Compositions and methods of modulating formation and / or composition of extracellular vesicles

Abstract

The present disclosure relates to the finding that certain Sigma1 inhibitors can be used to promote an antitumor immune response in a subject.

Description

[0001] Attorney Docket No. 205961-7069W01(00279)

[0002] TITLE OF THE INVENTION

[0003] Compositions and Methods of Modulating

[0004] Formation and / or Composition of Extracellular Vesicles

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Patent Application No. 63 / 747,714, filed January 21, 2025, which is incorporated herein by reference in its entirety.

[0006] BACKGROUND

[0007] Programmed death-ligand 1 (PD-L1, also known as B7-H1 or CD274) is a type I integral membrane glycoprotein that is inducibly expressed on the surface of many cell types, including immune cells, epithelial cells, and endothelial cells. Upon binding its cognate receptor, programmed death- 1 (PD-1, also known as CD279) expressed on T cells, PD-L1 acts as an inhibitory checkpoint molecule and suppresses T cell activation and subsequent proliferation. PD-1 / PD-L1 blockade of the host immune response normally serves to protect against autoimmunity and excessive tissue damage at the site of inflammation.

[0008] However, many cancers are able to co-opt this safety feature of the immune system in order to evade antitumor immune response. In the context of cancer, PD-L1 on the surface of cancer cells binds to PD-1 on tumor-infiltrating cytotoxic T lymphocytes (CTLs) and inactivates the host’s antitumor immune response. Immunomodulatory checkpoint inhibitor antibodies that block PD-L1 / PD-1 interactions have exhibited remarkable clinical efficacy in subsets of patients with melanoma, non-small cell lung cancer, metastatic bladder cancer, and renal cell carcinoma. However, in contrast to the robust and durable anti-tumor effects in responders, a relatively low percentage of patients respond to anti-PD-l / PD-Ll monotherapy.

[0009] As PD-L1 is a type I integral membrane glycoprotein, it is translated into, post-translationally modified in, and transported through the secretory pathway of cells. It was recently reported that PD-L1 is incorporated into cancer cell-derived extracellular vesicles, where it is capable of binding PD-1 on activated immune cells, thus creating a sort of immunosuppressive cloud around the tumor.

[0010] Extracellular vesicles (EVs) comprise a heterogeneous population of cellular membrane derived vesicles that vary in size, morphology, and content. EV-associated PD-L1 (evPD-Ll) has been shown to suppress activated T cells both in vitro and in vivo, thereby decreasing both T cell proliferation and production of cytotoxic mediators. Cancer patientsAttorney Docket No. 205961-7069W01(00279)

[0011] are reported to have elevated levels of circulating evPD-Ll, and higher circulating evPD-Ll levels correlate with poor response to anti-PD-1 therapy. Accordingly, EVs present a distinct mode of immune modulation.

[0012] Thus, there is a need in the art for methods and compositions to engineer exosomes and other extracellular vesicles that modulate the tumor microenvironment (TME) in order to promote antitumor immunity and / or prevent cancer cell adaptive immune resistance. The present disclosure addresses this need.

[0013] SUMMARY

[0014] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0015] Method relating to regulating Sigmal in subject

[0016] In some aspects, the present invention is directed to a method of modulating formation and / or composition of extracellular vesicles in a subject.

[0017] In some aspects, the present invention is directed to a method of suppressing and / or inhibiting formation of extracellular vesicles in a subject.

[0018] In some aspects, the present invention is directed to a method of activating and / or promoting a subject's antitumor immune response.

[0019] In some aspects, the present invention is directed to a method of preventing, minimizing, and / or reversing cancer cell adaptive immune resistance in a subject.

[0020] In some aspects, the present invention is directed to a method of preventing, minimizing, and / or reversing inactivation of T cells by cancer-derived extracellular vesicles in a subject.

[0021] In some aspects, the present invention is directed to a method of minimizing and / or suppressing inclusion of programmed death-ligand 1 (PD-L1) in extracellular vesicles in a subject.

[0022] In some embodiments, the method comprising administering to the subject a therapeutically effective amount of a compound that downregulates Sigmal expression and / or activity in a cell of the subject.

[0023] In some embodiments, the compound is a small molecule inhibitor of Sigmal.

[0024] In some embodiments, the compound is a protein inhibitor of Sigmal.

[0025] In some embodiments, the compound is a nucleic acid that downregulates Sigmal by RNA interference. In some embodiments, the compound is an expression vector expressing the nucleic acid.Attorney Docket No. 205961-7069W01(00279)

[0026] In some embodiments, the compound is a ribozyme that downregulates Sigmal. In some embodiments, the compound is an expression vector expressing the ribozyme.

[0027] In some embodiments, the compound is an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that down regulate Sigmal by CRISPR knockout or CRISPR knockdown.

[0028] In some embodiments, the compound is a trans -dominant negative mutant protein of Sigmal. In some embodiments, the compound is an expression vector that expresses the trans-dominant negative mutant protein of Sigmal.

[0029] Small molecule inhibitors of Sigmal

[0030] In some embodiments, the compound is IP AG (l-(4-Iodophenyl)-3-(2-adamantyl)guanidine);

[0031] In some embodiments, the compound is a compound of Formula (I):

[0032]

[0033] In some embodiments, ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclic heteroaryl ring. In some embodiments, the aryl or heteroaryl ring is optionally substituted with 0-4 R1groups.

[0034] In some embodiments, each occurrence of R1is independently selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce heteroalky 1, F, Cl, Br, I, -CN, -NO2, -OR3, -SR3, -S(=O)R3, -S(=O)2R3-NHS(=O)2R3-C(=O)R3, -OC(=O)R3, -CO2R3, -OCO2R3, -CH(R3)2, -N(R3)2, -C(=O)N(R3)2, -OC(=O)N(R3)2, -NHC(=O)NH(R3), -

[0035]

[0036] NHC(=O)R3. -NHC(=O)OR3, -C(OH)(R3)2. and -C(NH2)(R3)2.

[0037] In some embodiments, each occurrence of R2is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), wherein the alkyl, heteroalkyl or cycloalkyl group is optionally substituted with 0-5 R1groups, or X3and R2combine to form a (C3-C7)heterocycloalkyl group, optionally substituted with 0-2 R1groups.

[0038] In some embodiments, each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), wherein the alkyl, heteroalkyl, ary l, or cycloalkyl group is optionally substituted with 0-5 R1groups.

[0039] In some embodiments, X1is -CH2-, -S-, -O- or -(NR2)-.Attorney Docket No. 205961-7069W01(00279)

[0040] In some embodiments, X2is =CH2, =S, =O or =NR2.

[0041] In some embodiments, X3is -S-. -0-, or -NR2-.

[0042] In some embodiments, the compound is a compound of Formula (II):

[0043] RA-RB(II).

[0044]

[0045] In some embodiments, X4is selected from the group consisting of methoxy, F, Cl, Br, and I.

[0046] In some embodiments, RBis selected from the group consisting of:

[0047]

[0048] In some embodiments, the compound is a compound of formula (III):Attorney Docket No. 205961-7069W01(00279)

[0049]

[0050] In some embodiments, each occurrence of R1and R2is independently selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2. -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -N

[0051]

[0052] HC(=O)R5. -NHC(=O)OR5, -C(OH)(R5)2. and -C(NH2)(R5)2.

[0053] In some embodiments, R3is selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce alkoxy, F, Cl, Br, and I.

[0054] In some embodiments, R4is selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce alkoxy, F. Cl, Br, and I.

[0055] In some embodiments, each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted.

[0056] In some embodiments, X is selected from the group consisting of CH2, C=O, or O. In some embodiments, n is an integer from 1-3.

[0057] In some embodiments, x is an integer from 0-4.

[0058] In some embodiments, y is an integer from 0-4.

[0059] In some embodiments, the compound is a compound of formula (IV):

[0060]

[0061] In some embodiments, Z1is CRla; Z2is N; Z3is CRlcor N; Z4is CRld.

[0062] In some embodiments, Z1is N; Z2is CRlb; Z3is CRlc; Z4is CRldor N.

[0063] In some embodiments, Z1is CRla; Z2is N; Z3is CRlc; Z4is N.

[0064] In some embodiments, Z1is N; Z2is CRlb; Z3is N; Z4is CRld.

[0065] In some embodiments, X is selected from the group consisting of bond and O.

[0066] In some embodiments, each occurrence of Rla, Rlb, Rlc, and Rldis independently selected from the group consisting of C₁-C₆ alkyl, C₁-C₆ fluoroalkyl, C₁-C₆ heteroalkyl, F, CL Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -Attorney Docket No. 205961-7069W01(00279)

[0067] CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5. -NHC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2.

[0068] In some embodiments, each occurrence of R2a, R2b, R2c, and R2dis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5. -OCO2R5, -CH(R5)2. -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5. -NHC(=O)OR5, -C(OH)(R5)2. and -C(NH2)(R5)2.

[0069] In some embodiments, R3is selected from the group consisting of CN, F, Cl, Br, I, Ci-Ce haloalky 1, Ci-Ce haloalkoxy, and -C(O)OR6.

[0070] In some embodiments, R4is selected from the group consisting of CN, F, Cl, Br, I, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR7.

[0071] In some embodiments, each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, ary l, and -C1-C3 alky 1-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, ary 1, or cycloalkyl group is optionally substituted.

[0072] In some embodiments, R6is selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and C3-C6 cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group is optionally substituted.

[0073] In some embodiments, R7is selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalky 1, and C3-C6 cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group is optionally substituted.

[0074] In some embodiments, each occurrence of Ra, Rb, Rc, Rd, Re, and Rfis independently selected from the group consisting of H and CH3, with the proviso that, if Y is CRlcand Z is CRld, then at least one of Ra, Rb, Rc, Rd, Re, and Rfis CH3.

[0075] In some embodiments, the compounds is a salt, solvate, enantiomer, diastereoisomer, tautomer, or N-oxide thereof, or any combinations of the above.

[0076] In some embodiments, the compound is selected from the group consisting of: l-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A); l-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B); l-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C); l-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D); l-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine (Compound F); and 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound G). In some embodiments, the compound is a salt, solvate or N-oxide thereof, and any combinations of the compounds in this paragraph.

[0077] In some embodiments, the compound is selected from the group consisting of 1,3-Attorney Docket No. 205961-7069W01(00279)

[0078] bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E); l-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine) (Compound H). In some embodiments, the compound is a salt, solvate or N-oxide thereof, and any combinations of the compounds in this paragraph.

[0079] In some embodiments, in formula (IV), each occurrence of Rla, Rlb, Rlc, and Rldis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, and -OR5.

[0080] In some embodiments, in formula (IV), each occurrence of R2a, R2b, R2c, and R2dis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, and -OR5.

[0081] In some embodiments, in formula (IV), R3is CN, F. Cl. Br, I. -CF3. -CHF2. -OCF3. -OCHF2, -C(O)OH, or -C(O)OMe.

[0082] In some embodiments, in formula (IV), R4is CN, F, Cl, Br, I, -CF3, -CHF2, -OCF3, -OCHF2, -C(O)OH, or -C(O)OMe.

[0083] In some embodiments, the compound

[0084]

[0085] is l-(6-cyano-5-methoxypyridin-3-yl)-3-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or A -ox ide of the compound in this paragraph.

[0086] In some embodiments, the compound

[0087]

[0088] is l-(6-cyano-5-methoxypyridin-3-yl)-3-(2,2-dimethyl-3-(4- (trifluoromethyl)phenoxy)propyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or A'-oxide of the compound in this paragraph.

[0089] H

[0090] MeO N

[0091] In some embodiments, the compound i

[0092]

[0093] s N ' I-(5-cyano-4-methoxypyridin-2-yl)-3-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or A-oxide of the compound in this paragraph.Attorney Docket No. 205961-7069W01(00279)

[0094] In some embodiments, the compound is

[0095]

[0096] (3,4-dichlorophenyl)-3-(3-methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or A-oxide of the compound in this paragraph.

[0097] In some embodiments, the compound is

[0098]

[0099] (6-chloro-5-fluoropyridin-3-yl)-3-(3-methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or A-oxide of the compound in this paragraph.

[0100] H H

[0101] MeO NH

[0102] •> C In some embodiments, the compound i

[0103]

[0104] s N " 1- (5-cyano-4-methoxypyridin-2-yl)-3-(3-methyl-3-(4-(trifluoromethyl)phenyl)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or A-oxide of the compound in this paragraph.

[0105]

[0106] methyl 6-(3-(3-methyl-3-(4-(trifluoromethyl)phenyl)butyl)guanidino)nicotinate. In some embodiments, the compound is a salt, solvate, tautomer, or A-oxide of the compound in this paragraph.

[0107] In some embodiments, the compound

[0108]

[0109] isFmethyl 6-(3-(2.2-dimethyl-3-(4-(trifluoromethyl)phenoxy)propyl)guanidino)nicotinate. In some embodiments, the compound is a salt, solvate, tautomer, or A-oxide of the compound in this paragraph.Attorney Docket No. 205961-7069W01(00279)

[0110] In some embodiments, the compound

[0111]

[0112] is 1-(3-methyl-3-(4-(trifluoromethyl)phenoxy)butyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or / V-oxide of the compound in this paragraph.

[0113] In some embodiments, the compound

[0114]

[0115] is 1-(6-cyano-5-methoxypyridin-3-yl)-3-(3-methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or A-oxide of the compound in this paragraph.

[0116] In some embodiments, the subject has cancer.

[0117] In some embodiments, the compound is administered as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0118] In some embodiments, the subject is further administered at least one additional anticancer agent and / or at least one agent that treats one or more cancer symptoms or comorbidities.

[0119] In some embodiments, the compound is administered by a route comprising oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.

[0120] In some embodiments, the subject is a mammal.

[0121] In some embodiments, the subject is a human.

[0122] Method relating to regulating Sigma 1 in cell

[0123] In some aspects, the present invention is directed to a method of modulating formation of extracellular vesicles in a cell and / or composition of extracellular vesicles released by a cell.

[0124] In some aspects, the present invention is directed to a method of suppressing and / or inhibiting formation of extracellular vesicles in a cell.

[0125] In some aspects, the present invention is directed to a method of preventing, minimizing, and / or reversing a release of T cell-inactivating extracellular vesicles by a cell In some aspects, the present invention is directed to a method of minimizing and / or suppressing release of programmed death-ligand 1 (PD-L1) in extracellular vesicles by a cell.Attorney Docket No. 205961-7069W01(00279)

[0126] In some embodiments, the method comprising contacting the cell with a compound that downregulates Sigmal in the cell.

[0127] In some embodiments, the compound is a small molecule inhibitor of Sigmal.

[0128] In some embodiments, the compound is a protein inhibitor of Sigmal.

[0129] In some embodiments, the compound is a nucleic acid that down regulates Sigmal by RNA interference. In some embodiments, the compound is an expression vector expressing the nucleic acid.

[0130] In some embodiments, the compound is a ribozyme that downregulates Sigmal. In some embodiments, the compound is an expression vector expressing the ribozyme.

[0131] In some embodiments, the compound is an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate Sigmal by CRISPR knockout or CRISPR knockdown.

[0132] In some embodiments, the compound is a trans -dominant negative mutant protein of Sigmal. In some embodiments, the compound is an expression vector that expresses the trans-dominant negative mutant protein of Sigmal.

[0133] Small molecule inhibitors of Sigmal

[0134] In some embodiments, the compound is IP AG (l-(4-Iodophenyl)-3-(2-adamantyl)guanidine).

[0135] In some embodiments, the compound is a compound of Formula (I):

[0136]

[0137] In some embodiments, ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclic heteroaryl ring, and wherein the aryl or heteroaryl ring is optionally substituted with 0-4 R1groups.

[0138] In some embodiments, each occurrence of R1is independently selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR3, -SR3, -S(=O)R3, -S(=O)2R3, -NHS(=O)2R3, -C(=O)R3, -OC(=O)R3, -CO2R3, -OCO2R3, -CH(R3)2, -N(R3)2, -C(=O)N(R3)2, -OC(=O)N(R3)2, -NHC(=O)NH(R3), -NHC(=O)R3. -NHC(=O)OR3, -C(OH)(R3)2, and -C(NH2)(R3)2.

[0139] In some embodiments, each occurrence of R2is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and -C1-C3 alkyl-(C3-C6 cycloalkyl),Attorney Docket No. 205961-7069W01(00279)

[0140] wherein the alkyl, heteroalkyl or cycloalkyl group is optionally substituted with 0-5 R1groups, or X3and R2combine to form a (C3-C7)heterocycloalkyl group, optionally substituted with 0-2 R1groups.

[0141] In some embodiments, each occurrence of R3is independently selected from the group consisting of H, C₁-C₆ alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted with 0-5 R1groups.

[0142] In some embodiments, X1is -CH2-, -S-, -O- or -(NR2)-.

[0143] In some embodiments, X2is =CH2, =S, =O or =NR2.

[0144] In some embodiments, X3is -S-, -0-, or -NR2-.

[0145] In some embodiments, the compound is a compound of Formula (II):

[0146] RA-RB(II).

[0147] In some embodiments, RAis selected from the group consisting of X4

[0148]

[0149]

[0150] In some embodiments, X4is selected from the group consisting of methoxy, F, Cl, Br, and I.

[0151] In some embodiments, RBis selected from the group consisting of:Attomey Docket No. 205961-7069W01(00279)

[0152]

[0153] In some embodiments, the compound is a compound of formula (III):

[0154]

[0155] In some embodiments, each occurrence of R1and R2is independently selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5. -NHC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2.

[0156] In some embodiments, R3is selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce alkoxy, F, Cl, Br, and I.

[0157] In some embodiments, R4is selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce alkoxy, F. Cl, Br, and I.

[0158] In some embodiments, each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted.Attorney Docket No. 205961-7069W01(00279)

[0159] In some embodiments, X is selected from the group consisting of CH2, C=O, or O. In some embodiments, n is an integer from 1-3.

[0160] In some embodiments, x is an integer from 0-4.

[0161] In some embodiments, y is an integer from 0-4.

[0162] In some embodiments, the compound is a compound of formula (IV):

[0163] In some e

[0164]

[0165] mbodiments, Z1is CRla; Z2is N; Z3is CRlcorN; Z4is CRld.

[0166] In some embodiments, Z1is N; Z2is CRlb: Z3is CRlc; Z4is CRldor N.

[0167] In some embodiments, Z1is CRla; Z2is N: Z3is CRlc; Z4is N.

[0168] In some embodiments, Z1is N; Z2is CRlb; Z3is N; Z4is CRld.

[0169] In some embodiments, X is selected from the group consisting of bond and O.

[0170] In some embodiments, each occurrence of Rla, Rlb, Rlc, and Rldis independently selected from the group consisting of C₁-C₆ alkyl, C₁-C₆ fluoroalkyl, C₁-C₆ heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -NHC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2.

[0171] In some embodiments, each occurrence of R2a, R2b, R2c, and R2dis independently selected from the group consisting of C₁-C₆ alkyl, C₁-C₆ fluoroalkyl, C₁-C₆ heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2. -OC(=O)N(R5)2, -NHC(=O)NH(R5), -N

[0172]

[0173] HC(=O)R5. -NHC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2.

[0174] In some embodiments. R3is selected from the group consisting of CN, F. Cl, Br, I, C1-C6 haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR6.

[0175] In some embodiments, R4is selected from the group consisting of CN, F, Cl, Br, I, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR7.

[0176] In some embodiments, each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted.

[0177] In some embodiments, R6is selected from the group consisting of H, Ci-Ce alkyl, C₁-C₆ heteroalkyl, and C₃-C₆ cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group isAttorney Docket No. 205961-7069W01(00279)

[0178] optionally substituted.

[0179] In some embodiments, R7is selected from the group consisting of H, Ci-Ce alkyl, C₁-C₆ heteroalkyl, and C₃-C₆ cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group is optionally substituted.

[0180] In some embodiments, each occurrence of Ra, Rb, Rc, Rd, Re, and Rfis independently selected from the group consisting of H and CH₃, with the proviso that, if Y is CR1cand Z is CR1d, then at least one of Ra, Rb, Rc, Rd, Re, and Rfis CH₃.

[0181] In some embodiments, the compound is a salt, solvate, enantiomer, diastereoisomer, tautomer, or. V-oxide thereof, or any combinations of the compound above.

[0182] In some embodiments, the compound is selected from the group consisting of: l-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A); l-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B); l-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C); l-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D); l-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine (Compound F); and l-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound G). In some embodiments, the compound is a salt, solvate or N-oxide thereof, and any combinations of the compounds of this paragraph.

[0183] In some embodiments, the compound is selected from the group consisting of: 1,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E); l-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine) (Compound H). In some embodiments, the compound is a salt, solvate or N-oxide thereof, and any combinations of the compounds of this paragraph.

[0184] In some embodiments, in Formula (IV), each occurrence of Rla, Rlb, Rlc, and Rldis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, and -OR5.

[0185] In some embodiments, in Formula (IV), each occurrence of R2a, R2b, R2c, and R2dis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, and -OR5.

[0186] In some embodiments, in Formula (IV), R3is CN, F, Cl, Br. I, -CF₃, -CHF₂, -OCF₃, -OCHF₂, -C(O)OH, or -C(O)OMe.

[0187] In some embodiments, in Formula (IV), R4is CN, F, Cl, Br, I, -CF₃, -CHF₂, -OCF₃, -OCHF2, -C(O)OH, or -C(O)OMe.Attorney Docket No. 205961-7069W01(00279)

[0188] MeO

[0189] In some embodiments, the compound

[0190]

[0191] is l-(6-cyano-5-methoxypyridin-3-yl)-3-(3-methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or A-oxide of the compound of this paragraph.

[0192] H H

[0193] N^- YNNH

[0194] In some embodiments, the compound

[0195]

[0196] is 1-(6-cyano-5-methoxypyridin-3-yl)-3-(2,2-dimethyl-3-(4-(trifluoromethyl)phenoxy)propyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or A-oxide of the compound of this paragraph.

[0197]

[0198] l-(5-cyano-4-methoxypyridin-2-yl)-3-(3-methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or A-oxide of the compound of this paragraph.

[0199] In some embodiments, the compound is

[0200]

[0201] Cl 1-(3,4-dichlorophenyl)-3-(3-methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or 2V-oxide of the compound of this paragraph.

[0202] In some embodiments, the compound is

[0203]

[0204] 1-(6-chloro-5-fluoropyridin-3-yl)-3-(3-methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or V-oxide of the compound of this paragraph.

[0205] In some embodiments, the compound

[0206]

[0207] is 1-Attorney Docket No. 205961-7069W01(00279)

[0208] (5-cyano-4-methoxypyridin-2-yl)-3-(3-methyl-3-(4-(trifluoromethyl)phenyl)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or JV-oxide of the compound of this paragraph.

[0209] T H H

[0210] N^N N NH

[0211] In some embodiments, the compound

[0212]

[0213] is methyl 6-(3-(3-methyl-3-(4-(trifluoromethyl)phenyl)butyl)guanidino)nicotinate. In some embodiments, the compound is a salt, solvate, tautomer, or JV-oxide of the compound of this paragraph.

[0214] H H N NH

[0215] In some embodiments, the compound

[0216]

[0217] is methyl 6-(3-(2,2-dimethyl-3-(4-(trifluoromethyl)phenoxy)propyl)guanidino)nicotinate. In some embodiments, the compound is a salt, solvate, tautomer, or JV-oxide of the compound of this paragraph.

[0218] In some embodiments, the compound

[0219]

[0220] is (3-methyl-3-(4-(trifluoromethyl)phenoxy)butyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or JV-oxide of the compound of this paragraph.

[0221] In some embodiments, the compound

[0222]

[0223] is l-(6-cyano-5-methoxypyridin-3-yl)-3-(3-methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine. In some embodiments, the compound is a salt, solvate, tautomer, or JV-oxide of the compound of this paragraph.

[0224] In some embodiments, the cell is a cancer cell.

[0225] In some embodiments, the cancer cell is from a cold cancer.

[0226] In some embodiments, the cell is an isolated cell.Attorney Docket No. 205961-7069W01(00279)

[0227] In some embodiments, the cell is from a cell line.

[0228] In some embodiments, the cell is a primary cell.

[0229] In some embodiments, the cell is in a subject.

[0230] In some embodiments, the cell is a mammalian cell.

[0231] In some embodiments, the cell is a human cell.

[0232] BRIEF DESCRIPTION OF THE FIGURES

[0233] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.

[0234] Fig. 1 illustrates the mechanistic impact of administration of a Sigmal inhibitor. Figs. 2A-2C show that pharmacological inhibition of Sigmal does not prevent the formation of PD-L1 transcripts or STAT1 phosphorylation and translocation into the nucleus in response to IFN-y. Fig. 2A: qRT-PCR quantification of PD-L1 transcripts for MDA-MB-231, PC3, WM164, HT29, and MDA-MB-436 cell lines treated for 16 hours with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y and 10 pmol / L IP AG, or 10 pmol / L IP AG alone. Data represent mean values from three independent determinations and error bars represent SEM, except WM164, which has two independent determinations and error bars represent SD. Fig.

[0235] 2B: Immunoblot of total STAT1 and phospho-STATl Y701 from whole-cell protein extracts of MDA-MB-231, PC3, WM164, HT29, and MDA-MB-436 cell lines treated for 16 hours with DMSO. 10 ng / mL IFN-y. 10 ng / mL IFN-y and 10 pmol / L IP AG, or 10 pmol / L IP AG. Immunoblots were quantified by densitometry for each cell line. Data are presented as fold induction over IFN-y treated control samples and represent mean values from two independent determinations and error bars represent SD. Fig. 2C: Isolation of nuclear and cytosolic fractions of HT29 and MDA-MB-436 cells treated for 16 hours with DMSO. 10 ng / mL IFN-y, 10 ng / mL IFN-y and 10 pmol / L IPAG, or 10 pmol / L IP AG showing that the translocation of pSTATl into the nucleus is not inhibited by Sigmal modulation.

[0236] Immunoblot showing phospho-STATl Y701, RCC1 (nuclear marker), and GAPDH (cytosolic marker).

