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  • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
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  • C07C251/34—Oximes with oxygen atoms of oxyimino groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
  • C07C251/36—Oximes with oxygen atoms of oxyimino groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with the carbon atoms of the oxyimino groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C251/40—Oximes with oxygen atoms of oxyimino groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with the carbon atoms of the oxyimino groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of an unsaturated carbon skeleton
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  • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
  • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
  • C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
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• • A—HUMAN NECESSITIES
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  • A61K2239/55—Lung
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/57—Skin; melanoma

Definitions

• the present disclosure relates to methods of treating cancer with RARy agonists.
• Retinoids are a class of small molecules that interact with the nuclear receptors retinoic acid receptor (RAR) and retinoid X receptor (RXR). Retinoids that interact with RXR are termed rexinoids and, in some contexts, the term retinoid is used to indicate a compound that interacts with RAR. Both RAR and RXR have three subtypes, a, p, and y. Retinoids can act as either agonists or antagonists of these receptors. Some retinoids interact with both RAR and RXR and with each of the subtypes, while others will preferentially or specifically interact with a particular receptor type or subtype(s). Retinoids regulate development and the differentiation of multiple types of cells.
• Retinoids have pleotropic effects depending, in part, on the particular receptor types and subtypes that a particular retinoid activates or inhibits. Retinoids exhibit a degree of structural diversity. It is not possible to reliably predict which receptor type and subtype a particular retinoid will interact with or whether it will act as an agonist or antagonist.
• RARy agonists had the greatest antiproliferative effect against a neuroblastoma cell line. In both studies apoptosis was seen as the primary mechanism of inhibiting tumor cell proliferation. Cheung et al. (Biochem Biophys Res Commun. 229(1):349-54, 1996) studied the effects of RAR subtype specific agents by transfecting a neuroblastoma cell line with each individual RAR subtype and concluded the RARp expression was necessary for growth inhibition. Raffo et al. (Anticancer Res.
• Treatments for cancer are ever evolving, gaining in specificity and sophistication. Early non-surgical cancer treatments generally targeted rapidly dividing cells which were more sensitive to radiological and chemical assault. Over time, more specific and less generally toxic treatments have been developed. Some treatments appear to have broad applicability, for example immune checkpoint inhibitors. Others are targeted to cancers that express a particular antigen or other biomarker involved in the regulation of proliferation or differentiation; including many monoclonal antibodies and kinase inhibitors. Yet as the variety of cancer treatments has grown, it has become ever harder to determine which candidate treatments might be productively pursued and for what indications.
• TIL tumor-infiltrating lymphocytes
• R 7 to R 12 are independently: C 1-6 alkyl , a hydrogen atom, an alkoxy group, a halogen atom (for example F, Cl, or Br), a nitro group, a hydroxy group, OCF3, or COR 13 , where R 13 is C 1-6 alkyl or CF3.
• Y is oxygen, sulfur, or NR 14 , where R 14 is C 1-6 alkyl.
• R 16 is H or F.
• the RARy-selective agonist has the structure [0009]
• One aspect is a method of generating, differentiating, or expanding the number of TIL by contacting them with a RARy selective agonist.
• contacting comprises culturing the TIL in vitro in a media supplemented with the RARy selective agonist.
• contacting comprises administering the RARy selective agonist to a subject having a tumor.
• the RARy selective agonist is selective in that it has no or negligible agonistic activity for RARa at clinically relevant concentrations. In some embodiments the RARy selective agonist is selective in that it has no or negligible agonistic activity for both RARa and RARp at clinically relevant concentrations.
• R 7 to R 12 are independently: C 1-6 alkyl , a hydrogen atom, an alkoxy group, a halogen atom (for example F, Cl, or Br), a nitro group, a hydroxy group, OCF 3 , or COR 13 , where R 13 is C 1-6 alkyl or CF 3 .
• Y is oxygen, sulfur, or NR 14 , where R 14 is C 1-6 alkyl.
• R 16 is H or F.
• the RARy selective agonist is 4-((1E,3E)-3-(3-(terf-butyl)-2- ethyl-benzofuran-5-yl)-3-(hydroxyimino)prop-1-en-1-yl)-3-fluorobenzoic acid (GA1 E), having the structure or a pharmaceutically acceptable salt thereof.
• the RARy selective agonist is 4-((1 E,3Z)-3-(3-(tert-butyl)-2-ethyl-benzofuran-5-yl)-3-(hydroxyimino)prop-1-en-1- yl)-3-fluorobenzoic acid (GA1Z), having the structure or a pharmaceutically acceptable salt thereof.
• the RARy agonist is 3-fluoro-4-((1 E,3E)-3-(hydroxyimino)-3-(5, 5,8,8- tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-en-1-yl)benzoic acid (GA2E), having the structure or a pharmaceutically acceptable salt thereof.
• the RARy selective agonist 3-fluoro-4-((1 E,3Z)-3-(hydroxyimino)-3-(5, 5,8, 8-tetramethyl-5, 6,7,8- tetrahydronaphthalen-2-yl)prop-1-en-1-yl)benzoic acid (GA2Z), having the structure or a pharmaceutically acceptable salt thereof.
• the paraposition of the COOR group is preferred.
• both R 1 are CH3.
• both R 2 are H.
• X is C(R 5 )2.
• both R 5 are CH3.
• one R 3 is H and the other R 3 is F.
• both R 4 are H.
• all R 3 and R 4 are H.
• R is H.
• R is methyl or ethyl.
• the COOR group is in the para position. In some embodiments, the COOR group is in the meta position.
• Some embodiments specifically include one or more specific substituents at one or more of the variable positions. Some embodiments specifically exclude one or more specific substituents at one or more of the variable positions.
• the RARy selective agonist is CD437, CD2325, CD666, trifarotene, or BMS961 .
• One aspect is a method of treating cancer with RARy agonist-expanded TIL.
• the RARy agonist is administered to a subject having a cancer to be treated.
• T lymphocytes are expanded in in vitro culture by exposing the culture to the RARy agonist, and the RARy agonist-expanded T lymphocytes are administered to a subject having a cancer to be treated, as adoptive T cell therapy. That is, the culture media is supplemented with the RARy agonist.
• the concentration of RARy agonist in the culture media can be in a range of 1 pM to 1 mM. In some embodiments, the concentration of RARy agonist in the culture media is 0.5 nM.
• the T lymphocytes are TIL isolated from a tumor explant from a subject.
• the T lymphocytes are peripheral blood mononuclear cells (PBMC).
• the T lymphocyte culture further comprises lethally irradiated tumor cells.
• the tumor cells to be lethally irradiated are obtained from the subject to be treated.
• the culture media is supplemented with interleukin 2 (IL-2).
• the expanded T lymphocytes are administered to the subject by infusion, for example, by intravenous or intratumoral infusion.
• the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the RARy agonist is GA1 E. In some embodiments, the RARy agonist is GA2E.
• the cancer is breast cancer.
• the breast cancer is triple negative breast cancer.
• the cancer is Her2 + breast cancer.
• the cancer is lung cancer, for example, non-small cell lung cancer.
• the cancer is a solid tumor.
