Conjugates, compositions, and methods for restoring CAR-T cell activity

Abstract

Chimeric antigen receptor (CAR) T cell restoring conjugates and compositions and methods of use for restoring exhausted CAR T cells.

Description

[Technical field]

[0001] Priority This patent application relates to and claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 238300, filed August 30, 2021, the contents of which are hereby incorporated by reference into the present disclosure in their entirety.

[0002] The present disclosure relates to chimeric antigen receptor (CAR) T cells, conjugates comprising Toll-like receptor (TLR) 7 / 8 agonists (i.e., TLR7, TLR8, or TLR7 and TLR8), compositions comprising same, and methods of using same to restore activity to exhausted CAR T cells. [Background technology]

[0003] Chimeric antigen receptor (CAR) T cells have demonstrated considerable promise in the treatment of hematopoietic cancers. However, their utility in eradicating solid tumors is compromised by the development of a dysfunctional or exhausted phenotype that results in reduced proliferation and impaired effector function.

[0004] In view of the above, there is an unmet need to reactivate exhausted CAR T cells. Desirably, reactivation of CAR T cells is targeted, thereby minimizing, if not eliminating, toxicity to healthy cells.

[0005] It is an object of the present disclosure to provide conjugates, compositions, and methods for restoring activity to exhausted CAR T cells. This and other objects, as well as features and advantages of the invention, will be apparent from the description provided herein. Summary of the Invention

[0006] (a) A method is provided for restoring exhausted chimeric antigen receptor (CAR)-T cells in a subject with cancer who is being treated with a cancer-binding conjugate comprising (i) a vector comprising a promoter operably linked to a nucleic acid sequence encoding a CAR or (ii) a T cell expressing a CAR, and (b) a ligand that binds to cancer cells with specificity and is conjugated to a first targeting moiety. The CAR is conjugated to a first targeting moiety, a second targeting moiety, or both the first targeting moiety and the second targeting moiety, and the ligand that binds to cancer cells with specificity and the first targeting moiety can optionally be conjugated via a first linker. The method may include administering to a subject (e.g., having exhausted CAR T cells) a CAR T cell activity restoring conjugate that includes an agonist of toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), or toll-like receptor 7 and toll-like receptor 8 (TLR7 / 8), conjugated to a first targeting moiety or a second targeting moiety, where the agonist and either the first targeting moiety or the second targeting moiety are conjugated via a second linker.

[0007] The CAR T cell activity restoring conjugate has the structure:

[0008] [ka] [In formula: R 1 is acyclic alkyl or cyclic alkyl optionally substituted with one or more substituents independently selected from alkyl, halo, heteroalkyl, alkoxy, and cycloalkyl; R 2 -NR 2x R 2y , Hydrogen (H), -OR z , -SO2N(R z )2, or N3 (In the formula: R 2x and R 2y are each independently H, -N(R z)2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or alkyl optionally substituted by one or more substituents independently selected from oxo, halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; Each R Z is independently H or optionally substituted alkyl; or R 2x and R 2y taken together form an optionally substituted 3-10 membered monocyclic or bicyclic heterocycloalkyl. and; Each R 3 is independently H, halo, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, or alkoxy is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halo, or cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally substituted; X 1 , X 2 , and X 3 are independently N or CR q (In the formula, each R q are independently hydrogen, halo, or optionally substituted alkyl; Z is GL-, where L is a linker (e.g., a second linker) and G is a targeting moiety (e.g., a first targeting moiety or a second targeting moiety); In formula (I), n is 1 to 6; m in formula (I) is 0 to 4. or a pharma- ceutically acceptable salt thereof.

[0009] The CAR T cell activity restoring conjugate has the structure:

[0010] [ka] [In formula: R 1 , R 3 , R 4 , and R 5 each independently represents H, alkyl, alkoxy, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR 2x ,

[0011] [ka] (In the formula, R 2x and R 2y each of is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; Z is GL-, where L is a linker (e.g., L is a second linker) and G is a targeting moiety (e.g., G is a first targeting moiety or a second targeting moiety); X 1 , X 2 , and X 3 is an independent CR q or N (wherein each R q are independently H, halo, or optionally substituted alkyl; Y is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z)2, or -CON(R z )2(in the formula: R 2x and R 2y are independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl (e.g., optionally substituted with one or more substituents, each of which is independently oxo, halo, alkyl, heteroalkyl, alkoxy, or cycloalkyl); Each R z is independently H or optionally substituted alkyl; or R 2x and R 2y are taken together to form an optionally substituted heterocycloalkyl (e.g., the optionally substituted heterocycloalkyl is a monocyclic or bicyclic heterocycloalkyl and / or the optionally substituted heterocycloalkyl is a 3-10 membered heterocycloalkyl); In formula (II), n is 0 to 30. or a pharma- ceutically acceptable salt thereof.

[0012] In certain embodiments, the CAR T cell activity restoring conjugate has the structure:

[0013] [ka] [In formula: R 1 is acyclic alkyl or cyclic alkyl optionally substituted with one or more substituents independently selected from halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; Y is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3Rz , -N3, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z )2(in formula:R 2x and R 2y are independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl (e.g., optionally substituted with one or more substituents, each of which is independently oxo, halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl); Each R Z is independently H or optionally substituted alkyl; or R 2x and R 2y are taken together to form an optionally substituted heterocycloalkyl (e.g., the optionally substituted heterocycloalkyl is a monocyclic or bicyclic heterocycloalkyl and / or the optionally substituted heterocycloalkyl is a 3-10 membered heterocycloalkyl); Each R 3 is independently halo, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halogen, or cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally substituted; X 1 , X 2 , and X3 is an independent CR q or N (wherein each R q is independently H, halogen, or optionally substituted alkyl; Z is LG, where L is a linker (e.g., a second linker) and G is a targeting moiety (e.g., a first targeting moiety or a second targeting moiety); In formula (III), n is 0 to 30; m in formula (III) is 0 to 4. or a pharma- ceutically acceptable salt thereof.

[0014] In use, CAR T cells in a subject (e.g., exhausted CAR T cells) can bind and endocytose the CAR T cell activity restoring conjugate such that the T cells are restored to activity by an agonist of the CAR T cell activity restoring conjugate.

[0015] In various embodiments, X in formula (I), formula (II), or formula (III) 1 , X 2 , and X 3 Each of may be N.

[0016] The CAR T cell activity restoring conjugate has the structure:

[0017] [ka] or a pharma- ceutically acceptable salt of any of the foregoing structures. Alternatively, the CAR T cell activity restoring conjugate may have the structure:

[0018] [ka] or a pharma- ceutically acceptable salt of any of the preceding structures.

[0019] In certain embodiments, the agonist of the CAR T cell activity restoring conjugate is an agonist of TLR7 / 8 and has the structure:

[0020] [ka] or a pharma- ceutically acceptable salt of any of the preceding structures.

[0021] The first targeting moiety, the second targeting moiety, or both the first targeting moiety and the second targeting moiety have the structure:

[0022] [ka] It may be or may include a group having the following formula:

[0023] Alternatively, the first targeting moiety, the second targeting moiety, or both the first targeting moiety and the second targeting moiety have the structure:

[0024] [ka] It may be or may include a group having the following formula:

[0025] In certain embodiments, the CAR T cell activity restoring conjugate has the structure:

[0026] [ka] or a pharma- ceutically acceptable salt thereof. Alternatively, the CAR T cell activity restoring conjugate may have the structure:

[0027] [ka] [In the formula, n = 0 to 200] or a pharma- ceutically acceptable salt of any of the preceding structures.

[0028] Further alternatively, the CAR T cell activity restoring conjugate has the structure:

[0029] [ka] or a pharma- ceutically acceptable salt thereof. Furthermore, the CAR T cell activity restoring conjugate may have the following structure:

[0030] [ka] or a pharma- ceutically acceptable salt of any of the preceding structures. In certain embodiments, the CAR T cell activity restoring conjugate is

[0031] [ka] or a pharma- ceutically acceptable salt of any of the preceding structures. In certain embodiments, the CAR T cell activity restoring conjugate is

[0032] [ka] or a pharma- ceutically acceptable salt of any of the preceding structures. In certain embodiments, the CAR T cell activity restoring conjugate is

[0033] [ka] or may be a pharma- ceutically acceptable salt of any of the preceding structures.

[0034] The ligand that binds with specificity to cancer cells may be selected from the group consisting of folate, 5-methyltetrahydrofolate, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) ligands, neurokinin 1 receptor (NK-1R) ligands, carbonic anhydrase IX (CAIX) ligands, ligands for gamma glutamyl transpeptidase, ligands for luteinizing hormone releasing hormone (LHRHR), ligands for CD73, ligands for fibroblast activation protein, ligands for heat shock proteins (HSPs), ligands for glucose transporter 1 (glut-1), natural killer group 2D receptor (NKG2D) ligands, and cholecystokinin B receptor (CCKBR or CCK2) ligands.

[0035] The first targeting moiety and the second targeting moiety may be independently selected from the group consisting of 2,4-dinitrophenyl (DNP), L-rhamnose, tacrolimus (FK506), 2,4,6-trinitrophenol (TNP), biotin, rapamycin, digoxigenin, folate, 5-methyltetrahydrofolate, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centirin, and DARPin.

[0036] In certain embodiments, the first targeting moiety and the second targeting moiety are independently selected from the group consisting of DNP, FK506, TNP, biotin, rapamycin, digoxigenin, folate, 5-methyltetrahydrofolate, fluorescein, FITC, NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centirin, and DARPin; and the ligand that binds with specificity to cancer cells is selected from the group consisting of folate, DUPA ligand, NK-1R ligand, CAIX ligand, gamma glutamyl transpeptidase ligand, LHRHR ligand, CD73 ligand, HSP ligand, glut-1 ligand, fibroblast activation protein ligand, NKG2D ligand, and CCKBR or CCK2 ligand.

[0037] The first linker and the second linker may be independently releasable or non-releasable. The first linker and the second linker may be any of C1 to C6 20 Alkyl, alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, -SS, -CO-O-(CH2) n -SS-(n=2-6), -O-CO-O-(CH2) n -SS-(n=2-6), -S-CO-O-(CH2) n -SS-(n=2-6), -NH-CO-O-(CH2) n -SS- (n = 2 to 6), carboxylates, carbonates, carbamates, ureas, thioureas, polyethylene glycols (PEG) (e.g., PEG n , n=1-200), polyproline, oligo-(4-piperidine)carboxylic acid, oligopiperidine, amino acid (e.g., hydrophilic amino acid), peptide, saccharopeptide, sugar, peptidoglycan (e.g., non-natural peptidoglycan), polyvinylpyrrolidone, Pluronic F-127, or any combination of two or more of the foregoing. The first linker, the second linker, or both the first linker and the second linker may comprise PEG.

