cell suspension of human umbilical vein endothelial cells (HUVEC; C2519AS, Lonza) at 3×106 cells / ml was injected into the collagen-coated media channel. The channel was rotated twice to create a confluent hollow lumen 3D blood vessel. The chip was incubated downward with the cells for 15 minutes to attach the cells to the media-gel interface and form a monolayer. Next, 50 μL of the cell suspension was reinjected and the chip was flipped to cover the top of the 3D blood vessel channel. After incubating for 90 minutes in a humidified chamber at 37°C, cell culture media was gently added to both channels and further incubated to form a confluent monolayer. After angiogenesis, NK cells (labeled with a cell tracker) were added to the 3D blood vessel at an E:T ratio of 2:1. Treatment with a STING agonist (ADU-S100, TAK-676) was added to the fluid channel on the opposite side of the vascular barrier. NK cell migration + / - blood vessels was quantified over 24 hours. Image capture and analysis were performed using a fluorescence confocal microscope and processing software. The 3D blood vessel channel was rinsed in PBS and fixed with 4% PFA at room temperature for 15 minutes. The cell membrane was permeabilized with 0.1% Triton X-100 for 5 minutes at room temperature and washed twice with PBS. HUVEC cells were stained for F-actin with green phalloidin (Thermo Fisher Scientific A12379) and Hoechst 33342. Images were captured with a Nikon Eclipse 80i fluorescence microscope equipped with a Z-stack (Prior) and a CoolSNAP CCD camera (Roper Scientific).

[0135] Immune cell migration assays were performed as previously described (Kitajima et al., Cancer Discov. 9:34-45(2019)). Briefly, NCI-H226 cells were cultured at 5 × 10 per well of a 6-well plate. 5 Cells were plated at a density of 5 × 10 and treated with STING agonist (ADU-S100) at 50 μM for 24 h. Spheroids were grown at a density of 5 × 10 5 Spheroids were generated by seeding in ultra-low attachment dishes for 24 h and labeled with a fluorescent dye (cell proliferation dye eFluor 450, Invitrogen, 65-0842) according to the manufacturer's instructions. Spheroids were pelleted and then resuspended in type I rat tail collagen (Corning) at a final concentration of 2.5 mg / mL after adding 10x PBS containing phenol red on ice. The pH of the resulting spheroid suspension was adjusted to 7.0-7.5 using NaOH and confirmed using PANPEHA Whatman paper (Sigma-Aldrich). The spheroid-collagen suspension was then introduced into the central channel of a 3-D microfluidic cell culture chamber (AIM Biotech) of previously described design (Aref et al., Lab Chip 18:3129-3143(2018)). The collagen hydrogel containing the cancer cell spheroids was incubated for 40 min at 37 °C in a humidified chamber, after which RPMI-1640 medium containing NK cells at an effector-to-target (E:T) ratio of 2:1 was perfused through one of the side channels located next to the central channel. After co-culture of the cancer cell spheroids and NK cells for 3 days, the migration of NK cells into the collagen hydrogel was visualized through images taken with a Nikon Eclipse 80i fluorescence microscope equipped with a Z-stack (Prior) and a CoolSNAP CCD camera (Roper Scientific) and analyzed using the NIS-Elements AR software package. Quantification of immune cell infiltration into the central channel was performed using Cell 100 μM (C100 μM) located in the region of interest (ROI; 6 ROIs / microfluidic cell culture chamber). This was done by measuring the total area occupied by Tracker Red dye positive cells.

[0136] Statistical analysis. Statistical significance was evaluated using unpaired two-sided Student's t-test for pair-wise comparison, one-sample t-test against an expected value of 0% change or 100% control, or one-way ANOVA followed by Tukey's post hoc test. For non-parametric analysis of IHC scores and mRNA expression in MPM cell lines, the Kruskal-Wallis global test followed by Dunn's multiple comparison post hoc test was used (data obtained from the Cancer Cell Line Encyclopedia of the Broad Institute). P-values less than 0.05 were considered significant. Asterisks used to indicate significance correspond to *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Columns represent mean ± SD. In one-way ANOVA followed by post hoc test, asterisks are shown only in pairs of the items of interest. GraphPad Prism (version 9.2.0) was used for all statistical analyses.

[0137] Example 2: STING is primed for activation in malignant pleural mesothelioma MPM

[0138] This specification describes the discovery that malignant pleural mesothelioma strongly expresses tumor cell STING and responds to STING agonist treatment ex vivo. Dynamic single-cell RNA sequencing of explants treated with STING agonist revealed mainly CXCR3 chemokine activation in tumor cells and cancer-associated fibroblasts, as well as cytotoxicity of T cells. In contrast, primary NK cells were resistant to STING agonist-induced cytotoxicity. STING agonist enhanced the migration and killing of NK cells, especially anti-mesothelin chimeric antigen receptor (CAR)-NK cells, and improved therapeutic activity. These studies demonstrate the fundamental importance of using human tumor samples to evaluate innate and cellular immunotherapies. The clear results of STING agonist treatment in humans were revealed by functionally profiling mesothelioma tumor explants with elevated tumor cell STING expression, supporting combination with NK cell and CAR-NK cell therapies.