[0237] Figs. 3A-3C shows that pharmacological inhibition of Sigmal blocks post-translational modification and maturation of IFN-y-induced PD-L1. Fig. 3 A: Immunoblot of PD-L1 from whole-cell protein extracts of MDA-MB-231, PC3, WM164, HT29, and MDA-MB-436 cell lines treated for 16 hours with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y and 10 pmol / L IPAG, or 10 pmol / L IPAG. Immunoblots were quantified by densitometry for each cell line. Data are presented as fold induction over DMSO-treated control samples andAttorney Docket No. 205961-7069W01(00279)

[0238] represent mean values from at least three independent determinations and error bars represent SEM. * p<0.05, ** p<0.01, *** p<0.001, ns = no significance. Fig. 3B: Quantification of the upper molecular weight PD-L1 band ranging from 45-55 kDa versus the lower molecular weight PD-L1 band ranging from 45-35 kDa showing the accumulation of lower molecular weight PD-L1 when IFN-y treatment is combined with IP AG. Data are presented as fold induction over IFN-y treated samples and represent mean values from at least three independent determinations and error bars represent SEM. * p<0.05, ** p<0.01, *** p<0.001, ns = no significance. Fig. 3C: Immunoblot of Endoglycosidase H treated whole-cell protein extracts from HT29 and MDA-MB-436 cell lines treated for 16 hours with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y and 10 pmol / L IP AG, or 10 pmol / L IP AG showing the accumulated PD-L1 band at 40 kDa in the combination treatment is immaturely glycosylated.

[0239] Figs. 4A-4B show that pharmacological inhibition of Sigmal suppresses IFN-y induced PD-L1 cell surface expression. Fig. 4A: Flow cytometric analysis of cell surface PD-L1 in PC3 and MDA-MB-436 cell lines treated for 16 hours with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y and 10 pmol / L IP AG, or 10 pmol / L IP AG from at least four independent determinations and error bars represent SEM. * p<0.05, ** p<0.01, *** p<0.001. Fig. 4B: Cell surface biotinylation of PD-L1 MDA-MB-436 and PC3 cell lines showing that less PD-L1 is at the cell surface when IP AG is combined with IFN-y treatment.

[0240] Figs. 5A-5E show that pharmacological inhibition of Sigmal suppresses extracellular vesicle production. Fig. 5A: Representative nanoparticle tracking analysis of EV size profdes. Fig. 5B: Quantitation of the effect of Sigmal modulation on EV production and error bars representing SEM. **** p<0.0001, ns = no significance. Fig. 5C: Transmission electron microscopy (TEM) images of the EVs present in the 100k pellet from each treatment condition. Scale bar is 100 nm. FIGS. 5D-5E show evaluation of cell viability on PC3 cells treated with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y + 10 pmol / L IP AG, or 10 pmol / L IP AG for 24 hours. Trypan blue (Fig. 5D) and cleaved PARP (Fig. 5E) show no significant cell death or induction of apoptosis is occurring under the treatment conditions, indicating decreased EV production is not due to induction of cell death. ShRNA knockdown of p97 / V CP consistently induced apoptosis, including induction of cleaved PARP and was used as a positive control.

[0241] Figs. 6A-6B show that pharmacological inhibition of Sigmal suppresses PD-L1 content of extracellular vesicles in response to IFN-y. Fig. 6A: Immunoblot of PC3 whole-cell protein extracts and lysates of EVs from PC3 cells. 1 x 109EVs were loaded per lane. Fig. 6B: Immunoblots from at least 3 independent determinations were quantified byAttorney Docket No. 205961-7069W01(00279)

[0242] densitometry for protein content per EV relative to DMSO. Error bars represent SEM. ** p<0.01, *** p<0.001, ns = no significance.

[0243] Fig. 7 shows that pharmacological inhibition of Sigmal prevents PD-L1 incorporation into exosomes. Immunoblots of PC3 100K EV pellets separated on an iodixanol density gradient showing PD-L1 colocalizing in fractions with canonical exosome markers CD9, CD63, CD81, and TSG101.

[0244] Figs. 8A-8B show that pharmacological inhibition of Sigmal reduces the potency of evPD-Ll in an assay of IFN-y mediated T cell suppression. Engineered Jurkat cells expressing PD-1, aAPC-TCR, and NFAT-luciferase reporter (2,500 cells / well) were cocultured for 6 hours with engineered aAPC-expressing CHO cells (40,000 cells / well) in the presence or absence of 1 x 1010conditioned EVs collected from PC3 cells treated with DMSO, 10 ng / mL IFN-y, or 10 ng / mL IFN-y + 10 pmol / L IPAGfor 24 hours. Fig. 8A: Quantification of Jurkat luciferase signal relative to No EV control. Data represent six independent determinations and error bars represent SEM. ** p<0.01, *** p<0.001, ns = no significance. These data show that a Sigmal inhibitor can prevent IFN-y from upregulating the potency of evPD-Ll. Fig. 8B: Illustration summarizing the EV mediated T cell inactivation assay. An equal number of PC3-derived EVs isolated from the indicated treatment conditions were added to each well of the T cell inactivation assay. Note that EVs produced by PC3 cells that were co-treated with the small molecule Sigmal inhibitor (IP AG) and IFN-y had fewer PD-L1 incorporated per EV and thus lost the T cell inactivation potency gained by the EVs produced by IFN-y treated PC3 cells.

[0245] Figs. 9A-9C demonstrate that pharmacological inhibition of Sigmal does not prevent the formation of PD-L1 transcripts or STAT1 phosphorylation and translocation into the nucleus in response to IFN-y, in accordance with some embodiments. Fig. 9A: qRT-PCR quantification of PD-L1 transcripts for MDA-MB-231, PC3, WM164, HT29, and MDA-MB-436 cell lines treated for 16 hours with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y and 10 pmol / L IP AG, or 10 pmol / L IP AG alone. Data represent mean values from three independent determinations and error bars represent SEM. *p <.05, **p <.01, ns = no significance. Fig. 9B: Immunoblot of total STAT1 and phospho-STATl Y701 from whole-cell protein extracts of MDA-MB-231, PC3, WM164, HT29, and MDA-MB-436 cell lines treated for 16 hours with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y and 10 pmol / L IP AG, or 10 pmol / L IP AG. Immunoblots were quantified by densitometry for each cell line. Data are presented as fold induction over IFN-y treated control samples and represent mean values from two independent determinations and error bars represent SD. Fig. 9C: Isolation ofAttorney Docket No. 205961-7069W01(00279)

[0246] nuclear and cytosolic fractions of HT29 and MDA-MB-436 cells treated for 16 hours with DMSO, 10 ng / mL IFN-y, 10ng / mL IFN-y and 10 pmol / L IP AG, or 10 pmol / L IP AG showing that the translocation of pSTATl into the nucleus is not inhibited. Immunoblot showing phospho-STATl Y701, RCC1 (nuclear marker), and GAPDH (cytosolic marker).

[0247] Figs. 10A-10C demonstrate that pharmacological inhibition of Sigmal blocks post-translational modification and maturation of IFN-y-induced PD-L1. in accordance with some embodiments. Fig. 10A: Immunoblot of PD-L1 from whole-cell protein extracts of MDA-MB-231, PC3, WM164, HT29, and MDA-MB-436 cell lines treated for 16 hours with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y and 10 pmol / L IP AG, or 10 pmol / L IP AG.

[0248] Immunoblots were quantified by densitometry for each cell line. Data are presented as fold induction over dmso-treated control samples and represent mean values from at least three independent determinations and error bars represent SEM. *p <.05, **p <.01, ***p <.001, ns = no significance. Fig. 10B: Quantification of the upper molecular weight PD-L1 band ranging from 45-55 kDa versus the lower molecular weight PD-L1 band ranging from 35-45 kDa showing the accumulation of lower molecular weight PD-L1 when IFN-y treatment is combined with IP AG. Data are presented as fold induction over IFN-y treated samples and represent mean values from at least three independent determinations and error bars represent SEM. *p <.05, **p <.01, ***p <.001, ns = no significance. Fig. 10C: Immunoblot of endoglycosidase H treated whole-cell protein extracts from HT29 and MDA-MB-436 cell lines treated for 16 hours with DMSO. 10 ng / mL IFN-y. 10 ng / mL IFN-y and 10 umol / L IP AG, or 10 pmol / L IP AG showing that the accumulated PD-L 1 band at 40 kDa in the combination treatment is incompletely glycosylated.

[0249] Figs. 11A-1 IB demonstrate that pharmacological inhibition of Sigmal suppresses IFN-y induced PD-L1 cell surface expression, in accordance with some embodiments. Fig. 11A: Flow cytometric analysis of cell surface PD-L1 in PC3 and MDA-MB-436 cell lines treated for 16 hours with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y and 10 pmol / L IP AG, or 10 pmol / L IP AG from at least four independent determinations and error bars represent SEM. *p <.05, **p<.01, ***p<.001. Fig. 11B: Cell surface biotinylation of PD-L1 MDA-MB-436 and PC3 cell lines showing that less PD-L1 is at the cell surface when IP AG is combined with IFN-y treatment.

[0250] Figs. 12A-12G illustrate certain aspects of the isolated extracellular vesicles (EVs) from prostate cancer cells treated with IFN-y and Sigmal inhibitor, in accordance with some embodiments. PC3 cells were treated with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y + 10 pmol / L IP AG, or 10 pmol / L IP AG for 24 hours. Fig. 12 A: Representative zetaviewAttorney Docket No. 205961-7069W01(00279)

[0251] particle tracking analysis of EV size distribution. Fig. 12B: Relative number of EV particles (xlO9) per milligram (mg) of cell protein measured by zetaview nanoparticle analysis. This was determined for N = 3 to 4 batches of EV preparations. P-values for each comparison to the control condition are indicated. Although more EVs appeared to be produced by IFN-y treated PC3 cell, the difference did not reach significance in this study. Fig. 12C: Cryogenic electron microscopy (Cryo-EM) images of the EVs from each treatment condition. Scale bar represents 50 nm. Fig. 12D: Mean EV particle size in nanometers (nm). Error bars represent SEM. Fig. 12E: Number of cells collected from each treatment cell culture condition. Note that decreased numbers of cells in IFN-y and IFN-y + IP AG conditions reflect decreased proliferation, but not cell death. N = 4 per condition. P-values for each comparison to the control condition are indicated. There was no significant difference. Fig. 12F: The percentage of live cells from each culture condition determined by trypan blue exclusion assay. N = 4 per condition. Not significant (ns). Fig. 12G: Immunoblot of cleaved poly(adp-ribose) polymerase (cP ARP) as a marker of apoptosis. ShRNA knockdown of p97 / VCP consistently- induced apoptosis and was used as a positive control.

[0252] Figs. 13A-13B demonstrate that pharmacological inhibition of Sigmal prevents IFN-y induced PD-L1 incorporation into EVs, in accordance with some embodiments. PC3 cells were treated for 16 hours with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y and 10 pmol / L IP AG, or 10 pmol / L IP AG. Fig. 13 A: Immunoblot of EVs from PC3 cells. 1 * 109EVs were loaded per lane. Fig. 13B: Immunoblots of EVs separated on an iodixanol density gradient showing PD-L1 colocalizing in fractions with canonical tetraspanin EV markers CD9, CD63, CD81.

[0253] Figs. 14A-14F demonstrate that pharmacological inhibition of Sigmal reduces the potency of evPD-Ll in an assay of IFN-y mediated T cell inactivation. Fig. 14A: T cells isolated from normal human blood were either left unstimulated or activated by CD3 / CD28 cross linking for 48-72 hours with the latter then distributed equally into replicate cultures and incubated for 4 hours in the presence or absence of EV preparations from indicated treatments. Activated T cells are larger (> FSC) and more granular (>ssc) than unstimulated T cells by flow cytometry. Fig. 14B: Quantification of PD-1 on the surface of unstimulated (light gray) versus activated T (dark gray) cells. Fig. 14C: The activated, isolated T cell populations used in the EV co-culture assay expressed CD69 and PD-1. The flow' cytometry profile of unstained cells is shown in blue and double-stained cells in red. Fig. 14D: EV mediated T cell inactivation assay. PBMCs expressing PD-1 (2,500 cells / well) were cocultured for 4 hours with 1 * 1010conditioned EVs collected from PC3 cells treated withAttorney Docket No. 205961-7069W01(00279)

[0254] DMSO, 10 ng / mL IFN-y, and 10 ng / mL IFN-y + 10 pmol / L IP AG for 24 hours. An equal number of PC3-derived EVs isolated from the indicated treatment conditions were added to each well of the T cell inactivation assay. Data represent 3 to 5 independent determinations and error bars represent SEM. *p <.05, **p <.01, ***p <.001, ns = no significance. Fig. 14E: EV particle numbers and PD-L1 status were determined by NanoFCM. The relative number of PD-L1+ EVs particles indicated in red. DMSO (4.23 x 107PD-L1+ EVs of 4.51 x 109total EVs), IFN-y (1.81 x 108PD-L1+ EVs of 4.18 x 109total EVs), IFN-γ + IPAG (1.81 × 107PD-L1+ EVs of 4.69 x 109total EVs). Fig. 14F: Representative nano-fcm analysis of PD-L1 bearing EVs from PC3 cells treated with DMSO, 10 ng / mL IFN-y, and 10 ng / mL IFN-y + 10 pmol / L IP AG. CD9 (canonical EV marker) was used to identify number of exosome EV particles.

[0255] Figs. 15A-15B demonstrate that Sigmal inhibition prevents incorporation of other IFN-y / STATl induced immune suppressive factors into EVs, in accordance with some embodiments. PC3 cells were treated for 16 hours with DMSO, 10 ng / mL IFN-y, 10 ng / mL IFN-y and 10 pmol / L IP AG, or 10 pmol / L IP AG. Fig. 15A: Immunoblot of PC3 whole-cell protein extracts and lysates of EVs from PC3 cells. 1 x io9EVs were loaded per lane.

[0256] Indoleamine 2,3-dioxygenase 1 (IDO), STEAP1, EV markers HRS and CD9, GAPDH, and intracellular integral membrane protein Sigmal. Fig. 15B: Coomassie stain of SDS-PAGE resolved EV proteins.

[0257] Figs. 16A-16C illustrate certain aspects of the procedure for isolation of extracellular vesicles (EVs) from cell culture medium, in accordance with some embodiments. To isolate EVs from > 1 liter volumes of cell culture medium, the present study precleared precleared the medium by centrifugation to remove dead cells and debris (conditioned medium, CM), and subsequently the present study performed hydrostatic filtration dialysis (HFD) to initially concentrate EVs, followed by high speed / ultracentrifugation. Fig. 16A: Illustration of HFD set-up. Filtration membrane were fixed at onto funnels and assembled inside graduated cylinders. Conditioned medium poured into cylinder flowed through by gravity. The flow through fraction, HFDb, was not used for experiments. EVs were isolated from the retentate. HFDa, by ultracentrifugation. Fig. 16B: Ultracentrifugation work flow of HFDa retentate fraction. HFDa fraction was centrifuged at 18 - 20,000 x g for 35’ to isolate initial, crude “P20” (indicating EV pellet collected by centrifugation at 20,000 x g) pellet, then 2 mL of PBS filtered through 0.1 micron (urn) filter was added to the crude P20 pellet and subsequently centrifuged 17.000 - 27.000 x g for 45 minutes. The supernatant (SN20) was discarded, and the final, washed P20 EV pellet was used for experiments. Fig. 16C: Photo ofAttorney Docket No. 205961-7069W01(00279)

[0258] resulting EV-P20 pellet.

[0259] Figs. 17A-17C demonstrate that Sigmal inhibitor prevents epidermal growth factor receptor (EGFR) incorporation into extracellular vesicles (EV) without decreasing total cellular EGFR protein levels, in accordance with some embodiments. MDA-MB-231 cells were treated for 24 hours with DMSO or 10 pmol / L IP AG. Fig. 17A: Immunoblot of whole cell protein extracts and EVs from MDA-MB-231 cells. 1 x 109EVs were loaded per lane. Fig. 17B: Silver stain SDS-PAGE gel of EVs from DMSO and IPAG-treated MDA-MB-231 cells. 1 x 109EVs were loaded per lane. Fig. 17C: Immunoblots of EVs separated on an iodixanol density gradient showing EGFR colocalizing in fractions with canonical EV markers HRS, CD81, Syntenin, CD9, and CD63. Fraction 10 (most dense) to 1 (least dense) fractions.

[0260] Figs. 18A-18B illustrate certain aspects of the characterization of extracellular vesicles from MDA-MB-231 cells, in accordance with some embodiments. Overnight treatment with 10 pM prototypic Sigmal inhibitor (Sigmali, IPAG). Fig. 18A:

[0261] Representative nanoparticle tracking analysis of EV size profiles using Zetaview. Fig. 18B: Quantitation of the effect of Sigmal inhibition on EV production. EV particles counted by NTA (N = 1). Particle count normalized to protein content determined by BCA assay. Y-axis represents EV numbers x 109.

[0262] Figs. 19A-19B illustrate certain aspects of the protein analysis of whole cell and extracellular vesicle (EV) lysates from MDA-MB-231 cells, in accordance with some embodiments. IxlO9EVs loaded per lane. Overnight treatment with 10 pM prototypic Sigmal inhibitor (Sigmali, in this case, IPAG) or drug vehicle (DMSO). Fig. 19A: Silver stain of Vehicle and Sigmal inhibitor-treated MDA-MB-231 EV lysates. Fig. 19B:

[0263] Immunoblot of whole-cell protein extracts and lysates of EVs from MDA-MB-231 cells. 1 x 109EVs were loaded per lane.

[0264] Fig. 20 illustrates certain aspects of the iodixanol density gradient fractions of small EVs from MDA-MB-231 cells, in accordance with some embodiments. Fraction 10 (most dense) to 1 (least dense) fractions. Immunoblots of MDA-MB-231 EVs separated on iodixanol density gradients. From MDA-MB-231 cells treated with DMSO or IPAG (10 uM) for 24 hours. These samples correspond to the total EV and whole cell lysates in Figs. 19A-19B. Proteins of interest, EGFR and PD-L1, are show n in conjunction with canonical exosome markers, including syntenin, HRS, CD9, CD63, and CD81.

[0265] Figs. 21A-21B illustrate certain aspects of the protein analysis of cell lysates and extracellular vesicle (EV) lysates from PC3 prostate cancer cells, in accordance with someAttorney Docket No. 205961-7069W01(00279)

[0266] embodiments. IxlO9EVs loaded per lane. Overnight with 10 pM prototypic Sigmal inhibitor (Sigmali, in this case IP AG), Sigmal antagonist (Sigmala, in this case NE100) or drug vehicle (DMSO) treatment. Fig. 21 A: Immunoblot of whole-cell protein extracts and lysates of EVs from PC3 cells. 1 x 109EVs were loaded per lane. Fig. 21B: Coomassie stain of whole cell lysates and Silver stain of EVs produced by PC3 cells treated with Vehicle (DMSO), Sigmali (IPAG), or Sigmala (NE100).

[0267] Fig. 22 illustrates certain aspects of the decreased extracellular vesicles production from PC3 cells treated with other Sigmal inhibitors (Sigmali), in accordance with some embodiments. Overnight treatment with 10 pM novel Sigmal inhibitor (Sigmali) small molecule compounds Cmpd 110 (aka, CT-110, CT-189) and Cmpd 1107 (aka CT-1107). Data presented as relative EV numbers relative to treatment with drug Vehicle (DMSO).

[0268] Fig. 23 demonstrates that Sigmal knockout reduced the amount of protein in EVs, in accordance with some embodiments. Extracellular vesicles (EVs) were isolated as described Example 2 section from wild-ty pe (WT) and Sigmarl knockout (KO) mouse embry o fibroblasts (MEFs), and protein concentration per EV particle was calculated using Qubit protein assay. 9.57 x 10'8and 3.25 x 10’8micrograms protein per EV particle were detected in WT and KO MEF cell culture medium, respectively.

[0269] DETAILED DESCRIPTION OF THE INVENTION

[0270] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0271] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly' recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g, 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise. Likewise, the statement "about X, Y. or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise.Attorney Docket No. 205961-7069W01(00279)

[0272] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as " A, B, or A and B." In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting: information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0273] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0274] Definitions

[0275] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described.

[0276] As used herein, each of the following terms has the meaning associated with it in this section.

[0277] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0278] The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0279] The term "abnormal," when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g, age, treatment, time of day, etc.)Attorney Docket No. 205961-7069W01(00279)

[0280] from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics that are normal or expected for one cell or tissue type might be abnormal for a different cell or tissue type.

[0281] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

[0282] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subj ect. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

[0283] A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

[0284] In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0285] A disease or disorder is "alleviated" or "ameliorated" if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

[0286] As used herein, the term " Sigma" refers to the Sigmal receptor (Sigmal), Sigma2 receptor (Sigma2), any splice variant thereof or any isoform thereof.

[0287] As used herein, a " Sigma receptor modulator" is a compound that binds to the Sigma receptor and modifies the activity or biological function of the receptor as compared to the activity or biological function of the receptor in the absence of the modulator. In certain embodiments, the modulator can activate the receptor and thus cause a biological response that is enhanced over the baseline activity of the unbound receptor. In certain embodiments, the modulator cannot activate the receptor thoroughly and thus causes a biological response that is smaller in magnitude compared to those of full modulators. In certain embodiments,Attorney Docket No. 205961-7069W01(00279)

[0288] the modulator can bind to the receptor but does not activate it, resulting in receptor blockage and inhibiting the binding of other modulators. Such an modulator does not diminish the baseline intracellular response in the absence of an modulator. In certain embodiments, the modulator can function as a putative antagonist, agonist, or as an inverse agonist, which reduces the activity of the receptor by inhibiting its constitutive activity.

[0289] The terms "patient," "subject," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

[0290] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0291] As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., a compound of the disclosure (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a condition contemplated herein and / or a symptom of a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein and / or the symptoms of a condition contemplated herein. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

[0292] The phrase "therapeutically effective amount," as used herein, refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition associated with the Sigma receptor, including alleviating symptoms of such diseases.

[0293] As used herein, the terms "effective amount," "pharmaceutically effective amount" and "therapeutically effective amount" refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0294] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non- toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with anyAttorney Docket No. 205961-7069W01(00279)

[0295] of the components of the composition in which it is contained.

[0296] As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, acetic, hexafluorophosphoric, citric, gluconic, benzoic, propionic, butyric, sulfosalicylic, maleic, lauric, malic, fumaric, succinic, tartaric, amsonic, pamoic, p-tolunenesulfomc, and mesylic. Appropriate organic acids may be selected, for example, from aliphatic, aromatic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, camphorsulfonic, citric, fumaric, gluconic, isethionic, lactic, malic, mucic, tartaric, para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic (besylate), stearic, sulfanilic, alginic, galacturonic, and the like. Furthermore, pharmaceutically acceptable salts include, by way of non-limiting example, alkaline earth metal salts (e.g, calcium or magnesium), alkali metal salts (e.g., sodium-dependent or potassium), and ammonium salts.

[0297] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate bufferAttorney Docket No. 205961-7069W01(00279)

[0298] solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985. Easton, PA), which is incorporated herein by reference.

[0299] The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term "substantially free of' as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term "substantially free of' can mean having a trivial amount of. such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0300] As used herein, the term "alkyl," by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e. Ci-6 means one to six carbon atoms) and including straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. Most preferred is (Ci-Ce)alkyl, particularly ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

[0301] As used herein, the term "substituted alkyl" means alkyl as defined above, substituted by one, two or three substituents selected from the group consisting of halogen, -OH, alkoxy, -NH2, -N(CH3)2, -C(=O)OH. trifluoromethyl, -C=N, -C(=O)O(Ci-C4)alkyl. -C(=O)NH2, -SO2NH2, -C(=NH)NH2, and -NO2, preferably containing one or two substituents selectedAttorney Docket No. 205961-7069W01(00279)

[0302] from halogen, -OH, alkoxy, -NH2, trifluoromethyl, -N(CH3)2, and -C(=O)OH, more preferably selected from halogen, alkoxy and -OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxy cyclopentyl and 3-chloropropyl.

[0303] As used herein, the term "heteroalkyl" by itself or in combination w ith another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalky 1 group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -OCH2CH2CH3, -CH2CH2CH2OH, -CH2CH2NHCH3, -CH2SCH2CH3, and -CH2CH2S(=O)CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2NHOCH3, or -CH2CH2SSCH3 As used herein, the term "alkoxy" employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1 -propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers. Preferred are (C1-C3) alkoxy, particularly ethoxy and methoxy.

[0304] As used herein, the term "halo" or "halogen" alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.

[0305] As used herein, the term "cycloalkyl" refers to a mono cyclic or polycyclic nonaromatic radical, wherein each of the atoms forming the ring (z.e. skeletal atoms) is a carbon atom. In certain embodiments, the cycloalkyl group is saturated or partially unsaturated. In other embodiments, the cycloalkyl group is fused with an aromatic ring. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties:Attorney Docket No. 205961-7069W01(00279)

[0306] O

[0307]

[0308] O o CD CO CO Monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Dicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycyclic cycloalkyls include adamantine and norbomane. The term cycloalkyl includes "unsaturated nonaromatic carbocyclyl" or "nonaromatic unsaturated carbocyclyl" groups, both of which refer to a nonaromatic carbocycle as defined herein, which contains at least one carbon carbon double bond or one carbon carbon triple bond.

[0309] As used herein, the term "heterocycloalkyl" or "heterocyclyl" refers to a heteroalicyclic group containing one to four ring heteroatoms each selected from O, Sand N. In certain embodiments, each heterocycloalkyl group has from 4 to 10 atoms in its ring system, with the proviso that the ring of said group does not contain two adjacent O or S atoms. In other embodiments, the heterocycloalkyl group is fused with an aromatic ring. In certain embodiments, the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quatemized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure. A heterocycle may be aromatic or non-aromatic in nature. In certain embodiments, the heterocycle is a heteroaryl.

[0310] An example of a 3-membered heterocycloalkyl group includes, and is not limited to, aziridine. Examples of 4-membered heterocycloalkyl groups include, and are not limited to. azetidine and a beta lactam. Examples of 5-membered heterocycloalkyl groups include, and are not limited to, pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-membered heterocycloalkyl groups include, and are not limited to, piperidine, morpholine and piperazine. Other non-limiting examples of heterocycloalkyl groups are:Attorney Docket No. 205961-7069W01(00279)

[0311]

[0312] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane, 2,3 -dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-di oxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin, and hexamethyleneoxide.