[0038] A CAR T cell activity restoring conjugate comprising an agonist of TLR7, TLR8, or TLR7 / 8 conjugated via a linker to a targeting moiety that binds with specificity to the CAR of a CAR T cell, the conjugate having the structure:

[0039] [ka] [In formula: R 1 is acyclic alkyl or cyclic alkyl optionally substituted with one or more substituents independently selected from alkyl, halo, heteroalkyl, alkoxy, and cycloalkyl; R 2 -NR 2x R 2y , H, -OR z , -SO2N(R z )2, or N3 (wherein: R 2x and R 2y are each independently hydrogen, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or alkyl optionally substituted by one or more substituents independently selected from oxo, halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; Each R Z is independently H or optionally substituted alkyl; or R 2x and R 2y taken together form an optionally substituted 3-10 membered mono- or bicyclic heterocycloalkyl; Each R 3 is independently H, halo, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, or alkoxy is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halo, or cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally substituted; X 1 , X 2 , and X 3 are independently N or CR q (In the formula, each R q are independently H, halo, or optionally substituted alkyl; Z is GL-, where L is a linker and G is a targeting moiety; In formula (I), n is 1 to 6; m in formula (I) is 0 to 4. or a pharma- ceutically acceptable salt thereof; or structure:

[0040] [ka] [In formula: R 1 , R 3 , R 4 , and R 5 each independently represents H, alkyl, alkoxy, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR 2x ,

[0041] [ka] (In the formula, R 2x and R 2y each of is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; Z is GL-, where L is a linker and G is a targeting moiety; X 1 , X 2 , and X 3 is an independent CR q or N (wherein each R q are independently H, halo, or optionally substituted alkyl; Y is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -COR z , -COOR z , -CON(R z )2, -COSRz , -SO2N(R z )2, or -CON(R z )2(in the formula: R 2x and R 2y are independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl (e.g., optionally substituted with one or more substituents, each of which is independently oxo, halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl); Each R z is independently hydrogen or optionally substituted alkyl; or R 2x and R 2y are taken together to form an optionally substituted heterocycloalkyl (e.g., the optionally substituted heterocycloalkyl is a monocyclic or bicyclic heterocycloalkyl and / or the optionally substituted heterocycloalkyl is a 3-10 membered heterocycloalkyl); In formula (II), n is 0 to 30. or a pharma- ceutically acceptable salt thereof; or structure:

[0042] [ka] [In formula: R 1 is acyclic alkyl or cyclic alkyl optionally substituted with one or more substituents independently selected from halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; Y is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -CORz , -COOR z , -CONR z 2. -COSR z , -SO2N(R z )2, or -CON(R z )2(in the formula: Each R Z are independently H or optionally substituted alkyl; R 2x and R 2y are independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or alkyl optionally substituted by one or more substituents independently selected from oxo, halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; or R 2x and R 2y taken together form an optionally substituted 3-10 membered mono- or bicyclic heterocycloalkyl; Each R 3 is independently halo, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halogen, or cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally substituted; X 1 , X 2 , and X 3 is an independent CR q or N (wherein each R q are independently H, halogen, or optionally substituted alkyl; Z is LG, where L is a linker and G is a targeting moiety; In formula (III), n is 0 to 30; m in formula (III) is 0 to 4. or a pharma- ceutically acceptable salt thereof; G in formula (I), formula (II) or formula (III) has the structure:

[0043] [ka] Also provided is a CAR T cell activity restoring conjugate that is or comprises a group having the formula: 1 , X 2 , and X 3 Each of may be nitrogen (N).

[0044] The targeting moiety of the CAR T cell activity restoration conjugate may be selected from the group consisting of DNP, L-rhamnose, FK506, TNP, biotin, rapamycin, digoxigenin, folate, 5-methyltetrahydrofolate, fluorescein, FITC, NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centirin, and DARPin.

[0045] The linker of the CAR T cell activity restoring conjugate may be releasable. The linker of the CAR T cell activity restoring conjugate may be non-releasable. The linker may be any of the following: C1-C 20 Alkyl, alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, -SS, -CO-O-(CH2) n -SS-(n=2-6), -O-CO-O-(CH2) n -SS-(n=2-6), -S-CO-O-(CH2) n -SS-(n=2-6), -NH-CO-O-(CH2) n-SS- (n = 2 to 6), carboxylates, carbonates, carbamates, ureas, thioureas, PEG (e.g., PEG n , n=1-200), polyproline, oligo-(4-piperidine) carboxylic acid, oligopiperidine, amino acid (e.g., hydrophilic amino acid), peptide, saccharopeptide, sugar, peptidoglycan (e.g., non-natural peptidoglycan), polyvinylpyrrolidone, Pluronic F-127, or any combination of two or more of the foregoing. The linker may comprise PEG.

[0046] The CAR T cell activity restoring conjugate has the structure:

[0047] [ka] or a pharma- ceutically acceptable salt thereof. The CAR T cell activity restoring conjugate may have the formula:

[0048] [ka] [In the formula, n=0~200] or a pharma- ceutically acceptable salt of any of the preceding structures. The CAR T cell activity restoring conjugate may have a structure selected from:

[0049] [ka] or a pharma- ceutically acceptable salt thereof. Still further alternatively, the CAR T cell activity restoring conjugate may have a structure selected from:

[0050] [ka] or may be a pharma- ceutically acceptable salt of any of the preceding structures.

[0051] In certain embodiments, the CAR T cell activity restoring conjugate comprises:

[0052] [ka] or a pharma- ceutically acceptable salt of any of the preceding structures. Other structures of the CAR T cell activity restoring conjugates include:

[0053] [ka] and may be a pharma- ceutically acceptable salt of any of the foregoing structures. In certain embodiments, the CAR T cell activity restoring conjugate comprises a structure selected from

[0054] [ka] or may be a pharma- ceutically acceptable salt of any of the preceding structures.

[0055] Pharmaceutical compositions are also provided. The compositions can include a CAR T cell activity restoring conjugate and a pharma- ceutically acceptable carrier.

[0056] The embodiments of the present disclosure and other features, advantages, and aspects contained herein, as well as the accomplishments thereof, will become apparent in light of the following detailed description of various exemplary embodiments of the present disclosure, which will be better understood in conjunction with the accompanying drawings. [Brief description of the drawings]