• the cancer is a hematologic cancer.
• the cancer is a carcinoma, a sarcoma, a melanoma, a glioblastoma, a leukemia, a lymphoma, a myeloma, or a plasma cell cancer.
• the effective amount of the RARy agonist is about 0.01 to about 300 mg/m 2 /day; however, doses below or above this exemplary range are within the scope of the present disclosure.
• the daily dose can be about 0.5 to about 100 mg/m 2 /day, about 1 to about 90 mg/m 2 /day, about 5 to about 80 mg/m 2 /day; or at least about 0.02, 0.03, 0.05, 0.07, 0.1 , 0.2, 0.3, 0.5, 0.7, 1 , 2, 3, 5, 7, 10, 15, 20, 25, 30, 50, 70, or 100 mg/m 2 /day; or not more than about 0.1 , 0.2, 0.3, 0.5, 0.7, 1 , 2, 3, 5, 7, 10, 15, 20, 25, 30, 50, 60, 70.
• the effective amount for a human is about 0.006 to about 200 mg/day or about 1 to about 100 mg/day.
• the effective amount of the RARy agonist is about 0.27 pg/kg/day to about 8 mg/kg/day; however, doses below or above this exemplary range are within the scope of the present disclosure.
• the daily dose can be about 0.013 to about 2.7 mg/kg/day, about 0.025 to about 2.5 mg/kg/day, about 0.130 to about 22 mg/kg/day; or at least about 0.0005, 0.0008, 0.001 , 0.0013, 0.0020, 0.0025, 0.005, 0.0008, 0.0010, 0.0013, 0.0020, 0.0027, 0.005, 0.008, 0.010, 0.013, 0.020, 0.027, 0.05, 0.08, 0.1 , 0.13, 0.2, 0.5, 0.8, 1.0, 1.3, 1.8, 2.0, or 2.7 mg/kg/day; or not more than about 0.0027, 0.005, 0.008, 0.010, 0.013, 0.020, 0.027, 0.05, 0.08, 0.1
• the administering occurs periodically throughout an interval of treatment. That is, the RARy agonist is administered at regularly occurring time points within the interval of treatment. In some embodiments, periodically is twice daily, once daily, every other day, every third day, or twice weekly. In some embodiments, the administering occurs in repeated cycles throughout an interval of treatment. In some embodiments, a cycle comprises 1) administering the RARy selective agonist periodically over a first span of time and 2) suspending administration of the RARy selective agonist over a second span of time, after which a new cycle may be initiated.
• the first span of time (during which the RARy agonist is administered) is 10-15 days, or any integer number of days therein.
• the second span of time (during which administration of the RARy agonist is suspended) is two weeks to one month or any integer number of days therein.
• the interval of treatment extends from a first administration of the RARy agonist until, a complete response is achieved, stable disease is obtained, or disease progression again occurs after stable disease or regression.
• some embodiments further comprise administering an inhibitor of regulatory T cells (Tregs).
• the inhibitor of Tregs is a Treg-depleting antibody.
• the Treg-depleting antibody is anti-CD25 antibody, an anti-glucocorticoid- induced tumor necrosis factor-related protein (GITR) antibody, an anti-FoxP3 antibody, an anti-CCR4 antibody, or an anti-folate receptor 4 antibody.
• the inhibitor of Tregs comprises an RARa antagonist.
• RXR agonist having the structure where R is H or C 1-6 alkyl, or a pharmaceutically acceptable salt thereof.
• the RXR agonist is 3,7-dimethyl-6(S),7(S)-methano,7-[1 ,1 ,4,4-tetramethyl- 1 ,2,3,4-tetrahydron-aphth-7-yl]2(E), 4(E) heptadienoic acid (IRX4204).
• the RARy-selective agonist is a compound having a structure of GAG1 as defined above.
• some embodiments further comprise administering an anti-PD-1 or anti-PD-L1 antibody.
• some embodiments further comprise administering or supplementing culture media, as appropriate, with an anti-PD-1 or anti-PD-L1 antibody.
• One aspect is a method of potentiating chimeric antigen receptor-T (CAR-T) cancer immunotherapy comprising administering an effective amount RARy-selective agonist having the structure of GAG1 , as defined above, to a cancer patient who is receiving, has received, or is scheduled to receive, CAR-T cells.
• CAR-T chimeric antigen receptor-T
• One aspect is a compound having a structure of GAG1 as defined above.
• One embodiment is 4-((1E,3E)-3-(3-(terf-butyl)-2-ethyl-benzofuran-5-yl)-3-(hydroxyimino)prop-1- en-1-yl)-3-fluorobenzoic acid (GA1 E).
• Some embodiments are a pharmaceutically-acceptable salt of GA1 E.
• Some embodiments are a Ci- 6 ester of GA1 E.
• Some embodiments are a pharmaceutical composition comprising GA1 E, or a salt or ester thereof.
• One aspect is a method of general synthesis of GAG1 according to the synthetic scheme in Figure 1.
• a complete synthesis begins with Compound 1 and proceeds through intermediates, Compounds 2 through 5, to produce the species of GAG1 , the isomers GA1 E and GA1Z.
• Some embodiments are a method of synthesizing GAG1 species comprising producing intermediate Compound 4 by reacting Compound 3 with n-BuLi and N-methoxyl-N- methylacetamide.
• Some embodiments are a method of synthesizing GAG1 species comprising producing intermediate Compound 5 by reacting Compound 4 with methyl 3-fluoro- 4-formylbenzoate.
• Some embodiments are a method of synthesizing GAG1 species by reacting Compound 5 with hydroxylamine hydrochloride in the presence of an organic or inorganic base. In some embodiments, the base is pyridine.
• One aspect is intermediate Compound 4, having the structure
• a further aspect is a method of synthesizing Compound 4 by reacting Compound 3 with n- BuLi and N-methoxyl-N-methylacetamide.
• One aspect is intermediate Compound 5, having the structure
• a further aspect is a method of synthesizing Compound 5 by reacting Compound 4 with methyl 3-fluoro-4-formylbenzoate in the presence of a base, such as NaOH.
• Figure 1 presents a scheme for the synthesis of the species of GAG1 , GA1 E and GA1Z.
• Figure 2 presents a scheme for the synthesis of the species of GAG1 , GAG1-1 and GAG 1-2.
• Figures 3A-B depict the results of RARa and RARy transactivation assays of GA1 E ( Figure 3A) and GA2E ( Figure 3B).
• Figures 4A-B depict the results of treating a triple negative breast cancer in a syngeneic, immunocompetent mouse model with the RARy selective agonists tazarotenic acid and GA2E, versus vehicle control.
• Figure 4A shows tumor volume over the course of the experiment in each of the three arms of the study, and thus inhibition of tumor growth.
• Figure 4B shows body weight over the course of the experiment in each of the three arms of the study.
• Figures 5A-B depict the results of flow cytometric analysis of tumors excised at the end of the study described in Example 3, for each of the treatments, GA2E, tazarotenic acid, and vehicle control. Results are shown both as percentage (upper panels) and absolute number of cells/tumor (lower panels).