[0313] As used herein, the term "aromatic" refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e. having (4n + 2) delocalized π (pi) electrons, where n is an integer.

[0314] As used herein, the term "aryl." employed alone or in combination with other terms, means, unless otherw ise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl. Preferred examples are phenyl and naphthyl, most preferred is phenyl.

[0315] As used herein, the term "aryl-(Ci-C3)alkyl" means a functional group wherein a one-to three-carbon alkylene chain is attached to an aryl group, e.g., -CH₂CH₂-phenyl. Preferred is aryl-CFb- and aryl-CH(CH3)-. The term "substituted aryl-(Ci-C3)alkyl" means an aryl-(Ci-C3)alkyl functional group in which the aryl group is substituted. Preferred is substituted aryl(CH2)-. Similarly, the term "heteroaryl-(Ci-C3)alkyl" means a functional group wherein a one to three carbon alkylene chain is attached to a heteroaryl group.Attorney Docket No. 205961-7069W01(00279)

[0316] e.g., -CH₂CH₂-pyridyl. Preferred is heteroaryl-(CH2)-. The term "substituted heteroaryl-(Ci-C3)alkyl" means a heteroaryl-(Ci-C3)alkyl functional group in which the heteroaryl group is substituted. Preferred is substituted heteroaryl-(CH2)-.

[0317] As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocycle having aromatic character. A polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include the following moieties:

[0318]

[0319] Examples of heteroaryl groups also include pyridyl, pyrazinyl, pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (particularly

[0320] 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.

[0321] Examples of polycyclic heterocycles and heteroaryls include indolyl (particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl. quinolyl, tetrahydroquinolyl, isoquinolyl (particularly 1- and 5 -isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2- and 5 -quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

[0322] As used herein, the term "substituted" means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group. The term "substituted" further refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In certain embodiments, the substituents vary in number between one and four. In other embodiments, the substituentsAttorney Docket No. 205961-7069W01(00279)

[0323] vary' in number between one and three. In yet other embodiments, the substituents vary' in number between one and two.

[0324] As used herein, the term "optionally substituted" means that the referenced group may be substituted or unsubstituted. In certain embodiments, the referenced group is optionally substituted with zero substituents, i. e., the referenced group is unsubstituted. In other embodiments, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.

[0325] In certain embodiments, the substituents are independently selected from the group consisting of oxo, halogen, -CN, -NH2, -OH, -NH(CHs), -N(CH3)2, alkyl (including straight chain, branched and / or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoro alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, -S-alkyl, S(=O)2alkyl, -C(=O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -C(=O)N[H or alkyl]2, -OC(=O)N[substituted or unsubstituted alkyl]2, -NHC(=O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -NHC(=O)alkyl, -N[substituted or unsubstituted alkyl]C(=O)[substituted or unsubstituted alkyl], -NHC(=O) [substituted or unsubstituted alkyl], -C(OH) [substituted or unsubstituted alkyl]2, and -C(NH2)[substituted or unsubstituted alkyl]2. In other embodiments, by way of example, an optional substituent is selected from oxo, fluorine, chlorine, bromine, iodine, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -CH3, -CH2CH3. -CH(CH3)2, -CF3, -CH2CF3, -OCH3. -OCH2CH3, -OCH(CH3)2, -OCF3, - OCH2CF3, -S(=O)2-CH3, -C(=O)NH2, -C(=O)-NHCH3, -NHC(=O)NHCH3, -C(=O)CH3, and -C(=O)OH. In yet one embodiment, the substituents are independently selected from the group consisting of C1-6 alkyl, -OH, C1-6 alkoxy, halo, amino, acetamido, oxo and nitro. In yet other embodiments, the substituents are independently selected from the group consisting of C1-6 alkyl, C1-6 alkoxy, halo, acetamido, and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic, with straight being preferred.

[0326] As used herein, the term " IP AG" refers to l-(4-iodophenyl)-3-(2-adamantyl)guanidine).

[0327] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical valuesAttorney Docket No. 205961-7069W01(00279)

[0328] within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0329] Description

[0330] Interferon-gamma (IFN-y) is a pluripotent cytokine that plays a central role in the maintenance of immune system homeostasis and response to viral or bacterial infections. It can also promote anti-tumor immunity by inducing the proliferation of CTLs, such as CD8+ T cells and natural killer (NK) cells. IFN-y canonically signals through the JAK / STAT1 / 3 pathway and mediates an array of transcriptional responses. After STAT1 is phosphorylated by JAK1 / 2, it dimerizes and is translocated to the nucleus where it binds to the promoters of interferon stimulated genes, including PD-L1. Upregulation in response to IFN-y is a mechanism of adaptive immune resistance, which in the context of cancer is a process by which cancer cells evade anti-tumor immune response by changing phenotype in response to proinfl ammatory immune signals.

[0331] As PD-L1 is a ty pe I integral membrane glycoprotein, it is translated into, post-translationally modified in, and transported through, the secretory pathway of cells. In this pathway, the endoplasmic reticulum (ER) is the primary’ site of lipid and protein biosynthesis, folding, and assembly of secreted and integral membrane proteins. Integral membrane proteins are intrinsically dependent on ER protein homeostasis machinery’ to reach the cell surface. They are also dependent on the secretory pathway for controlled internalization of proteins and their subsequent endosomal trafficking and sorting.

[0332] Mechanistic studies of PD-L1 mediated immune suppression in the TME have largely focused on the actions of PD-L1 expressed on the surface of cancer cells. However, it was recently reported that PD-L1 is incorporated into cancer cell-derived extracellular vesicles, where it is capable of binding PD-1 on activated immune cells, thus creating a sort of immunosuppressive cloud around the tumor.

[0333] Extracellular vesicles (EVs) comprise a heterogeneous population of cellular membrane derived vesicles that vary' in size, morphology, and content. Based on current understanding of their biogenesis, EVs have been broadly divided into two main categories, exosomes and plasma membrane-derived EVs. The former originate from a sorting process through the endolysosomal pathway and the latter bud directly from the plasma membrane.Attorney Docket No. 205961-7069W01(00279)

[0334] EVs contain an array of proteins, lipids, and nucleotides, and the physical characteristics and biochemical composition of EVs determine their functions. An increasing number of biological processes and / or activities have been associated with EVs. However, a great deal remains unknow n regarding the underlying molecular mechanisms that govern EV biogenesis and regulation of EV content.

[0335] EV-associated PD-L1 (evPD-Ll) has been shown to suppress activated T cells both in vitro and in vivo, thereby decreasing both T cell proliferation and production of cytotoxic mediators. IFN-y has been reported to increase PD-L1 incorporation into cancer cell derived EVs, which subsequently bind and block the activation of T cells within the TME and remotely. Cancer patients are reported to have elevated levels of circulating evPD-Ll, and higher circulating evPD-Ll levels correlate with poor response to anti-PD-1 therapy. Thus, EVs present a distinct mode of immune modulation.

[0336] Extracellular vesicles are dependent on components of the secretory pathway, particularly the ER, which is an elaborate and extensive network of membrane cistemae and tubules that occupies much of the cytoplasmic space. A rapidly growing body of data demonstrates that the interconnected and contiguous ER network forms membrane contact sites (MCSs) with many organelles, including the plasma membrane (PM) and endosomes. The ER directly interacts with endosomes and influences their trafficking, cargo sorting, and fission through MCSs. ER-endosome MCSs can directly regulate the sorting of PM proteins that are internalized into vesicles that are guided and modified through the endolysosomal pathway, and these endosomes sort cargo during the routing and maturation process. The factors and mechanisms that govern selective sorting of EV cargo remain poorly defined.

[0337] Sigmal (gene name SIGMAR1,- also known as sigma- 1 receptor or SIR) is a unique pharmacologically responsive intracellular integral membrane scaffolding protein, which is enriched in the secretory pathway, particularly the ER of most cells. Sigmal itself has no known intrinsic signaling or enzymatic activity, rather it allosterically modulates the intracellular signaling and activities of its associated proteins. Sigmal physically associates with PD-L 1 and regulates its maturation, and degradation in a pharmacologically controllable manner (Mol Cancer Res 2018, 16(2):243-255). The multifunctionality of Sigmal enables it to regulate lipid and protein homeostasis at multiple levels and it contributes to protein synthesis, processing, trafficking, assembly, and quality control in the secretory pathway of cells. It has been further demonstrated that a selective small molecule inhibitor of Sigmal can block PD-Ll's cell surface expression and trigger lysosomal degradation.

[0338] Described herein is an examination of the mechanisms by w hich pharmacologicalAttorney Docket No. 205961-7069W01(00279)

[0339] inhibition of Sigmal can prevent the cell surface expression and incorporation of PD-L1 into EVs, both of which present opportunities to block antitumor immunity. A selective small molecule Sigmal inhibitor, IPAG, blocked IFN-y from upregulating PD-L1 at a post-translational step, by preventing complete N-linked glycosylation required for maturation and trafficking of functional PD-L1 to the cell surface. In multiple cancer cell lines pharmacological inhibition of Sigmal resulted in PD-L1 retention in the ER and cis-Golgi, as evidenced by Endo H sensitivity.

[0340] It has been hypothesized herein that pharmacological inhibition of Sigmal may block aspects of IFN-y mediated adaptive immune resistance. The present invention relates, in part, to the discovery' that a selective small molecule inhibitor of Sigmal blocked IFN-y-mediated upregulation of PD-L1 at a post-translational level, in part by blocking the maturation of PD-L1 glycosylation and transport to the plasma membrane. This resulted in decreased incorporation of PD-L1 into cancer cell derived EVs, specifically exosomes. Furthermore, the Sigmal inhibitor suppressed EV production, and the secreted EVs incorporated significantly less PD-L1 per exosome. As a result, the EVs produced by Sigmal inhibitor-treated cancer cells were significantly less potent in an assay of EV-mediated T cell inactivation. Thus, these data support the concept that Sigmal inhibitors play a key role in preventing cancer cell adaptive immune resistance.

[0341] The strategies of inhibiting Sigmal is not limited. In some embodiments, the compound that downregulates the expression level or the activity of Sigmal acts at the transcriptional level or the translational level. For example, the expression level of Sigmal can be down-regulated by gene knockdown, such as by RNA interference technique, ribozy me knockdown, or CRISPR knockdown.

[0342] In some embodiments, the compound that downregulates the expression level or the activity of Sigmal acts at the post-translational level. For example, the expression level of Sigmal can be down-regulated by' targeted protein degradation, such as proteolysis-targeting chimera (PROTAC) and other protein degradation strategies. For example, the activity of Sigmal can be down-regulated by small molecules inhibitors of Sigmal, antibodies that neutralizes Sigmal, and trans -dominant negative mutant of Sigmal.

[0343] Small molecule inhibitors, protein inhibitors, RNA interference molecules, CRISPR knockout / knockdown and other knockouts / knockdown techniques that can inhibit Sigmal are all well known in the art.

[0344] Small Molecule InhibitorsAttorney Docket No. 205961-7069W01(00279)

[0345] In some embodiments, the compound that downregulates Sigmal is a small molecule inhibitor. As used herein, the term “small molecule’7refers to a molecule having a size of less than 2000, 1800, 1600, 1400, 1200, 1000, 800, or 600 daltons.

[0346] The small molecule inhibitor contemplated within the disclosure may be synthesized using techniques well-known in the art of organic synthesis. The starting materials and intermediates required for the synthesis may be obtained from commercial sources or synthesized according to methods known to those skilled in the art.

[0347] In some embodiments, the compound is IP AG (l-(4-Iodophenyl)-3-(2-adamantyl)guanidine).

[0348] In one aspect, the compound contemplated within the disclosure is a compound of formula (I), or a salt, solvate, or A-oxide thereof

[0349]

[0350] wherein:

[0351] ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclic heteroaryl ring, and wherein the aryl or heteroaryl ring is optionally substituted with 0-4 R1groups;

[0352] each occurrence of R1is independently selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR3, -SR3, -S(=O)R3, -S(=O)2R3, -NHS(=O)2R3, -C(=O)R3, -OC(=O)R3, -CO2R3, -OCO2R3, -CH(R3)2, -N(R3)2, -C(=O)N(R3)2, -OC(=O)N(R3)2, -NHC(=O)NH(R3), -NHC(=O)R3. -NHC(=O)OR3, -C

[0353]

[0354] (OH)(R3)2, and -C(NH2)(R3)2;

[0355] each occurrence of R2is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl or cycloalkyl group is optionally substituted with 0-5 R1groups, or X3and R2combine to form a (C3-C7)heterocycloalkyl group, optionally substituted with 0-2 R1groups;

[0356] each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted with 0-5 R1groups;

[0357] X1is -CH2-, -S-, -O- or -(NR2)-;

[0358] X2is =CH2, =S, =0 or =NR2; and

[0359] X3is -S-. -O-, or -NR2-.Attorney Docket No. 205961-7069W01(00279)

[0360] In certain embodiments, ring A is a monocyclic aryl or monocyclic heteroaryl ring optionally substituted with 0-4 R1groups. In other embodiments, ring A is unsubstituted. In yet other embodiments, ring A is phenyl or substituted phenyl.

[0361] In a preferred embodiment, X1and X3are both -NH-, and X2is =NH.

[0362] In another aspect, the compound contemplated w ithin the disclosure is a compound of formula (II), or a salt, solvate, or A-oxide thereof:

[0363] RA-RB(II),

[0364] wherein:

[0365] RAis selected from the group consisting of

[0366]

[0367]

[0368] wherein

[0369] X4is selected from the group consisting of methoxy, F, Cl, Br, and I; and RBis selected from the group consisting of:

[0370]

[0371] Attorney Docket No. 205961-7069W01(00279)

[0372] In another aspect, the compound contemplated within the disclosure is a compound of formula (III), or a salt, solvate, or N-oxide thereof:

[0373] >2'

[0374] H H V NH

[0375]

[0376] (III), wherein within formula (III);

[0377] each occurrence of R1and R2is independently selected from the group consisting of -Ci-Ce alkyl, -C1-C6 fluoroalkyl. -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5. -SR5. -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -NHC(=O)OR5, -

[0378]

[0379] C(OH)(R5)2, and -C(NH2)(R5)2;

[0380] R3is selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce alkoxy, F, Cl, Br, and I;

[0381] R4is selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce alkoxy, F, Cl, Br, and I; each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted.

[0382] X is selected from the group consisting of CH2, C=O, or O;

[0383] n is an integer from 1-3;

[0384] x is an integer from 0-4; and

[0385] y is an integer from 0-4.

[0386] In certain embodiments, the compound contemplated within the disclosure is selected from the group consisting of:

[0387] l-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A; also known as JMS-51-58 or 51-58);

[0388] 1 -(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B);

[0389] l-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C);

[0390] l-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D);

[0391] l,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E);

[0392] 1 -(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine (Compound F);

[0393] 1 -(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound G);

[0394] 1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine (Compound H);Attorney Docket No. 205961-7069W01(00279)

[0395] a salt, solvate or N-oxide thereof; and any combinations thereof.

[0396] In one aspect, the compound contemplated within the disclosure is a compound of formula (IV), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or N-oxide thereof:

[0397] R2b R2a

[0398] Rbr Rf

[0399] R >e‘ R2c‘

[0400]

[0401] wherein:

[0402] one of the following applies:

[0403] (a) Z1is CRla; Z2is N; Z3is CR1Cor N; Z4is CRld;

[0404] (bl Z’ is N; Z2is CRlb; Z3is CR1C; Z4is CRldor N; (c) Z1is CRla; Z2is N; Z3is CR1C; Z4is N;

[0405] (d) Z1is N; Z2is CRlb; Z3is N; Z4is CRld;

[0406] X is selected from the group consisting of bond and O;

[0407] each occurrence of Rla, Rlb, Rlc. and Rldis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -N

[0408]

[0409] HC(=O)OR5. -C(OH)(R5)2, and -C(NH2)(R5)2; each occurrence of R2a, R2b, R2c, and R2dis independently selected from the group consisting of Ci-Cg alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -N

[0410]

[0411] HC(=O)OR5. -C(OH)(R5)2, and -C(NH2)(R5)2;

[0412] R3is selected from the group consisting of CN, F, Cl, Br, I, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR6;

[0413] R4is selected from the group consisting of CN, F, Cl, Br, I, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR7;

[0414] each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted;

[0415] R6is selected from the group consisting of H, Ci-Ce alkyl, C1-C6heteroalkyl, and C3-C6Attorney Docket No. 205961-7069W01(00279)

[0416] cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group is optionally substituted;

[0417] R7is selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and C3-C6 cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group is optionally substituted; and each occurrence of Ra, Rb, Rc, Rd, Re, and Rfis independently selected from the group consisting of H and CH3, with the proviso that, if Y is CRlcand Z is CRld, then at least one of Ra, Rb, Rc, Rd, Re, and Rfis CH3.

[0418] In certain embodiments, Z1is CRla; Z2is N; Z3is CRlc; Z4is CRld.

[0419] In certain embodiments, Z1is CRla; Z2is N; Z3is N; Z4is CRld.

[0420] In certain embodiments, Z1is N; Z2is CRlb; Z3is CRlc; Z4is CRld.

[0421] In certain embodiments, Z1is N; Z2is CRlb; Z3is CRlc; Z4is N.

[0422] In certain embodiments, Z1is CRla; Z2is N; Z3is CRlc; Z4is N.

[0423] In certain embodiments, Z1is N; Z2is CRlb; Z3is N; Z4is CRld.

[0424] In certain embodiments, X is bond. In certain embodiments, X is O.

[0425] In certain embodiments, each occurrence of Rla, Rlb, Rlc, and Rldis independently- selected from the group consisting of Ci-Ce alkyl. Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br. I. -CN. -NO2, and -OR5.

[0426] In certain embodiments, each occurrence of R2a, R2b, R2c, and R2dis independently selected from the group consisting of Ci-Ce alkyd, Ci-Ce fluoroalky l, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, and -OR5.

[0427] In certain embodiments, R3is CN. In certain embodiments. R3is F. In certain embodiments, R3is Cl. In certain embodiments, R3is Br. In certain embodiments, R3is I. In certain embodiments, R3is Ci-Ce haloalkyl. In certain embodiments, R3is -CFs. In certain embodiments, R3is -CHF2. In certain embodiments, R3is Ci-Ce haloalkoxy. In certain embodiments, R3is -OCF3. In certain embodiments, R3is -OCHF2. In certain embodiments, R3is -C(O)OR6. In certain embodiments, R3is -C(O)OH. In certain embodiments, R3is -C(O)O(optionally substituted Ci-Ce alkyl). In certain embodiments, R3is -C(O)O(optionally substituted Ci-Ce heteroalkyl). In certain embodiments, R3is -C(O)O(optionally substituted C3-C6 cycloalkyl). In certain embodiments, R3is -C(O)OMe. In certain embodiments. R3is -C(O)OEt. In certain embodiments, R3is -C(O)O(n-propyl). In certain embodiments, R3is -C(O)O(i-propyl). In certain embodiments, R3is -C(O)O(n-butyl). In certain embodiments, R3is -C(O)O(sec-butyl). In certain embodiments, R3is -C(O)O(iso-butyl). In certain embodiments, R3is -C(O)O(t-butyl).

[0428] In certain embodiments, R4is CN. In certain embodiments. R4is F. In certain embodiments, R4is Cl. In certain embodiments, R4is Br. In certain embodiments, R4is I.Attorney Docket No. 205961-7069W01(00279)

[0429] In certain embodiments, R4is C1-C6haloalkyl. In certain embodiments, R4is -CF3. In certain embodiments, R4is -CHF2. In certain embodiments, R4is C1-C6 haloalkoxy. In certain embodiments, R4is -OCF3. In certain embodiments, R4is -OCHF2. In certain embodiments, R4is -C(O)OR7. In certain embodiments, R4is -C(O)OH. In certain embodiments, R4is -C(O)O(optionally substituted Ci-Ce alkyl). In certain embodiments, R4is -C(O)O(optionally substituted Ci-Cs heteroalkyl). In certain embodiments, R4is -C(O)O(optionally substituted C3-C6 cycloalkyl). In certain embodiments, R4is -C(O)OMe. In certain embodiments, R4is -C(O)OEt. In certain embodiments, R4is -C(O)O(n-propyl). In certain embodiments, R4is -C(O)O(i-propyl). In certain embodiments, R4is -C(O)O(n-butyl). In certain embodiments, R4is -C(O)O(sec-butyl). In certain embodiments, R4is -C(O)O(iso-butyl). In certain embodiments, R4is -C(O)O(t-butyl).

[0430] In certain embodiments, R5is H. In certain embodiments, R5is Ci-Ce alkyl. In certain embodiments, R5is Ci-Ce heteroalkyl. In certain embodiments, R5is aryl. In certain embodiments, R5is -C1-C3 alkyl-(C3-Ce cycloalkyl). In certain embodiments, in R5the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted.

[0431] In certain embodiments, Rais H. In certain embodiments, Rais CH3.

[0432] In certain embodiments, Rbis H. In certain embodiments, Rbis CH3.

[0433] In certain embodiments, Rcis H. In certain embodiments, Rcis CH3.

[0434] In certain embodiments, Rdis H. In certain embodiments, Rdis CH3.

[0435] In certain embodiments, Reis H. In certain embodiments, Reis CH3.

[0436] In certain embodiments, Rfis H. In certain embodiments, Rfis CH3.

[0437] In certain embodiments, the compound of formula (IV) is selected from the group consisting of

[0438]

[0439] l-(6-cyano-5-methoxypyridin-3-yl)-3-(3-methyl- 3-(4-(trifluoromethyl)phenoxy)butyl)guanidine (Compound A);

[0440] H H V NH

[0441]

[0442] l-(6-cyano-5-methoxypyridin-3-yl)-3-(2,2-dimethyl-3-(4-(trifluoromethyl)phenoxy)propyl)guanidine (Compound B);Attomey Docket No. 205961-7069W01(00279) H H

[0443]

[0444] l-(5-cyano-4-methoxypyri din-2 -yl)-3-(3-methyl- 3-(4-(trifluoromethyl)phenoxy)butyl)guanidine (Compound C);

[0445]

[0446] CF3l-(3,4-dichlorophenyl)-3-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)guanidine (Compound D);

[0447]

[0448] l-(6-chloro-5-fluoropyridin-3-yl)-3-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)guanidine (Compound E);

[0449]

[0450] (4-(trifluoromethyl)phenyl)butyl)guanidine (Compound F);

[0451]

[0452] methyl 6-(3-(3-methyl-3-(4- (trifluoromethyl)phenyl)butyl)guanidino)nicotinate (Compound G);

[0453]

[0454] methyl 6-(3-(2,2-dimethyl-3-(4- (trifluoromethyl)phenoxy)propyl)guanidino)ni cotinate (Compound H);

[0455]

[0456] l-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)guanidine (CompoundAttorney Docket No. 205961-7069W01(00279)

[0457]

[0458] l-(6-cyano-5-methoxypyridin-3-yl)-3-(3-methyl- 3-(4-(trifluoromethyl)phenoxy)butyl)guanidine (Compound J).

[0459] The compounds of the disclosure may possess one or more stereocenters, and each stereocenter may exist independently in either the R or S configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In certain embodiments, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In other embodiments, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and / or separation of a mixture of enantiomers and / or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.

[0460] The methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and / or pharmaceutically acceptable salts of compounds having the structure of any compound of the disclosure, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tertbutyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like. In certain embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In other embodiments, the compounds described herein exist in unsolvated form.

[0461] In certain embodiments, the compounds of the disclosure may exist as tautomers. All tautomers are included within the scope of the compounds presented herein.

[0462] In certain embodiments, compounds described herein are prepared as prodrugs. A "prodrug" refers to an agent that is converted into the parent drug in vivo. In certainAttorney Docket No. 205961-7069W01(00279)

[0463] embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In other embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.

[0464] In certain embodiments, sites on, for example, the aromatic ring portion of compounds of the disclosure are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In certain embodiments, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.

[0465] Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to2H.3H. “C,13C,14C,36C1,18F,123I,125I,13N,15N,15O,17O,18O,32P, and35S. In certain embodiments, isotopically-labeled compounds are useful in drug and / or substrate tissue distribution studies. In other embodiments, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet other embodiments, substitution with positron emitting isotopes, such as11C,18F,15O and13N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.

[0466] In certain embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0467] The compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and as described, for example, in Fieser & Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplemental (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March. Advanced Organic Chemistry 4thEd., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry 4th Ed., Vols. A and B (Plenum 2000,2001), and Green & Wuts,Attorney Docket No. 205961-7069W01(00279)

[0468] Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for the preparation of compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein.

[0469] Compounds described herein are synthesized using any suitable procedures starting from compounds that are available from commercial sources, or are prepared using procedures described herein.

[0470] In certain embodiments, reactive functional groups, such as hydroxyl, amino, imino, thio or carboxy groups, are protected in order to avoid their unwanted participation in reactions. Protecting groups are used to block some or all of the reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed. In other embodiments, each protective group is removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal.

[0471] In certain embodiments, protective groups are removed by acid. base, reducing conditions (such as, for example, hydrogenolysis), and / or oxidative conditions. Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile. Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of amines that are blocked with acid labile groups, such as t-butyl carbamate, or with carbamates that are both acid and base stable but hydrolytically removable.

[0472] In certain embodiments, carboxylic acid and hydroxy reactive moieties are blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc. Carboxylic acid reactive moieties are protected by conversion to simple ester compounds as exemplified herein, which include conversion to alkyl esters, or are blocked with oxidatively-removable protective groups such as 2,4-dimethoxybenzyl, while coexisting amino groups are blocked with fluoride labile silyl carbamates.

[0473] Allyl blocking groups are useful in the presence of acid- and base- protecting groups since the former are stable and are subsequently removed by metal or pi-acid catalysts. For example, an allyl-blocked carboxylic acid is deprotected with a palladium-catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protectingAttorney Docket No. 205961-7069W01(00279)

[0474] groups. Yet another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, that functional group is blocked and does not react. Once released from the resin, the functional group is available to react.

[0475] Typically blocking / protecting groups may be selected from:

[0476] Cbz Alloc

[0477]

[0478] Other protecting groups, plus a detailed description of techniques applicable to the creation of protecting groups and their removal are described in Greene & Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994. which are incorporated herein by reference for such disclosure.