[0057] [Figure 1] Figure 1A is a diagram of the activity recovery strategy of chimeric antigen receptor (CAR) T cells. Figure 1A shows the cell killing effect of CAR T cells in the presence of adaptor compounds. Figure 1B shows the activity recovery strategy of chimeric antigen receptor (CAR) T cells. Figure 1B shows the activity recovery of CAR T cells in the presence of targeted activity recovery compounds. [Diagram 2]Figure 2A is a diagram of an embodiment of a CAR T cell activity restoration strategy using an anti-fluorescein CAR and a fluorescein-Toll-like receptor (TLR) 7a conjugate to selectively activate CAR-T cells in vivo. Figure 2A shows that upon administration of a fluorescein-folate conjugate (an embodiment of a "cancer-killing" conjugate) to a subject with a folate receptor-expressing tumor, the "cancer-killing" conjugate formed a cross-link between the anti-fluorescein CAR T cell (i.e., a CAR T cell having an anti-fluorescein cell surface receptor) and a folate receptor-expressing cancer cell, promoting cancer cell killing and CAR T cell proliferation. Figure 2B is a diagram of an embodiment of a CAR T cell activity restoration strategy using an anti-fluorescein CAR and a fluorescein Toll-like receptor (TLR) 7a conjugate to selectively activate CAR-T cells in vivo. Figure 2B shows that upon administration of a fluorescein-TLR7a conjugate (an embodiment of an "activity-restoring" conjugate) to a subject with a folate receptor-expressing tumor, the "activity-restoring" conjugate binds to the same anti-fluorescein receptor on the surface of the CAR T cell, thereby allowing endocytosis of TLR7a by the CAR T cell and restoration of activity of the CAR-T cell. The legend for Figure 2B applies to both illustrations in Figure 2A and Figure 2B. [Figure 3-1]Figure 3A supports that fluorescein-conjugated fluorescent dyes bind only to anti-fluorescein CAR-expressing T cells. Figure 3A demonstrates that fluorescein-near infrared (NIR) dye conjugates (either fluorescein-NIR dye or fluorescein-Alexafluor647) bound to anti-fluorescein CAR T cells in a manner that could be quantitatively blocked by the addition of 1000-fold excess fluorescein. Flow cytometry was performed on anti-fluorescein CAR T cells in the absence (blue histogram) or presence (green histogram) of fluorescein-NIR dye conjugates (10 nM), or in the presence of both fluorescein-dye conjugates and 1000-fold excess fluorescein (magenta histogram). Figure 3B supports that fluorescein-conjugated fluorescent dyes bind only to anti-fluorescein CAR-expressing T cells. FIG. 3B demonstrates that the CAR-negative cell lines, MDA-MB-231 cells and KB cells, did not express binding sites for the fluorescein-dye conjugate (10 nM). [Figure 3-2] Figure 3C supports that fluorescein-conjugated fluorescent dyes bind only to anti-fluorescein CAR-expressing T cells. Figure 3C shows confocal microscopy assessment of internalization of fluorescein-Alexafluor 647 conjugates by CAR T cells first incubated at 4°C for 1 hour. Bars = 10 μm. Figure 3D supports that fluorescein-conjugated fluorescent dyes bind only to anti-fluorescein CAR-expressing T cells. Figure 3D shows confocal microscopy assessment of internalization of fluorescein-Alexafluor 647 conjugates by CAR T cells after cells from Figure 3C were subsequently transferred to 37°C for 4 hours. Bars = 10 μm. [Figure 4-1]Figure 4A shows the activation of human CD3+ T cells upon administration of different concentrations of either TLR7-54 or TLR7-1a agonist. Figure 4A shows the percentage increase in CD69+ cells after isolated human peripheral blood CD3+ T cells were stimulated with anti-CD3+ mAb in the absence or presence of increasing concentrations of TLR-54 or TLR7-1a as measured by flow cytometry. Data shows the increase in these parameters above baseline levels (i.e., vehicle (DMSO treatment)). Bar graphs represent mean ± standard deviation (SD), n=3. Figure 4B shows the activation of human CD3+ T cells upon administration of different concentrations of either TLR7-54 or TLR7-1a agonist. Figure 4B shows the percent increase in CD25+ cells after isolated human peripheral blood CD3+ T cells were stimulated with anti-CD3+ monoclonal antibody (mAb) in the absence or presence of increasing concentrations of TLR-54 or TLR7-1a as measured by flow cytometry. Data show the increase in these parameters over baseline levels (i.e., vehicle (DMSO treatment)). Bar graphs represent the mean ± standard deviation (SD), n=3. [Figure 4-2] FIG. 4C shows activation of human CD3+ T cells upon administration of different concentrations of either TLR7-54 or TLR7-1a agonist. FIG. 4C shows levels of interferon gamma (INF-γ) in cell-free supernatants at 24 hours of stimulated T cells, as measured by enzyme-linked immunosorbent assay (ELISA). Data show increases in these parameters over baseline levels (i.e., vehicle (DMSO-treated)). Bar graphs represent mean ± standard deviation (SD), n=3. FIG. 4D shows activation of human CD3+ T cells upon administration of different concentrations of either TLR7-54 or TLR7-1a agonist. FIG. 4D shows levels of tumor necrosis factor alpha (TNF-α) in cell-free supernatants at 24 hours of stimulated T cells, as measured by ELISA. Data show increases in these parameters over baseline levels (i.e., vehicle (DMSO-treated)). Bar graphs represent mean ± standard deviation (SD), n=3. [Figure 5-1] Figure 5A shows the effect of CAR T cell targeted and non-targeted TLR7-1a agonists on CAR T cell exhaustion in vivo (TRo). Figure 5A shows the protocol for induction of CAR T cell exhaustion, which involves serial transfer of CAR T cells to fresh MDA-MB-231 human breast cancer cells in culture every 12 hours. Figure 5B shows the effect of CAR T cell targeted and non-targeted TLR7-1a agonists on CAR T cell exhaustion in vivo (TRo). Figure 5B shows the decreased ability of anti-fluorescein CAR T cells to kill MDA-MB-231 cells after three rounds of serial transfer. Figure 5C shows the effect of CAR T cell targeted and non-targeted TLR7-1a agonists on CAR T cell exhaustion in vivo (TRo). Figure 5C shows the effect of T cell targeted and non-targeted TLR7-1a agonists. Figure 5C shows increased expression of T cell exhaustion markers (programmed cell death protein (PD-1+), T cell immunoglobulin and mucin domain containing-3 (TIM3+), and lymphocyte activation 3 (LAG3+)) after three rounds of sequential transfer. [Figure 5-2] FIG. 5D shows the effect of CAR T cell targeted and non-targeted TLR7-1a agonists on CAR T cell exhaustion in vi (T Ro). FIG. 5D shows restoration of the ability of anti-fluorescein CAR T cells to kill MDA-MB-231 cells in culture after incubation with targeted or non-targeted TLR7-1a. Data show the change in these markers above baseline levels (i.e. vehicle (DMSO treated)). Bar graphs represent mean ± SD, n=3. FIG. 5E shows the effect of CAR T cell targeted and non-targeted TLR7-1a agonists on CAR T cell exhaustion in vi (T Ro). FIG. 5E shows the reduction in expression of cell surface exhaustion markers by active restored anti-fluorescein CAR T cells. Data show the change in these markers above baseline levels (i.e. vehicle (DMSO treated)). Bar graphs represent mean ± SD, n=3. [Figure 6-1]FIG. 6A shows the effect of anti-fluorescein CAR T cell therapy on the growth and immune characteristics of MDA-MB-231 and KB tumors. NOD SCID gamma (NSG) mice were implanted with 4 million MDA-MB-231 cells and 1 million KB cells in separate flanks of the same mouse, and 2 weeks later, either saline or 8×106 anti-fluorescein CAR T cells were injected (i.e., when MDA-MB-231 and KB tumors reached ∼160 or 80 mm3, respectively, to adjust for their different growth rates). 4 and 24 hours after CAR T cell injection, all mice were intravenously injected with 500 nmol / kg fluorescein-folate, which was then repeated once per week. Figure 6A shows the tumor volumes of cohorts that were either left untreated (dotted line) or treated with both anti-fluorescein CAR T cells and fluorescein-folate bispecific adaptors (solid line). Figure 6B shows the effect of anti-fluorescein CAR T cell therapy on the growth and immune characteristics of MDA-MB-231 and KB tumors. NOD SCID gamma (NSG) mice were implanted with 4 million MDA-MB-231 cells and 1 million KB cells in separate flanks of the same mice, and 2 weeks later, injected with either saline or 8x106 anti-fluorescein CAR T cells (i.e., when MDA-MB-231 and KB tumors reached ~160 or 80 mm3, respectively (i.e., to adjust for their different growth rates)). Four and 24 hours after CAR T cell infusion, all mice were injected intravenously with 500 nmol / kg fluorescein-folate, which was then repeated once per week. Figure 6B shows the percentage of CD3+ T cells (as a percentage of total cells in the tumor) on day 18, when tumors were excised and both cancer and stromal cells were released using a tumor dissociation kit prior to analysis by flow cytometry. Figure 6C shows the effect of anti-fluorescein CAR T cell therapy on the growth and immune characteristics of MDA-MB-231 and KB tumors.NOD SCID gamma (NSG) mice were implanted with 4 million MDA-MB-231 cells and 1 million KB cells in separate flanks of the same mouse and 2 weeks later, injected with either saline or 8x106 anti-fluorescein CAR T cells (i.e., when MDA-MB-231 and KB tumors reached ~160 or 80 mm3, respectively (i.e., to adjust for their different growth rates)). 4 and 24 hours after CAR T cell injection, all mice were injected intravenously with 500 nmol / kg fluorescein-folate, which was then repeated once / week. Figure 6C shows the exhaustion markers PD-1+ and TIM3+ (as a percentage of all human CD3+ T cells) on day 18, when tumors were excised and both cancer and stromal cells were released using a tumor dissociation kit prior to analysis by flow cytometry. [Figure 6-2]FIG. 6D shows the effect of anti-fluorescein CAR T cell therapy on the growth and immune characteristics of MDA-MB-231 and KB tumors. NOD SCID gamma (NSG) mice were implanted with 4 million MDA-MB-231 cells and 1 million KB cells in separate flanks of the same mouse, and 2 weeks later, either saline or 8×106 anti-fluorescein CAR T cells were injected (i.e., when MDA-MB-231 and KB tumors reached ∼160 or 80 mm3, respectively, to adjust for their different growth rates). 4 and 24 hours after CAR T cell injection, all mice were intravenously injected with 500 nmol / kg fluorescein-folate, which was then repeated once per week. Figure 6D shows the ratio of CD86+ F4 / 80+ CD11b+ to CD206+ F4 / 80+ CD11b+ myeloid cells on day 18 when tumors were excised and both cancer and stromal cells were released using a tumor dissociation kit before analysis by flow cytometry. n=5 mice / group. All data were analyzed by two-way analysis of variance (ANOVA) and plotted as mean±SEM (*p<0.05, **p<0.01, ***p<0.001). Data shown are representations of two independent experiments. Figure 6E shows the effect of anti-fluorescein CAR T cell therapy on the growth and immune characteristics of MDA-MB-231 and KB tumors. NOD SCID gamma (NSG) mice were implanted with 4 million MDA-MB-231 cells and 1 million KB cells in separate flanks of the same mouse and 2 weeks later, injected with either saline or 8x106 anti-fluorescein CAR T cells (i.e., when MDA-MB-231 and KB tumors reached ~160 or 80 mm3, respectively, to adjust for their different growth rates). 4 and 24 hours after CAR T cell injection, all mice were injected intravenously with 500 nmol / kg fluorescein-folate, which was then repeated once / week. Figure 6E shows representative images showing human CD3+ T cell infiltration into MDA-MB231 and KB cell solid tumors. Bars = 200 μm. [Figure 7]Figure 7 shows that fluorescein-conjugated fluorescent dye (fluorescein-NIR dye) is specifically targeted to anti-fluorescein CAR T cells in vivo. NSG mice were implanted with 1 million KB cells in one flank, and when the KB tumor volume reached ∼50 mm3, anti-fluorescein CAR T cells (8x106 cells containing ∼50% anti-fluorescein CAR T cells) were injected. 500 nmol / kg fluorescein-folate was injected both 4 and 24 hours later, and again once a week thereafter. 15 days after CAR T cell injection, mice were injected with 500 nmol / kg fluorescein-NIR dye via tail vein, and 4 hours later tumors were dissociated and analyzed by flow cytometry for fluorescein-NIR dye uptake. CD3+ T cells were detected by anti-human CD3 in the APC-Cy7 channel, and green fluorescent protein (GFP)-transfected anti-fluorescein CAR T cells were detected using the GFP channel. [Figure 8-1]Figures 8A-8F show the recovery of anti-fluorescein CAR T cell activity in vivo after intravenous injection of fluorescein-TLR7-1a conjugate. Figure 8A shows the scheme for the in vivo study. Mice were subcutaneously injected with 106 KB cells on day -7 and 8x106 anti-fluorescein CAR T cells on day 1. After 6 h, 24 h and 9 days, mice were intravenously injected with fluorescein-folate to induce binding of folate receptor positive KB cancer cells to CAR T cells. Mice were then intravenously injected with fluorescein-TLR7-1a on days 4-7 and 11-14. Tumor volume (Figure 8B) and animal weight change (Figure 8C) were measured every 3 days. Tumors were excised and dissociated into component cells on day 16, and human CD3+ T cells were determined as a percentage of all tumor cells in Figure 8D. PD-1+TIM3+ cells as a percentage of total human CD3+ T cells are shown in Figure 8E, and the ratio of mouse CD86+ cells to CD206+ cells, which also express myeloid markers F4 / 80 and CD11b, in the anti-fluorescein CAR T cell treated group is shown in Figure 8F. All data are plotted as mean ± SEM. Data shown are representative of at least two independent experiments. Data were analyzed by two-way ANOVA (**p<0.01; ns, not significant). [Figure 8-2] (As stated above.) [Figure 8-3] (As stated above.) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] While the present disclosure is susceptible to various modifications and alternative forms, illustrative embodiments thereof have been shown by way of example in the drawings and are herein described in detail.

[0059] The present disclosure is directed to the restoration of exhausted chimeric antigen receptor (CAR) T cells, which increases cell proliferation and restores impaired effector function, thereby making the restored CAR T cells useful for eradicating solid tumors.

[0060] CAR-T cells can become dysfunctional or "exhausted," or experience reduced proliferation when chronically exposed to tumor antigens or immune suppressors in the tumor microenvironment (e.g., myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), regulatory T cells (TRegs), and suppressive cytokines). To address these issues, in at least one embodiment, endocytosis of a recognition region (e.g., scFV fragment) that is part of the CAR is utilized to deliver an activity-restoring compound or conjugate, or a pharma- ceutically acceptable salt thereof, to the CAR-T cells.

[0061] The activity-restoring conjugate, or a pharma- ceutically acceptable salt thereof, comprises an active agent for CAR T cell activity restoration conjugated via a linker to a targeting moiety (e.g., selectively binds the CAR of the CAR T cell). The T cell activity-restoring conjugate, or a pharma- ceutically acceptable salt thereof, may be a compound, drug, or active agent formulated to restore activity of exhausted CAR-T cells. "CAR-T cell activity restoration" and variants thereof refer to activating CAR-T cells, increasing proliferation of CAR-T cells, blocking inhibitory signaling of exhausted or dysfunctional CAR-T cells, reactivating CAR-T cells by antigen-independent pathways, or increasing other functions of CAR-T cells. Embodiments of the conjugate may be particularly useful in preventing or reversing T cell exhaustion or dysfunction, reduced proliferation, and similar situations induced by the tumor microenvironment. In various embodiments, the activity restoring compound, or a pharma- ceutically acceptable salt thereof, is selected from the group consisting of Toll-like receptor (TLR) agonists (e.g., agonists of TLR7, TLR8, and / or TLR7 and TLR8 (TLR7 / 8)).

[0062] As described above, the T cell activity restoration conjugate further comprises a targeting moiety. The targeting moiety can selectively bind to the CAR of the CAR T cell. The terms "selectively binds", "binds with specificity", "binds with high affinity" or "specifically binds" when referring to a ligand / receptor, recognition region / targeting moiety, or other binding pair, refer to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologics. Thus, under specified conditions, a specified ligand or targeting moiety binds to a specific receptor (e.g., present on a cancer cell) or targeting moiety (e.g., present on a CAR recognition region), respectively, and does not bind in large amounts to other proteins present in a sample (e.g., associated with normal, healthy cells). Specific binding or binding with specificity or high affinity can also mean, for example, that a binding moiety or ligand binds to its target with an affinity that is often at least 25% greater, more often at least 50% greater, most often at least 100% (2-fold) greater, typically at least 10-fold greater, more usually at least 20-fold greater, and most usually at least 100-fold greater than the affinity for any other receptor.