• Figure 5A shows the number of live cells in the tumors and their percentage of the total cells (Live), the number of leukocytes based on CD45 staining and their percentage of the live cells (CD45), the total number of T cells based on CD3 staining and their percentage of the leukocytes (Tot T), and CD4 + T cell based on CD4 staining and their percentage of total T cells (CD4T).
• Figure 5B shows the number of memory T helper cells based on staining for CD4 and intracellular IFNy and their percentage of total CD4 + cells (TH I FNy), the number of Th17 cells based on staining for CD4 and intracellular IL-17 and their percentage of total CD4 + cells (TH IL17), the number of T regulatory cells based on staining for CD4, CD25, and FOXP3 and their percentage of total CD4 + cells (T reg), CD8 + T cell based on CD8 staining and their percentage of total T cells (CD8T), and memory cytolytic T cells based on staining for CD8 and intracellular IFNy and their percentage of CD8 + cells (TC IFNy).
• Figures 6A-B depict weight change in Balb/c mice implanted with EMT6 triple negative breast cancer cells in control mice ( ⁇ ) and GA2E treated mice at 10 mg/kg ( «),25 mg/kg (A), or 50 mg/kg (X) from the first day of treatment (Day 0) through the end of the study, reported as mass ( Figure 6A) or percent change ( Figure 6B).
• Figure 7 depicts tumor growth, in mm 3 , in Balb/c mice implanted with EMT6 triple negative breast cancer cells in control mice ( ⁇ ) and GA2E treated mice at 10 mg/kg ( ⁇ ) 25 mg/kg (A), or 50 mg/kg (X), from the first day of treatment (Day 0) through the end of the study.
• Figures 8A-C depict the production of IFNy (in pg/ml) by recall antigen-stimulated PBMC treated with vehicle control or a RARy selective agonist; Figure 8A GA2E; Figure 8B GA3Ep; Figure 8C tazarotenic acid.
• Figures 9A-C depict PBMC proliferation of recall antigen-stimulated CD8 + PBMC treated with vehicle control or a RARy selective agonist; Figure 9A GA2E; Figure 9B GA3Ep; Figure 9C tazarotenic acid.
• Figures 10A-B depict weight change in NSG-B2M mice implanted with a Her2 + breast cancer cell line (JIMT-1) and human PBMC in control ( ⁇ ) and GA2E treated ( ⁇ ) mice, from the first day of treatment (Day 0) through the end of the study, reported as mass ( Figure 10A) or percent change (Figure 10B).
• JIMT-1 Her2 + breast cancer cell line
• ⁇ human PBMC in control
• GA2E treated mice ⁇ mice
• Figure 11 depicts tumor growth, in mm 3 , in NSG-B2M mice implanted with a Her2 + breast cancer cell line (JIMT-1) and human PBMC in control ( ⁇ ) and GA2E treated ( ⁇ ) mice, from the first day of treatment (Day 0) through the end of the study.
• JIMT-1 Her2 + breast cancer cell line
• ⁇ human PBMC
• GA2E treated mice from the first day of treatment (Day 0) through the end of the study.
• Figure 12A-E depicts flow cytometric T subset analysis of CD8 + TIL from NSG-B2M mice implanted with a Her2 + breast cancer cell line (JIMT-1) and human PBMC in control ( ⁇ ) and GA2E treated ( ⁇ ) mice on Day 26 (termination) of the study.
• the subsets were total CD8 + T cells ( Figure 12A, TC), naive CD8 + T cells (Figure 12B, CD8 Naive), central memory CD8 + T cells ( Figure 12C, CD8 TCM), effector memory CD8 + T cells (Figure 12D, CD8 TEM), and terminally differentiated effector CD8 + T cells (Figure 12E, CD8 TEFF). Significance of the difference between treated and control: * indicates P ⁇ 0.05; *** indicates P ⁇ 0.001 ; no asterisks indicates P>0.05.
• Figure 13A-C depicts flow cytometric biomarker analysis of CD8 + Tl L from NSG-B2M mice implanted with a Her2 + breast cancer cell line (JIMT-1) and human PBMC in control ( ⁇ ) and GA2E treated ( ⁇ ) mice on Day 26 (termination) of the study.
• the subsets were PD-1 + CD8 + T cells (Figure 13A, TC PD1), CD180 + CD8 + T cells ( Figure 13B, TC CD18), and CD54 + CD8 + T cells ( Figure 13C, TC CD54).
• Significance of the difference between treated and control ** indicates P ⁇ 0.005; *** indicates P ⁇ 0.001 ; **** indicates P ⁇ 0.0001.
• Figure 14A-E depicts flow cytometric T subset analysis of CD4 + TIL from NSG-B2M mice implanted with a Her2 + breast cancer cell line (JIMT-1) and human PBMC in control ( ⁇ ) and GA2E treated ( ⁇ ) mice on Day 26 (termination) of the study.
• the subsets were total CD4 + T cells ( Figure 14A, TH), naive CD4 + T cells (Figure 14B, CD4 Naive), central memory CD4 + T cells ( Figure 14C, CD4 TCM), effector memory CD4 + T cells ( Figure 14D, CD4 TEM), and terminally differentiated effector CD4 + T cells ( Figure 14E, CD4 TEFF). Significance of the difference between treated and control: * indicates P ⁇ 0.05; *** indicates P ⁇ 0.001 ; no asterisks indicates P>0.05.
• Figure 15A-D depicts flow cytometric biomarker analysis of CD4 + TIL from NSG-B2M mice implanted with a Her2 + breast cancer cell line (JIMT-1) and human PBMC in control ( ⁇ ) and GA2E treated ( ⁇ ) mice on Day 26 (termination) of the study.
• the subsets were PD-1 + CD8 + T cells (Figure 15A, TH PD1), CD180 + CD4 + T cells ( Figure 15B, TH CD18), CD54 + CD4 + T cells ( Figure 15C, TH CD54), and Fox3P + CD4 + T cells (Figure 15D, Tregs). Significance of the difference between treated and control: * indicates P ⁇ 0.05; no asterisks indicates P>0.05.
• Figure 16 depicts dose-response curves for cell viability for EMT6 cells grown in the presence of three RARy agonists GA1 E ( ⁇ ), GA2E (•), and GA3Ep (A).
• Figure 17 depicts dose-response curves for cell viability for Lewis Lung carcinoma (LLC) cells grown in the presence of three RARy agonists GA1 E ( ⁇ ), GA2E (•), and GA3Ep (A).
• Figures 18A-B depict weight change in B57BL/6 mice implanted with LLC cells in control mice ( ⁇ ) and GA2E treated mice at 10 mg/kg ( ⁇ ) or 25 mg/kg (A), from the first day of treatment (Day 0) through the end of the study, reported as mass ( Figure 18A) or percent change (Figure 18B).
• Figure 19 depicts tumor growth, in mm 3 , in B57BL/6 mice implanted with LLC cells in control mice ( ⁇ ) and GA2E treated mice at 10 mg/kg ( ⁇ ) or 25 mg/kg (A), from the first day of treatment (Day 0) through the end of the study.