[0139] Advances in the study of human TIME using patient samples have enabled the development of immunotherapies by treating patient-derived organotypic tumor spheroids (PDOTS) and tissue fragment explants in short-term culture (Jenkins et al., Cancer Discov. 8:196-215 (2018); Voabil et al., Nat. Med. 27:1250-1261 (2021)). To date, these platforms have focused on anti-PD(L)-1 immune checkpoints, which can run in parallel with patient responses, but they also offer promise in designing new cancer immunotherapies. In contrast to patient-derived xenografts grown in humanized mouse models, these systems provide the ability to examine immune responses within the native human tumor immune constitution and have the potential to study cell therapies without interference from the murine microenvironment. Furthermore, insights gained from direct modeling of human TIME can also help close the translational gap for immunotherapies that are effective in syngeneic mouse models but fail in clinical trials.

[0140] Multiple human cancer types have recently been shown to silence STING and downstream interferon responses to evade immune detection (Ghosh et al., Cancer Cell 39:494-508(2021); Kitajima et al., Cancer Discov. 9:34-45(2019); Lau et al., Science 350:568-71(2015)), clearly demonstrating an important role for tumor cell STING signaling in human cancers. Higher STING expression also correlates with better responses to therapy across cancer types (Hayman et al., Nat. Commun. 12:2327(2021); Zugazagoitia et al., Clin. Cancer Res. 26:4360-4368(2020); Qi et al., Biosci. Rep. 40:BSR20202603(2020); Chon et al., J. Cancer 10:4932-4938(2019)). However, how STING agonists affect tumor cells and different cell types in the human TIME has not been carefully investigated and could inform the development of new treatment combinations, including cell therapies. This problem is addressed herein by pursuing large-scale studies using PDOTS and developing methodologies for performing dynamic single-cell RNA sequencing in tumor explants, as well as by scrutinizing STING agonist responses in inflammatory tissue types.

[0141] To identify human tumor histotypes with intact STING, we performed immunohistochemical profiling of 300 archival samples from diverse thoracic malignancies (Figure 1A). Among the malignancies evaluated, MPM expressed the highest levels of STING. MPM arises from the inner layer of the lung serosa and confers a poor prognosis despite the recent introduction of combinations of checkpoint immunotherapies to standard treatment (Janes et al., N. Engl. J. Med. 385:1207-1218(2021)e). MPM demonstrated near-universal high expression of STING protein in tumor and stromal cells, in contrast to non-small cell lung cancer (NSCLC), thymoma, and especially small cell lung cancer (SCLC) (Mahadevan et al., Cancer Discov. 11:1952-1969(2021); Canadas et al., Nat. Med. 24:1143-1150(2018)) (Figure 1A and Figure 7A). STING was also highly expressed in benign pleura, consistent with its baseline upregulation in the mesothelial cell type (Figure 7B). MPM-derived cell lines expressed high levels of STING protein, but neither cell lines nor tumors demonstrated baseline cGAS-STING pathway activation as measured by phosphoro-IRF3, CXCL10, and IFIT1 expression and secreted 2’3’-cGAMP (Figure 7C, Figure 8A–Figure 8D). Flow cytometry–based immunoprofiling of a large panel of resected MPM specimens further demonstrated strong immune infiltration in most tumors but features of exhaustion across multiple immune cell subsets, including heterogeneous expression of the checkpoint proteins PD-1, TIM-3, and LAG-3 (Figure 1B–Figure 1D and Figure 7D–Figure 7E) (Awad et al., Cancer Immunol. Res. 4:1038-1048(2016)). Characterization of T cells revealed terminal differentiation consistent with exhaustion; subtype characterization of monocytes / macrophages demonstrated abundance of intermediate cells; and characterization of NK cells demonstrated decreased cytotoxic capacity (increased CD56 bright / CD16 low compared with circulating NK cells; Figure 1D).Therefore, MPM expresses high levels of STING, causes inflammation, and exhibits a depleted TIME.