[0063] In certain embodiments, the conjugates herein may be useful for applications when administered in combination with a "cancer-killing" compound. Figure 1A shows the cell-killing effect of CAR T cells in the presence of a "cancer-killing" conjugate ("cancer-killing" is used herein for ease of reference and is distinct from "activity-restoring") that binds CAR T cells to cancer cells. Figure 1B shows the activity-restoring of CAR T cells in the presence of a cancer-killing conjugate and an "activity-restoring" conjugate, including an activity-restoring compound that binds to a receptor of the CAR T cell, e.g., the fluorescein receptor.

[0064] 2A shows that upon administration of a fluorescein-folate conjugate (an embodiment of a "cancer-killing" conjugate) to a subject having a folate receptor-expressing tumor and anti-fluorescein CAR T cells (i.e., CAR T cells having an anti-fluorescein cell surface receptor), the "cancer-killing" conjugate can form a cross-link between the anti-fluorescein CAR T cells and the folate receptor-expressing cancer cells, which promotes cancer cell killing and CAR T cell proliferation. Although the fluorescein receptor is referred to in various embodiments and examples herein, it will be understood that other receptors on CAR T cells may be targeted and / or bound by the activity-restoring conjugates herein. FIG. 2B shows that upon administration of a fluorescein-TLR7a conjugate (an embodiment of an "activity restoration" conjugate) to a subject with a folate receptor-expressing tumor and exhausted anti-fluorescein CAR T cells, the "activity restoration" conjugate binds to the anti-fluorescein receptor on the surface of the CAR T cells and the activity restoration conjugate is endocytosed by the CAR T cells, thereby resulting in activity restoration of the CAR T cells.

[0065] In view of the above, a method is provided for restoring the activity of exhausted CAR T cells in a subject with cancer treated by CAR T cell therapy. The CAR T cell can be a cytotoxic lymphocyte, such as a cytotoxic T cell. In certain embodiments, the CAR can be used in conjunction with NK cells. In certain embodiments, the CAR can be used in conjunction with lymphokine-activated killer (LAK) cells. In certain embodiments, the CAR can be used in conjunction with a combination of NK cells, LAK cells, and / or T cells. It will be understood that a variety of engineered cell therapies are currently known in the art, and in certain embodiments, the CAR T cell therapy can include any currently known or below-discovered engineered cell or cell therapy that is useful for treating or preventing cancer and that benefits from use in conjunction with the CAR T cell activity restoration conjugates herein.

[0066] In certain embodiments, the engineered cells are NK cells prepared from progenitor or stem cells. In certain embodiments, the engineered cells are T cells prepared from progenitor or stem cells.

[0067] In at least one embodiment, T lymphocytes (e.g., cytotoxic T lymphocytes) are engineered to express a CAR. In at least one embodiment, NK cells are engineered to express a CAR.

[0068] CAR is a fusion protein that includes a recognition domain, a costimulatory domain, and an activation signaling domain. In certain embodiments, CAR binds to cell surface antigens of immunosuppressive cells or cancer cells with high specificity.

[0069] In certain embodiments, the recognition region of the CAR can be an scFv, Fab fragment, etc. of an antibody that binds with specificity (e.g., high specificity) to a cell surface antigen (e.g., cluster of differentiation 19 (CD19)). When the recognition region of the CAR comprises an scFv region, the scFv region can be prepared from (i) antibodies known in the art that bind to a targeting moiety, (ii) antibodies prepared de novo using at least one targeting moiety, such as a hapten, and (iii) sequence variants derived from the scFv regions of such antibodies, e.g., scFv regions that have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the scFv region from which they are derived.

[0070] "Percent (%) sequence identity" is defined in reference to a polypeptide sequence or a nucleotide sequence as the percentage of amino acid or nucleic acid residues, respectively, of a candidate sequence that are identical to the residues of a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not to consider any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent sequence identity can be achieved in a variety of ways within the art, for example, using publicly available computer software. For example, the determination of percent identity or similarity between sequences can be performed, for example, by using the GAP program (Genetic Computer Group, software; currently available through Accelrys online), and alignment can be performed, for example, using the ClustalW algorithm (VNTI software, InforMax Inc., Bethesda, MD). Furthermore, sequence databases can be searched using the nucleic acid or amino acid sequence of interest. The algorithm for database searching is typically based on the BLAST software, but one skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, the percent identity may be determined along the entire length of the nucleic acid or amino acid sequence.

[0071] In certain embodiments, the activation signaling domain generates a lymphocyte activation signal upon binding of the CAR to the targeting moiety. Suitable activation signaling domains may include, but are not limited to, T cell CD3 chain, CD3 delta receptor protein, mbl receptor protein, B29 receptor protein, Fc receptor gamma, 4-1BB domain, CD28 activation domain, or IL-15 / IL-2 domain. Those skilled in the art will understand that sequence variants of these activation signaling domains may be used if the variants have the same or similar activity as the model domain. In various embodiments, such variants have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with the amino acid sequence of the domain from which they are derived.

[0072] The construct encoding the CAR can be prepared using genetic engineering techniques. Such techniques are described in detail in Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3rd Edition, Cold Spring Harbor Laboratory Press, (2001), and Green and Sambrook, "Molecular Cloning: A Laboratory Manual," 4th Edition, Cold Spring Harbor Laboratory Press, (2012), both of which are incorporated herein by reference in their entirety (collectively, "Protocols").

[0073] By way of non-limiting example, a plasmid or viral expression vector (e.g., lentiviral vector, retroviral vector, sleeping beauty, and piggyback (a transposon / transposase system including a non-viral mediated CAR gene delivery system)) is prepared encoding a fusion protein comprising a recognition region, one or more costimulatory domains, and an activating signaling domain linked in-frame in a 5' to 3' direction.

[0074] Other configurations are also permissible, including a recognition region, an activating signaling domain, and one or more costimulatory domains.

[0075] The term "vector" refers to any nucleic acid that functions to carry, carry, or express a nucleic acid of interest. A nucleic acid vector may have a specific function, such as expression, packaging, pseudotyping, or transduction. A vector may also have an operational function when applied for use as a cloning or shuttle vector. The structure of a vector can include any desired form that can be made and is desirable for a particular use. Such forms can include, for example, circular forms, such as plasmids and phagemids, as well as linear or branched forms. For example, circular forms, such as plasmids and phagemids, as well as linear or branched forms. A nucleic acid vector may be composed of, for example, DNA or RNA, and may contain partially or completely nucleotide derivatives, analogs, or mimetics. Such vectors may be obtained from natural sources, or may be produced by recombinant or chemical synthesis.

[0076] The location of the recognition region of the fusion protein will usually be such that presentation of the region on the outside of the cell is achieved. If desired, the CAR may also contain additional elements, such as a signal peptide (e.g., CD8α signal peptide) that ensures correct transport of the fusion protein to the cell surface, a transmembrane domain (e.g., CD8α transmembrane domain, CD28 transmembrane domain, or CD3ζ transmembrane domain) that ensures that the fusion protein is maintained as an integral membrane protein, and a hinge domain (e.g., CD8α hinge) that provides flexibility to the recognition region and allows strong binding to the targeting moiety.

[0077] Cytotoxic lymphocytes (e.g., cytotoxic T lymphocytes or NK cells) can be engineered to express a CAR construct by transfecting a population of lymphocytes with an expression vector encoding the CAR construct. Suitable methods for preparing a transduced population of lymphocytes expressing a selected CAR construct are well known to those of skill in the art.

[0078] In one embodiment, the cells used in the methods described herein may be autologous cells, although xenogeneic cells may also be used, such as when the patient being treated has received high doses of chemotherapy or radiation treatments that destroy the patient's immune system. In one embodiment, allogeneic cells may be used.

[0079] In the context of the methods herein, CAR T cell therapy can include therapy in which a subject receives T cells expressing a CAR. In certain embodiments, CAR T cell therapy can be administered to a subject via a vector that includes a promoter operably linked to a nucleic acid sequence encoding a CAR. The CAR can bind to a first targeting moiety, a second targeting moiety, or both the first targeting moiety and the second targeting moiety. In certain embodiments, the CAR can bind to a first targeting moiety, a second targeting moiety, or both the first targeting moiety and the second targeting moiety with specificity.

[0080] CAR T cell therapy further includes a subject receiving a cancer-binding conjugate, comprising at least a ligand conjugated with a first targeting portion of a CAR. The ligand can bind to a receptor of a cancer cell (e.g., the ligand can bind to a cancer cell receptor with specificity). In certain embodiments, the ligand and the first targeting portion are conjugated via a first linker. As used herein, the term "ligand" refers to a molecule, ion, or atom that is attached to the central atom or ion (e.g., a drug) of a conjugate.

[0081] The method further comprises administering the CAR T cell activity restoring conjugate to a subject (e.g., having exhausted CAR T cells). The CAR T cell activity restoring conjugate may comprise an agonist of toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), or toll-like receptor 7 and toll-like receptor 8 (TLR7 / 8) conjugated to either a first targeting moiety or a second targeting moiety (e.g., such that the CAR can bind to the first targeting moiety and / or the second targeting moiety). In certain embodiments, the agonist and either the first targeting moiety or the second targeting moiety are optionally conjugated via a second linker.

[0082] The CAR T cell activity restoring conjugate has the structure:

[0083] [ka] [In formula: R 1 is acyclic alkyl or cyclic alkyl optionally substituted with one or more substituents independently selected from alkyl, halo, heteroalkyl, alkoxy, and cycloalkyl; R 2 -NR 2x R 2y , H, -OR z , -SO2N(R z )2, or N3 (wherein: R 2x and R 2y are each independently hydrogen, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or alkyl optionally substituted by one or more substituents independently selected from oxo, halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; Each R Z is independently hydrogen or optionally substituted alkyl; or R 2x and R 2y taken together form an optionally substituted 3-10 membered mono- or bicyclic heterocycloalkyl; Each R 3 are independently hydrogen, halo, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, or alkoxy is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halo, or cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally substituted; X 1 , X 2 , and X 3 are independently N or CR q (In the formula, each R q are independently hydrogen (H), halo, or optionally substituted alkyl; Z is GL-, where L is a linker and G is a first targeting moiety or a second targeting moiety (e.g., to which CAR binds with specificity); In formula (I), n is 1 to 6; m in formula (I) is 0 to 4. or a pharma- ceutically acceptable salt thereof.

[0084] In certain embodiments, the CAR T cell activity restoring conjugate has the structure:

[0085] [ka] [In formula: R 1 , R 3 , R 4 , and R 5 each independently represents H, alkyl, alkoxy, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR 2x ,

[0086] [ka] (In the formula, R 2x and R 2y each of is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; Z is GL-, where L is a linker and G is a first targeting moiety or a second targeting moiety (e.g., capable of binding to CAR with specificity); X 1 , X 2 , and X 3 is an independent CR q or N (wherein each R q are independently H, halo, or optionally substituted alkyl; Y is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -COR z , -COOR z, -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z )2(in the formula: R 2x and R 2y are independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl (e.g., optionally substituted with one or more substituents, each of which is independently oxo, halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl); Each R z is independently H or optionally substituted alkyl; or R 2x and R 2y are taken together to form an optionally substituted heterocycloalkyl (e.g., the optionally substituted heterocycloalkyl is a monocyclic or bicyclic heterocycloalkyl and / or the optionally substituted heterocycloalkyl is a 3-10 membered heterocycloalkyl); In formula (II), n is 0 to 30. or a pharma- ceutically acceptable salt thereof.