• Figure 20 depicts the results of flow cytometric analysis of LLC tumors excised at the end of the study described in Example 10, for each of the treatments, vehicle control and GA2E at dosages of 10 and 25 mg/kg in columns 1 , 2, and 3, respectively, in each panel. Results are shown both as percentage of total T cells (upper panels) and as absolute number of cells pre mg/tumor (lower panels). Depicted are the number of live cells in the tumors and their percentage of the total cells (Live), the number of leukocytes based on CD45 staining and their percentage of the live cells (CD45), and the total number of T cells based on CD3 staining and their percentage of the leukocytes (Tot T).
• Figure 21 depicts the results of flow cytometric analysis of LLC tumors excised at the end of the study described in Example 10, for each of the treatments, vehicle control and GA2E at dosages of 10 and 25 mg/kg in columns 1 , 2, and 3, respectively, in each panel. Results are shown both as percentage of total T cells (upper panels) and as absolute number of cells pre mg/tumor (lower panels). Depicted are the number of CD4 + T cell based on CD4 staining and their percentage of total T cells (CD4+), T helper cells based on positive staining for CD4 and intracellular IFN-y (Thelperl), and T regulatory cells based on positive staining for CD4, CD25, and FOXP3.
• Figure 22 depicts the results of flow cytometric analysis of LLC tumors excised at the end of the study described in Example 10, for each of the treatments, vehicle control and GA2E at dosages of 10 and 25 mg/kg in columns 1 , 2, and 3, respectively, in each panel. Results are shown both as percentage of total T cells (upper panels) and as absolute number of cells pre mg/tumor (lower panels).
• CD8 + T cell based on CD8 staining and their percentage of total T cells (CD8+), cytotoxic T cells based on positive staining for CD8 and intracellular IFN-y (Cytotoxic CD8), naive CD8 + T cells based on positive staining for CD8 and CD62L, and negative staining for CD44 (CD8 naive), CD8 + central memory T cells (CD8 Tern) based on positive staining for CD8, CD62L, and CD44, and CD8 + effector memory T cells (CD8 T em) based on positive staining for CD8 and CD44 and negative staining for CD62L.
• CD8+ cytotoxic T cells based on positive staining for CD8 and intracellular IFN-y
• CD8 naive CD8 + T cells based on positive staining for CD8 and CD62L
• CD44 CD8 naive
• CD8 + central memory T cells CD8 Tern
• CD8 T em CD8 +
• TIL tumor-infiltrating lymphocytes
• RARy selective agonist refers to RARy agonists that have no or negligible agonistic activity for RARa at some clinically relevant concentrations.
• the RARy selective agonist is selective in that it has no or negligible agonistic activity for both RARa and RARp at some clinically relevant concentrations.
• “Clinically relevant concentrations” refers to the concentration in the blood (typically determined by assaying plasma or serum) of a subject receiving efficacious doses of the RARy selective agonist, or the concentration used in in vitro culture for generating, differentiating, or expanding TIL, as relevant to the method in which the RARy selective agonist is being used.
• the EC50 for activation of RARy is at least 100-fold less, or at least 50-fold less, than for the activation of RARa, or RARa and RARp.
• R 7 to R 12 are independently: C 1-6 alkyl, a hydrogen atom, an alkoxy group, a halogen atom (for example F, Cl, or Br), a nitro group, a hydroxy group, OCF3, or COR 13 , where R 13 is C 1-6 alkyl or CF3. Y is oxygen, sulfur, or NR 14 , where R 14 is C 1-6 alkyl.
• R 16 is H or F. In some embodiments, R is methyl or ethyl. In some embodiments, R is H. In some embodiments, R 16 is F.
• Y is O:
• the RARy-selective agonist having a structure of GAG1 has the structure
• alkyl means a moiety composed of carbon and hydrogen containing no double or triple bonds.
• Alkyl may be linear alkyl, branched alkyl, cycloalkyl, or a combination thereof.
• C 1-6 alkyl means an alkyl group having 1 to 6 carbon atoms and includes, but is not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, /so-butyl, f-butyl, pentyl isomers, hexyl isomers, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and combinations thereof having anywhere from 1-6 carbon atoms. These considerations apply equally to the alkyl portion or an alkoxy group.
• One aspect disclosed herein is a method of generating, differentiating, or expanding TIL by contacting them with a RARy selective agonist.
• RARy agonists stimulate T cell effector function, but not Treg cells, and thus can promote an anti-cancer immune response.
• the RARy selective agonists described herein can promote expansion of CD8 + TIL.
• IL-2 has been used previously for expanding TIL. In vitro culture can result in changes to the T cell receptor repertoire and the in v/tro-expanded TIL are typically infused with further IL-2 as a growth factor.
• IL-2 is also associated with a variety of toxicities, some associated with capillary leak syndrome presented by edema, hypotension and reduced urine output within hours of infusion, but also fevers, rigors, myalgia and nausea.
• An alternative agent to expand TIL without producing such substantial toxicity is a long-felt, unmet need.
• TIL refers primarily to the lymphocytes that can be found within a tumor.
• TIL can be isolated from explanted tumor tissue and expanded in vitro. Yet TIL infiltrate the tumor from outside the tumor thus can also be found outside the tumor. Therefore, it is also possible to culture PBMC in vitro in the presence of tumor antigen (for example, lethally irradiated tumor cells) to generate or expand T cells capable of infiltrating into tumors. Methods of generating, differentiating, or expanding TIL encompass such use of PBMC.
• tumor antigen for example, lethally irradiated tumor cells
• contacting TIL with a RARy selective agonist comprises culturing the TIL in vitro in a media supplemented with the RARy selective agonist.
• Procedures for culturing TIL are known to those of skill in the art. To summarize a basic procedure, resected tumor tissue is minced into approximate 1-3 mm 3 fragments and placed in culture (for example in 24- or 48-well plates with 2 or 1 mL of culture media, respectively) and the TIL allowed to extravasate from the tissue. Alternatively, the tumor tissue may be subjected to enzymatic digestion and/or mechanical disaggregation to obtain a single cell suspension.
• the TIL can be cultured in vitro for 3-6 weeks in an appropriate medium, replacing half the media every 2-3 days and splitting at 80% confluence. T umor cells disappear from the culture over the first 1-3 weeks.
• anti-CD3 antibody and 100 to 200-fold excess of irradiated feeder cells can be added to the culture.
• the TIL can be transferred to a bioreactor to support high cell density and achieve a population of from 10 9 to 2x10 11 cells.
• the RARy selective agonist is used continually in the culture.
• the PBMC are obtained from the subject to be treated (autologous). In other embodiments, the PBMC are obtained from an HLA-matched donor. In some embodiments, tumor cells are obtained from the subject to be treated, while in other embodiments they are obtained from a different individual. In some embodiments, the PBMC and the tumor cells are obtained from the same individual. In some embodiments, that individual is the subject to be treated. In some embodiments, the PBMC and the tumor cells are obtained from different individuals, for example, the PBMC can be autologous but cultured with tumor cells from an HLA-matched or allogeneic donor. In some embodiments, the PBMC (or TIL) are cultured with lethally- irradiated tumor cells, but in alternative embodiments the culture contains a tumor cell lysate, or purified or synthetic tumor antigens.
• contacting comprises administering the RARy selective agonist to a subject having a tumor.