[0142] Despite lacking baseline STING pathway activation, multiple MPM cell lines treated with the clinical STING agonist ADU-S100 (Corrales et al., Cell Rep. 11:1018-30(2015); Amouzegar et al., Cancers(Basel) 13:2695(2021)) showed robust pathway activation and secreted high levels of CXCL10 (Figs. 8A-8D). MPM cell lines with minimal response to clinical STING agonists in vitro showed reduced IRF3 transcription factor expression (Figs. 8A, 8D, and 8E). However, overall, high levels of inactive basal STING and significant induction of CXCL10 release by STING agonism suggest that STING signaling is primed to respond in MPM. Next, STING agonism in human tumor specimens was analyzed using a newly resected MPM tumor explant model that retains the relevant TIME (Fig. 2A) (Jenkins et al., Cancer Discov. 8:196-215(2018)). After treatment, PDOTS of 40-100 μm (S2) were suspended in collagen and treated for 6 days, and the response was evaluated by live / dead immunofluorescence and cytokine production (Jenkins et al., Cancer Discov. 8:196-215(2018)). ADU-S100 induced CXCL10 release and strong killing of PDOTS 6 days after treatment in specific samples (Figs. 2A-2B and 9A-9B). Fig. 2B shows the quantification of the cell area and survival / death ratio of each stain. t-test vs dH20 control: **p<0.01, ****p<0.0001. Scale bar = 100 μm. Importantly, STING activation in MPM cell lines cultured in vitro did not cause cytotoxicity and suggested a contribution from the TIME (Fig. 8F). Overall, treatment of 35 patient specimens with ADU-S100 showed statistically significant cell death >20% above the control in 12 / 35 (34%) patient specimens (p<0.05), and 7 specimens approximated clinical criteria for response with a >30% decrease in cell area (p<0.05) (Figs. 2C-2D and 9C).Figure 2C shows responses based on the criteria of a live cell area (≥30% decrease) and a ≥20% increase in cell death, with p < 0.05 by t-test between the treated triplicate wells, ADU-S100 vs dH2O control. Figure 2D shows responses by decrease in live cell area (I), epithelioid MPM (E), biphasic MPM (B), yes / no (Y / N) neoadjuvant treatment, and male / female (M / F). There was a non-significant trend towards a higher response in patients who received neoadjuvant chemotherapy (HR 1.4, 95% CI 0.23 - 7.27, chi-square p = 0.72), and the response was not correlated with histology (testing epithelioid and biphasic specimens), age, or gender (Table 2). Additionally, treatment with TAK-676, a next-generation systemic STING agonist at the second clinical stage, also showed responses in 4 / 13 (31%) of patient specimens and promising activity ex vivo (Figure 2F). A potential correlation was observed between tumor CD8 abundance and ex vivo treatment response to ADU-S100 (R2 = 0.35, p < 0.05; Figure 10A), prompting testing of the effect of CD8 neutralization. Indeed, anti-CD8 antibody treatment partially rescued ADU-S100 cytotoxicity in three patient specimens (Figures 2E - 2G and Figures 10A - 10B), motivating the development of a higher-resolution approach to understand the effect of STING agonism on different cells within the TIME.

[0143]

Table 2

[0144] Example 3: Dynamic scRNAseq of MPM explants.

[0145] Using a validated methodology (Sehgal et al., J. Clin. Invest. 131:e135038 (2021)) for performing dynamic single-cell RNA sequencing (scRNAseq), we focused on specimens that showed modest dose-dependent killing in response to ADU-S100 after 24-hour STING agonist treatment, which was rescued by CD8 neutralization (Figures 3A–3D, 10B, 11A–11D, and 12A–12C). The fraction bar graph in Figure 3D shows each cluster by treatment, normalized to the number of cells per sample. Pre-treatment flow cytometry profiling demonstrated the mean proportion of T cells and a monocyte / macrophage population above the mean (Figures 1B and 10C). IRF3 immunofluorescence also showed nuclear translocation after ADU-S100 treatment, confirming effective STING activation in PDOTS (Figure 10E). For this short-term scRNAseq analysis, tumor fragments larger than 100 μm suspended in media were used, and size filtration was confirmed not to change the leukocyte composition of each fraction (Jenkins et al., Cancer Discov. 8:196–215 (2018)) (Figure 10D). UMAP clustering validated the broad representation of tumor cells, fibroblasts, and immune cell populations (Figures 3A, 11A–11B). Interestingly, ADU-S100 strongly induced mainly CXCR3 ligand expression (CXCL9, CXCL10, CXCL11) in subsets of mesothelin (MSLN)-positive tumor cells (cluster 1) and cancer-associated fibroblasts (CAF; clusters 4, 5) compared to the myeloid cell population (clusters 7, 8; Figures 3B–3C). This analysis also revealed a strong and unique STING agonist-induced IL-33 expression in CAF, while other ISGs such as IFIT1 showed more widespread expression across cell populations, confirming broad target engagement (Figure 3C). Differential expression analysis showed that the subset of MPM cells (cluster 1) that most highly expressed CXCR3 ligands in response to ADU-S100 also showed increased numbers of ISGs and downregulated TGFB1 expression compared to other MPM cells (clusters 0, 3) (Figures 11C, Tables 3–4).Multiple granzyme- and perforin-positive T cells and NK cells were identified within cluster 2 (Figure 11D), consistent with the effects of CD8 neutralization and the potential contribution of T cell- and / or NK cell-mediated killing. However, analysis of the abundance of cells from each individual sample (contour and fraction plots in Figures 3D and 12A–12C) demonstrated depletion of the CD8-positive cell population, including Tregs, after high-dose STING agonist treatment, in contrast to the increased STING agonist response in MPM cluster 1. Analysis of NK cell ligands in tumor cells showed a dose-dependent increase in the ADU-S100 expression of HLA-A / B / C (inhibitory), conjugated with an increase in NECTIN2 (CD112; activating), in parallel with a decrease in the expression of the predominantly inhibitory ligand PVR (CD155) (Lupo et al., J. Hematol. Oncol. 13:76 (2020)) (Figure 12B). These data reveal a prominent role of tumor cells and CAFs as targets of STING agonism, promotion of the release of T cell- and NK cell chemotactic factors, and changes in the tumor / CAF cell state. However, these results are also consistent with reports that excessive STING activity can be toxic to T cells (Cerboni et al., J. Exp. Med. 214:1769-1785 (2017); Larkin et al., J. Immunol. 199:397-402 (2017); Gulen et al., Nat Commun. 8:427 (2017)), suggesting that STING-mediated enhancement of tumor CXCR3 chemokine release can be counteracted by cytotoxicity in immune effector cells.