[0479] RNA Interference Molecules

[0480] In some embodiments, the compound that downregulates Sigma 1 is a nucleic acid that downregulates Sigmal by means of RNA interference.

[0481] In some embodiments, the nucleic acid that downregulates Sigmal by means of RNA interreference includes an isolated nucleic acid. In other embodiments, the modulator is an RNAi molecule (such as but not limited to siRNA and / or shRNA and / or miRNAs) or antisense molecule, which inhibits Sigmal expression and / or activity. In yet other embodiments, the nucleic acid comprises a promoter / regulatory sequence, such that the nucleic acid is preferably capable of directing expression of the nucleic acid. Thus, the instant specification provides expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual,Attorney Docket No. 205961-7069W01(00279)

[0482] Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein.

[0483] In certain embodiments, siRNA is used to decrease the level of Sigmal. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary’ mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U. S. Patent No. 6,506,559; Fire etal., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432: 173-178) describes a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G / C content, C / T content at the termini, Tm and the nucleotide content of the 3' overhang. See, for instance. Schwartz el al., 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, the instant specification also includes methods of decreasing levels of Sigmal using RNAi technology.

[0484] In certain embodiments, the instant specification provides a vector comprising an siRNA or antisense polynucleotide. In other embodiments, the siRNA or antisense polynucleotide inhibits the expression of Sigmal. The incorporation of a desired polynucleotide into a vector and the choice of vectors is well-known in the art.

[0485] In certain embodiments, the expression vectors described herein encode a short hairpin RNA (shRNA) inhibitor. shRNA inhibitors are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzy mes (e.g, dicer) that cleaves the shRNA to form siRNA.

[0486] The siRNA, shRNA, or antisense polynucleotide can be cloned into a number of types of vectors as described elsewhere herein. For expression of the siRNA or antisense polynucleotide, at least one module in each promoter functions to position the start site forAttorney Docket No. 205961-7069W01(00279)

[0487] RNA synthesis.

[0488] In order to assess the expression of the siRNA, shRNA, or antisense polynucleotide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected using a viral vector. In certain embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.

[0489] Following the generation of the siRNA polynucleotide, a skilled artisan will understand that the siRNA polynucleotide has certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, in some embodiments, the siRNA polynucleotide is further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Agrwal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al., 1985 Tetrahedron Lett. 26:2191-2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed.. Macmillan Press, London, pp. 97-117 (1989)).

[0490] Any polynucleotide may be further modified to increase its stability’ in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and / or the inclusion of nontraditional bases such as inosine, queosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0491] In certain embodiments, an antisense nucleic acid sequence expressed by a plasmid vector is used to inhibit Sigmal protein expression. The antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of Sigmal.

[0492] Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specificAttorney Docket No. 205961-7069W01(00279)

[0493] mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes.

[0494] The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U. S. Patent No. 5,190,931.

[0495] Alternatively, antisense molecules of the instant specification may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the instant specification include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U. S. Patent No. 5,023,243).

[0496] Ribozyme

[0497] In some embodiments, the compound that down regulates Sigmal includes a ribosome that inhibits Sigmal protein expression.

[0498] A ribozyme is used to inhibit Sigmal protein expression. Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure which are complementary, for example, to the mRNA sequence encoding Sigmal. Ribozymes are antisense RNAs which have a catalytic site capable of specifically cleaving complementary RNAs. Therefore, ribozy mes having sequence complementary to Sigmal mRNA sequences are capable of downregulating the expression of Sigmal by reduces the level of Sigmal mRNA. Ribozymes targeting Sigmal, may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them. In some embodiments, the DNA encoding the ribozymes are incorporated in a vector, which is described in the "‘Vector’ section elsewhere in the instant specification.

[0499] CRISPR knockout / knockdown and other knockouts / knockdown techniques

[0500] In some embodiments, the compound that dow n regulates the activity or expression level of Sigmal comprises a nucleic acid that down regulates the expression level of Sigmal by the means of CRISPR knockout.

[0501] In some embodiments, the compound down regulates the activity or expression levelAttorney Docket No. 205961-7069W01(00279)

[0502] of Sigmal comprises a CRISPR / Cas9 system for knocking out Sigmal.

[0503] The CRISPR / Cas9 system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a "seed" sequence within the guide RNA (gRNA) and a conserved di -nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The CRISPR / Cas9 system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and CAR T cells. The CRISPR / Cas9 system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergistic activation of target genes.

[0504] The Cas9 protein and guide RNA form a complex that identifies and cleaves target sequences. Cas9 is comprised of six domains: REC I, REC II Bridge Helix, PAM interacting, HNH, and RuvC. The Reel domain binds the guide RNA, while the Bridge helix binds to target DNA. The HNH and RuvC domains are nuclease domains. Guide RNA is engineered to have a 5' end that is complementary to the target DNA sequence. Upon binding of the guide RNA to the Cas9 protein, a conformational change occurs activating the protein. Once activated, Cas9 searches for target DNA by binding to sequences that match its protospacer adjacent motif (PAM) sequence. A PAM is a two or three nucleotide base sequence within one nucleotide downstream of the region complementary to the guide RNA. In one nonlimiting example, the PAM sequence is 5’-NGG-3'. When the Cas9 protein finds its target sequence with the appropriate PAM, it melts the bases upstream of the PAM and pairs them with the complementary region on the guide RNA. Then the RuvC and HNH nuclease domains cut the target DNA after the third nucleotide base upstream of the PAM.

[0505] One non-limiting example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in U. S. Patent Appl. Publ. No. US2014 / 0068797. CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes.

[0506] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene. In certainAttorney Docket No. 205961-7069W01(00279)

[0507] embodiments, the CRISPR / Cas system comprises an expression vector, such as, but not limited to, an pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, T7, Cas3, Cas8a, Cas8b, CaslOd, Csel, Csyl, Csn2, Cas4, CaslO, Csm2, Cmr5, Fokl, other nucleases known in the art, and any combinations thereof.

[0508] In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector includes an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, for example doxycycline). However, it should be appreciated that other inducible promoters can be used. The inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector.

[0509] In certain embodiments, guide RNA(s) and Cas9 can be delivered to a cell as a ribonucleoprotein (RNP) complex. RNPs are comprised of purified Cas9 protein complexed with gRNA and are well known in the art to be efficiently delivered to multiple types of cells, including but not limited to neurons, stem cells and immune cells (Addgene, Cambridge, MA, Minis Bio LLC, Madison, WI).

[0510] The guide RNA is specific for a genomic region of interest and targets that region for Cas endonuclease-induced double strand breaks. The target sequence of the guide RNA sequence may be within a loci of a gene or within anon-coding region of the genome. In certain embodiments, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.

[0511] Guide RNA (gRNA), also referred to as "short guide RNA" or "sgRNA", provides both targeting specificity and scaffolding / binding ability for the Cas9 nuclease. The gRNA can be a synthetic RNA composed of a targeting sequence and scaffold sequence derived from endogenous bacterial crRNA and tracrRNA. gRNA is used to target Cas9 to a specific genomic locus in genome engineering experiments. Guide RNAs can be designed using standard tools well known in the art.

[0512] In the context of formation of a CRISPR complex, "target sequence" refers to a sequence to which a guide sequence is designed to have some complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there isAttorney Docket No. 205961-7069W01(00279)

[0513] sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In certain embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be within an organelle of a eukary otic cell, for example, mitochondrion or nucleus. Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. As with the target sequence, it is believed that complete complementarity is not needed, provided this is sufficient to be functional.

[0514] In certain embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In certain embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).

[0515] In certain embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylaseAttorney Docket No. 205961-7069W01(00279)

[0516] activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity’ and nucleic acid binding activity. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in U. S. Patent Appl. Publ. No. US20110059502, incorporated herein by reference. In certain embodiments, a tagged CRISPR enzy me is used to identify the location of a target sequence.

[0517] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian and non-mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery' systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery' systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell (Anderson, 1992, Science 256:808-813; and Yu, et al., 1994, Gene Therapy 1:13-26).

[0518] In certain embodiments, the CRISPR / Cas is derived from a type II CRISPR / Cas system. In other embodiments, the CRISPR / Cas system is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species.

[0519] In general, Cas proteins comprise at least one RNA recognition and / or RNA binding domain. RNA recognition and / or RNA binding domains interact with the guiding RNA. Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains. The Cas proteins can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. In certain embodiments, the Cas-like protein of the fusion protein can be derived from a wild ty pe Cas9 protein or fragment thereof. In other embodiments, the Cas can be derived from modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, and so forth) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein. In general, a Cas9 protein comprises at least two nuclease (i.e.. DNase) domains. For example, a Cas9 protein can comprise a RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC andAttorney Docket No. 205961-7069W01(00279)

[0520] HNH domains work together to cut single strands to make a double-stranded break in DNA. (Jinek, et al., 2012, Science, 337:816-821). In certain embodiments, the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC-like or a HNH-like nuclease domain). For example, the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent). In some embodiments in which one of the nuclease domains is inactive, the Cas9-derived protein is able to introduce a nick into a doublestranded nucleic acid (such protein is termed a "nickase"), but not cleave the double-stranded DNA. In any of the above-described embodiments, any or all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and / or substitution mutations using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.

[0521] In one non-limiting embodiment, a vector drives the expression of the CRISPR system. The art is replete with suitable vectors that are useful in the instant specification. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The vectors of the instant specification may also be used for nucleic acid standard gene delivery protocols. Methods for gene delivery- are known in the art (U. S. Patent Nos. 5,399,346.

[0522] 5,580,859 & 5.589,466, incorporated by reference herein in their entireties).

[0523] Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4thEdition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New- York, 2012), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis virus, gammaretrovirus and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U. S. Patent No. 6.326.193).

[0524] In some embodiments, the compound that down regulates the activity or expression level of Sigmal comprises a nucleic acid that dow n regulates the expression level of Sigmal by the means of CRISPR knockdown. CRISPR knockdown includes, but not limited to, CRISPRCasl3 knockdown. (See e.g., Mendez-Mancilla et al., Cell Chemical Biology 29, 1-7, 2021 Jul 27, and Kushawah et al., Dev Cell. 2020 Sep 28;54(6):805-817. The entireties ofAttorney Docket No. 205961-7069W01(00279)

[0525] which are incorporated herein by reference).

[0526] In some embodiments, the present invention includes any other methods for effecting gene knockdown and / editing, which allow for deletion and / or inactivation of Sigmal, such as but not limited to those described in WO 2018 / 236840 (which is incorporated herein in its entirety by reference).

[0527] Downregulating Sigmal by inactivating and / or sequestering

[0528] In some embodiments, the compound that downregulates the activity or expression level of Sigmal includes a protein that downregulates the activity of Sigmal by inactivating and / or sequestering Sigmal. In some embodiment, the compound includes a nucleic acid that express the protein that downregulates the activity of Sigmal by inactivating and / or sequestering Sigmal. In some embodiments, the compound includes an expression vector that express the protein that downregulates the activity of Sigmal by inactivating and / or sequestering Sigmal (see “Vector’ section for descriptions on vectors).

[0529] In some embodiments, the compound that downregulates the expression level of Sigmal is a trans-dominant negative mutant of Sigmal, and / or a nucleic acid or a vector expressing the trans-dominant negative mutant of Sigmal.

[0530] Methods

[0531] The present disclosure provides methods of modulating formation and / or composition of extracellular vesicles formed by a cell. The present disclosure further provides methods of suppressing and / or inhibiting formation of extracellular vesicles by a cell. In certain embodiments, the cell is ex vivo. In certain embodiments, the cell is in vivo.

[0532] The present disclosure provides methods of modulating formation and / or composition of extracellular vesicles in a subject.

[0533] The present disclosure provides methods of activating and / or promoting a subject's antitumor immune response.

[0534] The present disclosure provides methods of preventing, minimizing, and / or reversing cancer cell adaptive immune resistance in a subject.

[0535] The present disclosure provides methods of preventing, minimizing, and / or reversing inactivation of T cells in a subject. In some embodiments, the inactivation of T cells is caused by a cancer. In some embodiments, the cancer is a so-called “cold cancer.” In some embodiments, the inactivation of T cells is caused by a tumor microenvironment (TME). In some embodiments, the TME is created by a so-called “cold cancer.”Attorney Docket No. 205961-7069W01(00279)

[0536] Non-limiting examples of cold cancers include pancreatic ductal adenocarcinoma (PDAC), prostate cancer, glioblastoma (GBM), ovarian cancer, cholangiocar cinoma, hepatocellular carcinoma, adrenocortical carcinoma, uveal melanoma, colorectal cancer (such as microsatellite stable (MSS) colorectal cancer), breast cancer (such as ER+ / HER2- breast cancer), non-small cell lung cancer, head and neck squamous cell carcinoma, gastric cancer, esophageal cancer, bladder cancer, neuroblastoma, medulloblastoma, Ewing sarcoma, rhabdomyosarcoma, and low-grade gliomas.

[0537] The present disclosure provides methods of minimizing and / or suppressing PD-L1 inclusion in extracellular vesicles in a subject.

[0538] In certain embodiments, the method comprises contacting the cell with a Sigma inhibitor. In other embodiments, the method comprises administering to the subject a therapeutically effective amount of a Sigma inhibitor.

[0539] In certain embodiments, the subject has cancer. In some embodiments, the subject has a cold cancer.

[0540] In certain embodiments, the subject is further administered at least one additional anticancer agent and / or at least one additional agent that treats, ameliorates, and / or prevents one or more cancer symptoms or co-morbidities. In other embodiments, the compound and the at least one additional agent are co-administered to the subject. In yet other embodiments, the compound and the at least one additional agent are co-formulated.

[0541] In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

[0542] In some aspects, the present invention is directed to a method of modulating formation of extracellular vesicles in a cell and / or composition of extracellular vesicles released by a cell.

[0543] In some aspects, the present invention is directed to a method of suppressing and / or inhibiting formation of extracellular vesicles in a cell.

[0544] In some aspects, the present invention is directed to a method of preventing, minimizing, and / or reversing a release of T cell-inactivating extracellular vesicles by a cell.

[0545] In some aspects, the present invention is directed to a method of minimizing and / or suppressing release of programmed death-ligand 1 (PD-L1) in extracellular vesicles by a cell.

[0546] In some embodiments, the cell is a cancer cell.

[0547] In some embodiments, the cancer cell is from a cold cancer.

[0548] In some embodiments, the cell is an isolated cell.

[0549] In some embodiments, the cell is from a cell line.Attorney Docket No. 205961-7069W01(00279)

[0550] In some embodiments, the cell is a primary' cell.

[0551] In some embodiments, the cell is in a subject.

[0552] In some embodiments, the cell is a mammalian cell.

[0553] In some embodiments, the cell is a human cell.

[0554] Vectors

[0555] Vectors can increase the stability of the nucleic acids, make the delivery easier, or allow the expression of the nucleic acids or protein products thereof in the cells.

[0556] Therefore, in some embodiments, the protein inhibitors or the nucleic acids that that down regulates the activity or expression level of Sigmal is incorporated into a vector.

[0557] In some embodiments, the instant specification relates to a vector, including the nucleic acid sequence of the instant specification or the construct of the instant specification. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In certain embodiments, the vector of the instant specification is an expression vector. Suitable host cells include a wide variety’ of prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Prokary ote- and / or eukaryote-vector based systems can be employed for use with the instant specification to produce polynucleotide, or their cognate polypeptides. Many such systems are commercially and widely available.

[0558] In some embodiments, the vector is a viral vector. Viral vector technology is well known in the art and is described, for example, in virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g, WO 01 / 96584; WO 01 / 29058; and U. S. Pat. No. 6,326,193.

[0559] In some embodiments, the viral vector is a suitable adeno-associated virus (AAV), such as the AAV1-AAV8 family of adeno-associated viruses. In some embodiments, the viral vector is a viral vector that can infect a human. The desired nucleic acid sequence, such as the nucleic acids that down regulates Sigmal described above, can be inserted between the inverted terminal repeats (ITRs) in the AAV. In various embodiments, the viral vector is an AAV2 or an AAV 8. The promoter can be a thyroxine binding globulin (TBG) promoter. In various embodiments, the promoter is a human promoter sequence that enables the desiredAttorney Docket No. 205961-7069W01(00279)

[0560] nucleic acid expression in the brain. The AAV can be a recombinant AAV, in which the capsid comes from one AAV serotype and the ITRs come from another AAV serotype. In various embodiments, the AAV capsid is selected from the group consisting of a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and a AAV8 capsid. In various embodiments, the ITR in the AAV is at least one ITR selected from the group consisting of a AAV1, AAV2, AAV3. AAV4, AAV5, AAV6, AAV7, and an AAV8 ITR. In various embodiments, the instant specification contemplates an AAV8 viral vector (recombinant or nonrecombinant) containing a desired nucleic acid expression sequence and at least one promoter sequence that, when administered to a subject, causes elevated systemic expression of the desired nucleic acid. In some embodiments, the viral vector is a recombinant or nonrecombinant AAV2 or AAV5 containing any of the desired nucleic acid expression sequences described herein. In some embodiments, the AAV is an engineered AAVs for delivering nucleic acid across the blood brain barrier to the central and peripheral nervous systems, such as those as described by Chan et al., NatNeurosci. 2017 Aug; 20(8): 1172— 1179. The entirety of this reference is incorporated herein by reference.

[0561] In some embodiments, the vector in which the nucleic acid sequence is introduced is a plasmid that is or is not integrated in the genome of a host cell when it is introduced in the cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the instant specification or the gene construct of the instant specification can be inserted include a tet-on inducible vector for expression in eukaryote cells.

[0562] The vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.

[0563] In certain embodiments, the recombinant expression vectors may also contain nucleic acid molecules which encode a peptide or peptidomimetic inhibitor of the instant specification, described elsewhere herein.

[0564] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either dow nstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with aAttorney Docket No. 205961-7069W01(00279)

[0565] polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0566] It will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression. The promoters employed may be constitutive, tissue-specific, inducible, and / or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0567] The recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer resistance to certain drugs, p-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

[0568] Combination Therapies

[0569] The compounds contemplated within the disclosure are intended to be useful in combination with one or more additional compounds. These additional compounds may comprise compounds of the present disclosure and / or at least one additional anticancer agent and / or at least one additional agent that treats one or more cancer symptoms or comorbidities.Attorney Docket No. 205961-7069W01(00279)

[0570] A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emaxequation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.

[0571] Enzyme Regul. 22:27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

[0572] Administration / Dosage / Formulations

[0573] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations contemplated within the disclosure may be administered to the subject either prior to or after the onset of a coronavirus infection. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations contemplated within the disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0574] Administration of the compositions contemplated within the disclosure to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a coronavirus infection in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated within the disclosure to treat a coronavirus infection in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound contemplated within the disclosure is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0575] Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within the disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient.Attorney Docket No. 205961-7069W01(00279)

[0576] composition, and mode of administration, without being toxic to the patient.

[0577] In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

[0578] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds contemplated within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0579] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms contemplated within the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a coronavirus infection in a patient.

[0580] In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.

[0581] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manyAttorney Docket No. 205961-7069W01(00279)

[0582] cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. In certain embodiments, the pharmaceutically acceptable carrier is not DMSO alone.

[0583] In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.

[0584] Compounds of the disclosure for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg. about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg. about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0585] In some embodiments, the dose of a compound of the disclosure is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 10.000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg. or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, orAttorney Docket No. 205961-7069W01(00279)

[0586] less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg. or less than about 10 mg, or less than about 5 mg. or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0587] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of Sigma-receptor related disorders or diseases in a patient.

[0588] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0589] Routes of administration of any of the compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans )urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0590] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.Attorney Docket No. 205961-7069W01(00279)

[0591] Oral Administration

[0592] For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose: granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

[0593] For oral administration, the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g, magnesium stearate, talc, or silica); disintegrates (e.g, sodium starch glycollate); or wetting agents (e.g, sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point. Pa. (e.g, OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g, sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).

[0594] The present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the disclosure, and a further layer providing for the immediate release of another medication. Using a wax / pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.

[0595] Parenteral Administration

[0596] For parenteral administration, the compounds of the disclosure may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection orAttorney Docket No. 205961-7069W01(00279)

[0597] infusion, or for administration in a bolus dose and / or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and / or dispersing agents may be used.

[0598] Additional Administration Forms

[0599] Additional dosage forms of this disclosure include dosage forms as described in U. S. Patents Nos. 6,340,475; 6,488.962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790.

[0600] Additional dosage forms of this disclosure also include dosage forms as described in U. S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466;

[0601] 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757.

[0602] Controlled Release Formulations and Drug Delivery Systems

[0603] In certain embodiments, the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

[0604] The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may. although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

[0605] For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

[0606] In certain embodiments of the disclosure, the compounds of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0607] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

[0608] The term pulsatile release is used herein in its conventional sense to refer to a drugAttorney Docket No. 205961-7069W01(00279)

[0609] formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

[0610] The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

[0611] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

[0612] As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

[0613] Dosing

[0614] The therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of Sigma-receptor related disorders or diseases in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

[0615] A suitable dose of a compound of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

[0616] It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every’ day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

[0617] In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the modulator of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarilyAttorney Docket No. 205961-7069W01(00279)

[0618] suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%. 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%. 90%. 95%. or 100%.

[0619] Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the viral load, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and / or infection.

[0620] The compounds for use in the method of the disclosure may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g, about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0621] Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50(the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50and ED50. Capsid assembly modulators exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such capsid assembly modulators lies preferably within a range of circulating concentrations that include the ED50with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

[0622] Those skilled in the art recognizes, or is able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of thisAttorney Docket No. 205961-7069W01(00279)

[0623] disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size / volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing / oxi dizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

[0624] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

[0625] The examples described herein illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings or disclosure of the present disclosure as set forth herein.

[0626] The examples described herein are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the disclosure should in no way be construed as being limited to the examples described herein, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0627] Without further description, it is believed that one of ordinary skill in the art can. using the description and illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The working examples therefore, specifically point out selected embodiments of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

[0628] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

[0629] While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0630] EXAMPLES

[0631] The disclosure is now described with reference to the following Examples. TheseAttorney Docket No. 205961-7069W01(00279)

[0632] Examples are provided for the purpose of illustration only, and the disclosure is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

[0633] Example 1-1: Materials and Methods

[0634] Chemicals

[0635] IPAG (1-(4-Iodophenyl)-3-(2-adamantyl) guanidine) was purchased from Tocris. Recombinant human Interferon-gamma (IFN-y) was purchased from Gibco. Endoglycosidase H (Endo H) was purchased from New England Biolabs.

[0636] Cell lines

[0637] PC3, MDA-MB-231, MDA-MB-436, and HT-29 cells were purchased from ATCC. WM164 cells were purchased from Rockland, Inc. Cells lines were authenticated by short tandem repeat profiling. All cancer cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (Coming) at 37°C with 5% CO2.

[0638] Immunoblotting and antibodies

[0639] Cell lysis, SDS-PAGE, and immunoblotting were performed. Immunoblotted proteins were revealed using Luminata Western HRP Substrate Chemiluminescence Kit (Millipore). The rabbit anti-PD-Ll (E1L3N XP). rabbit anti-STATl (D1K9Y), rabbit anti-phospho-STAT1 Y701 (58D6), rabbit anti-RCCl (D15H6), rabbit anti-Calnexin (C5C9), rabbit anticleaved PARP (D64E10), rabbit anti-Sigmal (D4J2E), and rabbit anti-HRS (D7T5N) antibodies were purchased from Cell Signaling Technology. The mouse anti-CD9 (C-4), mouse anti-CD81 (B-11) and mouse anti-GAPDH (6C5) antibodies were purchased from Santa Cruz Biotechnology. The rabbit anti-TSG101 (EPR7130(B)) and rabbit anti-CD63 antibodies were purchased from Abeam. The rabbit anti-Na(+) / K(+) ATPase antibody was purchased from Proteintech. Where indicated, cell lysates were treated with endoglycosidase H (New England Biolabs) according to the manufacturer's instructions before separation by SDS-PAGE.

[0640] PD-L1 transcript analysis by quantitative reverse transcriptase PCR (qRT-PCR)

[0641] PD-L1 mRNA transcript levels were quantified. Briefly, cells were treated approximately 24 hours after seeding with 10 ng / mL IFN-y for 16 hours in the presence or absence of 10 pmol / L of the Sigmal inhibitor, IPAG. Total cellular RNA was extracted fromAttorney Docket No. 205961-7069W01(00279)

[0642] cells using the RNeasy Kit (Qiagen) per the manufacturer’s protocol. Taq-Man primer probe sets were purchased from Life Technologies, and the genes and catalog numbers used for the qRT-PCR experiments are the following: PD-L1 (Hs00204257_ml) and GAPDH (Hs99999905-m1). The reactions were performed in triplicate using the Brilliant II qRT-PCR Master Mix 1-Step Kit (Agilent Technologies) following the manufacturer's instructions. qRT-PCR was performed using the QuantStudio 12KFlex Real Time PCR System (Applied Biosystems). PD-L1 transcripts were normalized to GAPDH transcripts.

[0643] Nuclear and cytosolic fractionation

[0644] Cells were seeded approximately 24 hours prior to treatment with 10 ng / mL IFN-y for 16 hours in the presence or absence of 10 pmol / L of the Sigmal inhibitor, IP AG. Cells were detached from the plate using 0.25% trypsin-EDTA (Coming), centrifuged at 500 x g for 5 minutes and then resuspended DPBS. 5 × 106cells were transferred to an Eppendorf tube and the nuclear and cytosolic fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Scientific) per the manufacturer’s instructions.

[0645] Flow cytometry

[0646] Cells were seeded, and 24 hours later, treated with 10 ng / mL IFN-y for 16 hours in the presence or absence of 10 pmol / L of the Sigmal inhibitor, IP AG. Cells were detached from the plate using 2 mmol / L EDTA (Coming). Cells were washed in Dulbecco’s modified phosphate buffered saline (DPBS) (Coming) and aliquoted with 1 x 106cells per tube. Cell were then fixed in 4% methanol-free formaldehyde (Sigma- Aldrich) for 15 minutes at 37°C. Cells were washed twice in staining buffer (0.5% bovine serum albumin in PBS). Cells were stained with the rabbit anti-PD-Ll extracellular domain-specific (D8T4X) Alexa Fluor 488 (Cell Signaling Technology) at 1:100 for 60 minutes at 37°C. Cells were washed and resuspended in staining buffer. Fluorescence of each cell population was analyzed on the BD LSR II flow cytometer using FACS Diva Software (BD Biosciences) and reported as mean fluorescence intensity (MFI) relative to control.