• One aspect disclosed herein is a method of treating cancer comprising providing RARy selective agonist-expanded TIL to a patient in need thereof.
• the RARy selective agonist is administered to a subject having a cancer to be treated.
• TIL are expanded in culture by exposing the culture to the RARy selective agonist, and the RARy selective agonist-expanded TIL are administered to a subject having a cancer to be treated as adoptive T cell therapy.
• the subject is a mammal.
• the subject is a human.
• the RARy selective agonist is GAG1.
• the RARy selective agonist is GAG2.
• the cancer is a solid tumor, a carcinoma, a sarcoma, or a hematologic cancer.
• the cancer is a melanoma, a glioblastoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer, pancreatic cancer ovarian cancer, a leukemia, a lymphoma, a myeloma, or a plasma cell cancer.
• the cancer is breast cancer.
• the breast cancer is triple negative breast cancer.
• the cancer is Her2 + breast cancer.
• the cancer is lung cancer, for example, non-small cell lung cancer.
• the subject in need thereof is human.
• Triple negative breast cancer is typically more aggressive and has a poorer prognosis than other types of breast cancer, in part due to there being fewer treatment options. T riple negative breast cancer is also more likely to metastasize and more likely to recur after treatment.
• Triple negative breast cancers are graded on their resemblance to normal healthy breast cells on a 3 point scale, with higher grades indicating less resemblance to normal cells.
• Triple negative breast cancers are often grade 3.
• Triple negative breast cancers are usually “basal-like” meaning they resemble the basal cells lining the breast ducts. Basal-like cancers tend to be more aggressive and higher grade.
• the onset of triple negative breast cancer tends to occur at an earlier age, for example, under 50 as compared to other types of breast cancer which are more commonly diagnosed in persons over 60 years of age. About 70% of cancers having a BRCA mutation are triple negative. Altogether, these factors make triple negative breast cancer a particularly difficult disease to treat.
• neoadjuvant chemotherapy chemotherapy prior to surgical removal of the tumor
• PARP poly ADP- ribose polymerase
• PD-1 blockade such as by atezolizumab, in combination with albumin-bound paclitaxel
• the effective amount is about 0.01 to about 300 mg/m 2 /day; however, doses below or above this exemplary range are within the scope of the present disclosure.
• the daily dose can be about 0.5 to about 100 mg/m 2 /day, about 1 to about 90 mg/m 2 /day, about 5 to about 80 mg/m 2 /day; or at least about 0.02, 0.03, 0.05, 0.07, 0.1 , 0.2, 0.3, 0.5, 0.7, 1 , 2, 3, 5, 7, 10, 15, 20, 25, 30, 50, 70, or 100 mg/ m 2 /day; or not more than about 0.1 , 0.2, 0.3, 0.5, 0.7, 1 , 2, 3, 5, 7, 10, 15, 20, 25, 30, 50, 60, 70.
• the effective amount of the RARy agonist is about 0.27 pg/kg/day to about 8 mg/kg/day; however, doses below or above this exemplary range are within the scope of the present disclosure.
• the daily dose can be about 0.013 to about 2.7 mg/kg/day, about 0.025 to about 2.5 mg/kg/day, about 0.130 to about 22 mg/kg/day; or at least about 0.0005, 0.0008, 0.001 , 0.0013, 0.0020, 0.0025, 0.005, 0.0008, 0.0010, 0.0013, 0.0020, 0.0027, 0.005, 0.008, 0.010, 0.013, 0.020, 0.027, 0.05, 0.08, 0.1 , 0.13, 0.2, 0.5, 0.8, 1.0, 1.3, 1.8, 2.0, or 2.7 mg/kg/day; or not more than about 0.0027, 0.005, 0.008, 0.010, 0.013, 0.020, 0.027, 0.05, 0.08, 0.1
• the effective amount for a human is about 0.006 to about 200 mg/day or about 1 to about 100 mg/day.
• the daily dose is given in a single administration. In other embodiments, the daily dose is split into multiple administrations, for example two administrations, 9-15, 10-14, 11-13, or 12 hours apart.
• the RARy selective agonist be present at a therapeutic level in the subject throughout treatment. This is because the primary therapeutic effect of the RARy selective agonist is indirect, promoting an immune response, the expansion of TIL, which persists in the absence of the RARy selective agonist. Because it takes time for the immune response to develop, the beneficial effect may not be observed until administration of the RARy selective agonist is paused. Indeed, in some embodiments, it may be beneficial to have a drug holiday during which administration of the RARy selective agonist suspended. To the extent that administration of the RARy selective agonist is associated with a toxicity or other undesirable side-effect, a suspension of administration allows time for the subject to recover from the ill effects, rather than for them to continue to worsen.
• the administering occurs periodically throughout an interval of treatment. That is, the RARy agonist is administered at regularly occurring time points within the interval of treatment. In some embodiments, periodically is twice daily, once daily, every other day, every third day, or twice weekly. In some embodiments, the administering occurs in repeated cycles throughout an interval of treatment.
• a cycle comprises 1) administering the RARy selective agonist periodically over a first span of time and 2) suspending administration of the RARy selective agonist over a second span of time, after which a new cycle may be initiated.
• the first span of time (during which the RARy agonist is administered) is 10-15 days, or any integer number of days therein.
• the second span of time (during which administration of the RARy agonist is suspended) is two weeks to one month or any integer number of days therein.
• the interval of treatment extends from a first administration of the RARy agonist until a complete response is achieved, stable disease is obtained, or disease progression occurs.
• the RARy selective agonist may be administered to a subject by any suitable route of administration.
• the RARy a selective agonist is administered orally.
• the RARy selective agonist is administered by injection or infusion, for example, intravenously, subcutaneously, or intratumorally.
• the RARy selective agonist is administered intranasally.
• the RARy selective agonist is administered by nasal or oral (pulmonary) inhalation.
• Ex vivo expanded TIL are generally administered by infusion, for example intravenous or intratumoral infusion. However, in some embodiments, ex vivo expanded TIL are administered in a single bolus or split into multiple boluses, intravenously, or intratumorally. In some embodiments, the RARy selective agonist is included in the bolus or infusion with the TIL. In various embodiments, infusion may extend for half an hour, for an hour, for several hours, for a day, or for several days.
• TIL about 10 9 to >10 11 TIL are administered, for example, about 1x10 10 to 2x10 11 .
• the RARy selective agonist is used continually in the culture and may be included with the infused, expanded TIL.
• the RARy agonist may also be administered to the patient subsequent to TIL infusion for a set period of time or until tumor is eliminated or no further benefit from the TIL is observed.
• the RARy selective agonist is used as monotherapy. In other embodiments, it is used in combination with one of the current therapies, either before, during, or after, the other treatment.
• RARy selective agonists promote expansion of TIL that are CD8 + effector cells. Consistent with what is known about CD8 + effector cells generally, these cell express PD-1 , which can act as a negative regulator of CD8 + effector cell activity. Thus, in some embodiments, treatment with a RARy selective agonist is combined with PD-1 blockade. Treatment with RARy selective agonists can also increase the presence of PD-1 + T cells among CD8 + TIL. For this reason as well, in some embodiments, treatment with a RARy selective agonist is combined with PD-1 blockade. Several products for PD-1 blockade, also called immune checkpoint inhibition, are undergoing clinical evaluation.