[0146]

Table 3

[0147]

Table 4

[0148] Example 4: STING agonists are toxic to human T cells.

[0149] To further explore this observation, STING-induced cytotoxicity in T cells (Cerboni et al., J. Exp. Med. 214:1769-1785 (2017); Larkin et al., J. Immunol. 199:397-402 (2017); Gulen et al., Nat. Commun. 8:427 (2017)), as well as cytotoxicity in other immune cell types, was evaluated using the models described herein. ADU-S100 treatment was cytotoxic to T cells as measured by flow cytometry in MPM tumor explants, in contrast to downstream IFNβ exposure, which increased over 24 to 72 hours of STING agonist treatment (Figures 4A and 13A). T cells purified from peripheral blood with or without expression of the B cell maturation antigen chimeric antigen receptor (BCMA CAR) also showed dose-dependent cytotoxicity after STING agonist treatment (Figures 4B, 13B), and as recently proposed, it is highly likely to limit the combination of STING agonists and CAR T cells (Xu et al., J. Exp. Med. 218:e20200844 (2021); Smith et al., J. Clin. Invest. 127:2176-2191 (2017)).

[0150] NK cells were also sensitive to STING agonism (Figure 4A), but human NK cells did not show significant cytotoxicity from STING agonist treatment, regardless of culture in IL-2 or co-culture with TILs (Figures 4A-4C and 13A-13D). Figure 4C shows flow cytometry after treatment with 50 μM ADU-S100, 10 μM TAK-676, or dH20 control + / - 200 U / mL IL-2 for 72 hours, gated on live cells out of a total of 10,000 events expressing CD8 or CD56. Batch 3 primary NK cells expanded from PBMCs and TILs from a 66-year-old male with stage I NSCLC. One-way ANOVA with corrected pairwise comparisons p<0.01: **p<0.01, ***p<0.001. These findings were maintained across human tumors and expanded primary NK cells even after 72 hours of high-dose ADU-S100 or TAK-676 exposure. NK cells rely primarily on metabolism via oxidative phosphorylation (Keppel et al., J. Immunol. 194:1954-62 (2015)) and require ongoing autophagic flux (Wang et al., Nat. Commun. 7:11023 (2016)), while T cells rely on glycolysis and tolerate defective autophagy (Clarke et al., Nat. Rev. Immunol. 19:170-183 (2019)). Indeed, consistent with their elevated autophagic flux after ADU-S100 treatment (Figure 13E), STING protein levels were lower in NK cells, and STING was rapidly degraded within 3-6 hours of STING agonism (Figures 4D and 13F-13G). In contrast, T cell STING was phosphorylated and activated by ADU-S100 but minimally degraded (Figures 4D and 13F). Treatment with chloroquine (CLQ), which blocks the flow of autophagy by inhibiting autophagosome-lysosome fusion (Mauthe et al., Autophagy 14:1435-1455 (2018)), prevented ADU-S100-induced STING degradation, confirming autophagy-dependent STING recycling in NK cells (Figure 13G).Therefore, STING agonist treatment induces the cytotoxicity of effector T cells, while most of the primary NK cells remain intact.