[0647] Biotinylation of cell surface proteins

[0648] Cells were seeded approximately 24 hours prior to treatment with 10 ng / mL IFN-y for 16 hours in the presence or absence of 10 pmol / L of the Sigmal inhibitor, IP AG. Cells were detached from the plate using a cell scraper and then washed in ice cold DPBS, pH 8.0 three times to remove any contaminating proteins or broken cells. Cells were resuspended in iceAttorney Docket No. 205961-7069W01(00279)

[0649] cold DPBS, pH 8.0 at a concentration of 25 x 106cells / mL and freshly prepared 10 mM Sulfo-NHS-SS-Biotin (ThermoFisher) was added per the manufacturer's instructions. Cells were incubated at room temperature for 30 minutes. Cells were washed once in ice cold 50 mM Tris, pH 8.0 to quench any non-reacted biotin reagent and then an additional 3 times with ice cold DPBS, pH 8.0.

[0650] Isolation of extracellular vesicles

[0651] 5 x 106PC3 cells were plated in 15-cm dishes and allowed to grow for 48 hours in RPMI-1640 supplemented with 10% fetal bovine serum. Cells were then rinsed 3 times with DPBS to remove all cellular and bovine EVs. Serum free medium was added to the cells containing 10 ng / mL IFN-y in presence or absence of the Sigmal inhibitor. IP AG. Twenty four hours later, the supernatant was collected from the cells and processed by differential centrifugation. The cells were collected and counted using a Countess II automated cell counter (ThermoFisher) to determine vesicle production per cell.

[0652] First, the supernatants were cleared of dead cells and debris by centrifuging at 2,000 x g for 20 minutes at 4 °C. The supernatant was carefully transferred to a clean ultracentrifuge tube, so as not to disturb the pellet, and centrifuged at 10,000 x g for 35 minutes at 4 °C to pellet and remove large microvesicles. The supernatant was again carefully transferred to a clean ultracentrifuge tube and centrifuged at 100,000 x g for 90 minutes at 4 °C to pellet small EVs (" OOK EV pellef ’). All 100K EV pellets for each condition, washed in DPBS, and pelleted at 100,000 x g for 90 minutes at 4 °C. The 100K EV pellet was resuspended in DPBS and an aliquot was immediately analyzed by nanoparticle tracking analysis (NTA). Other aliquots were frozen at -80 °C for storage. The pellets were lysed in RIPA buffer (Pierce) and sonicated for 15 seconds prior to analysis by immunoblot.

[0653] Density gradient separation of EV populations

[0654] The 100K EV pellets were further purified using an iodixanol density gradient. 30%, 20%, and 10% w / v iodixanol solutions were prepared by diluting a 60% w / v iodixanol stock solution (OptiPrep, Sigma-Aldrich) with a 0.25 M sucrose / 10 mM Tris / 1 mM EDTA buffer solution at pH 7.4. The EVs suspended in the buffer solution were combined with iodixanol to make 1,600 pL of a 30% w / v suspension and loaded into the bottom of a SW 55 Ti rotor tube (Beckman). Then, 700 pL of the 20% w / v iodixanol and 700 pL of 10% w / v iodixanol solutions were sequentially layered on top of the 30% EV-iodixanol suspension to create a discontinuous gradient. After a 15-minute rest period, the tubes were spun at 350,000 x g forAttorney Docket No. 205961-7069W01(00279)

[0655] 1 hour at 4 °C using a SW 55 Ti swing rotor (Beckman) in a L8-70M ultracentrifuge (Beckman). Ten equal fractions were collected from the bottom of the tube and their densities determined using an ABBE-3L refractometer (Fisher Scientific). The fractions were then diluted to 1 mL using DPBS to wash away the iodixanol, and the EVs were pelleted at 100,000 x g for 90 minutes at 4 °C using a TLA-100.2 rotor (Beckman) in an Optima TL tabletop ultracentrifuge (Beckman). EV pellets were resuspended in 30 pL of DPBS and stored at -80°C to await further use.

[0656] Characterization of extracellular vesicles by nanoparticle tracking analysis (NTA) Aliquots of the isolated EVs were diluted 1: 100 in DPBS and were then analyzed by NTA to determine particle size and particle concentration using a NanoSight NS300 (Malvern Instruments). Three aliquots per treatment were analyzed and averaged together. The instrument collected three 30-second videos per aliquot at 30 frames per second to capture the EV particles moving under Brownian motion. The NanoSight NS300 software then analyzed the videos to determine the particle size profile and particle concentration of each sample.

[0657] Transmission electron microscopy

[0658] Of the isolated 100K EV pellet suspended in DPBS, 10 pL were loaded onto 400 square mesh carbon-coated copper grids (Electron Microscopy Sciences), which were glow discharged for 60 seconds at 15 mA, 0.3 mbar using an PELCO easiGlow glow discharger (Ted Pellla, Inc.) immediately before loading. The grids were then washed in ultrapure water for 60 seconds, negatively stained for 60 seconds using 1 % uranyl acetate (Electron Microscopy Sciences), then washed for 60 seconds in ultrapure water. The grids were then air dried for 5 minutes before being imaged using a FEI Tecnai 12 Transmission Electron Microscope, equipped with an XR111 11-megapixel CCD (Advanced Microscopy Techniques) at 80.0 kV.

[0659] PD-L1 / PD-1 blockade assay

[0660] The PD-L1 / PD-1 Blockade Assay Kit (Promega, J1250) was used. Briefly, 4 x 105engineered artificial antigen presenting cell (aAPC) antigen expressing and PD-L1 negative CHO cells were added per well to Wallac B& W 96-well isoplates with white w alls and bottoms. Approximately 16 hours later, 2.5 x 103engineered nuclear factor of activated T cells (NFAT)-luciferase Jurkat cells expressing the aAPC T-cell receptor (TCR) and PD-1Attorney Docket No. 205961-7069W01(00279)

[0661] were added to each well in the presence or absence of 1 x 1010freshly isolated drug-treated cancer cell-derived EVs. as described above. Six hours later, Promega Bio-Gio reagent was added to each well and allowed to incubate at room temperature for 5 minutes before reading the plate on a GloMax Discover microplate luminometer (Promega).

[0662] When the NFAT-luciferase Jurkat cells are cocultured with the aAPC / PD-Ll negative CHO cells, the aAPC-TCR on the Jurkat cells becomes activated when it binds to the aAPC antigen on the CHO cells. In the absence of PD-L1 binding to PD-1, the TCR signaling cascade results in downstream activation of the NF AT response element. In these engineered Jurkat cells, a luciferase gene is added downstream of the NF AT response element, resulting in only activated T cells showing luminescent signal. This activation of the NF AT response element can be inhibited by PD-1 / PD-L1 interactions. When PD-1 on the Jurkat cells binds evPD-Ll on the cancer cell EVs, PD-1 will suppress TCR signaling, the NF AT response element will become inactive, and the luminescence will be suppressed.

[0663] Trypan blue assay

[0664] Cells were detached from the plate using 0.25% trypsin-EDTA (Coming). The trypsin was washed away, and the cells were resuspended in DPBS (Coming). Cell suspensions were mixed in a 1:1 ratio with trypan blue and the percent live cells were determined in quadruplicate using a Countess II automated cell counter (ThermoFisher).

[0665] Statistical analysis

[0666] To determine the statistical significance of single comparisons, an unpaired Student’s t test was used. To determine the statistical significance of multiple comparisons, a one-way ANOVA with Bonferroni post-test was performed using Prism software (GraphPad).

[0667] Example 1-2: Pharmacological inhibition of Sigmal does not block IFN-y mediated STAT1 phosphorylation and translocation to the nucleus and does not suppress PD-L1 transcript levels

[0668] IFN-y induces STAT1 phosphorylation, which subsequently dimerizes and translocates to the nucleus where it mediates transcriptional upregulation of PD-L1. To determine if pharmacological inhibition of Sigmal blocks this signaling cascade, experiments were conducted using IP AG ( 1 -(4-Iodophenyl)-3-(2-adamantyl)guanidine). a selective small molecule Sigmal inhibitor. The Sigmal specificity of IP AG has been demonstrated by several orthogonal studies in multiple publications.Attorney Docket No. 205961-7069W01(00279)

[0669] Rather than focusing on a specific cancer type, cancer cell lines that either constitutively express high levels of PD-L1. or inducible lines that express low or undetectable basal levels of PD-L1 in the absence of IFN-y, were examined. MDA-MB-231 and PC3 cells were chosen to examine the effect of IP AG on cell lines with high levels of constitutive PD-L1 expression. PC3 cells were also chosen because of their ability to produce relatively large quantities of EVs with associated PD-L1. The low basal PD-L1 expressing WM164, HT29, and MDA-MB-436 cells were chosen to examine the effects of IPAG on cell lines with IFN-y inducible PD-L1. The lack of high basal PD-L1 expression on the latter group of cell lines permitted examination of the effects of IPAG on nascent PD-L1.

[0670] Consistent with earlier reports, IFN-y induced PD-L1 transcripts in all cell lines evaluated herein (Fig. 2A). Co-treatment with IPAG did not block IFN-y mediated upregulation of PD-L1 transcripts, and in the majority of cell lines, there were more PD-L1 transcripts, up to two-fold more, compared to IFN-y treatment alone (Fig. 2A). IFN-y treatment resulted in increased STAT1 protein levels and increased phospho-STATl levels (Fig. 2B). IPAG did not significantly reduce IFN-y induced phospho-STATl levels (Fig. 2B). Additionally, IPAG did not prevent phospho-STATl from translocating into the nucleus of the cells (Fig. 2C), which is required for IFN-y mediated transcriptional induction of PD-L1. Therefore, IPAG did not prevent IFN-y mediated transcriptional upregulation of PD-L1.

[0671] Example 1-3: Pharmacological inhibition of Sigmal blocks post-translational modification and maturation of IFN-y-induced PD-L1

[0672] Sigmal physically associates with nascent PD-L1. However, it was unclear whether IPAG, would prevent nascent PD-L1 from maturing and progressing through the secretory pathway.

[0673] In all of the cell lines tested, IFN-y upregulated PD-L1 protein levels, consistent with the increased levels of PD-L1 mRNA transcripts (Fig. 2A). When IPAG was combined with IFN-y, PD-L1 protein levels decreased, as previously reported (Mol Cancer Res 2018, 16(2):243-355), in cells that constitutively expressed high levels of PD-L1 (Fig. 3A). In low basal expressing, IFN-y inducible PD-L1 cell lines (WM164, HT29, and MDA-MB-436), it was observed that PD-L1 protein was still produced (Fig. 3A), indicating that PD-L1 transcripts were translated into PD-L1 protein, thus IPAG did not block PD-L1 translation. However, in the low basal expressing lines, the combination of IFN-y and IPAG resulted in accumulation of PD-L1 at a lower molecular weight band, at approximately 40 kDa, that was not seen with IFN-y alone (Fig. 3A). Quantification of the banding pattern (Fig. 3B)Attorney Docket No. 205961-7069W01(00279)

[0674] confirmed that there was a higher percentage of the PD-L1 migrating at approximately 40 kDa in the IFN-y and IPAG combination treatment condition compared to treatment with IFN-y alone. Additionally, others have reported that differential banding patterns of PD-L1 could be the result of differential N-linked glycosylation (Nat Commun 2016, 7:12632).

[0675] To determine whether the accumulated lower molecular weight PD-L1 band reflected inhibition of proper glycosylation, cell lysates were treated with endoglycosidase H (Endo H). This enzyme cleaves all immature, high-mannose N-linked glycans in the ER and cis Golgi before glycoproteins are processed by the enzyme Golgi alpha-mannosidase II in the medial Golgi, after which the glycans are no longer susceptible to cleavage by Endo H. It was observed that the ~40 kDa PD-L1 band that accumulated in the IFN-y and IPAG combination treatment condition was Endo H sensitive (Fig. 3C). As the lower molecular weight band did not migrate at 33 kDa (which represents the non-glycosylated PD-L1), but rather ~40 kDa, it appears that IPAG did not prevent the initial attachment of the N-linked glycans, but rather prevented its proper trimming and maturation. Thus, IPAG prevented the maturation of nascent PD-L1 by disrupting N-linked glycosylation.

[0676] Example 1-4: Pharmacological inhibition of Sigmal decreases cell surface PD-L1 levels IFN-y mediated upregulation of PD-L1 also increases PD-L1 cell surface expression. It has been demonstrated herein that IFN-y induced cell surface PD-L1 levels in all cancer cell lines tested. Flow cytometry experiments demonstrated IPAG prevented constitutively expressed and IFN-y induced PD-L1 expression at the surface of these cells (Fig. 4A).

[0677] Further, cell surface protein biotinylation studies were performed to confirm this effect using an orthogonal method which has the added advantage of detecting distinct banding patterns and thus PD-L1 protein post-translational modifications (Fig. 4B). In MDA-MB-436 cells. IFN-y increased cell surface PD-L1 levels, however, the lower ~40 kDa band produced in response to IFN-y and IPAG combination was not biotinylated, thus indicating that the Endo H sensitive band of PD-L1 was not present at the cell surface (Fig. 4B). The cell compartment control calnexin (ER-membrane protein) was not biotinylated, while the Na+ / K+-ATPase (cell surface protein) was biotinylated (Fig. 4B), providing further support for the concept that pharmacological inhibition of Sigmal did not impact all cell surface and integral membrane proteins. Thus, IPAG selectively blocked IFN-y mediated cell surface expression of PD-L1.

[0678] Example 1-5: Pharmacological inhibition of Sigmal suppresses the production ofAttorney Docket No. 205961-7069W01(00279)

[0679] extracellular vesicles

[0680] There is a growing body of evidence suggesting that Sigmal is a multifunctional chaperone or scaffolding protein that contributes to the maintenance of ER protein homeostasis in cancer cells and supports their increased utilization of the secretory pathway. Extracellular vesicle production, and the physical and biochemical characteristics thereof, are dependent on the secretory pathway.

[0681] To determine whether IPAG would alter the production and characteristics of EVs, nanoparticle tracking analysis (NTA) was performed. NTA experiments showed no salient differences in the overall size profile of EVs produced by cells treated with vehicle (DMSO), IFN-y, IFN-y with IP AG, or IP AG alone (Fig. 5A, Table 1). However, the number of EVs secreted per cell significantly decreased (approximately 75%) in IP AG treated cell culture (Fig. 5B). Additional analysis of the 100 K EV pellets by transmission electron microscopy (TEM) confirmed the presence of vesicles mostly ranging in size from 50-200 nm (Fig. 5C).

[0682] Table 1. Size profile of extracellular vesicles3

[0683] Treatment Mode ± SEM Mean ±SEM DMSO 139.2 ± 2.7 nm 181.1 ± 3.1 nm IFN-y 142.0 ± 3.0 nm 188.8 ± 3.4 nm IFN-y + IP AG 150.2 ± 3.8 nm 190.0 ± 4.4 nm IPAG 145.9 ± 4.8 nm 185.3 ± 4.2 nm

[0684]

[0685] aMean and mode of each treatment condition compiled from 27 independent determinations ± SEM

[0686] Decreased EV production by IP AG was not due to cell death or cells undergoing apoptosis, which w as confirmed by a trypan blue exclusion assay and the absence of cleaved PARP, respectively (Figs. 5D-5E). Therefore, pharmacological inhibition of Sigmal suppressed the number of EVs released by the cancer cells.

[0687] Although IP AG induces an unfolded protein response (UPR) and autophagy, these properties alone may not explain its EV decreasing effects. General ER stress does not necessarily decrease EV production. Indeed, thapsigargin and tunicamycin, which both trigger ER stress manifested as UPR, do not decrease, but rather increase, EV production.

[0688] Thus, the discovery’ that a pharmacological inhibitor of Sigmal that also triggers UPR and autophagy decreased EV production was unexpected. Furthermore, previous studies have shown that thapsigargin and tunicamycin did not trigger degradation of PD-L1. Altogether,Attorney Docket No. 205961-7069W01(00279)

[0689] these data suggest a novel and distinct mechanism of action of Sigmal inhibitors, one in which UPR and autophagy are necessary’, but not sufficient, to suppress the production of EVs.

[0690] Example 1-6: Pharmacological inhibition of Sigmal suppresses extracellular vesicle associated PD-L1 (evPD-Ll)

[0691] To be packaged into EVs, PD-L1 needs to first be expressed on the cell surface. PD-L1 may subsequently enter the endosomal sorting pathway where it can be selectively packaged into exosomes or recycled to the cell surface. Since IP AG prevented PD-L1 from reaching the cell surface, it was hypothesized that IP AG would inhibit PD-L 1 incorporation into EVs.

[0692] It has been demonstrated herein that IFN-y promotes PD-L1 incorporation into EVs. Furthermore, increased levels of PD-L1 in EVs produced by IFN-y treated PC3 cells were observed (Figs. 6A-6B). IP AG prevented IFN-y-induced PD-L1 from being incorporated into the EVs (Figs. 6A-6B). These data support the hypothesis that targeting Sigmal pharmacologically can selectively suppress the packaging of PD-L 1 into EVs.

[0693] Interestingly, while IP AG prevented PD-L1 from being incorporated into EVs, it did not suppress other EV associated proteins, including CD9, CD63, HRS, and TSG101 (Figs.

[0694] 6A-6B). TSG101 is a component of the ESCRT complex that plays a crucial role in exosome assembly and production. Tetraspanins, including CD9. and CD63. are scaffolding proteins that mediate selective EV cargo packaging. They are also integral membrane glycoproteins and as such are processed through the secretory pathway. However, in contrast to PD-L1, CD9 incorporation into EVs was not altered by IP AG, and CD63 incorporation into EVs was enriched with IPAG.

[0695] Additionally, IPAG treatment increased packaging of HRS into EVs. HRS is another component of EVs and a ubiquitin binding protein that has been reported to facilitate packing of ubiquitinated proteins into EVs. It has been reported that IPAG induces protein ubiquitination leading to alterations in the fate of Sigmal associated proteins (Nat Cell Biol 2019, 21(1):9-17). As demonstrated herein, IPAG treatment alone or in combination with IFN resulted in increased HRS in EVs. Without wishing to be bound by theory, these data suggest that proteins that are ubiquitinated in response to IPAG treatment may be targeted and sorted into EVs. These data indicate a selective targeting of EV-associated proteins by a Sigmal modulator. Taken together, these data suggest that targeting Sigmal pharmacologically can be used to selectively package multiple proteins into EVs.Attorney Docket No. 205961-7069W01(00279)

[0696] PC3 cells secreted PD-L1 on exosomes (Cell 2019, 177(2):414-427). However, it was unclear whether the Sigmal inhibitor blocked evPD-Ll that was specifically exosomal. Separation of the 100K EVs by iodixanol density gradient fractionation revealed PD-L1 in vesicles with densities consistent with the published densities of exosomes (Cell 2019, 177(2):428-445) (Fig. 7). Furthermore, PD-L1 co-isolated into fractions containing CD9, CD81, CD63, and TSG101, all of which are canonical markers of exosomes (Fig. 7). These results suggest that the evPD-Ll described here are in exosomes.

[0697] Example 1-7: Pharmacological inhibition of Sigmal results in the production of extracellular vesicles with significantly diminished potency in an assay of T cell inactivation

[0698] The data above demonstrate that IP AG can decrease the number of EVs produced by cancer cells as well as decrease the amount of PD-L1 per EV. These results suggest that pharmacological inhibition of Sigmal can reduce the density as well as the potency of the tumor protective cloud of evPD-Ll and thus may decrease the chance that T cells entering the TME will be inactivated. To determine if PD-L1 -containing EVs produced in response to IP AG treatment have diminished potency in blocking T cell inactivation in vitro, a modified version of a Nuclear Factor of Activated T cell (NF AT) transcriptional response element luciferase reporter co-culture assay (NFAT-luciferase) was used. Using the modified version of this assay, as described herein, Chinese hamster ovary (CHO) activator cells that express the artificial antigen presenting cell (aAPC) antigen (an engineered cell surface protein designed to activate cognate TCRs in an antigen-independent manner), but do not express PD-L1, were co-cultured with an immortalized luciferase reporter transfected T cell line (Jurkat) that expresses both the T cell receptor (TCR) for the aAPC antigen and PD-1 (Fig.

[0699] 8A). When these cells were co-cultured, the activator cells activate the reporter T cells via aAPC-TCR interactions, and because the activator cells lack PD-L1, there is no suppression of NF AT luciferase signaling. Under these conditions, the maximum relative luciferase signal is generated in the absence of cancer cell derived EVs in the cell culture medium (Fig. 8A). However, when cancer cell derived PD-L1 expressing EVs (evPD-Ll) are added to this coculture, evPD-Ll is able to bind PD-1 on the reporter T cells and block NF AT activation (Figs. 8A-8B). PC3 cells treated with IFN-y did not increase the overall amount of EVs produced, however, it did produce EVs with increased amounts of PD-L1. It was found that this increased the efficacy of the PD-L1 -containing EVs in the T cell inactivation assay (Fig.

[0700] 8 A). When IP AG was combined with IFN-y, this not only decreased the number of EVsAttorney Docket No. 205961-7069W01(00279)

[0701] produced by PC3 cells, it also decreased the concentration of EV associated PD-L1. In this assay, equal numbers of EVs were added to each co-culture condition (Fig. 8B). The PD-L1-containing EVs produced by IPAG co-treated cells were significantly less potent in their ability to inactivate T cells. Thus, IPAG was able to inhibit the ability of cancer cells to inactivate T cells by way of evPD-Ll. The implications of these data are that pharmacological inhibition of Sigmal may be used not only to prevent cancer cells from inactivating T cells in direct contact but can also be used to prevent the cloud of immunosuppressive EVs.

[0702] Example 2: Sigmal inhibitor suppression of adaptive immune resistance mechanisms mediated by cancer cell derived extracellular vesicles

[0703] Adaptive immune resistance in cancer describes the various mechanisms by which tumors adapt to evade anti-tumor immune responses. IFN-y induction of programmed deathligand 1 (PD-L1) was the first defined and validated adaptive immune resistance mechanism. The endoplasmic reticulum (ER) is central to adaptive immune resistance as immune modulatory secreted and integral membrane proteins are dependent on ER. Sigmal is a unique ligand-regulated integral membrane scaffolding protein enriched in the ER of cancer cells. PD-L1 is an integral membrane glycoprotein that is translated into the ER and processed through the cellular secretory pathway. At the cell surface, PD-L1 is an immune checkpoint molecule that binds PD-1 on activated T-cells and blocks anti-tumor immunity. PD-L1 can also be incorporated into cancer cell-derived extracellular vesicles (EVs), and EV-associated PD-L1 can inactivate T-cells within the tumor microenvironment. Here, the present study demonstrates that a selective small molecule inhibitor of Sigmal can block IFN-y mediated adaptive immune resistance in part by altering the incorporation of PD-L1 into cancer cell-derived EVs. Sigmal inhibition blocked post-translational maturation of PD-L1 downstream of IFN-y / STATl signaling. Subsequently, EVs released in response to IFN-y stimulation were significantly less potent suppressors of T-cell activation. These results suggest that by reducing tumor derived immune suppressive EVs, Sigmal inhibition may promote antitumor immunity. Sigmal modulation presents a novel approach to regulating the tumor immune microenvironment by altering the content and production of EVs. Altogether, these data support the notion that Sigmal may play a role in adaptive immune resistance in the tumor microenvironment.

[0704] Example 2-1:Attorney Docket No. 205961-7069W01(00279)

[0705] Programmed death-ligand 1 (PD-L1, also known as B7-H1 or CD274) is a type I integral membrane glycoprotein that can be induced to express on the surface of many cell types.1 Upon binding its cognate receptor, programmed death- 1 (PD-1, also known as CD279), PD-L1 acts as an inhibitory checkpoint molecule and suppresses T cell activation and subsequent proliferation.1 PD-1 / PD-L1 blockade of the host immune response normally serves to protect against autoimmunity and excessive tissue damage at the site of inflammation. However, many cancers co-opt this protective feature of the immune system to evade antitumor immunity. In the context of cancer, PD-L1 on the surface of cancer cells binds to PD-1 on tumor-infiltrating cytotoxic T lymphocytes (CTLs) and inactivates these contributors to the host’s antitumor immune response.1 Immunomodulatory checkpoint inhibitor antibodies that block PD-L1 / PD-1 interactions have exhibited remarkable clinical efficacy in subsets of patients with melanoma, non-small cell lung cancer, metastatic bladder cancer, and renal cell carcinoma. However, only a relatively low percentage of patients respond to anti-PD-l / PD-Ll monotherapy. Furthermore, in many patients who initially respond to immune checkpoint blockade such as anti-PD-l / PD-Ll therapy, tumors can adapt and become resistant to this immunotherapy thus limiting the duration and magnitude of their anti-tumor response. The mechanisms underlying resistance to checkpoint blockade are not well understood.

[0706] Emerging data suggest that chronic upregulation of interferon-gamma (IFN-y) response pathways is a key driver of acquired resistance. So-called adaptive immune resistance (AIR) in cancer describes the various mechanisms by which tumors adapt to evade anti -tumor immune responses. IFN-y induction of PD-L1 was the first defined and therapeutically validated AIR mechanism. PD-L1 upregulation in response to IFN-y is a key mechanism of AIR. IFN-y is a pluripotent cytokine that plays a central role in the maintenance of immune system homeostasis. It can promote anti-tumor immunity by inducing the proliferation of CTLs, such as CD8+ T cells and natural killer (NK) cells. IFN-y plays a central role in coordinating and balancing innate and adaptive immunity by activating the immune responses to eliminate pathogens and tumors and preventing immune overactivation with associated, nonspecific tissue damage. The complex, context-dependent mechanisms that maintain this balance remain poorly defined.