[0087] In certain embodiments, the CAR T cell activity restoring conjugate has the structure:

[0088] [ka] [In the formula, R 1 is acyclic alkyl or cyclic alkyl optionally substituted with one or more substituents independently selected from halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; Y is H, -OR z , -NR2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z )2(in the formula: R 2x and R 2y are independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or alkyl optionally substituted by one or more substituents independently selected from oxo, halo, alkyl, heteroalkyl, alkoxy, or cycloalkyl; Each R z is independently hydrogen or optionally substituted alkyl; or R 2x and R 2y taken together form an optionally substituted 3-10 membered mono- or bicyclic heterocycloalkyl; Each R 3 is independently halo, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halogen, or cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally substituted; X 1 , X 2 , and X 3 is an independent CRq or N (wherein each R q are independently H, halogen, or optionally substituted alkyl; Z is LG-, where L is a linker and G is a first targeting moiety or a second targeting moiety; In formula (III), n is 0 to 30; m in formula (III) is 0 to 4. or a pharma- ceutically acceptable salt thereof.

[0089] In certain embodiments, CAR T cells (e.g., exhausted CAR T cells) in a subject bind and endocytose the CAR T cell activity-restoring conjugate, and the CAR T cells are restored to activity by the agonist. In various embodiments, the X of Formula (I), Formula (II), and / or Formula (III) 1 , X 2 , and X 3 Each of may be nitrogen (N).

[0090] The CAR T cell activity restoring conjugate has the structure:

[0091] [ka] or a pharma- ceutically acceptable salt thereof. Alternatively, the CAR T cell activity restoring conjugate may have the structure:

[0092] [ka] or a pharma- ceutically acceptable salt of any of the foregoing structures. Also, or alternatively, the CAR T cell activity restoring conjugate may have the structure:

[0093] [ka] or a pharma- ceutically acceptable salt thereof.

[0094] The first targeting moiety, the second targeting moiety, or the first targeting moiety and the second targeting moiety have the structure:

[0095] [ka] or a pharma- ceutically acceptable salt thereof.

[0096] Alternatively, the first targeting moiety, the second targeting moiety, or the first targeting moiety and the second targeting moiety have the structure:

[0097] [ka] or a pharma- ceutically acceptable salt thereof.

[0098] The CAR T cell activity restoring conjugate has the structure:

[0099] [ka] or a pharma- ceutically acceptable salt thereof. Alternatively, the CAR T cell activity restoring conjugate may have the formula:

[0100] [ka] [In the formula, n = 0 to 200] or a pharma- ceutically acceptable salt of any of the preceding structures. In yet another alternative, the CAR T cell activity restoring conjugate may have a structure selected from:

[0101] [ka] or a pharma- ceutically acceptable salt thereof. Even further alternatively, the CAR T cell activity restoring conjugate may have a structure selected from:

[0102] [ka] or a pharma- ceutically acceptable salt of any of the preceding structures. Other structures of the CAR T cell activity restoring conjugates include:

[0103] [ka] or a pharma- ceutically acceptable salt thereof. Yet another structure of the CAR T cell activity restoring conjugate is:

[0104] [ka] or a pharma- ceutically acceptable salt of any of the preceding structures.

[0105] In certain embodiments, the CAR T cell activity restoring conjugate comprises:

[0106] [ka] or a pharma- ceutically acceptable salt of any of the preceding structures.

[0107] In some embodiments, as described above, the CAR T cell therapy comprises a cancer-binding conjugate comprising a ligand and a first targeting moiety, wherein the ligand and the first targeting moiety are optionally conjugated via a first linker.

[0108] The ligand may be bound by (or has affinity for or specificity to bind to) a cancer (e.g., a cancer cell). The ligand bound by (or has affinity for or specificity to bind to) a cancer cell may be selected from the group consisting of folate, 5-methyltetrahydrofolate, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) ligand, neurokinin 1 receptor (NK-1R) ligand, carbonic anhydrase IX (CAIX) ligand, gamma glutamyl transpeptidase ligand, luteinizing hormone releasing hormone (LHRHR) ligand, CD73 ligand, fibroblast activation protein ligand, heat shock protein (HSP) ligand, glucose transporter 1 (glut-1) ligand, natural killer group 2D receptor (NKG2D) ligand, and cholecystokinin B receptor (CCKBR or CCK2) ligand.

[0109] "Folate" can be, for example, folic acid, a folic acid analog, or another folate receptor binding molecule, including, but not limited to, analogs and derivatives of folic acid, such as folic acid (e.g., leucovorin), pteroylpolyglutamic acid, pteroyl-D-glutamic acid, and folate receptor binding pterdines, such as tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid (e.g., 5-methyltetrahydrofolic acid (5-MTHF)), and their deaza and dideaza analogs.

[0110] An "analog" or "derivative" with respect to a peptide, polypeptide, or protein, refers to another peptide, polypeptide, or protein that has a similar or identical function as the original peptide, polypeptide, or protein, but does not necessarily contain a similar or identical amino acid sequence or structure of the original peptide, polypeptide, or protein. An analogue preferably satisfies at least one of the following: (a) a proteinaceous agent having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the original amino acid sequence; (b) a proteinaceous agent encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding the original amino acid sequence; or (c) a proteinaceous agent encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to a nucleotide sequence encoding the original amino acid sequence.

[0111] The terms "deaza" and "dideaza" analogs refer to art-recognized analogs that have a carbon atom replacing one or two nitrogen atoms of naturally occurring folic acid structure, or its analog or derivative.For example, deaza analogs can include 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs of folic acid, folinic acid, pteropolyglutamic acid, and folate receptor-binding pteridines such as tetrahydropterin, dihydrofolic acid, and tetrahydrofolic acid.Dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs.The aforementioned folic acid analogs are conventionally called "folates" to reflect their ability to bind to folate receptors. Other folate receptor binding analogs include aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-deaminohydroxyfolate, deaza analogs such as 1-deazamethopterin or 3-deazamethopterin, and 3',5'-dichloro-4-amino-4-deoxy-N 10 -Contains methylpteroylglutamic acid (dichloromethotrexate).

[0112] The foregoing analogs and / or derivatives are also referred to as "a folate," "the folate," or "folates," reflecting their ability to bind to the folate receptor. Such molecules, when conjugated to an exogenous molecule, may be effective in enhancing transmembrane transport, such as via folate-mediated endocytosis. The foregoing may be used in the folate receptor binding ligands described herein.

[0113] The first targeting moiety and the second targeting moiety (e.g., having affinity for binding to a CAR) can be independently selected from the group consisting of 2,4-dinitrophenyl (DNP), L-rhamnose, tacrolimus (FK506), 2,4,6-trinitrophenol (TNP), biotin, rapamycin, digoxigenin, folate, 5-methyltetrahydrofolate, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centirin, DARPin, affibody, affilin, anticalin, atrimer, avimer, bicyclic peptide, FN3 scaffold, cys-knot, finomer, Kunitz domain, or Obody.

[0114] The first linker and the second linker, if applicable, may be independently releasable or non-releasable. The first linker and the second linker may be any of C1 to C6 20 Alkyl, alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, -SS, -CO-O-(CH2) n -SS-(n=2-6), -O-CO-O-(CH2) n -SS-(n=2-6), -S-CO-O-(CH2) n -SS-(n=2-6), -NH-CO-O-(CH2) n -SS- (n = 2 to 6), carboxylates, carbonates, carbamates, ureas, thioureas, polyethylene glycols (PEG) (e.g., PEG n , n=1-200), polyproline, oligo-(4-piperidine)carboxylic acid, oligopiperidine, amino acid (e.g., hydrophilic amino acid), peptide, saccharopeptide, sugar, peptidoglycan (e.g., non-natural peptidoglycan), polyvinylpyrrolidone, Pluronic F-127, or any combination of two or more of the foregoing.

[0115] The first linker, the second linker, or both the first and second linkers can comprise PEG.

[0116] As shown above, the various substituents of the various formulas are "optionally substituted". As used herein, the term "substituted" means that any one or more hydrogens of the specified atom or group are replaced by a selection from the indicated group of the substituent, as long as the normal valence of the specified atom is not exceeded and the replacement results in a stable compound. If the replacement is oxo (keto, i.e., =0), two hydrogens of the atom are replaced. The present disclosure includes all isotopes (including radioisotopes) of atoms occurring in the present conjugates.

[0117] When the conjugates are further substituted, they may be substituted at one or more available positions, typically from one to three or four positions, with one or more suitable groups such as those disclosed herein. Suitable groups that may be present in the "substituted" groups include, for example, but are not limited to, halogen; cyano; hydroxyl; nitro; azide; alkanoyl (such as C1-6 alkanoyl groups such as acyl); carboxamide; alkyl groups (including cycloalkyl groups having 1 to about 8 carbon atoms); alkenyl and alkynyl groups (including groups having one or more unsaturated bonds and 2 to about 8 carbon atoms); alkoxy groups having one or more oxygen linkages and 1 to about 8 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those having one or more thioether linkages and 1 to about 8 carbon atoms; alkylsulfinyl groups including those having one or more sulfonyl linkages and 1 to about 8 carbon atoms; alkylsulfonyl groups including those having one or more sulfonyl linkages and 1 to about 8 carbon atoms; aminoalkyl groups including those having one or more N atoms and groups having 1 to about 8 carbon atoms.

[0118] As used herein, "alkyl" includes both branched and straight chain aliphatic saturated hydrocarbon groups having the specified number of carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, and s-pentyl. Preferred alkyl groups are C1-6 alkyl groups. Particularly preferred alkyl groups may be methyl, ethyl, propyl, butyl, and 3-pentyl.

[0119] In general, the term "acyl" or "acyl substituent" refers to those resulting from removal of one or more hydroxyl groups from an oxoacid, including inorganic acids, which contains a double-bonded oxygen atom and an alkyl group.

[0120] The term "alkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, such as, but not limited to, -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms.

[0121] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable linear or branched chain, or combination or combinations thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH2=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3 and -CN. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3.

[0122] Similarly, the term "heteroalkylene," by itself or as part of another substituent, means (unless otherwise stated) a divalent radical derived from a heteroalkyl, such as, but not limited to, -CH-CH-S-CH-CH and -CH-S-CH-CH-NH-CH. For heteroalkylene groups, heteroatoms can occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-. As noted above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule by a heteroatom, e.g., -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or SOR'. When "heteroalkyl" is described, a description of the specific heteroalkyl group, e.g., -NR'R'', etc., follows, and it will be understood that the terms heteroalkyl and -NR'R'' are not overlapping or mutually exclusive. Rather, the specific heteroalkyl group is described to add clarity. Thus, the term "heteroalkyl" should not be construed herein to exclude specific heteroalkyl groups, such as -NR'R'', etc.