[0151] Example 5: STING agonists enhance NK cell therapy.

[0152] NK cells are generally scarce in MPM specimens (Figure 1B) and can be potentially inhibited by inhibitory signals on tumor cells such as MHC-I, which can increase after STING agonist treatment (Figure 12B). Next, STING agonism combined with adoptive transfer of primary or engineered NK cells was investigated to determine whether this represents a promising therapeutic strategy by coupling tumor CXCR3 chemokine release to effector cell types resistant to STING agonist cytotoxicity. Addition of primary NK cells alone to the processing channels of the microfluidic device was unable to kill MPM PDOTS, but co-treatment with ADU-S100 significantly enhanced the primary NK cell response using cells from 2 out of 3 donors (Figure 5A and Figure 14A). Furthermore, ADU-S100-mediated enhancement of NK cell cytotoxicity in MPM PDOTS was impaired by CXCR3 neutralization (Figure 5B). To further overcome the potential inhibitory effect of MHC-I, the next focus was on an anti-MSLN CAR strategy clinically developed for MPM that utilizes NK cells instead of T cells as an alternative vector for the anti-MSLN CAR (Janes et al., N. Engl. J. Med. 385:1207-1218 (2021)) (Figure 16A). Indeed, using brightly MSLN-positive MPM specimens, it was found that anti-MSLN CAR NK cells significantly increased ADU-S100 activity on day 6 in PDOTS samples that were minimally responsive to ADU-S100 treatment alone (Figure 14B). Furthermore, combined addition of NK cell therapy, particularly CAR-NK cell therapy, and ADU-S100 promoted deep growth inhibition of MLSN+PDOTS over time in culture, in contrast to the 10-day rebound that occurred after monotherapy (Figure 5C; Figure 14C). These data confirm that continuous STING agonist exposure is not toxic to NK cell therapy and suggest that it can enhance activity, particularly in combination with anti-MSLN CAR NK cells.

[0153] To separate the roles of tumor cells and further validate these findings, MPM cell lines (H2591, H226, MS428) that highly express STING and secrete CXCL10 over time during STING agonist treatment, or MPM cell lines that uniquely lack STING expression and do not respond to STING agonism (H2461; Figures 8A, 15A) were used to compare NK cell migration and killing - / + ADU - S100 treatment in vitro (Figures 6, 15A - 15F, 16A - 16C). STING agonism enhanced granzyme release by NK cells (Figure 15B) and apoptosis of tumor cells (Figures 6A - 6D, 15C, 16B - 16C) only in co - culture with MPM cells expressing STING. These findings were consistent across E:T ratios (Figures 15C and 16B), 2D and 3D cultures (Figures 6A - 6D), and, similar to experiments using patient specimens (Figures 5A - 5D, 6A - 6D, 14A - 14C, 15C, 16C), varied somewhat by NK cell donor. Similar to the results seen with NK cell cytotoxicity in MPM patient specimens (Figure 5B), the increase in NK cell migration to tumor cells induced by STING agonists was rescued by treatment with a CXCR3 - neutralizing antibody (Figures 6B, 15E). To model NK cell migration across the vascular barrier, human umbilical vein endothelial cells (HUVEC) were cultured in 3D to form blood vessels, and then physiological NK cell migration exiting the blood vessels and passing through collagen to reach MPM tumor cell lines was evaluated (Figure 15F). ADU - S100, particularly TAK - 676, enhanced NK cell migration in the presence and absence of the vascular barrier, and a decrease in total migration through the blood vessels was expected (Figure 6C).

[0154] Finally, since PDOTS data suggested that mesothelin CAR construct expression could enhance adoptive NK cell therapy in MPM when combined with a STING agonist (Figure 5C), the combination of this treatment was evaluated in vitro to assess cytotoxicity. Anti-mesothelin CAR expression further enhanced NK cell killing in vitro and, in combination with ADU-S100 treatment, caused the most tumor cell death (Figures 6D and 16A-16C). Collectively, these data demonstrate that STING agonism in STING-positive human tumor models activates the anti-viral signaling program, promotes the release of CXCR3 ligand chemokines from tumor cells, enhances NK cell recruitment and cytotoxicity, and can have potent combinatorial activity with NK cell therapy.

[0155] Evaluating human tumors in short-term cultures that preserve the tumor immune microenvironment can potentially inform clinical trials of combinations of next-generation immunotherapies, including cell therapies, by overcoming some of the limitations of mouse models, patient-derived xenografts, and passaged organoids. Described herein is the dynamic single-cell RNA sequencing of ADU-S100-treated human tumor explants to interrogate the mechanism of action of a clinical-stage STING agonist. STING agonism engages its target in most cells of the TIME but predominantly drives CXCR3 chemokine activation in tumor cells and cancer-associated fibroblasts while causing T cell cytotoxicity. The dampening of effector T cell activity is an unexpected result that may contribute to the off-target clinical activity of STING agonists to date in humans. However, these studies have revealed that this drawback can be overcome by the addition of NK cell therapy, which benefits from STING agonist enhancement of NK cell migration and killing (Myers et al., Nat. Rev. Clin. Oncol. 18:85-100 (2021)).