• anti-PD-1 antibodies pembrolizumab, nivolumab, cemiplimab, dostarlimab, tislelizumab, spartalizumab, camrelizumab, sintilimab, toripalimab, JTX-4014, INCMGA00012, AMP-514, and budigalimab
• anti-PD-L1 antibodies atezolizumab, avelumab, durvalumab, envafolimab, CK-301 , CS-1001 , KN035SHR-1316, CBT-502, BGB-A333, and BMS-936559.
• AMP- 224 is a fusion protein of PD-L2 (also known as B7-DC) with an antibody Fc region, which is being developed as an anti-PD-1 checkpoint inhibitor.
• B7-DC a fusion protein of PD-L2
• a growing mode of cancer treatment is chimeric antigen receptor T cell (CAR-T) therapy.
• CAR-T cells are effectively artificial TIL.
• treatment with a RARy selective agonist, as described herein is combined with CAR-T therapy.
• RARy-selective agonists can potentiate chimeric antigen receptor-modified immune cells (CAR-MIC) therapy, as described in US2019001563A1 and US20180338940A1 (each of which is incorporated by reference for all that they teach about potentiation of CAR-MIC with RARy agonists).
• some embodiment are methods of potentiating CAR-MIC cancer immunotherapy comprising administering a RARy-selective agonist having the structure of GAG1 to a cancer patient who is receiving, has received, or is scheduled to receive, CAR-MIC.
• the CAR- MIC is a CAR-T cell.
• some embodiments further comprise administering an inhibitor of regulatory T cells (Tregs).
• the inhibitor of Tregs is a Treg-depleting antibody.
• the Treg-depleting antibody is anti-CD25 antibody, an anti-GITR antibody, an anti-FoxP3 antibody, an anti-CCR4 antibody, or an anti-folate receptor 4 antibody.
• the inhibitor of Tregs comprises a RARa antagonist.
• R is H or C 1-6 alkyl , or a pharmaceutically acceptable salt thereof.
• R is methyl or ethyl.
• R is H; that is the RXR agonist is 3,7-dimethyl-6(S),7(S)-methano,7-[1 ,1 ,4,4-tetramethyl-1 ,2,3,4-tetrahydron-aphth-7-yl]2(E), 4(E) heptadienoic acid, also known as IRX4204.
• RARy selective agonists can have undesirable side effects, such as weight loss that can become excessive, especially at higher dosages, as seen in some of the Examples below.
• Compounds with the structure of XAG also have an anticachectic effect, as described in US 20070185055A1 (which is incorporated by reference for all that it teaches about the treatment of cachexia with RXR agonists), which could counteract or mitigate the side effects of the RARy agonist contributing to potentially excessive weight loss. Accordingly, for either or both of these reasons, in some embodiments treatment with a RARy selective agonist, as described herein, is combined with treatment with an appropriate RXR agonist, such as IRX4204.
• an appropriate RXR agonist such as IRX4204.
• the average surface area of a human body is generally accepted to be 1.9 m 2 for an adult male, 1.6 m 2 for an adult female, and 1.33 m 2 for a 12-13 year old child. These values can be used to calculate dose ranges for doses of the RARy selective agonist.
• the total daily dosage of RARy selective agonist active agents can be administered as a single dose or as two doses administered with a 24 hour period spaced 8 to 16, or 10 to 14, hours apart. For repeated administrations over several days or longer, the treatment can be repeated until a desired suppression of disease or disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the disclosure.
• the desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
• the RARy selective agonist can be administered to a mammal using standard administration techniques, including parenteral, oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
• parenteral includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration.
• the RARy selective agonist preferably is suitable for oral administration, for example as a pill, tablet or capsule.
• Administration may be continuous or intermittent.
• the dosage may also be determined by the timing and frequency of administration.
• the RARy selective agonist disclosed herein can be given on a daily, weekly, biweekly, or monthly basis for a period of time, followed by an optional drug holiday (drug free period) and that this drug administration/drug holiday cycle can be repeated as necessary.
• the RARy selective agonist could be administered daily for a week, and then not administered for the rest of a month, and then administered for another week, etc.
• the total daily dosage of RARy agonist can be administered as a single dose or as two doses administered with a 24-hour period spaced 8 to 16, or 10 to 14, hours apart.
• a lump or tumor involving some lymph nodes can be felt and measured externally by physical examination.
• a tumor marker test can be done for certain cancers.
• test whether blood test, cell count, or tumor marker test, it is repeated at specific intervals so that the results can be compared to earlier tests of the same type.
• Partial response the cancer has shrunk by a percentage but disease remains. Levels of a tumor marker (if applicable) may have fallen (or increased, based on the tumor marker, as an indication of decreased tumor burden) but evidence of disease remains. • Stable disease - the cancer has neither grown nor shrunk; the amount of disease has not changed. A tumor marker (if applicable) has not changed significantly.
• Other measures of the efficacy of cancer treatment include intervals of overall survival (that is time to death from any cause, measured from diagnosis or from initiation of the treatment being evaluated)), cancer-free survival (that is, the length of time after a complete response cancer remains undetectable), and progression-free survival (that is, the length of time after disease stabilization or partial response that resumed tumor growth is not detectable).
• treating broadly include any kind of treatment activity, including the mitigation, or prevention of disease, or an aspect thereof, in man or other animals, or any activity that otherwise affects the structure or any function of the body of man or other animals.
• Treatment activity includes the administration of the medicaments, dosage forms, and pharmaceutical compositions described herein to a patient, especially according to the various methods of treatment and methods of generating, differentiating or expanding TIL disclosed herein, whether by a healthcare professional, the patient his/herself, or any other person.
• Treatment activities include the orders, instructions, and advice of healthcare professionals such as physicians, physician’s assistants, nurse practitioners, and the like, that are then acted upon by any other person including other healthcare professionals or the patient him/herself.
• the orders, instructions, and advice aspect of treatment activity can also include encouraging, inducing, or mandating that a particular medicament, or combination thereof, be chosen for treatment of a condition - and the medicament is actually used - by approving insurance coverage for the medicament, denying coverage for an alternative medicament, including the medicament on, or excluding an alternative medicament, from a drug formulary, or offering a financial incentive to use the medicament, as might be done by an insurance company or a pharmacy benefits management company, and the like.
• treatment activity can also include encouraging, inducing, or mandating that a particular medicament be chosen for treatment of a condition - and the medicament is actually used - by a policy or practice standard as might be established by a hospital, clinic, health maintenance organization, medical practice or physicians group, and the like. All such orders, instructions, and advice are to be seen as conditioning receipt of the benefit of the treatment on compliance with the instruction.
• a financial benefit is also received by the patient for compliance with such orders, instructions, or advice.
• a financial benefit is also received by the healthcare professional for compliance with such orders, instructions, or advice.
• R 7 to R 12 are independently: C 1-6 alkyl, a hydrogen atom, an alkoxy group, a halogen atom (for example F, Cl, or Br), a nitro group, a hydroxy group, OCF 3 , or COR 13 , where R 13 is C 1-6 alkyl or CF 3 .