[0707] Mechanistic studies of PD-L1 mediated immune suppression in the TME have largely focused on the actions of PD-L1 expressed on the surface of cancer cells. However, PD-L1 is also incorporated into cancer cell-derived extracellular vesicles (EVs) that create a sort of immunosuppressive barrier to the tumor. EVs comprise heterogeneous populations of cellularAttorney Docket No. 205961-7069W01(00279)

[0708] membrane derived vesicles that vary in size, morphology, density, and content. EVs contain an array of proteins, lipids, and nucleotides, and the physical characteristics and biochemical composition of EVs determine their functions. There is a rapidly growing number of biological activities associated with EVs; however, much remains unknown regarding the underlying molecular mechanisms that govern EV biogenesis and regulation of EV content and their biological actions. EV-associated PD-L1 (evPD-Ll) has been shown to suppress activated T cells both in vitro and in vivo, thereby decreasing both T cell proliferation and production of cytotoxic mediators. IFN-y has been reported to increase PD-L1 incorporation into cancer cell derived EVs and IFN-y enhanced PD-L1 expression by EVs has been reported to contribute to the immune suppressive tumor microenvironment (TME). Cancer patients are reported to have elevated levels of circulating evPD-Ll, and these higher circulating evPD-Ll levels have been shown to correlate with a poor response to anti-PD-1 therapy.

[0709] EVs are dependent on components of the secretory pathway, particularly the endoplasmic reticulum (ER). The ER is an elaborate and extensive network of membrane cistemae and tubules that occupies much of the cytoplasmic space. A rapidly growing body of data demonstrates that the interconnected and contiguous ER network forms membrane contact sites (MCSs) with many organelles, including the plasma membrane (PM) and endosomes. The ER directly interacts with endosomes and influences their trafficking, cargo sorting, and fission through MCSs. ER-endosome MCSs can directly regulate the sorting of PM proteins that are internalized into vesicles guided and modified through the endolysosomal sorting pathway, and these endosomes sort cargo during the routing and maturation process. The factors and mechanisms that govern the selective sorting of EV cargo are complex and remain poorly defined. The cellular factors that regulate the production and activities of EVs in AIR are also poorly understood.

[0710] The ER is central to AIR as immune modulatory secreted and integral membrane proteins are dependent on ER. Sigmal (gene name SIGMAR1; also known as sigma- 1 receptor) is a unique pharmacologically responsive intracellular integral membrane scaffolding protein. Sigmal is enriched in the secretory pathway, particularly the ER of most cells. Sigmal itself has no known intrinsic signaling or enzymatic activity, rather it allosterically modulates the intracellular signaling and activities of its associated proteins. The multifunctionality of Sigmal enables it to regulate lipid and protein homeostasis at multiple levels and it contributes to protein synthesis, processing, trafficking, assembly, and quality control in the secretory pathway of cells. We, and others, have shown that inhibitionAttorney Docket No. 205961-7069W01(00279)

[0711] of Sigmal disrupts ER protein and membrane dynamics through the secretory pathway.

[0712] While it has been shown that Sigmal inhibition can induce autophagic degradation of PD-L1, here, it is hypothesized that pharmacological inhibition of Sigmal could block aspects of IFN-y mediated adaptive immune resistance through effects on PD-L1 expression and incorporation into immune modulatory EVs. The present study also discovered that a selective small molecule inhibitor of Sigmal blocked IFN-y-mediated upregulation of PD-L1 at a post-translational level. Furthermore, Sigmal inhibitor treatment decreased incorporation of PD-L1 into cancer cell derived EVs. The EVs produced by Sigmal inhibitor-treated cancer cells interfered with IFN-y-induced EV mediated T cell inactivation. Altogether, these data support the notion that Sigmal could play a role in adaptive immune resistance in the TME through both intracellular and extracellular mechanisms.

[0713] Example 2-2: Materials and methods

[0714] Chemicals

[0715] IPAG (1-(4-Iodophenyl)-3-(2-adamantyl) guanidine) was purchased from Tocris. Recombinant human Interferon-gamma (IFN-y) was purchased from Gibco. Endoglycosidase H (Endo H) was purchased from New England Biolabs.

[0716] Cell lines

[0717] PC3 (androgen receptor negative prostate adenocarcinoma). MDA-MB-231 (triple negative breast cancer), MDA-MB-436 (triple negative breast cancer), and HT-29 (colon cancer) cells were purchased from ATCC. WM164 (melanoma) cells were purchased from Rockland, Inc. Cells lines were authenticated by short tandem repeat profiling. All cancer cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (Coming) at 37°C with 5% CO2.

[0718] Immunoblotting and antibodies

[0719] Cell lysis, SDS-PAGE. and immunoblotting were performed. Immunoblotted proteins were revealed using Luminata Western HRP Substrate Chemiluminescence Kit (Millipore). The rabbit anti-PD-Ll (E1L3N XP, catalog # 13684), rabbit anti-STATl (D1K9Y, catalog # 14994), rabbit anti-phospho-STATl Y701 (58D6, catalog # 9167), rabbit anti-RCCl (D15H6, catalog # 5134), rabbit anti-Calnexin (C5C9, catalog # 2679), rabbit anti-cleaved PARP (D64E10, catalog # 5625), rabbit anti-Sigmal (D4J2E. catalog # 61994), and rabbit anti-HRS (D7T5N, catalog # 15087) antibodies were purchased from Cell SignalingAttorney Docket No. 205961-7069W01(00279)

[0720] Technology. The mouse anti-CD9 (C-4, catalog # sc -13,118), mouse anti-CD81 (B-ll, catalog # sc -166,029) and mouse anti-GAPDH (6C5. catalog # sc -32,233) antibodies were purchased from Santa Cruz Biotechnology. The rabbit anti-CD63 antibody (catalog # ab68418) was purchased from Abeam. The rabbit anti-Na(+) / K(+) ATPase antibody (catalog # 14418-1 -AP) was purchased from Proteintech. Where indicated, cell lysates were treated with endoglycosidase H (New England Biolabs) according to the manufacturer’s instructions before separation by SDS - PAGE.

[0721] PD-L1 transcript analysis by quantitative reverse transcriptase PCR (qRT-pcr)

[0722] PD-L1 mRNA transcript levels were quantified. Briefly, cells were treated approximately 24 hours after seeding with 10 ng / mL IFN-y for 16 hours in the presence or absence of 10 pmol / L of the Sigmal inhibitor, IPAG. Total cellular RNA was extracted from cells using the RNeasy Kit (Qiagen) per the manufacturer’s protocol. Taq-Man primer probe sets were purchased from Life Technologies, and the genes and catalog numbers used for the qRT-PCR experiments are the following: PD-L1 (Hs00204257_ml) and GAPDH (Hs99999905-m1). The reactions were performed in triplicate using the Brilliant II qRT-PCR Master Mix 1-Step Kit (Agilent Technologies) following the manufacturer’s instructions. qRT-PCR was performed using the QuantStudio 12KFlex Real Time PCR System (Applied Biosystems). PD-L1 transcripts were normalized to GAPDH transcripts.

[0723] Nuclear and cytosolic fractionation

[0724] Cells were seeded approximately 24 hours prior to treatment with 10 ng / mL IFN-y for 16 hours in the presence or absence of 10 pmol / L of the Sigmal inhibitor, IPAG. Cells were detached from the plate using 0.25% trypsin-EDTA (Coming), centrifuged at 500 x g for 5 minutes and then resuspended DPBS. 5 × 106cells were transferred to an Eppendorf tube and the nuclear and cytosolic fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Scientific) per the manufacturer’s instructions.

[0725] Flow cytometry

[0726] Cells were seeded, and 24 hours later, treated with 10 ng / mL IFN-y for 16 hours in the presence or absence of 10 pmol / L of the Sigmal inhibitor, IPAG. Cells were detached from the plate using 2 mmol / L EDTA (Coming). Cells were washed in Dulbecco’s modified phosphate buffered saline (DPBS) (Coming) and aliquoted with 1 × 106cells per tube. Cells were then fixed in 4% methanol-free formaldehyde (Sigma-Aldrich) for 15 minutes at 37°C.Attorney Docket No. 205961-7069W01(00279)

[0727] Cells were washed twice in staining buffer (0.5% bovine serum albumin in PBS). Cells were stained with the rabbit anti-PD-Ll extracellular domain-specific (D8T4X) Alexa Fluor 488 (Cell Signaling Technology, catalog # #86744) at 1: 100 for 60 minutes at 37°C. Cells were washed and resuspended in staining buffer. Fluorescence of each cell population was analyzed on the BD LSR II flow cytometer using FACS Diva Software (BD Biosciences) and reported as mean fluorescence intensity (MFI) relative to control.

[0728] Biotinylation of cell surface proteins

[0729] Cells were seeded approximately 24 hours prior to treatment with lOng / mL IFN-y for 16 hours in the presence or absence of 10 pmol / L of the Sigmal inhibitor, IP AG. Cells were detached from the plate using a cell scraper and then washed in ice cold DPBS, pH 8.0 three times to remove any contaminating proteins or broken cells. Cells were resuspended in ice cold DPBS, pH 8.0 at a concentration of 25 x 106cells / mL, and freshly prepared 10 mm Sulfo-NHS-SS-Biotin (ThermoFisher) was added per the manufacturer’s instructions. Cells were incubated at room temperature for 30 minutes. Cells were washed once in ice cold 50 mm Tns, pH 8.0 to quench any non-reacted biotin reagent and then an additional three times with ice cold DPBS, pH 8.0.

[0730] Isolation of extracellular vesicles (EVs)

[0731] Five million PC3 cells were plated in 15-cm dishes and allowed to grow for 48 hours in RPMI-1640 supplemented with 5% fetal bovine serum. Cells were then rinsed with DPBS to remove all cellular and bovine EVs. Serum-free medium was added to the cells containing 10 ng / mL IFN-y in the presence or absence of the Sigmal inhibitor, IP AG (10 pmol / L). Twenty-four hours later, the supernatant was collected from the cells and processed by filtration and differential centrifugation, described below. The cells were collected and counted using a Countess II automated cell counter (ThermoFisher) to determine vesicle production per cell. First, the supernatants were cleared of dead cells and cell debris by centrifuging at 2,000 RCF for 30 minutes at 4°C. Subsequently. EV isolation was performed by hydrostatic filtration dialysis (HFD) first followed by differential centrifugation. HFD was performed. Cellulose ester (CE) dialysis membrane with molecular weight cutoff (MWCO) 1000 kDa width 16 mm, 0.79 mL / cm (Repligen # 131486) and / or width 31 mm 3.1 mL / cm (Repligen # 131492) were used to concentrate 300 mL and 1,200 mL of CM media respectively. The system setup was set according to. All the parts were abundantly sprayed with 70% (v / v) ethanol and let dry for at least 1 hour in a biological safety cabinet. TheAttorney Docket No. 205961-7069W01(00279)

[0732] samples were first concentrated by gravity to a final volume of 10 mL; the filtration happened inside the biological safety cabinet. The concentrated CM (10 mL) was recovered from the dialysis by opening the clips on top of a 50 mL tube (referred to as HFDa). Thereafter, the concentrated CM was transferred to a 70 mL capped polycarbonate bottle (Beckman Coulter, catalog # 355655) and centrifuged at 14,000 rpm for 35 minutes at 4°C using a 45 Ti fixed angle rotor (k factor 133 at max speed; Beckman Coulter) installed in an Optima L-90K Ultracentrifuge (Beckman-Coulter). The supernatant (SN) was poured into a clean polycarbonate bottle and centrifuged at 40,000 rpm for 35 minutes at 4°C using a 45 Ti fixed angle rotor. All the pellets obtained were resolubilized in 1 ml of DPBS filtered (DPBSO.lpm) with 0.1 pm syringe filter (Minisart® PES syringe filter, Sartorious) and transferred to a 1 mL thick-wall polycarbonate tube (Beckman Coulter, catalog # 362305). Tubes were centrifuged at 25,000 rpm for 35 minutes using a TLA-110 fixed angle rotor (k factor 13 at max speed; Beckman Coulter) installed in an Optima TLX-120 benchtop ultracentrifuge (Beckman-Coulter). Pellets were resolubilized in 200 pL DPBSO.lpm. This procedure is illustrated in Figs. 15A-15B.

[0733] Nanoparticle tracking analysis (NTA)

[0734] NTA was performed using the ZetaView PMX-220 Twin (Particle Metrix) configured with 488 nm and 640 nm lasers with long wave-pass (LWP) cutoff filters (500 nm and 660 nm respectively) and a sensitive CMOS camera 640 x 480 pixels. Samples were diluted in 2 mL of 0.1 pm filtered (Minisart® high flow hydrophilic 0.1 pm syringe filter Sartorious) deionized water (DI 18 MQ / cm) to obtain a particle concentration between 1 x 107and 1 × 108particles / mL (50-200 particles). The instrument was set to a sensitivity of 80, a shutter speed of 100, and a frame rate of 30 frames per second (fps). Each sample was measured at 11 different positions throughout the cell, with 1 cycle of reading at each position to have a minimum of 1000 traces. If the number of traces was below 1000 counts some additional sample was flushed inside the cell and the acquisition was repeated. Postacquisition parameters were set to a minimum brightness of 20, a maximum size area of 1000 pixels, a minimum size area of 10 pixels, and a trace length of 15 frames. Automated cell quality control was checked using high quality deionized water (DI). Camera alignment and focus optimization were performed using polystyrene NanosphereTM lOOnm size standard beads (Thermo Scientific, catalog # 3100A). Data analysis was performed with ZetaView 8.05.14 software provided by the manufacturer. Automated reports of the particles recordingAttorney Docket No. 205961-7069W01(00279)

[0735] across the 11 positions were manually checked, and any outlier position was removed to calculate particle concentration and distribution.

[0736] Discontinuous density gradient separation of EV populations

[0737] The EV pellets obtained after the first round of centrifugation after HFD concentration were re-solubilized in 1.600 pL of iodixanol (Optiprep 60% (w / v) stock solution in water, SIGMA, catalog # D1556-250 ML) diluted with a 0.25 M sucrose / 10 mm Tris / 1 mm EDTA buffer solution at pH 7.4 for a final concentration of 30% (w / v) iodixanol and placed at the bottom of a 3.5 mL thick-wall polycarbonate tube (13 x 51 mm tube, Beckman Coulter, catalog # 349622) A discontinuous iodixanol gradient was generated by layering with a syringe and G21 needle successive 700 pL of 20%, and 700 pL of 10% (w / v) iodixanol in filtered with 0.1 pm syringe filter (Minisart® PES syringe filter, Sartorious) 0.25 M sucrose / 10 mm Tris / 1 mm EDTA buffer solutions layered on top of the discontinuous gradient. Tubes were spun at 53,700 rpm for 1 hour at 4°C using a SW55 Ti rotor (k factor at 48 at max speed; Beckman Coulter) installed in an Optima L-90K Ultracentrifuge (Beckman-Coulter). Ten 300 pL gradient fractions were collected by a syringe and G21 needle from top to bottom of the discontinuous density gradient and the densities of blank (no sample) discontinuous gradient were determined using an ABBE-3 L refractometer (Fisher Scientific). The fractions were then diluted to 1 mL using DPBSO.lpm, transferred to 1 mL thick-wall polycarbonate tube (Beckman Coulter, catalog # 362305), and centrifuged at 80,000 rpm for 60 minutes for all the density fractions obtained from the P20 pellets using a TLA-110 fixed angle rotor (k factor 13 at max speed; Beckman Coulter) installed in an Optima TLX- 120 benchtop ultracentrifuge (Beckman-Coulter). Pellets were resolubilized for immunoblotting in RIP A buffer supplemented with a cocktail of protease and phosphate inhibitors.

[0738] Cryogenic electron microscopy (Cryo-EM)

[0739] Of the isolated EV pellets suspended in DPBS, 3 pL w ere applied onto Quantifoil holey carbon grids and plunge frozen in liquid ethane using a Vitrobot Mark IV. Images were taken on a Titan Krios G3i equipped with a K.3 Bioquantum.

[0740] Immunofluorescent staining for Nano-fcm analysis

[0741] Mouse anti -human CD9 FITC-conjugated antibody (clone HI9a, Biolegend, catalog # 312104). mouse anti-human CD63 AF-647-conjugated antibody (clone H5C6, Biolegend, catalog # 353016), and mouse anti-human PD-L1 PE-conjugated antibody (clone B7-H1,Attorney Docket No. 205961-7069W01(00279)

[0742] Biolegend, catalog # 329705) were used for immunofl uorescent staining of EVs. One hundred ng corresponding to 1 pL anti-CD9 and 0.5 pL of anti-CD63 were added to 5 pL of P20-EV sample with a particle concentration of approximately 5 x 1010particles / ml. Two hundred ng corresponding to 1 pL of anti -human PD-L1 was added to 5 pL of P20-EV sample with a particle concentration of approximately 5 x 1010particles / ml. The mixture was incubated at room temperature for 60 minutes. Buffer and EVs plus antibodies were diluted 100 times with DPBS filtered with 0.02 pm Whatman™ Anotop™ syringe filter (Cytiva, catalog # 6809-3002). The flow nano-analyzer nFCM (NanoFCM Co. Ltd) was used to measure particle concentration and size of particles following the manufacturer’s instructions. Briefly, two single photon-counting avalanche photodiodes (APDs) platform was used for the simultaneous detection of side scatter (SSC; SPCM-1 detector, bandpass filter 488 / 10) and fluorescence of individual particles (FITC, SPCM-2 detector, bandpass filter 525 / 40; AF-647, SPCM-3 detector, bandpass filter 670 / 30 and PE, SPCM-3 detector, bandpass filter 580 / 40). HPLC-Ultrapure water (SIGMA, catalog # 900682-4 L) served as the sheath fluid via gravity feed. The instrument was aligned and calibrated with 200 nm fluorescence reference beads for concentration and a cocktail of silica beads (68, 91, 113, and 151 nm) for size, respectively (NanoFCM Co. Ltd). Using the calibration curve, the flow rate and side scattering intensity' were converted into corresponding particle number and size. Unstained samples were analyzed to determine the concentration and the size of the EVs. The particle number in the 1-minute acquisition was targeted to be in the 2000-12,000 events / min range recommended by the company.

[0743] T cell activation assay

[0744] Human T cells were isolated from whole blood using Lymphoprep (Stemcell) according to the manufacturer’s protocol. Briefly, whole blood was diluted with an equal volume of PBS + 2% FBS, layered onto Lymphoprep, and centrifuged at 800 g for 25 minutes at room temperature. The mononuclear cell layer was collected, pooled, and washed with PBS + 2% FBS. Cells were resuspended at a concentration of 5 × 107per mL in PBS and subjected to magnetic separation using the Easy Sep direct human T cell isolation kit (Stemcell) according to the manufacturer’s protocol. T cells were either left unstimulated or activated with 25ul Immunocult Human CD3 / CD28 / CD2 (Stemcell) and incubated at 37C and 5% CO2 for 3 days.

[0745] T cell co-culture with EVs and flow cytometryAttorney Docket No. 205961-7069W01(00279)

[0746] T cells were collected, washed, and plated at 25,000 cells per well in 100 pl Immunocult-XF T cell expansion media. EVs (2.8 × 1010particles / ml) were added at 10 pl per well in triplicate. Cultures were incubated for 4 hours at 37C and 5% CO2. Replicate wells were pooled and washed twice with FACS buffer. Cells were stained for 1 hour with antibodies specific for human CD4 (Miltenyi, 130-113-228) and CD8 (Miltenyi, 130-110-680). T cells were analyzed on a Guava 8 hT flow cytometer (EMD Millipore). Data analysis was performed using FlowJo software (Tree Stars Inc.).

[0747] Trypan blue assay

[0748] Cells were detached from the plate using 0.25% trypsin-EDTA (Coming). The try psin was washed away, and the cells were resuspended in DPBS (Coming). Cell suspensions were mixed in a 1: 1 ratio with trypan blue, and the percent live cells were determined in quadruplicate using a Countess II automated cell counter (ThermoFisher).

[0749] Statistical analysis

[0750] To determine the statistical significance of single comparisons, an unpaired Student’s t-test was used. To determine the statistical significance of multiple comparisons, a one-way ANOVA with Bonferroni posttest was performed using Prism software (GraphPad).

[0751] Example 2-3: Pharmacological inhibition of Sigmal does not suppress PD-L1 transcript levels and does not block IFN-y mediated STAT1 phosphorylation and translocation to the nucleus

[0752] IFN-y canonically signals through the JAK / STAT1 / 3 pathway and mediates an array of transcriptional responses. After STAT1 is phosphorylated, it dimerizes and is translocated to the nucleus, where it binds to the promoters of interferon stimulated genes, including PD-Ll. The present study asked whether pharmacological inhibition of Sigmal suppresses PD-L1 by blocking the upstream JAK / STAT1 / 3 signaling cascade that drives transcription regulation of PD-L1 or whether the effects of Sigmal modulation occur primarily downstream at a post-translation step, the present study used IP AG (l-(4-Iodophenyl)-3-(2-adamantyl)guanidine), a selective small molecule Sigmal inhibitor. The Sigmal specificity of IP AG has been demonstrated.

[0753] The present study, rather than focusing on a specific cancer type, focused on cancer cell lines that either constitutively express high levels of PD-L1 or inducible lines that express low or undetectable basal levels of PD-L1 in the absence of IFN-y. The present studyAttorney Docket No. 205961-7069W01(00279)

[0754] chose MDA-MB-231 and PC3 cells to examine the effect of IP AG on cell lines with high levels of constitutive PD-L1 expression. PC3 cells were also chosen because of their ability to produce relatively large quantities of EVs with associated PD-L1. The low basal PD-L1 expressing WM164, HT29, and MDA-MB-436 cells were chosen to examine the effects of IP AG on cell lines with IFN-y inducible PD-L1. The lack of high basal PD-L1 expression in the latter group of cell lines permitted examination of the effects of IP AG on nascent PD-L1.

[0755] IFN-y induced PD-L1 transcripts in all of the cell lines tested here (Fig. 9A). Cotreatment with IP AG did not block IFN-y mediated upregulation of PD-L1 transcripts, and in the majority of cell lines, there were more PD-L1 transcripts, up to two-fold more, compared to IFN-y treatment alone (Fig. 9A). IFN-y treatment resulted in increased STAT1 protein levels and increased phospho-STATl levels (Fig. 9B). IPAG did not significantly reduce IFN-y induced phospho-STATl levels (Fig. 9B). IPAG also did not prevent phospho-STATl from translocating into the nucleus of the cells (Fig. 9C), which is required for IFN-y mediated transcriptional induction of PD-L1. Therefore, IPAG did not prevent IFN-y mediated transcriptional upregulation of PD-L1.

[0756] While most publications have focused on the immunomodulatory actions of PD-L1, relatively little is known about the intrinsic cancer cell signaling activities of PD-L1. PD-L1 has a conserved cytoplasmic sequence that is involved in apoptotic resistance to both type I and type II interferon cytotoxicity. Additionally, human cancer samples that acquire enhancing mutations within this region have been shown to have increased resistance to pro-apoptotic signaling of interferons. Thus, the ability of IP AG to prevent PD-L1 from progressing through the secretory pathway and reaching the cell surface would have a detrimental effect on cancer cell survival in the presence of sustained IFN-y signaling. This might create a feedback loop wherein the cell increases the production of new PD-L1 to compensate for the loss of PD-L1 signaling and potentially explain why multiple cell lines show elevated PD-L1 transcript levels following combined treatment with IFN-y and IPAG (Fig. 9A).

[0757] Example 2-4: Pharmacological inhibition of Sigmal blocks post-translational modification and maturation of IFN-y-induced PD-L1

[0758] It has been demonstrated that Sigmal physically associates with PD-L1 and triggers its lysosomal degradation. Here, the present study asked whether the Sigmal inhibitor, IPAG, would prevent nascent PD-L1 from maturing and progressing through the secretory pathway in cell lines from multiple types of cancers. In all the cell lines tested, IFN-y upregulated PD-Attorney Docket No. 205961-7069W01(00279)

[0759] L1 protein levels, consistent with the increased levels of PD-L1 mRNA transcripts (Fig. 9A). When IP AG was combined with IFN-y in cells that constitutively express high levels of PD-Ll, PD-L1 protein levels decreased (Fig. 10A). In the low basal expressing, IFN-y inducible PD-L1 cell lines (WM164, HT29, and MDA-MB-436), PD-L1 protein was still produced in the presence of IP AG (Fig. 10A), indicating that IP AG did not block PD-L1 translation. However, in the low basal expressing lines, the combination of IFN-y and IP AG resulted in the accumulation of PD-L1 at a lower molecular weight band, at approximately 40 kDa, that was not seen with IFN-y alone (Fig. 10A). Quantification of the banding pattern (Fig. 10B) confirmed that there was a higher percentage of the PD-L1 migrating at approximately 40 kDa in the IFN-y and IP AG combination treatment condition compared to treatment with IFN-y alone in the low basal PD-L1 expressing lines.

[0760] Sigmal is associated with nascent PD-L1, and others have reported that differential banding patterns of PD-L1 could be the result of differential N-linked glycosylation.

[0761] Therefore, the present study asked whether the accumulated lower molecular weight PD-L1 band reflected inhibition of proper glycosylation. To answer this question, the present study treated cell lysates with endoglycosidase H (Endo H), which cleaves all immature, high-mannose N-linked glycans in the ER and cis Golgi before glycoproteins are processed by the enzyme Golgi alpha-mannosidase II in the medial Golgi, after which the glycans are no longer susceptible to cleavage by Endo H. The present study found that the ~ 40 kDa PD-L1 band that accumulated in the IFN-y and IP AG combination treatment condition was Endo H sensitive (Fig. 10C). As the lower molecular weight band did not migrate at 33 kDa (which would represent the non-glycosylated PD-L1), but rather ~ 40 kDa, it appears that IP AG did not prevent the initial attachment of the N-linked glycans, rather prevented its proper trimming and maturation. Thus, IP AG did not prevent the maturation of nascent PD-L1 by disrupting N-linked glycosylation. Rather, IPAG prevented the maturation of nascent PD-L1 by disrupting its proper trimming and maturation.