[0156] More generally, data available from mouse models and clinical trials of injectable STING agonists support the complex interplay of STING activation in the TIME. Indeed, cell types other than CD8 T cells, such as monocytes and NK cells, may be involved in the low-frequency clinical responses to STING agonists reported in patients (Harrington et al., Annals of Oncology 29:viii712(2018); Meric-Bernstam et al., Journal of Clinical Oncology 37:2507(2019)). Furthermore, recent studies in syngeneic mouse models have revealed an important function of NK cells in tumor control mediated by the endogenous STING agonist ligand 2’3’-cGAMP (Marcus et al., Immunity 49:754-763(2018); Nicolai et al., Sci. Immunol. 5:eaaz2738(2020)). These data indicate that NK cells are involved in the in vivo response to mouse STING agonists, which would otherwise be difficult to model using artificial humanized mouse xenografts. In the relevant literature describing the preclinical activity of TAK-676, the inventors have observed enhanced trafficking and activation of NK cells following systemic administration of TAK-676 in mouse models. TAK-676 treatment was also particularly potent in overcoming the human vascular barrier in our ex vivo model.

[0157] In addition to the complexity of injectable STING agonist trials, there is a potential threshold effect on cytokine release, whereby tumor cell STING activation crosses over from metastasis promotion (Chen et al., Nature 533:493-498 (2016); Bakhoum et al., Nature 553:467-472 (2018)) to immune rejection. In MPM, minimal baseline phosphorylation of downstream IRF3 was observed in patient specimens (Figure 7C), extracellular 2’3’-cGAMP released from cell lines was negligible (Figure 8C), suggesting a potentially low contribution from basal cGAS-STING signaling to the observed immune exhaustion (Figures 1A–1D). However, these data instead suggest that increased tumor cell STING expression in MPM confers a specific vulnerability to therapeutic STING agonism, particularly when combined with NK cell therapy. This vulnerability may also extend to other tumor types with high basal STING expression or STING-silenced tumors treated with epigenetic inhibitors (Falahat et al., Proc. Natl. Acad. Sci. U.S.A. 118:e2013598118 (2021)).

[0158] The clinical development of STING agonists is further limited by the narrow therapeutic concentration range of injectable agents that are rapidly eliminated (Harrington et al., Annals of Oncology 29:viii712(2018); Meric-Bernstam et al., Journal of Clinical Oncology 37:2507(2019)). Novel sustained-release and systemic formulations of STING agonists can address some of these issues (Amouzegar et al., Cancers(Basel) 13:2695(2021)), but the data disclosed herein also show that certain exposures can potentially kill endogenous effector T cells and limit combinations with adoptive transfer transgenic TCR-T or CAR T cell therapies (Xu et al., J. Exp. Med. 218:e20200844(2021); Smith et al., J. Clin. Invest. 127:2176-2191(2017)). Instead, the findings described herein that NK cells are resistant to certain high-dose STING agonist exposures, are actually activated, and are mobilized to kill MPM cells support this new immunobiology and provide a direct combinatorial approach with NK cell therapies for clinical development. Furthermore, the advantage of adding STING agonists to NK cell therapies may not necessarily rely on CAR constructs and allows for combinations with various freshly generated NK effector cells (Myers et al., Nat. Rev. Clin. Oncol. 18:85-100(2021)). Treatments to enhance natural NK cell activation may also be effective in combination with STING agonists. Interestingly, single-cell RNA sequencing data show NK inhibitory MHC I upregulation, but they also reveal specific regulation of CD112 and CD155 that can converge to activate NK cells, particularly in combination with adoptive NK cell therapy (Figures 5A-5D and Figures 6A-6D) (Figure 12B).

[0159] Burst-dose STING agonism (concurrent with NK cell infusion) may enable subsequent cross-priming of T cells via NK-to-dendritic cell-to-T cell crosstalk that prevents T cell cytotoxicity and enhances antitumor immunity, and thus the timing and sequencing of combination immunotherapies remain important. Potent / specific TBK1 inhibitors described in Jenkins et al., Cancer Discov. 8:196-215 (2018) activate T cells and may be used in combination therapy with the treatment of the present invention described herein.

[0160] Example 6: CXCR3 overexpression in CAR-NK cells stimulates migration and homing to the tumor microenvironment.