• Y is oxygen, sulfur, or NR 14 , where R 14 is C 1-6 alkyl.
• R is methyl or ethyl.
• R is H.
• Some embodiments are a pharmaceutical composition or formulation comprising a compound having the structure of GAG1 or a pharmaceutically acceptable salt thereof.
• the RARy selective agonist is a compound having the structure of GAG1 or a pharmaceutically acceptable salt thereof.
• Y is O:
• the RARy selective agonist is 4- ((1E,3E)-3-(3-(terf-butyl)-2-ethyl-benzofuran-5-yl)-3-(hydroxyimino)prop-1-en-1-yl)-3- fluorobenzoic acid (GA1E), having the structure
• R is methyl or ethyl.
• R is H.
• R is methyl or ethyl.
• the RARy selective agonist is 3-fluoro-4-((1 E,3E)-3- (hydroxyimino)-3-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-en-1- yl)benzoic acid (GA2E), having the structure
• the RARy selective agonist is 3-fluoro-4-((1 E,3Z)-3-(hydroxyimino)-3- (5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-en-1-yl)benzoic acid (GA2Z), having the structure
• the paraposition of the COOR group is preferred.
• both R 1 are CH3.
• both R 2 are H.
• X is C(R 5 )2.
• both R 5 are CH3.
• one R 3 is H and the other R 3 is F.
• both R 4 are H.
• all R 3 and R 4 are H.
• R is H.
• R is methyl or ethyl.
• the para- position of the COOR group is preferred.
• Some embodiments specifically include one or more specific substituents at one or more of the variable positions. Some embodiments specifically include one or more of the alternatives, while other embodiments specifically exclude one or more of these alternatives.
• the RARy selective agonist has the structure and other embodiments have the structure
• the RARy selective agonist has the structure
• the RARy selective agonist is a compound with the structure and in other embodiments, the RARy selective agonist is a compound with the structure
• R is H.
• the alkyl part of the ester has anywhere from 1 to 6 carbon atoms and includes, but is not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, /so-butyl, f-butyl, pentyl isomers, hexyl isomers, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and combinations thereof having anywhere from 1-6 carbon atoms, etc.
• the various subsets (including individual species) and combinations of these esters are contemplated as further distinct embodiments, including straight-chain, branched, and/or cyclic moieties of any length or set of lengths within the Ci-6 alkyl genus.
• the RARy selective agonist is tazarotenic acid (selective for RARy over RARa, but not over RARP), CD437, CD2325, CD666, trifarotene, or BMS961.
• the RARy agonist is a RARy-selective agonist in that it has no or only negligible agonistic activity with RARa and RARp at clinically relevant concentrations. In some embodiments, the RARy agonist is a RARy-selective agonist in that it has no or only negligible agonistic activity with RARa at clinically relevant concentrations. In various embodiments, a RARy selective agonist has no or only negligible agonistic activity with RARa or RARp at some clinically relevant concentrations when the agonist’s KD for RARy is greater than 10-, 20-, 50-, or 100-fold lower than for the other RAR(s).
• a RARy selective agonist has no or only negligible agonistic activity with RARa or RARp at some clinically relevant concentrations when the agonist’s EC50 with RARy is greater than 10-, 20-, 50-, or 100-fold lower than for the other RAR(s).
• EC50 is typically determined in an activation assay, for example, a transactivation assay.
• the RARy selective agonist meets at least one of these criteria, for example the least stringent.
• RARy agonists constitute means for activating RARy, or means for generating, differentiating, or expanding TIL. Some embodiments specifically include one or more of the disclosed genera, sub-genera, or species of these RARy agonists. Some embodiments specifically exclude one or more of the disclosed genera, sub-genera, or species of these RARy agonists.
• GA1 E 1 HNMR (DMSO-D6): 5 1.31 (triplet, 3H), 1.50 (s, 9H), 2.97 (q, 2H), 6.95 (d, 1 H), 7.38 (d, 1 H), 7.57 (d, 1 H), 7.71 (d, 1 H), 7.82 (d, 1 H), 7.86 (s, 1 H), 7.90 (d, 1 H), 7.97 (t, 1 H).
• Transactivation assays were conducted to test the specificity of binding activity of GA1 E and GA2E for RARa and RARy. Retinoic acid receptor transactivation activity and binding efficiencies were determined essentially as described in U.S. Pat. Nos.: 5,298,429 and 5,071 ,773, incorporated by reference herein for all they teach regarding transactivation assays. Transactivation assays employed expression plasmids encoding the full-length receptors RARa and RARy.
• Reporter plasmids containing the herpes virus thymidine kinase promoter and the appropriate retinoic acid receptor response element (RAREs) were positioned upstream of an open coding region encoding firefly luciferase.
• GA1 E and GA2E were tested over a concentration range of 0.5 to 625 nM (GA2E) or .05 to 3320 nM (GA1 E).
• GA1 E induced less than 10% transactivation for RARa across the whole concentration range, but over 50% transactivation for RARy at concentrations >25nM and over 70% at concentrations >125 nM ( Figure 3A).
• GA2E induced more than 85% transactivation at all tested concentrations and less than 10% transactivation of RARa at concentrations of ⁇ 25 nM ( Figure 3B).
• mice were treated with 21 daily oral doses 10 mg/kg of 1 mg/ml tazarotenic acid or GA2E, or vehicle control, starting on Day 0. Tumor volume was measured, and the mice weighed, on days 0, 1 , 3, 6, 8, 10, 13, 15, 17, 20, and 21.
• Figure 5A-B present the results of the flow cytometry both as a percentage of cells and as the number of cells per tumor. Generally, only a minority of cells in the tumor were alive, as revealed by staining with the dye FVS700, which penetrates and stains necrotic cells (Figure 5A, “Live” panel). Of these live cells, a substantial proportion were leukocytes ( Figure 5A, “CD45” panels).
• T memory cells (expected to be predominantly TH1 memory cells) based on intracellular staining for IFNy ( Figure 5B, “TH IFGy” panels), TH17 cells based in intracellular staining for IL-17 ( Figure 5B, “TH IL17” panels), or Treg cell based on staining for CD25 and FoxP3 ( Figure 5B, “Treg” panels).
• T cells about 25-30% of the T cells were CD8 + ( Figure 5B, “CD8 T” panels), expected to be predominantly cytotoxic cells.
• PBMC Peripheral blood mononuclear cells from a cytomegalovirus (CMV)-positive donor were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with CMV recall antigens at 1 mg/ml.
• CMV cytomegalovirus
• CFSE carboxyfluorescein succinimidyl ester
• DMSO carboxyfluorescein succinimidyl ester
• CFSE is a fluorescent reactive cell membrane permeable dye. After it is taken up, CFSE covalently modifies proteins and is retained in cells for long periods of time. The CFSE- modified proteins are generally evenly divided between daughter cells at each division so that fluorescent intensity of the cells falls by have at each division. Thus diminution of the dye’s fluorescent signal can be used as a measure of proliferation.