[0762] Example 2-5: Pharmacological inhibition of Sigmal decreases cell surface PD-L1 levels IFN-y mediated upregulation of PD-L1 also increases PD-L1 cell surface expression. Here, the present study confirmed that IFN-y induced cell surface PD-L1 levels in representative examples of high (PC3) and low basal PD-L1 expressing (MDA-MB-436) cell lines (Figs. 11 A-l IB). By flow cytometry, the present study found that IPAG prevented constitutively expressed and IFN-y induced PD-L1 expression at the surface of these cells (Fig. HA). The present study performed cell surface protein biotinylation studies to confirmAttorney Docket No. 205961-7069W01(00279)

[0763] this effect using an orthogonal method which has the added advantage of detecting distinct banding patterns and thus PD-L1 protein post-translational modifications (Fig. 1 IB). In MDA-MB-436 cells, IFN-y increased cell surface PD-L1 levels; however, in response to IFN-y and IP AG combination, the lower ~ 40 kDa band produced was not biotinylated. This indicates that the Endo H sensitive band of PD-L1 was not present at the cell surface (Fig. 1 IB). The cell compartment control calnexin (ER-membrane protein) was not biotinylated, while the Na+ / K+-ATPase (cell surface protein) was biotinylated (Fig. 1 IB). Of note, this further supports the concept that pharmacological inhibition of Sigmal did not impact all cell surface and integral membrane proteins. Thus, IP AG selectively decreased IFN-y mediated cell surface expression of PD-L1.

[0764] Example 2-6: Pharmacological inhibition of Sigmal suppresses EV-associated PD-L1 (evPD-Ll)

[0765] A growing body of evidence demonstrates that Sigmal is a multifunctional chaperone or scaffolding protein that contributes to the maintenance of ER protein homeostasis in cancer cells and supports their increased utilization of the secretory pathway. EV production and its physical and biochemical characteristics are dependent on the secretory pathway. Therefore, the present study asked whether IPAG would alter the production and characteristics of EVs. Zetaview nanoparticle tracking analysis (NTA) showed no salient differences in the overall size profile of EVs produced by cells treated with vehicle (DMSO), IFN-y, IFN-y with IP AG, or IP AG alone (Figs. 12A-12B). Analysis of the EV pellets by cryogenic electron microscopy (Cryo-EM) confirmed the presence of vesicles mostly ranging in size from 50-200 nm (Fig. 12C). Under these conditions, IP AG treatment also did not induce cell death or apoptosis, which the present study confirmed by trypan blue exclusion assay and the absence of cleaved PARP, respectively (Figs. 12D-12E).

[0766] Next, the present study asked whether pharmacological inhibition of Sigmal would also decrease PD-L1 incorporation into EVs as well as decrease the number of EVs. To be packaged into EVs, PD-L1 needs to first be expressed on the cell surface. From there, PD-L1 can enter the endosomal sorting pathway where it can be packaged into EVs or recycled to the cell surface. Since IP AG prevented PD-L1 from reaching the cell surface, the present study hypothesized that it would inhibit PD-L1 incorporation into EVs. Others have reported that IFN-y promotes PD-L1 incorporation into EVs. Here, the present study reproduced these results and found increased levels of PD-L1 in EVs produced by IFN-y treated PC3 cells (Figs. 13A-13B). IPAG prevented IFN-y-induced PD-L1 from being incorporated into theAttorney Docket No. 205961-7069W01(00279)

[0767] EVs (Figs. 13A-13B).

[0768] Interestingly, while IP AG prevented PD-L1 from being incorporated into EVs, it did not suppress other EV-associated proteins, including CD9, CD81, and CD63 (Figs. 13A-13B). Tetraspanins, including CD9 and CD63, are scaffolding proteins that are thought to mediate selective EV cargo packaging. They are also integral membrane glycoproteins and as such are processed through the secretory pathway. However, in contrast to PD-L1, CD9 incorporation into EVs was not altered by IP AG, and CD63 incorporation into EVs was enriched with IPAG. These data indicate a selective targeting of EV-associated proteins by a Sigmal modulator.

[0769] Separation of the EVs by iodixanol density gradient fractionation revealed PD-L1 coisolated into fractions containing CD9, CD81. and CD63. all of which are canonical markers of exosomal EVs (Figs. 13A-12B). This suggested that some of the Sigmal inhibitor blocked evPD-Ll that was exosomal.

[0770] Example 2-7: Pharmacological inhibition of Sigmal results in the production of EVs with significantly diminished potency in an assay of T cell inactivation

[0771] The data above demonstrate that IPAG can decrease the number of evPD-Ll available for the inactivation of T cells entering the TME. Here, the present study hypothesized that the capacity of EVs produced during IPAG treatment may also have diminished potency in blocking T cell inactivation in vitro, as expected by their reduced PD-L1 content. The present study incubated activated PD-1 -positive normal human T cells with EVs prepared from PC3 prostate cancer cells. The present study compared equal numbers of EVs from untreated PC3 cells with those from PC3 cultured with or without IFN-y plus or minus the Sigmal inhibitor IPAG. Cultures of T cells isolated from normal human blood were activated by CD3 / CD28 cross linking for 48-72 hours, then distributed equally into replicate cultures and incubated for 4 hours in the presence or absence of the various EV preparations (Figs. 14A-14F). The cells were then analyzed by flow cytometry and gated for activated T cells, which are delineated by the circular gate in Fig. 14 A. Activated cells consisted of over 80% CD4 T cells and 10% CD8 T cells with minimal double positives and cells negative for both antigens with these ratios being unchanged by exposure to the EVs (data not shown). Unstimulated cultures generally contained less than 20% of the cells gating as activated and expressing PD-1. In contrast, the majority' of the T cells in stimulated cultures exhibited a distinct elevated SSC / FSC scatter and expressed PD-1 (Figs. 14A-14B). The majority of the activated T cells also expressed the activation marker CD69 in addition to PD-1 (Figs. 14C). The inclusion ofAttorney Docket No. 205961-7069W01(00279)

[0772] EVs from untreated PC3 cells resulted in a moderate reduction in the recovery of activated T cells, which was further reduced when the EVs were obtained from IFN-γ-treated PC3 cells. The inhibitory effect on T cell activation was significantly attenuated in the presence of EVs from PC3 cells treated with both IFN-y and IP AG compared to EVs from cells treated with IFN-y alone (Fig. 14D). The magnitude of the effect on T cell activation by EVs obtained from PC3 cells treated with IFN-y was consistent with the nanoFCM profiling where the percentage of PD-L1 positive EVs is in the range of 4% of total EVs compared to less than 1% in control (DMSO) and IFN-y and IP AG combination treatments (Fig. 14E). Considering the large number of EVs used in this assay, with relatively few evPD-Ll, it is possible that other factors, unaffected by Sigmal inhibitors, may be contributing to the immune modulatory effects, and this may explain why the IFN-y + IP AG treatment condition does not completely abrogate the T cell inhibition effect of the EVs.

[0773] Example 2-8: Sigmal targeting selectively sorts proteins into EVs

[0774] Whereas PD-L1 cell surface levels and incorporation into EVs were decreased by¬ IP AG, other transmembrane glycoproteins such as CD9 and CD63 were not excluded from EVs, with the former remaining unchanged between conditions and the latter increasing in EVs (Figs. 13A-13B). It remains unclear why the relative level of CD63 was increased in EVs. One recently reported explanation for this phenomenon is that inhibited endocytosis can induce a selective increase in vesicular secretion of CD63. Another integral membrane protein called STEAP1 (six-transmembrane epithelial antigen of the prostate 1), a cell surface antigen for therapeutic targeting in prostate cancer, which has been reported in some PC3 cell lines and PC3 derived EVs, did not change with Sigmal inhibitor treatment (Figs. 15A-15B). This is evidence of another EV-associated cell surface protein that is synthesized, processed, matured, and transported through the ER but not excluded from EVs by Sigmal targeting. This is potentially useful information for future clinical studies as STEAP1 targeting bispecific antibody therapeutics and chimeric antigen receptor T cell (CAR-T) immune therapies are emerging as novel approaches to treating advanced prostate cancers as well as potential diagnostic and prognostic biomarker strategies.

[0775] Sigmal inhibition suppressed the packaging of other adaptive immune resistance factors into EVs. Indoleamine 2,3 -dioxygenase 1 (IDO) is induced by IFN-y and packaged into EVs. Interestingly, the Sigmal inhibitors did not block intracellular levels of IFN-y induced IDO; however, Sigmal inhibition prevented the packaging of IFN-y induced IDO into EVs (Figs. 15A-15B). This is noted as IDO may contribute to the resistance to immuneAttorney Docket No. 205961-7069W01(00279)

[0776] checkpoint inhibitors.

[0777] The present study also found that Sigmal inhibition prevented the incorporation of epidermal growth factor receptor (EGFR) into EVs, and in this case, EGFR protein levels did not change, whereas packaging into EVs decreased (Figs. 17A-17B). Altogether, these data suggest that pharmacological targeting of Sigmal can selectively alter the sorting of proteins into EVs independent of cellular protein levels. Thus, Sigmal targeting can selectively regulate protein transport through the ER-associated secretory pathway.

[0778] Example 2-9:

[0779] evPD-Ll has been reported to suppress anti -tumor immunity within the TME and at distant sites. The present study examined the mechanisms by which pharmacological inhibition of Sigmal can interfere with the IFN-y / STATl axis induced cell surface expression and incorporation of PD-L1 into EVs, two locations where PD-L1 can block antitumor immune responses. A prototypical small molecule Sigmal inhibitor, IP AG, blocked IFN-y from upregulating PD-L1 at a post-translational step downstream of STAT1 by preventing complete N-linked glycosylation required for maturation and trafficking of functional PD-L1 to the cell surface. In multiple cancer cell lines, pharmacological inhibition of Sigmal resulted in PD-L1 retention in the ER and cis-Golgi, as evidenced by Endo H sensitivity.

[0780] The present study discovered that pharmacological inhibition of Sigmal prevented PD-L1 packaging into cancer cell-derived EVs. Of note, cancer cells producing EVs devoid of PD-L1 facilitate tumor growth inhibition in vivo even when the tumors were able to express cell-surface PD-L1.

[0781] Several standard of care cancer treatments have been reported to cause a significant increase in tumor-derived EV production and alter EV content to promote tumor survival, metastasis, immune evasion, and resistance to therapy. Here, the present study evaluated not only the quantity of EV particles produced by Sigmal inhibitor treated cancer cells but also the relative potency of these EVs (Figs. 12A-12G & 14A-14F). In the experiments, EV particle numbers did not significantly increase in response to Sigmal inhibitor treatment (Fig. I2B). To test the potency of these particles, the present study used equal numbers of EVs in the T cell inactivation assay (Fig. 14B). Under these conditions, the EVs produced by IP AG co-treated cells were significantly less potent in their ability to inactivate T cells (Figs. 14A-14B). The implications of these data are that pharmacological inhibition of Sigmal may be used not only to prevent cancer cells from inactivating T cells in direct contact but also to prevent the immunosuppressive actions of EVs in the TME.Attorney Docket No. 205961-7069W01(00279)

[0782] A major challenge is to better understand the mechanisms and cellular factors that promote immune resistance in the TME and how to sensitize immunologically unresponsive so-called '‘cold” tumors to anti-tumor immune responses. The ER plays a central role in regulating the production, processing, and activity of secreted and cell surface proteins as well as the content and production of EVs, and emerging evidence suggests that targeting the ER can modulate tumor immunity. Here, the present study shows that Sigmal targeting can regulate the production and content of immune modulatory EVs.

[0783] Some Sigmal selective small molecule compounds that have traditionally been categorized as antagonists trigger the unfolded protein response and autophagy and inhibit the growth and proliferation of cancer cells. In contrast, other putative antagonists of Sigmal, including BD1063. NE100, and BD1047, do not induce these effects or do so only modestly. It has been demonstrated that some compounds, which are referred to as Sigmal inhibitors, phenocopy key effects of shRNA-mediated knockdown and knockout of Sigmal (Oyer et al., Front Pharmacol. 2019; 10: 1141. doi: 10.3389 / fphar.2019.01141; Kim et al., Handb Exp Pharmacol. 2017. doi: 10.1007 / 164_2017_38; Maher et al., Mol Cancer Res.

[0784] 2018; 16(2):243— 255. doi: 10.1158 / 1541-7786. MCR-17-0166; Thomas etal., Cancer Res. 2017). IP AG fits this category of Sigmal inhibitor compounds, whereas BD1063 and NE100 do not. Although IP AG induces UPR and autophagy, these properties alone may not explain its evPD-Ll suppressive effects. Indeed, thapsigargin and tunicamycin, which both trigger ER stress manifested as UPR, did not trigger degradation of PD-L1 and did not decrease PD-L1 levels. Altogether, these data suggest a novel and distinct mechanism of action of Sigmal inhibitors, in which UPR and / or autophagy may be required but are not sufficient to suppress the production of evPD-Ll.

[0785] EVs alter signaling within the TME in multiple ways, beyond modulation of antitumor immunity. Cancer cell EVs can promote the growth, transformation, and survival of originating and neighboring cells with the tumor by way of autocrine and paracrine transfer of oncogenic proteins, mRNAs, miRNAs, and other metabolites and signaling molecules. Cancer cells can also promote their own growth, survival, and dissemination throughout the body by secreting EVs that manipulate stromal and vascular tissue within the TME and at distant metastatic sites. Cancer cell-derived EVs also have been shown to modulate the immune response through enhanced production of immune-suppressing cytokines and the induction of regulatory T cells within the TME. Therefore, Sigmal inhibitor-mediated decrease in cancer cell-derived EVs would likely alter the TME in multiple ways; beyond just decreasing the production of immunosuppressive evPD-Ll, it may also decrease EV-Attorney Docket No. 205961-7069W01(00279)

[0786] mediated pro-tumorigenic paracrine signaling and TME remodeling.

[0787] Pharmacological targeting of Sigmal may be used to selectively alter the sorting of proteins into EVs. For example, whereas PD-L1 cell surface levels and incorporation into EVs were decreased by IPAG, other transmembrane glycoproteins such as STEAP1, CD9, and CD63 were not excluded from EVs (Figs. 13A-13B & 15A-15B). Small molecules specifically targeting their cognate receptors can be used to alter their packaging into EVs. These differ from directly targeting cell surface receptors to prevent their packaging into EVs. The present study targets an ER scaffolding or chaperone protein that supports the maturation and trafficking of multiple proteins through the ER-associated secretory pathway and their packaging in EVs. As an example, the present study also found that Sigmal inhibition prevented the incorporation of EGFR into EVs. and in this case, EGFR protein levels did not change, whereas packaging into EVs decreased (Figs. 17A-17B). This is further evidence that Sigmal inhibition can regulate components of the endolysosomal sorting machinery to control protein packaging into EVs. Thus, Sigmal modulation has a broader impact on content and potential use as Sigmal is expressed in a broad range of tissues, and it is an approach that is not dependent on a single receptor. In another example of small molecule mediated EV cargo sorting, the treatment of promyelocytic leukemia cells with all -trans retinoic acid has been reported to increase the production and secretion of EVs and increase packaging of IL-8, VEGF, tissue factor, and mRNA of multiple angiogenesis related transcripts into EVs. This too differs from Sigmal mediated sorting, as this describes retinoid-induced transcriptomic changes in target cells, which causes the cell to differentiate into a new cell type with a distinctly altered secretory profile, and not by physically sorting proteins within the secretory pathway.

[0788] In conclusion, the data presented here illustrate a novel pharmacological mechanism to promote antitumor immunity using a selective small-molecule modulator of Sigmal. By suppressing both cell surface and evPD-Ll, Sigmal modulators could inhibit adaptive immune resistance by cancer cells, allowing the immune system to be more effective at targeting cancer cells. Furthermore, by altering the content of cancer cell EVs, Sigmal modulators could prove to be an effective mechanism for preventing pro-tumorigenic remodeling of the TME. These data support the evaluation of Sigmal small molecule modulators in in vivo models of cancer to determine how they reshape the relationship between the immune system and the TME.

[0789] Example 3: Sigmarl Knockout reduced protein concentration in EVsAttorney Docket No. 205961-7069W01(00279)

[0790] Referring to Fig. 23, using wild-type (WT) and Sigmarl knockout (KO) mouse embryo fibroblasts (MEFs), the present study confirmed that the genetic removal of Sigmarl (the gene encoding Sigmal receptor) reduced the protein concentration per EV particle.

[0791] The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art. and that such modifications and variations are considered to be within the scope of embodiments of the present application.

[0792] Enumerated Embodiments

[0793] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0794] Embodiment 1: A method of:

[0795] (a) modulating formation and / or composition of extracellular vesicles in a subject; (b) suppressing and / or inhibiting formation of extracellular vesicles in a subject; (c) activating and / or promoting a subject's antitumor immune response;

[0796] (d) preventing, minimizing, and / or reversing cancer cell adaptive immune resistance in a subject;

[0797] (e) preventing, minimizing, and / or reversing inactivation of T cells by cancer- derived extracellular vesicles in a subject; and / or

[0798] (f) minimizing and / or suppressing inclusion of programmed death-ligand 1 (PD- Ll) in extracellular vesicles in a subject;

[0799] the method comprising administering to the subject a therapeutically effective amount of a compound that downregulates Sigmal expression and / or activity in a cell of the subject.

[0800] Embodiment 2: The method according to Embodiment 1, wherein the compound that downregulates the Sigmal expression and / or activity comprises at least one selected from the group consisting of:

[0801] a small molecule inhibitor of Sigmal,

[0802] a protein inhibitor of Sigmal,

[0803] a nucleic acid that downregulates Sigmal by RNA interference, and / or an expressionAttorney Docket No. 205961-7069W01(00279)

[0804] vector expressing the nucleic acid,

[0805] a ribozyme that downregulates Sigmal, and / or an expression vector expressing the ribozyme,

[0806] an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that down regulate Sigmal by CRISPR knockout or CRISPR knockdown, and

[0807] a trans-dominant negative mutant protein of Sigmal, and / or an expression vector that expresses the trans-dominant negative mutant protein of Sigmal.

[0808] Embodiment 3: The method of any one of Embodiments 1-2, wherein the compound that downregulates Sigmal is a small molecule inhibitor selected from the group consisting of:

[0809] (i) IP AG (l-(4-Iodophenyl)-3-(2-adamantyl)guanidine);

[0810] (ii) a compound of Formula (I):

[0811]

[0812] wherein:

[0813] ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclic heteroaryl ring, and wherein the aryl or heteroaryl ring is optionally substituted with 0-4 R1groups;

[0814] each occurrence of R1is independently selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR3, -SR3, -S(=O)R3. -S(=O)2R3. -NHS(=O)2R3, -C(=O)R3, -OC(=O)R3, -CO2R3, -OCO2R3, -CH(R3)2, -N(R3)2. -C(=O)N(R3)2. -OC(=O)N(R3)2. -NHC(=O)NH(R3), -NHC(=O)R3.

[0815]

[0816] -NHC(=O)OR3, -C(OH)(R3)2, and -C(NH2)(R3)2;

[0817] each occurrence of R2is independently selected from the group consisting of H, C₁-C₆ alkyl, Ci-Ce heteroalkyl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl or cycloalkyl group is optionally substituted with 0-5 R1groups, or X3and R2combine to form a (C3-C7)heterocycloalkyl group, optionally substituted with 0-2 R1groups;

[0818] each occurrence of R3is independently selected from the group consisting ofH, Ci-Ce alkyd, Ci-Ce heteroalkyl, aryl, and -C1-C3 alky 1-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, ary l, or cycloalkyl group is optionally substituted with 0-5 R1groups;

[0819] X1is -CH2-, -S-, -O- or -(NR2)-;

[0820] X

[0821]

[0822] 2is =CH2, =S, =0 or =NR2; andAttorney Docket No. 205961-7069W01(00279)

[0823] X3is -S-, -0-, or -NR2-;

[0824] (iii) a compound of Formula (II):

[0825] RA-RB(II),

[0826] wherein:

[0827] H N NH RAis selected from the group consisting

[0828]

[0829] of *4

[0830]

[0831] X4is selected from the group consisting of methoxy, F, Cl, Br, and I; and

[0832] RBis selected from the group consisting of:

[0833]

[0834] Attorney Docket No. 205961-7069W01(00279)

[0835] wherein:

[0836] each occurrence of R1and R2is independently selected from the group consisting of -C1-C6 alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -NHC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2;

[0837] R3is selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce alkoxy, F, Cl, Br, and I;

[0838] R4is selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce alkoxy, F, Cl, Br, and I;

[0839] each occurrence of R5is independently selected from the group consisting ofH, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl. heteroalkyl, aryl, or cycloalkyl group is optionally substituted;

[0840] X is selected from the group consisting of CH2, C=O, or O;

[0841] n is an integer from 1-3;

[0842] x is an integer from 0-4; and

[0843] y is an integer from 0-4;

[0844] (v) a compound of formula (IV):

[0845]

[0846] wherein:

[0847] one of the following applies:

[0848] (a) Z1is CRla; Z2is N; Z3is CRlcor N; Z4is CRld;

[0849] (ty Z’ is N; Z2is CRlb; Z3is CRlc; Z4is CRldorN; (c) Z1is CRla; Z2is N; Z3is CRlc; Z4is N;

[0850] (

[0851]

[0852] d) Z1is N; Z2is CRlb; Z3is N; Z4is CRld;

[0853] X is selected from the group consisting of bond and O;

[0854] each occurrence of Rla, Rlb, Rlc, and Rldis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -C

[0855]

[0856] H(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -Attorney Docket No. 205961-7069W01(00279)

[0857] NHC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2;

[0858] each occurrence of R2a, R2b, R2c. and R2dis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -N

[0859]

[0860] HC(=O)OR5. -C(OH)(R5)2, and -C(NH2)(R5)2;

[0861] R3is selected from the group consisting of CN, F, Cl, Br, I, Ci-Cg haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR6;

[0862] R4is selected from the group consisting of CN, F, Cl, Br, I, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR7;

[0863] each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, C1-C6 heteroalkyl, aryl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted;

[0864] R6is selected from the group consisting of H, C₁-C₆ alkyl, Ci-Ce heteroalkyl, and C3-C6 cycloalkyl, wherein the alkyl, heteroalkyd, or cycloalkyl group is optionally substituted;

[0865] R7is selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and C3-C6 cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group is optionally substituted; and each occurrence of Ra, Rb, Rc, Rd, Re, and Rfis independently selected from the group consisting of H and CH3, with the proviso that, if Y is CRlcand Z is CRld, then at least one of Ra, Rb, Rc, Rd, Re, and Rfis CH3;

[0866] or a salt, solvate, enantiomer, diastereoisomer, tautomer, or A-oxide thereof, or any combinations thereof.

[0867] Embodiment 4: The method of any one of Embodiments 1-3, wherein the compound is selected from the group consisting of:

[0868] 1 -(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A);

[0869] 1 -(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B);

[0870] l-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C);

[0871] l-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D);

[0872] l-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine (Compound F); and 1 -(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound G);

[0873] a salt, solvate or N-oxide thereof, and any combinations thereof.

[0874] Embodiment 5: The method of any one of Embodiments 1-4, wherein the compound is selected from the group consisting of:

[0875] l,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E);Attorney Docket No. 205961-7069W01(00279)

[0876] l-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine) (Compound H);

[0877] a salt, solvate or N-oxide thereof, and any combinations thereof.

[0878] Embodiment 6: The method of Embodiment 3, wherein in (V) each occurrence of Rla, Rlb, Rlc, and Rldis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl. F, Cl, Br, I, -CN, -NCh, and -OR5.

[0879] Embodiment 7: The method of Embodiment 3, wherein in (V) each occurrence of R2a, R2b, R2c, and R2dis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, and -OR5.

[0880] Embodiment 8: The method of Embodiment 3, wherein in (V) R3is CN, F, Cl, Br, I, -CF3, -CHF2, -OCFs, -OCHF2, -C(O)OH, or -C(O)OMe.

[0881] Embodiment 9: The method of Embodiment 3, wherein in (V) R4is CN, F, Cl, Br, I, -CF3, -CHF2, -OCF3, -OCHF2, -C(O)OH, or -C(O)OMe.

[0882] Embodiment 10: The method of any one of Embodiments 1-3, wherein the compound is selected from the group consisting of:

[0883]

[0884] l-(6-cyano-5-methoxypyridin-3-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;

[0885] H H V NH

[0886]

[0887] l-(6-cyano-5-methoxypyridin-3-yl)-3- (2,2-dimethyl-3-(4-(trifluoromethyl)phenoxy)propyl)guanidine;

[0888]

[0889] l-(5-cyano-4-methoxypyridin-2-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;

[0890]

[0891] l-(3,4-dichlorophenyl)-3-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)guanidine;Attorney Docket No. 205961-7069W01(00279)

[0892] H H

[0893] F-. O

[0894]

[0895] CI^N^NHCF3 1-(6-chloro-5-fluoropyridin-3-yl)-3-(3-methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;

[0896] CF3

[0897] H H

[0898] MeO

[0899] NH

[0900]

[0901] N^Cl-(5-cyano-4-methoxypyridin-2-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenyl)butyl)guanidine;

[0902]

[0903] methyl 6-(3-(3-methyl-3-(4-(trifluoromethyl)phenyl)butyl)guanidino)nicotinate;

[0904] F

[0905] F

[0906]

[0907] methyl 6-(3-(2,2-dimethyl-3-(4- (trifluoromethyl)phenoxy)propyl)guanidino)nicotinate;

[0908] H H

[0909] N.

[0910] F

[0911] F3C

[0912] F

[0913]

[0914] l-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)guanidine; and H H

[0915] MeO N.

[0916] NH F NG

[0917] F

[0918]

[0919] l-(6-cyano-5-methoxypyridin-3-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;

[0920] or a salt, solvate, tautomer, or N-oxide thereof.

[0921] Embodiment 11: The method of any one of Embodiments 1-10, wherein the subj ect has cancer.

[0922] Embodiment 12: The method of any one of Embodiments 1-11, wherein the compound is administered as a pharmaceutical composition further comprising aAttorney Docket No. 205961-7069W01(00279)

[0923] pharmaceutically acceptable carrier.

[0924] Embodiment 13: The method of any one of Embodiments 1-12. wherein the subject is further administered at least one additional anticancer agent and / or at least one agent that treats one or more cancer symptoms or co-morbidities.

[0925] Embodiment 14: The method of any one of Embodiments 1-13, wherein the compound is administered by a route comprising oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.

[0926] Embodiment 15: The method of any one of Embodiments 1-14, wherein the subject is a mammal, optionally a human.

[0927] Embodiment 16: A method of:

[0928] (a) modulating formation of extracellular vesicles in a cell and / or composition of extracellular vesicles released by a cell;

[0929] (b) suppressing and / or inhibiting formation of extracellular vesicles in a cell; (c) preventing, minimizing, and / or reversing a release of T cell-inactivating extracellular vesicles by a cell; and / or

[0930] (f) minimizing and / or suppressing release of programmed death-ligand 1 (PD-L1) in extracellular vesicles by a cell,

[0931] the method comprising contacting the cell with a compound that downregulates Sigmal in the cell.