[0161] As shown in FIGS. 17A-18B, CXCR3 was degraded from the cell surface of primary NK cells and NK cell lines NK92 and JURKAT, both of which express CXCR3, at different time points after stimulation with 200 ng of recombinant human C-X-C motif chemokine ligand 10 (hCXCL10), measured by flow cytometry, and represented as the median fluorescence intensity (MFI) of the CXCR3 receptor. CXCR3 was also degraded from CAR- and CAR-CXCR-expressing cNK cells. FIGS. 18C-18D show CXCR3 surface expression measured by flow cytometry of primary NK cells (cNK) expressing CAR, CAR-CXCR3, or a control, stimulated with 200 ng of recombinant human CXCL10 stimulation at different time points (0 minutes and 60 minutes). CXCR3 was degraded from the cNK NT, CAR-cNK, and CAR-NK CXCR+ cell surfaces 1 hour after hCXCL10 treatment (FIG. 19C). CXCR3 was also degraded from cytokine-induced memory-like (CIML) NK NT, CIML CAR-NK, CIML CAR-NK CXCR+ 1 hour after hCXCL10 treatment (FIG. 19D). These data demonstrate that CXCR3 is lost from the cell surface after hCXCL10 treatment and that CXCR3 overexpressing cell lines (NK92, Jurkat) have higher CXCR3 surface expression and, as a result, lower CXCR3 receptor internalization compared to endogenous NK CXCR3 expression.

[0162] The immune cell migration cNK cell assay was performed on control cNK cells and cNK cells overexpressing CXCR3. CXCR3 overexpression resulted in increased NK cell migration to H226 MPM cells (FIGS. 20A-20B) and H2591 MPM cells (FIGS. 21A-21B).

[0163] The effects of CXCR3 and ADU-S100 (abbreviated as ADU) on NK cell migration were then tested. ADU-S100 increased the migration of cNK cells but decreased the migration of cNK overexpressing CXCR3 to H226 MPM cells (FIGS. 22A-22B) and H2591 MPM cells (FIGS. 22A-23B).

[0164] Furthermore, CXCR3 overexpression increases CAR-NK cell migration and cytotoxicity. CAR-NK control cells or CAR-NK cells overexpressing CXCR3 (CAR-NK CXCR+) were tested for migration towards H226 MPM cells and H226 cell killing. CAR-NK CXCR+ cells migrated (Figs. 24A–24B) and killed more H226 cells than NK control cells (Figs. 24C–24D). In Fig. 24D, cNK cells are labeled red, all cells (live and dead) are labeled blue with DAPI, and dead cells are labeled yellow with Draq7; the scale bar represents 150 μm.

[0165] The STING agonist ADU-S100 enhances CAR-NK migration. CAR-NK control cells and CAR-NK CXCR+ cells were tested for migration with and without ADU-S100. ADU-S100 did not affect CAR-NK control cell migration towards H226 MPM cells; CAR-NK CXCR+ cell migration increased after ADU-S100 treatment (Figs. 25A–25B).

[0166] CAR expression was confirmed in cNK and CIML NK cells isolated and generated from two donors. Non-transduced (abbreviated as UNT) cNK and CIML NK cells showed no binding to anti-APC-HA antibody, while cNK and CIML NK cells transduced with anti-Mesothelin CAR constructs (containing human agglutinin (HA) tag; abbreviated as CAR) with or without CXCR3 overexpression constructs increased binding to anti-APC-HA antibody (abbreviated as CAR-CXCR3) (Figures 26A - 27B). The fraction of CAR-high cNK cells was 23.9 ± 6.8% in cells expressing CAR-CXCR compared to 44.8% in cells expressing CAR only for donor 27 and 0.02% in non-transduced cells (Figure 26A). The fraction of CAR-high cNK cells was 21.7 ± 1.8 in cells expressing CAR-CXCR compared to 44% in cells expressing CAR only for donor 28 and 0.06% in non-transduced cells (Figure 27A). The fraction of CAR-high CIML cells was 49.9 ± 8.3% in cells expressing CAR-CXCR compared to 64.5% in cells expressing CAR only for donor 27 and 0.01% in non-transduced cells (Figure 26B). The fraction of CAR-high CIML cells was 39 ± 7.3 in cells expressing CAR-CXCR, while it was 53.7 in cells expressing CAR only and 0.02% in non-transduced cells for donor 28 (Figure 27B).