[0123] In view of the above, a CAR T cell activity restoring conjugate is provided. The activity restoring conjugate may include a targeting moiety that binds to the CAR of a CAR T cell with specificity and an agonist of TLR7, TLR8, or TLR7 / 8 conjugated via a linker (e.g., a second linker), and the CAR T cell activity restoring conjugate has the structure:

[0124] [ka] [In formula: R 1is acyclic alkyl or cyclic alkyl optionally substituted with one or more substituents independently selected from alkyl, halo, heteroalkyl, alkoxy, and cycloalkyl; R 2 -NR 2x R 2y , H, -OR z , -SO2N(R z )2, or N3 (wherein: R 2x and R 2y are each independently hydrogen, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or alkyl optionally substituted by one or more substituents independently selected from oxo, halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; Each R Z is independently H or optionally substituted alkyl; or R 2x and R 2y taken together form an optionally substituted 3-10 membered mono- or bicyclic heterocycloalkyl; Each R 3 are independently hydrogen, halo, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, or alkoxy is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halo, or cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally substituted; X 1 , X 2 , and X 3 are independently N or CR q(In the formula, each R q are independently H, halo, or optionally substituted alkyl; Z is GL-, where L is a linker (e.g., a second linker) and G is a targeting moiety (e.g., a first targeting moiety, a second targeting moiety, or both a first targeting moiety and a second targeting moiety); n is 1 to 6; m is 0 to 4. or a pharma- ceutically acceptable salt thereof.

[0125] In certain embodiments, the activity restoring conjugate comprises a targeting moiety that binds (e.g., with specificity) to the CAR of a CAR T cell and an agonist of TLR7, TLR8, or TLR7 / 8 conjugated via a linker (e.g., a second linker), and the CAR T cell activity restoring conjugate has the structure:

[0126] [ka] [In formula: R 1 , R 3 , R 4 , and R 5 each independently represents hydrogen, alkyl, alkoxy, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR 2x ,

[0127] [ka] (In the formula, R 2x and R 2y each of is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; Z is GL-, where L is a linker (e.g., a second linker) and G is a targeting moiety (e.g., a first and / or second targeting moiety); X 1 , X 2 , and X 3 is an independent CR q or N (wherein each R q are independently hydrogen, halo, or optionally substituted alkyl; Y is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z )2(in the formula: R 2x and R 2y are independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl (e.g., optionally substituted with one or more substituents, each of which is independently oxo, halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl); Each R z is independently hydrogen or optionally substituted alkyl; or R 2x and R 2y are taken together to form an optionally substituted heterocycloalkyl (e.g., the optionally substituted heterocycloalkyl is a monocyclic or bicyclic heterocycloalkyl and / or the optionally substituted heterocycloalkyl is a 3-10 membered heterocycloalkyl); In formula (II), n is 0 to 30. or a pharma- ceutically acceptable salt thereof.

[0128] In certain embodiments, the activity restoring conjugate comprises a targeting moiety that binds (e.g., with specificity) to the CAR of a CAR T cell and an agonist of TLR7, TLR8, or TLR7 / 8 conjugated via a linker (e.g., a second linker), and the CAR T cell activity restoring conjugate has the structure:

[0129] [ka] [In the formula, R 1 is acyclic alkyl or cyclic alkyl optionally substituted with one or more substituents independently selected from halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; Y is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -COR z , -COOR z , -CONR z 2. -COSR z , -SO2N(R z )2, or -CON(R z )2(in the formula: Each R Z are independently H or optionally substituted alkyl; R 2x and R 2y are independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or alkyl optionally substituted by one or more substituents independently selected from oxo, halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; or R 2x and R 2y taken together form an optionally substituted 3-10 membered mono- or bicyclic heterocycloalkyl; Each R 3 is independently halo, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halogen, or cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally substituted; X 1 , X 2 , and X 3 is an independent CR q or N (wherein each R q are independently H, halogen, or optionally substituted alkyl; Z is LG, where L is a linker (e.g., a second linker) and G is a targeting moiety (e.g., a first and / or second targeting moiety); n is 0 to 30; m is 0 to 4. or a pharma- ceutically acceptable salt thereof.

[0130] G (the targeting moiety) of formula (I), formula (II) or formula (III) has the structure:

[0131] [ka] or a group having a pharma- ceutically acceptable salt of any of the preceding structures. X in formula (I), (II), and / or (III) may be or may contain a group. 1 , X 2 , and X 3Each of may be nitrogen (N).

[0132] The CAR T cell activity restoring conjugate has the structure:

[0133] [ka] or a pharma- ceutically acceptable salt thereof.

[0134] Alternatively, the CAR T cell activity restoring conjugate comprises:

[0135] [ka] [In the formula, n=0~200] or a pharma- ceutically acceptable salt of any of the preceding structures. In yet another alternative, the CAR T cell activity restoring conjugate may have a structure selected from:

[0136] [ka] or a pharma- ceutically acceptable salt of any of the preceding structures. In certain embodiments, the CAR T cell activity restoring conjugate is

[0137] [ka] or a pharma- ceutically acceptable salt of any of the preceding structures. Other structures of the CAR T cell activity restoring conjugates include:

[0138] [ka] or a pharma- ceutically acceptable salt of any of the foregoing. In certain embodiments, the CAR T cell activity restoring conjugate comprises the following:

[0139] [ka] or may be a pharma- ceutically acceptable salt of any of the preceding structures.

[0140] In certain embodiments, the CAR T cell activity restoring conjugate comprises:

[0141] [ka] or a pharma- ceutically acceptable salt of any of the foregoing structures.

[0142] As described above, the CAR T cell activity restoration conjugate may include at least one targeting moiety (e.g., targeting the CAR). With respect to formulas (I)-(III), the targeting moiety may be selected from the group consisting of DNP, L-rhamnose, FK506, TNP, biotin, rapamycin, digoxigenin, folate, 5-methyltetrahydrofolate, fluorescein, FITC, NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centirin, and DARPin.

[0143] Further embodiments may include a linker disposed between the targeting moiety and the active agent (i.e., the TLR7, TLR8, or TLR7 / 8 agonist). With respect to formulae (I)-(III), the term "linker" includes a chain of atoms that is biofunctionally adapted to form a chemical bond with the TLR7, TLR8, or TLR7 / 8 agonist conjugated to either the first targeting moiety or the second targeting moiety, and may connect two or more parts of a molecule to form a compound. Illustratively, the chain of atoms may be selected from carbon (C), nitrogen (N), oxygen (O), sulfur (S), silicon (Si), and phosphorus (S), or C, N, O, S, and P, C, N, O, and S. The chain of atoms may covalently connect different functional capabilities, such as small molecule ligands and targeting moieties of a conjugate. The linker (e.g., the first or second linker) may include a wide range of links, such as from about 2 to about 2000 atoms of contiguous backbone, and may include releasable or non-releasable linkers. In certain embodiments, the linker may be optional, if desired.

[0144] The linker is C1 to C 20 Alkyl, alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, -SS, -CO-O-(CH2) n -SS-(n=2-6), -O-CO-O-(CH2) n -SS-(n=2-6), -S-CO-O-(CH2) n -SS-(n=2-6), -NH-CO-O-(CH2) n -SS- (n = 2 to 6), carboxylates, carbonates, carbamates, ureas, thioureas, PEG (e.g., PEG n , n=1-200), polyproline, oligo-(4-piperidine)carboxylic acid, oligopiperidine, amino acid (e.g., hydrophilic amino acid), peptide, saccharopeptide, sugar, peptidoglycan (e.g., non-natural peptidoglycan), polyvinylpyrrolidone, Pluronic F-127, or any combination of two or more of the foregoing.

[0145] The linker may comprise PEG. Illustratively, the linker has the formula:

[0146] [ka] [In the formula, n is an integer of 0 to 200] In another embodiment, n may include a structure having the formula: 1, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 15-31, 15-32, 15-33, 15-34, 15-35, 15-36, 15-37 , 15-38, 15-39, 15-40, 15-50, 15-60, 15-70, 15-80, 15-90, 15-100, 15-110, 15-120, 15-130, 15-140, 15-150, or n can be an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 It can be 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 108, 110, 120, 130, 140, or 150.

[0147] The linker may be a direct bond (e.g., reaction between an isothiocyanate group of FITC and a free amine group of the ligand) or the bond may be via an intermediate linker. In one embodiment, the intermediate linker, if present, may be any biocompatible linker known in the art, such as a bivalent linker. In one exemplary embodiment, the linker may be bivalent and may contain about 1 to about 30 carbon atoms (e.g., 1 to 30 carbon atoms). In another exemplary embodiment, the bivalent linker may contain about 2 to about 20 carbon atoms (e.g., 2 to 20 carbon atoms). In other embodiments, low molecular weight bivalent linkers (i.e., those having an approximate molecular weight of about 30 Daltons to about 300 Daltons) are used. In another embodiment, suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more atoms.

[0148] Examples of bivalent linkers are shown in Table 1. * ) indicates the point of attachment to a ligand, targeting moiety, or activity-restoring compound (e.g., a TLR7 / 8 agonist).

[0149] [Table 1-1]

[0150] [Table 1-2]

[0151] Alternative structures for the linker moiety are:

[0152] [ka] [In the formula, n is an integer of 0 to 200] may include.

[0153] A linker can be releasable or non-releasable. The term "releasable" in the context of a linker refers to a linker that includes at least one bond that can be broken (e.g., chemically or enzymatically hydrolyzed) under physiological conditions, such as, for example, by drug labile, pH labile, acid labile, base labile, oxidatively labile, metabolically labile, biochemically labile, enzymatically labile, or by reduction of a p-aminobenzyl-based polyvalent releasable bond. It is recognized that physiological conditions that result in bond breaking do not necessarily include biological or metabolic processes, but instead can include standard chemical reactions, such as hydrolysis reactions at physiological pH or as a result of compartmentalization into a cellular organelle such as an endosome that has a pH lower than the cytoplasmic pH. The cleavable bond can connect two adjacent atoms in a releasable linker, and / or can connect other linker moieties or targeting moieties and / or active components (e.g., TLR7, TLR8, or TLR7 / 8 agonists), for example, at either or both ends of the releasable linker, as described herein. In some cases, the releasable linker is broken into two or more fragments. In some cases, the releasable linker is separated from the targeting moiety. In certain embodiments, the CAR T cell activity restoring conjugate comprising a releasable linker, when administered, causes the targeting moiety and the agonist to be released from each other around the time the conjugate enters the CAR expressing cell.

[0154] Conversely, the term "non-releasable" in the context of a linker refers to a linker that includes at least one bond that is not easily or readily broken under physiological conditions. In some embodiments, a non-releasable linker includes a backbone that is stable under physiological conditions (e.g., the backbone is not susceptible to hydrolysis (aqueous or enzymatic hydrolysis)). In some embodiments, the TLR7, TLR8, or TLR7 / 8 agonist of a CAR T cell activity restoration conjugate that includes a non-releasable linker is not released from the targeting moiety. In some embodiments, a non-releasable linker lacks a backbone disulfide bond (e.g., SS) or ester. In some embodiments, a conjugate includes a targeting moiety and an active ingredient (e.g., an agonist) connected by a backbone that is substantially stable throughout the circulation of the conjugate. A non-releasable linker may include: an amide, an ester, an ether, an amine, and / or a thioether (e.g., thio-maleimide). Although specific examples are provided herein, it will be understood that any molecule may be used in a non-releasable linker so long as it forms at least one bond that is not easily or quickly broken under physiological conditions.