• the NSG-B2M mouse model is a severely immunocompromised animal, combining the lesions non-obese diabetic (NOD), severe combined immunodeficiency (SCID), IL-2 receptor Y null, and p2-microglobulin null.
• NOD non-obese diabetic
• SCID severe combined immunodeficiency
• IL-2 receptor Y null IL-2 receptor Y null
• p2-microglobulin null Human tumor cells readily engraft in this model and cells from the human immune system can also be engrafted in order to study various modes of cancer immunotherapy.
• one can model a human immune response to a human tumor in this system.
• mice Female NSG-B2M mice were injected in the mammary fat pad with 5 x10 6 JIMT-1 cells (human breast carcinoma cell line) suspended in 0.1 mL of phosphate-buffered saline (PBS) with an equal volume of Matrigel®. Once tumors reached a volume of 100-200 mm 3 , the mice were implanted with 1 x10 7 human PBMC by subcutaneous injection into the right flank (pre-randomization). PBMC were obtained from two donors and distributed evenly between groups. When tumors reached an average tumor volume of 200-250 mm 3 (about 3 days post-PBMC injection) animals were matched by tumor volume into treatment and control groups, and dosing initiated. This first day of dosing was defined as Day 0. The mice were administered a daily oral dose of 10 mL/kg of 10% dimethyl sulfoxide (DMSO) in PBS as vehicle control or 10 mg/kg of GA2E dissolved in 10% DMSO in PBS as the treatment.
• DMSO dimethyl s
• Tumor volume was measured twice weekly and on the day a study endpoint was reached. The mice were also weighed twice weekly and on the day a study endpoint was reached. Animals exhibiting a weight loss from Day 0 of >10% were provided a food supplement ad libitum. Animals exhibiting a weight loss of >20% in any 7-day period or >30% from Day 0 were considered moribund and euthanized.
• Study endpoint was defined as when the mean tumor volume of the control group (uncensored) reaches 1500 mm 3 . If this occurs before Day 28, treatment groups and individual mice were permitted to be dosed and measured up to Day 28. If the mean tumor volume of the control group (uncensored) did not reach 1500 mm 3 by Day 28, then the endpoint for all animals was the day when the mean tumor volume of the control group (uncensored) reached 1500mm 3 up to a maximum of Day 60.
• mice were sacrificed on Day 26 of the study and the tumors collected and analyzed by flow cytometry. Statistical analysis was by ordinary one-way ANOVA with T ukey’s test for significance comparison between groups. Gates were set to identify single cells by forward light scatter, live cells as single cells based on side scatter and staining with the vital stain Fixable Viability Dye eFluorTM 780, white blood cells from live cells based on side scatter and staining with anti-CD45, lymphocytes from white blood cells based on side and forward light scatter, and total T lymphocytes (tot T) from lymphocytes based on side scatter and staining with anti-CD3. Total T lymphocytes were divided into CD4 + (TH) and CD8 + (TC) lymphocytes based on staining with and anti-CD4 and CD8 antibodies and subsets assessed. The following observations were made on a per-mg of tumor tissue basis.
• CD8 + T effector cells expected to be cytolytic, are increased amongst TIL in shrinking tumors, is consistent with the interpretation that anticancer activity of RARy agonists, such as GA2E, arises through an immunologic mechanism rather than a direct effect.
• concentration of CD8 + TIL expressing the T cell markers PD- 1 , CD18P, and CD54 was also increased in the GA2E-treated tumors as compared to controls ( Figure 13A, P ⁇ 0.0001 ; Figure 13B, P ⁇ 0.001 ; and Figure 13C, P ⁇ 0.01 ; respectively).
• CD18 and CD54 are both involved in cell-cell adhesion and immune surveillance.
• TIL TNF-activated immunologic synapse and antigen recognition by the CD8 + T effector cells.
• PD-1 is involved in immune homeostasis, facilitating down-regulation of immune responses, for example, through apoptosis of the effector cells expressing PD-1.
• the increase in PD-1 + cells reflects an upregulated response.
• Antagonists of the PD-1/PD-L axis such as anti-PD-1 and anti-PD-L1 antibodies (immune checkpoint inhibitors) may thus further augment the immune response promoted by RARy agonists, such as GA2E.
• the tumor cells were also assessed by flow cytometry for expression of PD-L1 , CD18P, CD58 (LFA-3), and Her2. Treatment with GA2E did not significantly alter the proportion of cells expressing these markers, consistent with RARy agonists not having a direct effect on the tumor cells.
• EMT6 cells were cultured in vitro for 5 days the presence of a multiple concentrations of the RARy agonists GA1 E, GA2E, and GA3Ep and viability assessed by the Cell Titer Gio assay® (Promega). The compounds had no effect at concentrations below 1 pM, but rapidly became toxic as concentrations exceeded 10 pM ( Figure 16).
• the IC50 for GA1 E, GA2E, and GA3Ep were 52 pM, 19 pM, and 101 pM for GA1 E, GA2E, and GA3Ep, respectively.
• Lewis lung carcinoma (LLC) cells were cultured in vitro the presence of a multiple concentrations of the RARy agonists GA1 E, GA2E, and GA3Ep and viability assessed by the Cell Titer Gio assay® .
• the compounds had no effect at concentrations below 1 pM, but rapidly became toxic as concentrations exceeded 10 pM ( Figure 17).
• the IC50 for GA1 E, GA2E, and GA3Ep were 96 pM, 46 pM, and 45 pM for GA1 E, GA2E, and GA3Ep, respectively.
• the LLC model is based on an epidermoid carcinoma of the lung that spontaneously arose in a C57BL/6 mouse some 70 years ago, Syngeneic models have been useful in predicting clinical benefit.
• Tumor volumes and weight were measured on days 0, 2, 4, 6, 9, 11 , and 13. This study was ended on Day 13, as the average tumor volume was >1500 mm 3 , and the tumors analyzed by flow cytometry. Two animals in the 10 mg/kg group and one animal in the 25 mg/kg group were terminated early due to ulceration of the tumor.
• mice were sacrificed on Day 13 of the study and the tumors collected and analyzed by flow cytometry. Statistical analysis was by ordinary one-way ANOVA with T ukey’s test for significance comparison between groups. Gates were set to identify single cells by forward light scatter, live cells as single cells based on side scatter and staining with the vital stain Fixable Viability Dye eFluorTM 780, white blood cells from live cells based on side scatter and staining with anti-CD45, lymphocytes from white blood cells based on side and forward light scatter, and total T lymphocytes (tot T) from lymphocytes based on side scatter and staining with anti-CD3.
• T lymphocytes were divided into CD4 + (TH) and CD8 + (TC) lymphocytes based on staining with and anti-CD4 and CD8 antibodies and subsets assessed (Figure 20). The following observations were made on a per-mg of tumor tissue basis.
• the Examples demonstrate that RARy agonists inhibit tumor growth in multiple cancer models including breast cancer (Her2 + and triple negative) and lung cancer cell lines and promote TIL with mouse and human lymphocytes. As the RARy agonists did not appear to have a tumor growth inhibitory effect in vitro, it is apparent that the tumor growth inhibitory effect observed in vivo is due to increased production and promotion anti-tumor activity of TIL.

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