[0167] Similar to the confirmation of CAR expression, CXCR3 overexpression was confirmed by flow cytometry in untransduced or transduced cNK (Figure 28A, Figure 29A) and CIML NK cells (Figure 28B, Figure 29B) with or without the anti-methotrexate CAR construct with CXCR3 overexpression construct (Figure 28A - Figure 29B). The percentage of CXCR3-high cNK cells increased to 57.6 ± 3.3% in cells expressing CAR-CXCR compared to 36.7% in cells expressing only CAR for donor 27 (Figure 28A). The percentage of CXCR3-high cNK cells increased to 89.9 ± 0.3 in cells expressing CAR-CXCR compared to 77.1 in cells expressing only CAR for donor 28 (Figure 29A). The percentage of CXCR3-high CIML cells increased to 87.3 ± 1.2% in cells expressing CAR-CXCR compared to 64.3% in cells expressing only CAR for donor 27 (Figure 28B). The percentage of CXCR3-high CIML cells increased to 97.2 ± 0.4 in cells expressing CAR-CXCR compared to 89.8 in cells expressing only CAR for donor 28 (Figure 29B).

[0168] More generally, combination immunotherapy remains challenging to translate to the clinic, and studying the effect of activating one pathway for innate / adaptive immune crosstalk and the broader TIME using patient-derived tumor samples may provide the best approach to enhance adoptive cell therapy and overcome immune exhaustion.

[0169] All patent publications and non-patent publications indicate the technical level of those skilled in the art to which the present disclosure pertains. All of these publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.

[0170] The disclosure of this specification has been described with reference to specific embodiments, but it should be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. Therefore, it should be understood that numerous modifications can be made to the exemplary embodiments and other configurations can be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.

Claims

1. A nucleic acid construct comprising: a first nucleic acid comprising a first promoter operably linked to a nucleic acid encoding C-X-C motif chemokine receptor 3 (CXCR3); and a second nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a ligand binding domain comprising a single-chain antibody fragment that binds to an antigen on a tumor cell, a transmembrane domain, and an intracellular domain comprising a signaling domain.

2. The second nucleic acid is operably linked to a second promoter, which may be the same as or different from the first promoter; or a third nucleic acid encoding a self-cleaving peptide is disposed between the first nucleic acid and the second nucleic acid, and the first promoter drives the expression of the first, second, and third nucleic acids, The nucleic acid construct according to claim 1.

3. The nucleic acid construct according to claim 1, wherein the first promoter is configured to overexpress CXCR3.

4. The nucleic acid construct according to claim 1, wherein the antigen on the tumor cell is a malignant pleural mesothelioma (MPM) antigen.

5. The nucleic acid construct according to claim 4, wherein the MPM antigen is mesothelin.

6. The nucleic acid construct according to claim 5, wherein the ligand binding domain is derived from a portion of the anti-mesothelin ScFv YP218 antibody.

7. The nucleic acid construct according to claim 1, wherein the signaling domain comprises a primary signaling domain, a co-stimulatory signaling domain, or both a primary signaling domain and a co-stimulatory signaling domain.

8. The nucleic acid construct according to claim 7, wherein the signaling domain comprises a CD3ζ primary signaling domain and a 4-1BB co-stimulatory signaling domain, or a CD28 co-stimulatory signaling domain, or both a 4-1BB and a CD28 co-stimulatory signaling domain.

9. A vector comprising the nucleic acid construct according to claim 1.

10. The vector according to claim 9, which is a lentiviral vector.

11. The vector according to claim 10, wherein the lentiviral vector is a baboon envelope pseudotyped lentiviral vector.

12. A genetically modified immune cell containing one or more vectors comprising the nucleic acid construct according to claim 1.

13. The genetically modified immune cell according to claim 12, which is a natural killer (NK) cell, a T cell, or a combination thereof.

14. A pharmaceutical composition comprising an effective amount of the genetically modified immune cell according to claim 12 and a pharmaceutically acceptable carrier.

15. A method of treating cancer in a subject, comprising: administering an effective amount of the pharmaceutical composition according to claim 14 to a subject in need thereof.

16. The method according to claim 15, further comprising administering an effective amount of a STING agonist to the subject before, substantially simultaneously with, or after administration of the pharmaceutical composition.

17. The method according to claim 16, wherein the STING agonist comprises ADU-S100, TAK-676, BI-STING, BMS-986301, GSK532, JNJ-4412, MK-1454, SB11285, 3’3’-scylic AIMP, ALG-031048, E7766, JNJ-’6196, MK-2118, MSA-1, MSA-2, SNX281m, SR-717, KAT676, TTI-10001, XMT-2056, CRD-5500, or a combination thereof.

18. The method according to claim 17, wherein the STING agonist comprises ADU-S100 or TAK-676.

19. The method according to claim 16, wherein the STING agonist is delivered by intratumoral or intravenous injection.

20. The method according to claim 15, wherein the cancer is a solid tumor.

21. The method according to claim 20, wherein the cancer is MPM, melanoma, gastric cancer, liver cancer, lung cancer, bladder cancer, colorectal cancer, or breast cancer.

22. The method according to claim 21, wherein the cancer is MPM.

Images