[0155] Both releasable and non-releasable linkers may be engineered to optimize the biodistribution, bioavailability, and PK / PD (e.g., of the respective conjugate and / or its active ingredient) and / or to increase uptake (e.g., of the respective conjugate and / or its active ingredient) into targeted cells according to methods generally known in the art or developed below, such as by PEGylation.

[0156] The conjugate, and its pharma-ceutically acceptable salts, can be prepared by conventional methods of organic synthesis carried out by those skilled in the art. The description of the conjugate is limited by the principles of chemical bonding known to those skilled in the art. Thus, the group may be substituted by one or more number of substituents, and such substitutions are selected to provide a compound known to those skilled in the art that complies with the principles of chemical bonding and is not inherently unstable and / or is likely to be unstable under ambient conditions, such as aqueous, neutral, and some known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the rest of the molecule via a ring heteroatom according to the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0157] The conjugates, and their pharmaceutically acceptable salts, may exist as stereoisomers. Thus, various embodiments may include pure stereoisomers or mixtures of stereoisomers of the conjugates. The conjugates, and their pharmaceutically acceptable salts, may also exist in unsolvated and solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are included within the scope of the present disclosure.

[0158] The term "pharmaceutical acceptable salts" refers to those salts whose counterions can be used in pharmacy. In various embodiments, such salts include, but are not limited to, 1) acid addition salts that can be obtained by reacting the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, or malonic acid; or 2) salts formed when an acidic proton present in the parent compound is either replaced by a metal ion, such as an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucamine, and the like. Pharmaceutically acceptable salts are well known to those skilled in the art, and any such pharmaceutical acceptable salts are discussed in connection with the embodiments described herein.

[0159] In various embodiments, suitable acid addition salts are formed from acids that form non-toxic salts. Illustrative examples include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate, and trifluoroacetate.

[0160] In various embodiments, suitable base addition salts are formed from bases that form non-toxic salts. Illustrative examples include arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, such as hemisulfate and hemicalcium salts.

[0161] In view of the above, a pharmaceutical composition is further provided.In certain embodiments, the pharmaceutical composition comprises a CAR T cell activity restoring conjugate and a pharma- ceutical acceptable carrier.

[0162] As used herein, the term "composition" refers to any product that contains more than one component, typically including the conjugates described herein. The composition may be prepared from an isolated compound (i.e., a conjugate; "compound" and "conjugate" are used interchangeably herein) or from salts, solutions, hydrates, solvates, and other forms of the CAR T cell activity restoration conjugate. Certain functional groups, such as hydroxy, amino, and such groups, may form complexes with water and / or various solvents in various physical forms of the compound. It will be understood that in certain circumstances, the compound (and compositions that include the compound) may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and / or other morphological forms of the compound, and the composition may be prepared from various hydrates and / or solvates of the compound. Thus, a pharmaceutical composition that represents a conjugate includes each of the various morphological forms and / or solvates or hydrate forms of the compound, or any combination thereof, or individual forms thereof.

[0163] The compounds and compositions may be administered in unit dosage forms and / or compositions containing one or more pharma- ceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles, as well as combinations thereof. The term "administering" and its formulations generally refers to any and all means of introducing the compounds and compositions described herein into a cell, tissue, organ, or biological fluid of a subject.

[0164] As used herein, a "subject" is a mammal, preferably a human, but may be a non-human animal (including, but not limited to, laboratory, agricultural, domestic, or wild animals). Thus, the methods, compounds, and compositions are applicable to both human and veterinary diseases and applications. In various aspects, the subject may be a laboratory animal, such as a rodent (e.g., mouse, rat, hamster, etc.), rabbit, monkey, chimpanzee, domestic animal, such as dog, cat, or rabbit, agricultural animal, such as cow, horse, pig, sheep, or goat, or captive wild animal, such as bear, panda, lion, tiger, leopard, elephant, zebra, giraffe, gorilla, dolphin, or whale. In certain embodiments, the subject is a "patient", i.e., a living human or animal undergoing medical treatment for a disease or condition, including a human or animal with no defined disease being evaluated for symptoms of pathology. In certain embodiments, subjects that may be treated using the methods herein include subjects identified or selected as having cancer or at risk of having cancer. Such identification and / or selection may be made by clinical or diagnostic evaluation.

[0165] The compound can be formulated as a pharmaceutical composition and / or administered to a subject, for example, a human patient, in various forms adapted to the selected route of administration.In fact, any suitable method of administration known in the art can be used.In one aspect, the compound, or its pharmaceutically acceptable salt, or the pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier can be administered in a unit dosage form and / or formulation that contains conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0166] In addition, the compound or its pharmaceutically acceptable salt can be administered directly into the bloodstream, into muscle, or into internal organs.In various embodiments, suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​substernal, intracranial, intratumoral, intramuscular, and subcutaneous delivery.In one embodiment, means for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.The compounds and compositions can be similarly formulated for the desired mode of administration.

[0167] For example, parenteral formulations are typically aqueous solutions, which may contain carriers or excipients such as salts, carbohydrates and buffers (preferably at a pH of 3 to 9), but may also be formulated as sterile non-aqueous solutions, used in combination with a suitable vehicle such as sterile, pyrogen-free water or sterile saline, or as dry forms, if appropriate. In other embodiments, any of the liquid formulations described herein may be applied for parenteral administration. Preparation under sterile conditions by lyophilization to produce a sterile lyophilized powder for parenteral formulations can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art. The solubility of the compounds, or pharma-ceutically acceptable salts thereof, used in the preparation of parenteral formulations may be increased by the use of suitable formulation techniques, such as the incorporation of solubility enhancers.

[0168] Pharmaceutical dosage forms of the compound suitable for injection or infusion can include sterile aqueous solutions, dispersions or powders containing the active ingredient that are applied for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, and can be optionally encapsulated in liposomes. In all cases, the final dosage form should be sterile, liquid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be, for example, a solvent or liquid dispersion medium, including but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid PEG, etc.), vegetable oils, non-toxic glyceryl esters, and / or suitable mixtures thereof. In at least one embodiment, the proper fluidity can be maintained by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The action of microorganisms can be prevented by the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In certain cases, it may be desirable to include one or more isotonic agents, such as sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents formulated to delay absorption, for example, aluminum monostearate and gelatin.

[0169] Sterile injectable solution can be prepared by incorporating active ingredient in the required amount of suitable solvent containing one or more other ingredients mentioned above, and then sterilizing by filtration if necessary.In the case of sterile powder for preparing sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces powder of active ingredient and any additional desired ingredients present in the solution previously sterile filtered.

[0170] Useful dosages of compounds can be determined by comparing their in vitro and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice and other animals to human subjects are known in the art. In practice, dosages of compounds can vary significantly depending on the condition of the subject, the type of cancer being treated, how advanced the pathology is, the route of administration and tissue distribution of the compound, and the possibility of co-utilization of other therapeutic treatments (e.g., radiation therapy or additional drugs in combination therapy). The amount of composition and / or compound required for use in treatment (e.g., therapeutically or prophylactically effective amount or dose) will vary not only depending on the specific application, but also on the selected salt (if applicable) and the characteristics of the subject (e.g., age, condition, sex, subject's body surface area and / or weight, tolerance to drugs), and will ultimately be at the discretion of the attending physician, clinician, or other. A "therapeutically effective amount" or a "prophylactically effective amount" is defined as the amount of a reagent or pharmaceutical composition that is sufficient to induce the desired response.

[0171] The amount of the compound or its pharmaceutically acceptable salt administered to a subject may vary significantly depending on the cancer to be treated, the route of administration of the compound or its pharmaceutically acceptable salt, and tissue distribution. The amount administered to a subject may be based on body surface area, body weight, and physician assessment.

[0172] In various embodiments, the amount administered may be, for example, about 0.05 mg to about 30 mg, about 0.05 mg to about 25.0 mg, about 0.05 mg to about 20.0 mg, about 0.05 mg to about 15.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 9.0 mg, about 0.05 mg to about 8.0 mg, about 0.05 mg to about 7.0 mg, about 0.05 mg to about 6.0 mg, about 0.05 mg to about 5.0 mg, about 0.05 mg to about 4. The dosage may range from about 0 mg, about 0.05 mg to about 3.0 mg, about 0.05 mg to about 2.0 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.4 mg, about 0.05 mg to about 0.3 mg, about 0.05 mg to about 0.2 mg, about 0.05 mg to about 0.1 mg, about 0.1 mg to about 2 mg, about 0.3 mg to about 10 mg, about 0.1 mg to about 20 mg, or about 0.8 mg to about 3 mg. One of ordinary skill in the art will readily recognize that dosages may vary within the various ranges provided above based on the factors listed above and at the physician's discretion.

[0173] In other embodiments, the dose of the compound, or a pharma- ceutically acceptable salt thereof, is, for example, about 50 nmole / kg to about 3,000 nmole / kg of the subject's body weight, about 50 nmole / kg to about 2,800 nmole / kg of the subject's body weight, about 50 nmole / kg to about 2,600 nmole / kg, about 50 nmole / kg to about 2,400 nmole / kg, about 50 nmole / kg to about 2,200 nmole / kg, about 50 nmole / kg to about 2,100 nmole / kg About 50 nmole / kg to about 2,000 nmole / kg, about 50 nmole / kg to about 1,000 nmole / kg, about 50 nmole / kg to about 900 nmole / kg, about 50 nmole / kg to about 800 nmole / kg, about 50 nmole / kg to about 700 nmole / kg, about 50 nmole / kg to about 600 nmole / kg, about 50 nmole / kg to about 500 nmole / kg, about 50 nmole / kg to about 400 nmole / kg 50 nmole / kg, about 50 nmole / kg to about 300 nmole / kg, about 50 nmole / kg to about 200 nmole / kg, about 50 nmole / kg to about 100 nmole / kg, about 100 nmole / kg to about 300 nmole / kg, about 100 nmole / kg to about 500 nmole / kg, about 100 nmole / kg to about 1,000 nmole / kg, or about 100 nmole / kg to about 2,000 nmole / kg.In other embodiments, the dose is about 1 nmole / kg, about 5 nmole / kg, about 10 nmole / kg, about 20 nmole / kg, about 25 nmole / kg, about 30 nmole / kg, about 40 nmole / kg, about 50 nmole / kg, about 60 nmole / kg, about 70 nmole / kg, 80 nmole / kg, about 90 nmole / kg, about 100 nmole / kg, about 150 nmole / kg, about 200 nmole / kg of the subject's body weight. , about 250 nmole / kg, about 300 nmole / kg, about 350 nmole / kg, about 400 nmole / kg, about 450 nmole / kg, about 500 nmole / kg, about 600 nmole / kg, about 700 nmole / kg, about 800 nmole / kg, about 900 nmole / kg, about 1,000 nmole / kg, about 2,000 nmole / kg, about 2,500 nmole / kg or about 3,000 nmole / kg. In yet other embodiments, the dose is about 0.1 nmole / kg, about 0.2 nmole / kg, about 0.3 nmole / kg, about 0.4 nmole / kg, or about 0.5 nmole / kg, about 0.1 nmole / kg to about 1,000 nmole / kg, about 0.1 nmole / kg to about 900 nmole / kg, about 0.1 nmole / kg to about 850 nmole / kg, about 0.1 nmole / kg to about 800 nmole / kg, about 0.1 nmole / kg to about 700 nmole / kg, about 0.1 nmole / kg to about 1,0 ... e / kg to about 600 nmole / kg, about 0.1 nmole / kg to about 500 nmole / kg, about 0.1 nmole / kg to about 400 nmole / kg, about 0.1 nmole / kg to about 300 nmole / kg, about 0.1 nmole / kg to about 200 nmole / kg, about 0.1 nmole / kg to about 100 nmole / kg, about 0.1 nmole / kg to about 50 nmole / kg, about 0.1 nmole / kg to about 10 nmole / kg, or about 0.1 nmole / kg to about 1 nmole / kg.In other embodiments, the dose is from about 0.3 nmole / kg to about 1,000 nmole / kg, from about 0.3 nmole / kg to about 900 nmole / kg, from about 0.3 nmole / kg to about 850 nmole / kg, from about 0.3 nmole / kg to about 800 nmole / kg, from about 0.3 nmole / kg to about 700 nmole / kg, from about 0.3 nmole / kg to about 600 nmole / kg, from about 0.3 nmole / kg to about 500 nmole / kg of the subject's body weight. nmole / kg, about 0.3 nmole / kg to about 400 nmole / kg, about 0.3 nmole / kg to about 300 nmole / kg, about 0.3 nmole / kg to about 200 nmole / kg, about 0.3 nmole / kg to about 100 nmole / kg, about 0.3 nmole / kg to about 50 nmole / kg, about 0.3 nmole / kg to about 10 nmole / kg, or about 0.3 nmole / kg to about 1 nmole / kg.