[0932] Embodiment 17: The method according to Embodiment 16. wherein the compound that downregulates the Sigmal comprises at least one selected from the group consisting of:

[0933] a small molecule inhibitor of Sigmal,

[0934] a protein inhibitor of Sigmal,

[0935] a nucleic acid that downregulates Sigmal by RNA interference, and / or an expression vector expressing the nucleic acid,

[0936] a ribozyme that downregulates Sigmal, and / or an expression vector expressing the ribozyme,

[0937] an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate Sigmal by CRISPR knockout or CRISPR knockdown, and

[0938] a trans-dominant negative mutant protein of Sigmal, and / or an expression vector that expresses the trans-dominant negative mutant protein of Sigmal.

[0939] Embodiment 18: The method of any one of Embodiments 16-17, wherein the compound that downregulates Sigmal is a small molecule inhibitor selected from the groupAttorney Docket No. 205961-7069W01(00279)

[0940] consisting of:

[0941] (i) IP AG (l-(4-Iodophenyl)-3-(2-adamantyl)guanidine);

[0942] (ii) a compound of Formula (I):

[0943] (R1)x

[0944]

[0945] X2(I),

[0946] wherein:

[0947] ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclic heteroaryl ring, and wherein the aryl or heteroaryl ring is optionally substituted with 0-4 R1groups;

[0948] each occurrence of R1is independently selected from the group consisting of -C₁-C₆ alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR3, -SR3, -S(=O)R3, -S(=O)2R3, -NHS(=O)2R3, -C(=O)R3, -OC(=O)R3, -CO2R3, -OCO2R3, -CH(R3)2, -N(R3)2. -C(=O)N(R3)2, -OC(=O)N(R3)2, -NHC(=O)NH(R3), -NHC(=O)R3, -NHC(=O)OR3, -C(OH)(R3)2, and -C(NH2)(R3)2;

[0949] each occurrence of R2is independently selected from the group consisting ofH, Ci-Ce alkyl, Ci-Ce heteroalkyl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), wherein the alky 1, heteroalkyl or cycloalkyl group is optionally substituted with 0-5 R1groups, or X3and R2combine to form a (C3-C7)heterocycloalkyl group, optionally substituted with 0-2 R1groups;

[0950] each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Cg cycloalkyl), wherein the alkyl, heteroalkyd, ary 1, or cycloalkyl group is optionally substituted with 0-5 R1groups;

[0951] X1is -CH2-, -S-, -O- or -(NR2)-;

[0952] X2is =CH2, =S, =0 or =NR2; and

[0953] X3is -S-, -O-, or -NR2-;

[0954] (iii) a compound of Formula (II):

[0955] RA-RB(II),

[0956] wherein:

[0957] H N NH RAis selected from the group consisting of

[0958]

[0959] Attorney Docket No. 205961-7069W01(00279)

[0960]

[0961] X4is selected from the group consisting of methoxy, F, Cl, Br, and I; and

[0962] RBis selected from the group consisting of:

[0963]

[0964] wherein:

[0965] each occurrence of R1and R2is independently selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Cs heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5. -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -NHC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2;

[0966] R3is selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-CeAttorney Docket No. 205961-7069W01(00279)

[0967] alkoxy, F, Cl, Br, and I;

[0968] R4is selected from the group consisting of -Ci-Ce alkyl. -Ci-Ce alkoxy. F, Cl, Br, and I;

[0969] each occurrence of R5is independently selected from the group consisting ofH, C₁-C₆ alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted;

[0970] X is selected from the group consisting of CH2, C=O, or O;

[0971] n is an integer from 1-3;

[0972] x is an integer from 0-4; and

[0973] y is an integer from 0-4;

[0974] (v) a compound of formula (IV):

[0975]

[0976] wherein:

[0977] one of the following applies:

[0978] (a) Z1is CRla; Z2is N; Z3is CRlcor N; Z4is CRld;

[0979] (b) Z1is N; Z2is CRlb; Z3is CRlc; Z4is CRldorN; (c) Z1is CRla; Z2is N; Z3is CRlc; Z4is N:

[0980] (d) Z1is N; Z2is CRlb; Z3is N; Z4is CRld;

[0981] X is selected from the group consisting of bond and O;

[0982] each occurrence of Rla, Rlb, Rlc, and Rldis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br. I, -CN, -NO2, -OR5, -SR5. -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5. -CO2R5, -OCO2R5. -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -N

[0983]

[0984] HC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2; each occurrence of R2a, R2b, R2c, and R2dis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br. I, -CN. -NCh, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -N

[0985]

[0986] HC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2;

[0987] R3is selected from the group consisting of CN, F, Cl, Br, I, Ci-Ce haloalkyl, Ci-CeAttorney Docket No. 205961-7069W01(00279)

[0988] haloalkoxy, and -C(O)OR6;

[0989] R4is selected from the group consisting of CN, F. Cl, Br, I, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR7;

[0990] each occurrence of R3is independently selected from the group consisting of H, C₁-C₆ alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted;

[0991] R6is selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and C3-C6 cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group is optionally substituted;

[0992] R7is selected from the group consisting of H, Ci-Ce alkyd, Ci-Ce heteroalkyl, and Cs-Ce cycloalkyl, wherein the alkyl, heteroalky 1, or cycloalkyl group is optionally substituted; and each occurrence of Ra, Rb, Rc, Rd, Re, and Rfis independently selected from the group consisting of H and CH3, with the proviso that, if Y is CRlcand Z is CRld, then at least one of Ra, Rb, Rc, Rd, Re, and Rfis CH3;

[0993] or a salt, solvate, enantiomer, diastereoisomer, tautomer, or A-oxide thereof, or any combinations thereof.

[0994] Embodiment 19: The method of any one of Embodiments 16-18, wherein the compound is selected from the group consisting of:

[0995] 1 -(3-(4-fluorophenoxy )propyl)-3-(4-iodophenyl)guanidine (Compound A);

[0996] 1 -(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B);

[0997] l-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C);

[0998] l-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D);

[0999] l-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine (Compound F); and 1 -(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound G);

[1000] a salt, solvate or N-oxide thereof, and any combinations thereof.

[1001] Embodiment 20: The method of any one of Embodiments 16-19, wherein the compound is selected from the group consisting of:

[1002] l,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E);

[1003] l-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine) (Compound H);

[1004] a salt, solvate or N-oxide thereof, and any combinations thereof.

[1005] Embodiment 21: The method of Embodiment 18, wherein in (V) each occurrence of

[1006]

[1007] Ria RlbR1CR1d is independently selected from the group consisting of C₁-C₆ alkyl, Ci- Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, and -OR5.

[1008] Embodiment 22: The method of Embodiment 18, wherein in (V) each occurrence ofAttorney Docket No. 205961-7069W01(00279)

[1009] R2a, R2b, R2c, and R2dis independently selected from the group consisting of C₁-C₆ alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F. Cl. Br, I. -CN, -NO2, and -OR3.

[1010] Embodiment 23: The method of Embodiment 18, wherein in (V) R3is CN, F, Cl, Br, I, -CF3, -CHF2, -OCF3, -OCHF2, -C(O)OH, or -C(O)OMe.

[1011] Embodiment 24: The method of Embodiment 18, wherein in (V) R4is CN, F, Cl, Br, I, -CF3. -CHF2. -OCF3. -OCHF2. -C(O)OH, or -C(O)OMe.

[1012] Embodiment 25: The method of any one of Embodiments 16-18, wherein the compound is selected from the group consisting of:

[1013]

[1014] l-(6-cyano-5-methoxypyridin-3-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;

[1015] H H V NH

[1016] •CF-

[1017]

[1018] l-(6-cyano-5-methoxypyridin-3-yl)-3- (2,2-dimethyl-3-(4-(trifluoromethyl)phenoxy)propyl)guanidine;

[1019]

[1020] l-(5-cyano-4-methoxypyridin-2-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;

[1021]

[1022] Cl3l-(3,4-dichlorophenyl)-3-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)guanidine;

[1023]

[1024] 3l-(6-chloro-5-fluoropyridin-3-yl)-3-(3- methyl -3 -(4-(trifluoromethyl)phenoxy)butyl)guanidine;

[1025] ^^, CF3

[1026]

[1027] l-(5-cyano-4-methoxypyridin-2-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenyl)butyl)guanidine;

[1028] - n i -Attorney Docket No. 205961-7069W01(00279)

[1029]

[1030] methyl 6-(3-(3-methyl-3-(4- (trifluoromethyl)phenyl)butyl)guanidino)nicotinate;

[1031] F

[1032]

[1033] Fmethyl 6-(3-(2,2-dimethyl-3-(4-(trifluoromethyl)phenoxy)propyl)guanidino)nicotinate;

[1034] F

[1035]

[1036] F l-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)guanidine; and

[1037]

[1038] l-(6-cyano-5-methoxypyridin-3-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;

[1039] or a salt, solvate, tautomer, or / -oxide thereof.

[1040] Embodiment 26: The method of any one of Embodiments 16-25, wherein the cell is a cancer cell.

[1041] Embodiment 27: The method of Embodiment 26, wherein the cancer cell is from a cold cancer.

[1042] Embodiment 28: The method of any one of Embodiments 16-27, wherein

[1043] (a) the cell is an isolated cell;

[1044] (b) the cell is from a cell line;

[1045] (c) the cell is a primary cell;

[1046] (d) the cell is in a subject;

[1047] (e) the cell is a mammalian cell;

[1048] (f) the cell is a human cell.

Claims

Attorney Docket No. 205961-7069W01(00279)CLAIMSWhat is claimed is:

1. A method of:(a) modulating formation and / or composition of extracellular vesicles in a subject; (b) suppressing and / or inhibiting formation of extracellular vesicles in a subject; (c) activating and / or promoting a subject's antitumor immune response;(d) preventing, minimizing, and / or reversing cancer cell adaptive immune resistance in a subject;(e) preventing, minimizing, and / or reversing inactivation of T cells by cancer- derived extracellular vesicles in a subject; and / or(f) minimizing and / or suppressing inclusion of programmed death-ligand 1 (PD- Ll) in extracellular vesicles in a subject;the method comprising administering to the subject a therapeutically effective amount of a compound that downregulates Sigmal expression and / or activity in a cell of the subject.

2. The method according to claim 1, wherein the compound that downregulates the Sigmal expression and / or activity comprises at least one selected from the group consisting of:a small molecule inhibitor of Sigmal,a protein inhibitor of Sigmal,a nucleic acid that downregulates Sigmal by RNA interference, and / or an expression vector expressing the nucleic acid,a ribozy me that downregulates Sigmal, and / or an expression vector expressing the ribozyme,an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate Sigmal by CRISPR knockout or CRISPR knockdown, anda trans-dominant negative mutant protein of Sigmal, and / or an expression vector that expresses the trans-dominant negative mutant protein of Sigmal.

3. The method of any one of claims 1-2, wherein the compound that downregulates Sigmal is a small molecule inhibitor selected from the group consisting of:(i) IP AG (l-(4-Iodophenyl)-3-(2-adamantyl)guanidine);Attorney Docket No. 205961-7069W01(00279)(ii) a compound of Formula (I):wherein:ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclic heteroaryl ring, and wherein the aryl or heteroaryl ring is optionally substituted with 0-4 R1groups;each occurrence of R1is independently selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl. -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR3. -SR3. -S(=O)R3, -S(=O)2R3, -NHS(=O)2R3, -C(=O)R3, -OC(=O)R3, -CO2R3, -OCO2R3, -CH(R3)2, -N(R3)2, -C(=O)N(R3)2, -OC(=O)N(R3)2, -NHC(=O)NH(R3), -NHC(=O)R3, -NHC(=O)OR3, -C(OH)(R3)2, and -C(NH2)(R3)2;each occurrence of R2is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl or cycloalkyl group is optionally substituted with 0-5 R1groups, or X3and R2combine to form a (C3-C7)heterocycloalkyl group, optionally substituted with 0-2 R1groups;each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Cs cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted with 0-5 R1groups;X1is -CH2-, -S-, -O- or -(NR2)-;X2is =CH2, =S, =0 or =NR2; andX3is -S-. -O-, or -NR2-;(iii) a compound of Formula (II):RA-RB(II),wherein:RAis selected from the group consisting of- l iAttorney Docket No. 205961-7069W01(00279)X4is selected from the group consisting of methoxy, F, Cl, Br, and I; andRBis selected from the group consisting of:(iv) a compound of formula (III):wherein:each occurrence of R1and R2is independently selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5. -S(=O)2R5. -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2. -C(=O)N(R5)2. -OC(=O)N(R5)2. -NHC(=O)NH(R5), -NHC(=O)R5. -NHC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2;Attorney Docket No. 205961-7069W01(00279)R3is selected from the group consisting of -C₁-C₆ alkyl. -Ci-Ce fluoroalkyl, -Ci-Ce alkoxy. F, Cl, Br, and I;R4is selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce alkoxy, F, Cl, Br, and i;each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted;X is selected from the group consisting of CH2, C=O, or O;n is an integer from 1-3;x is an integer from 0-4; andy is an integer from 0-4;(v) a compound of formula (IV):wherein:one of the following applies:(a) Z1is CRla; Z2is N; Z3is CRlcor N; Z4is CRld;(b) Z1is N; Z2is CRlb; Z3is CR1C; Z4is CRldor N; (c) Z1is CRla; Z2is N; Z3is CRlc; Z4is N;(d) Z1is N; Z2is CRlb; Z3is N; Z4is CRld;X is selected from the group consisting of bond and O;each occurrence of Rla, Rlb, Rlc. and Rldis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, - CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -NHC(=O)OR5. -C(OH)(R5)2, and -C(NH2)(R5)2; each occurrence of R2a, R2b, R2c, and R2dis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -Attorney Docket No. 205961-7069W01(00279)NHC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2;R3is selected from the group consisting of CN, F. Cl, Br, I, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR6;R4is selected from the group consisting of CN, F, Cl, Br, I, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR7;each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Cs cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted;R6is selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and Cs-Ce cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group is optionally substituted;R7is selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and C3-C6 cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group is optionally substituted; and each occurrence of Ra, Rb, Rc, Rd, Re, and Rfis independently selected from the group consisting of H and CH3, with the proviso that, if Y is CRlcand Z is CRld, then at least one of Ra, Rb, Rc, Rd, Re, and Rfis CH3;or a salt, solvate, enantiomer, diastereoisomer, tautomer, or N-oxide thereof, or any combinations thereof.

4. The method of any one of claims 1-3, wherein the compound is selected from the group consisting of:1 -(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A);1 -(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B);l-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C);l-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D);l-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine (Compound F); and 1 -(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound G);a salt, solvate or N-oxide thereof, and any combinations thereof.

5. The method of any one of claims 1-4, wherein the compound is selected from the group consisting of:l,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E);l-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine) (Compound H);a salt, solvate or N-oxide thereof, and any combinations thereof.Attorney Docket No. 205961-7069W01(00279)6. The method of claim 3, wherein in (V) each occurrence of Rla, Rlb. Rlc, and Rldis independently selected from the group consisting of Ci-Cs alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, and -OR5.

7. The method of claim 3, wherein in (V) each occurrence of R2a, R2h, R2c, and R2dis independently selected from the group consisting of Ci-Cs alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, and -OR5.

8. The method of claim 3, wherein in (V) R3is CN, F, Cl, Br, I, -CF3, -CHF2, -OCF3, - OCHF2, -C(O)OH. or -C(O)OMe.

9. The method of claim 3, wherein in (V) R4is CN, F, Cl, Br, I, -CF3, -CHF2, -OCF3, - OCHF2, -C(O)OH, or -C(O)OMe.

10. The method of any one of claims 1-3, wherein the compound is selected from the group consisting of:l-(6-cyano-5-methoxypyridin-3-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;H H V NH CF3l-(6-cyano-5-methoxypyridin-3-yl)-3- (2,2-dimethyl-3-(4-(trifluoromethyl)phenoxy)propyl)guanidine;l-(5-cyano-4-methoxypyridin-2-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;Cl3l-(3,4-dichlorophenyl)-3-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)guanidine;Attomey Docket No. 205961-7069W01(00279)H H,'1 CF3 l-(6-chloro-5-fluoropyridin-3-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;l-(5-cyano-4-methoxypyridin-2-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenyl)butyl)guanidine;methyl 6-(3-(3-methyl-3-(4-(trifluoromethyl)phenyl)butyl)guanidino)nicotinate;methyl 6-(3-(2,2-dimethyl-3-(4- (trifluoromethyl)phenoxy)propyl)guanidino)nicotinate;H H IT. NFFl-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)guanidine; and H Hl-(6-cyano-5-methoxypyridin-3-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;or a salt, solvate, tautomer, or N-oxide thereof.

11. The method of any one of claims 1-10, wherein the subject has cancer.

12. The method of any one of claims 1-11, wherein the compound is administered as aAttomey Docket No. 205961-7069W01(00279)pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

13. The method of any one of claims 1-12, wherein the subject is further administered at least one additional anticancer agent and / or at least one agent that treats one or more cancer symptoms or co-morbidities.

14. The method of any one of claims 1-13, wherein the compound is administered by a route comprising oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.

15. The method of any one of claims 1-14, wherein the subject is a mammal, optionally a human.

16. A method of:(a) modulating formation of extracellular vesicles in a cell and / or composition of extracellular vesicles released by a cell;(b) suppressing and / or inhibiting formation of extracellular vesicles in a cell; (c) preventing, minimizing, and / or reversing a release of T cell-inactivating extracellular vesicles by a cell; and / or(I) minimizing and / or suppressing release of programmed death-ligand 1 (PD-L1) in extracellular vesicles by a cell,the method comprising contacting the cell with a compound that down regulates Sigmal in the cell.

17. The method according to claim 16, wherein the compound that downregulates the Sigmal comprises at least one selected from the group consisting of:a small molecule inhibitor of Sigmal,a protein inhibitor of Sigmal,a nucleic acid that downregulates Sigmal by RNA interference, and / or an expression vector expressing the nucleic acid,a ribozyme that downregulates Sigmal, and / or an expression vector expressing the ribozyme,an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate Sigmal by CRISPR knockout or CRISPR knockdown, andAttorney Docket No. 205961-7069W01(00279)a trans-dominant negative mutant protein of Sigmal, and / or an expression vector that expresses the trans-dominant negative mutant protein of Sigmal.

18. The method of any one of claims 16-17, wherein the compound that down reg ulates Sigmal is a small molecule inhibitor selected from the group consisting of:(i) IP AG (l-(4-Iodophenyl)-3-(2-adamantyl)guanidine);(ii) a compound of Formula (I):wherein:ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclic heteroaryl ring, and wherein the aryl or heteroaryl ring is optionally substituted with 0-4 R1groups;each occurrence of R1is independently selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR3, -SR3, -S(=O)R3. -S(=O)2R3. -NHS(=O)2R3. -C(=O)R3, -OC(=O)R3, -CO2R3. -OCO2R3, -CH(R3)2. -N(R3)2. -C(=O)N(R3)2. -OC(=O)N(R3)2. -NHC(=O)NH(R3), -NHC(=O)R3.-NHC(=O)OR3, -C(OH)(R3)2, and -C(NH2)(R3)2;each occurrence of R2is independently selected from the group consisting of H, C₁-C₆ alkyl, Ci-Ce heteroalkyl, and -C1-C3 alkyl-(C3-C6 cycloalkyl), w-herein the alkyl, heteroalkyl or cycloalkyl group is optionally substituted with 0-5 R1groups, or X3and R2combine to form a (C3-C7)heterocycloalkyl group, optionally substituted with 0-2 R1groups;each occurrence of R3is independently selected from the group consisting ofH, C₁-C₆ alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted with 0-5 R1groups;X1is -CH2-, -S-, -O- or -(NR2)-;X2is =CH2, =S, =0 or =NR2; andX3is -S-, -O-, or -NR2-;(iii) a compound of Formula (II):RA-RB(II),Attorney Docket No. 205961-7069W01(00279)wherein:X4is selected from the group consisting of methoxy, F, Cl, Br, and I; andRBis selected from the group consisting of:(iv) a compound of formula (III):(III), wherein:each occurrence of R1and R2is independently selected from the group consisting of -Attorney Docket No. 205961-7069W01(00279)Ci-Ce alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5. -S(=O)2R5. -NHS(=O)2R5. -C(=O)R5, -OC(=O)R5, -CO2R5. -OCO2R5, -CH(R5)2. -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -NHC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2;R3is selected from the group consisting of -C₁-C₆ alkyl, -Ci-Ce fluoroalkyl, -Ci-Ce alkoxy. F, Cl, Br, and I;R4is selected from the group consisting of -Ci-Ce alkyl, -Ci-Ce alkoxy, F, Cl, Br, and I;each occurrence of R5is independently selected from the group consisting ofH, C₁-C₆ alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted;X is selected from the group consisting of CH2, C=O, or O;n is an integer from 1-3;x is an integer from 0-4; andy is an integer from 0-4;(v) a compound of formula (IV):wherein:one of the following applies:(a) Z1is CRla; Z2is N; Z3is CR10or N; Z4is CRld;(b) Z1is N; Z2is CRlb; Z3is CRlc; Z4is CRldor N; (c) Z1is CRla; Z2is N; Z3is CRlc; Z4is N;(d) Z1is N; Z2is CRlb; Z3is N; Z4is CRld;X is selected from the group consisting of bond and O;each occurrence of Rla, Rlb, Rlc. and Rldis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -NHC(=O)OR5. -C(OH)(R5)2, and -C(NH2)(R5)2;Attomey Docket No. 205961-7069W01(00279)each occurrence of R2a, R2b, R2c, and R2dis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, C1-C6 heteroalkyl. F, Cl, Br. I, -CN. -NO2, -OR5, -SR5, -S(=O)R5, -S(=O)2R5, -NHS(=O)2R5, -C(=O)R5, -OC(=O)R5, -CO2R5, -OCO2R5, -CH(R5)2, -N(R5)2, -C(=O)N(R5)2, -OC(=O)N(R5)2, -NHC(=O)NH(R5), -NHC(=O)R5, -NHC(=O)OR5, -C(OH)(R5)2, and -C(NH2)(R5)2;R3is selected from the group consisting of CN, F, Cl, Br, I, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR6;R4is selected from the group consisting of CN, F, Cl, Br, I, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, and -C(O)OR7;each occurrence of R5is independently selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, aryl, and -C1-C3 alkyl-(C3-Ce cycloalkyl), wherein the alkyl, heteroalkyl, aryl, or cycloalkyl group is optionally substituted;R6is selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and C3-C6 cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group is optionally substituted;R7is selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce heteroalkyl, and C3-C6 cycloalkyl, wherein the alkyl, heteroalkyl, or cycloalkyl group is optionally substituted; and each occurrence of Ra, Rb, Rc, Rd, Re, and Rfis independently selected from the group consisting of H and CH3, with the proviso that, if Y is CRlcand Z is CRld, then at least one of Ra, Rb, Rc, Rd, Re, and Rfis CH3;or a salt, solvate, enantiomer, diastereoisomer, tautomer, or A-oxide thereof, or any combinations thereof.

19. The method of any one of claims 16-18, wherein the compound is selected from the group consisting of:1 -(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A);1 -(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B);l-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C);l-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D);l-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine (Compound F); and 1 -(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound G);a salt, solvate or N-oxide thereof, and any combinations thereof.

20. The method of any one of claims 16-19. wherein the compound is selected from the group consisting of:Attorney Docket No. 205961-7069W01(00279)l,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E):l-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine) (Compound H);a salt, solvate or N-oxide thereof, and any combinations thereof.

21. The method of claim 18, wherein in (V) each occurrence of Rla, Rlb, Rlc, and Rldis independently selected from the group consisting of Ci-Cs alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, and -OR5.

22. The method of claim 18, wherein in (V) each occurrence of R2a, R21’, R2c, and R2dis independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce fluoroalkyl, Ci-Ce heteroalkyl, F, Cl, Br, I, -CN, -NO2, and -OR5.

23. The method of claim 18, wherein in (V) R3is CN, F, Cl, Br, I, -CF3, -CHF2, -OCF3, - OCHF2, -C(O)OH, or -C(O)OMe.

24. The method of claim 18, wherein in (V) R4is CN, F, Cl, Br, I, -CFs, -CHF2, -OCFs, - OCHF2, -C(O)OH, or -C(O)OMe.

25. The method of any one of claims 16-18. wherein the compound is selected from the group consisting of:l-(6-cyano-5-methoxypyridin-3-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;(2,2-dimethyl-3-(4-(trifluoromethyl)phenoxy)propyl)guanidine;l-(5-cyano-4-methoxypyridin-2-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;Attorney Docket No. 205961-7069W01(00279)ClCF3 l-(3,4-dichlorophenyl)-3-(3-methyl-3-(4- (tnfluoromethy l)phenoxy )buty Dguani dine;3l-(6-chloro-5-fluoropyridin-3-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;^^,-CF3l-(5-cyano-4-methoxypyridin-2-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenyl)butyl)guanidine;H Hl\L _Nmethyl 6-(3-(3-methyl-3-(4- (trifluoromethyl)phenyl)butyl)guanidino)nicotinate;H Hl\L -NN NHmethyl 6-(3-(2,2-dimethyl-3-(4- (trifluoromethyl)phenoxy)propyl)guanidino)nicotinate;l-(3-methyl-3-(4- (trifluoromethyl)phenoxy)butyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)guanidine; andl-(6-cyano-5-methoxypyridin-3-yl)-3-(3- methyl-3-(4-(trifluoromethyl)phenoxy)butyl)guanidine;or a salt, solvate, tautomer, or A-oxide thereof.Attorney Docket No. 205961-7069W01(00279)26. The method of any one of claims 16-25, wherein the cell is a cancer cell.

27. The method of claim 26, wherein the cancer cell is from a cold cancer.

28. The method of any one of claims 16-27, wherein(a) the cell is an isolated cell;(b) the cell is from a cell line;(c) the cell is a primary' cell;(d) the cell is in a subject;(e) the cell is a mammalian cell;(f) the cell is a human cell.

Images