[0174] In various other embodiments, the dose of the compound, or a pharma- ceutically acceptable salt thereof, is, for example, about 10 nmole / kg to about 10,000 nmole / kg, about 10 nmole / kg to about 5,000 nmole / kg, about 10 nmole / kg to about 3,000 nmole / kg, about 10 nmole / kg to about 2,500 nmole / kg, about 10 nmole / kg to about 2,000 nmole / kg, about 10 nmole / kg to about 1,000 nmole / kg, about 10 nmole / kg to about 900 nmole / kg, or about 10 nmole / kg to about 1500 nmole / kg. le / kg, about 10 nmole / kg to about 800 nmole / kg, about 10 nmole / kg to about 700 nmole / kg, about 10 nmole / kg to about 600 nmole / kg, about 10 nmole / kg to about 500 nmole / kg, about 10 nmole / kg to about 400 nmole / kg, about 10 nmole / kg to about 300 nmole / kg, about 10 nmole / kg to about 200 nmole / kg, about 10 nmole / kg to about 150 nmole / kg, about 10 nmole / kg to about 100 nmole / kg, mole / kg, about 10 nmole / kg to about 90 nmole / kg, about 10 nmole / kg to about 80 nmole / kg, about 10 nmole / kg to about 70 nmole / kg, about 10 nmole / kg to about 60 nmole / kg, about 10 nmole / kg to about 50 nmole / kg, about 10 nmole / kg to about 40 nmole / kg, about 10 nmole / kg to about 30 nmole / kg, about 10 nmole / kg to about 20 nmole / kg, about 200 nmole / kg to about 900 nmole / kg, about 200 nmole / kg to about 800 nmole / kg, about 200 nmole / kg to about 700 nmole / kg, about 200 nmole / kg to about 600 nmole / kg, about 200 nmole / kg to about 500 nmole / kg, about 250 nmole / kg to about 600 nmole / kg, about 300 nmole / kg to about 600 nmole / kg, about 300 nmole / kg to about 500 nmole / kg, or about 400 nmole / kg to about 600 nmole / kg.

[0175] In various other embodiments, the dose of the compound, or a pharma- ceutically acceptable salt thereof, is, for example, about 1 nmole / kg to about 10,000 nmole / kg, about 1 nmole / kg to about 5,000 nmole / kg, about 1 nmole / kg to about 3,000 nmole / kg, about 1 nmole / kg to about 2,500 nmole / kg, about 1 nmole / kg to about 2,000 nmole / kg, about 1 nmole / kg to about 1,000 nmole / kg, about 1 nmole / kg to about 900 nmole / kg, about 1 nmole / kg to about 800 nmole / kg, about 1 nmole / kg to about 700 nmole / kg, about 1 nmole / kg to about 600 nmole / kg, about 1 nmole / kg to about 500 nmole / kg, or about 1 nmole / kg to about 200 nmole / kg. g, about 1 nmole / kg to about 400 nmole / kg, about 1 nmole / kg to about 300 nmole / kg, about 1 nmole / kg to about 200 nmole / kg, about 1 nmole / kg to about 150 nmole / kg, about 1 nmole / kg to about 100 nmole / kg, about 1 nmole / kg to about 90 nmole / kg, about 1 nmole / kg to about 80 nmole / kg, about 1 nmole / kg to about 70 nmole / kg, about 1 nmole / kg to about 60 nmole / kg, about 1 nmole / kg to about 50 nmole / kg, about 1 nmole / kg to about 40 nmole / kg, about 1 nmole / kg to about 30 nmole / kg, or about 1 nmole / kg to about 20 nmole / kg.

[0176] In another embodiment, about 20 μg / kg body weight to about 3 mg / kg body weight of the compound or its pharma- ceutically acceptable salt may be administered to the subject.In another aspect, the amount may be about 0.2 mg / kg body weight to about 0.4 mg / kg body weight, or about 50 μg / kg body weight.

[0177] Unless otherwise specified, in all dosage embodiments described herein, "kg" refers to kilograms of the subject's body weight.

[0178] A single dose or multiple doses of the compound, or a pharma- ceutically acceptable salt thereof, may be administered to a subject.

[0179] Any applicable dosing schedule known in the art can be used for the administration of the compound, its pharma- ceutically acceptable salt, or pharmaceutical composition comprising it. For example, once a day (aka qd), twice a day (aka bid), three times a day (aka tid), twice a week (aka BIW), three times a week (aka TIW), once a week, etc. can be used. In one aspect, the selected dosing schedule can take into account the concentration of the compound / composition administered (including, for example, the number of CAR-T cells administered).

[0180] "Cancer," when read in light of this specification, has its plain and ordinary meaning and may include, but is not limited to, a group of diseases involving abnormal cell proliferation with the ability to invade or spread to other parts of the body. Many types of cancers may be treated using the compositions, compounds, and methods described herein, including, but not limited to, carcinoma, sarcoma, osteosarcoma, lymphoma, melanoma, mesothelioma, nasopharyngeal carcinoma, leukemia, adenocarcinoma, and myeloma. Other, and perhaps more specifically, examples of cancers that may be treated by the methods and / or use of the compounds and compositions herein include, but are not limited to, lung cancer (including but not limited to non-small cell lung cancer), bone cancer (including but not limited to osteosarcoma), pancreatic cancer, skin cancer (including but not limited to cutaneous melanoma), head cancer, neck cancer, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer, cancer of the fallopian tubes, cancer of the cervix, cancer of the vagina, cancer of the vulva, Hodgkin's disease, cancer of the esophagus, small intestine cancer, and ovarian cancer. Includes intestinal cancer, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the prostate, leukemia (including but not limited to chronic leukemia, acute leukemia, acute myeloid leukemia, lymphocytic lymphoma, myeloid leukemia, myelomonocytic leukemia, and hairy cell leukemia), pleural mesothelioma, cancer of the bladder, Burkitt's lymphoma, cancer of the ureter, cancer of the kidney (including but not limited to renal cell carcinoma), cancer of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, and adenocarcinoma of the gastroesophageal junction.

[0181] In some aspects of these embodiments, the cancer is a cancer that expresses a folate receptor, such as, but not limited to, a cancer that expresses the folate receptor alpha. In other embodiments, the cancer is a cancer that expresses the folate receptor beta. In some aspects of these embodiments, the cancer is endometrial cancer, non-small cell lung cancer, ovarian cancer, or triple negative breast cancer.

[0182] The cancer to be treated can be a tumor.In another embodiment, the cancer can be a malignant tumor.In another embodiment, the cancer is acute myeloid leukemia, for example, the acute myeloid leukemia in which the cancer expresses folate receptor-β.

[0183] All patents, patent application publications, journal articles, textbooks, and other articles mentioned herein are indicative of the level of skill of those of ordinary skill in the art to which this disclosure pertains, and all such articles are incorporated herein by reference to the same extent as if each individual article was specifically and individually indicated to be incorporated by reference.

[0184] In the above description, numerous specific details are set forth to provide a thorough understanding of the present disclosure, it being understood that certain embodiments may be practiced without some or all of these specific details, and that the present disclosure is not limited to a particular biological system, a particular cancer, or a particular organ or tissue, which may, of course, vary, but remain applicable in light of the data provided herein.

[0185] Furthermore, various techniques and mechanisms of the present disclosure sometimes describe a connection or coupling between two components. Words such as attached, coupled, joined, connected with inflectional morphemes, and similar terms are used interchangeably unless a difference is stated or otherwise made clear from the context. These words and expressions do not necessarily mean a direct connection, but include a connection through the intermediation of a component. It should be noted that a connection between two components does not necessarily mean a direct, unobstructed connection, if various other components may be between the two components described. As a result, a connection does not necessarily mean a direct, unobstructed connection, unless stated otherwise.

[0186] Moreover, it will be understood that the present disclosure is presented in this manner solely for illustrative purposes, and that the principles and embodiments described herein may be applied to compounds and / or composition components having configurations other than those specifically described herein. Indeed, it is specifically contemplated that the components of the compositions and compounds of the present disclosure may be tailored to facilitate their desired application.

[0187] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of chemistry and biology. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the subject matter of this application, and preferred methods and materials are described herein. Furthermore, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, if a compound / composition is substituted by "an" alkyl or aryl, the compound / composition is optionally substituted by at least one alkyl and / or at least one aryl.

[0188] When ranges are used herein for physical properties, e.g., molecular weight, or chemical properties, e.g., chemical formula, all combinations and subcombinations of the ranges and specific embodiments thereof are intended to be included.

[0189] Additionally, when the term "about" refers to a number, numerical value, or range (e.g., including whole numbers, fractions, and percentages), the referenced number or numerical range is an approximation within experimental variability (or within statistical experimental error), and thus, a numerical value or range may vary between 1% and 15% of the stated number or numerical range (e.g., + / - 5% to 15% of the stated value) as long as one of ordinary skill in the art would consider it to be equivalent to the stated value (e.g., having the same function or result). The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude certain other embodiments described herein, such as any compound, composition of matter, composition, method, or process, which may "consist of" or "consist essentially of" the described features. The term "substantially" may allow for some variability in values ​​or ranges of a stated value or within a stated range limit, for example, within 90%, within 95%, or within 99%.

[0190] It will be understood that a method of treatment may include administering more than one treatment, compound, or composition to a subject, with the order, timing, number, concentrations, and volumes of administration being limited only by the medical needs and limitations of the treatment (i.e., for example, two treatments may be administered to a subject simultaneously, sequentially, sequentially, alternatively, or according to any other regimen).

[0191] Furthermore, in describing representative embodiments, the present disclosure may present a method and / or process as a specific order of steps. Unless the method or process relies on a specific order of steps described herein, the method or process should not be limited to the specific order of steps described. As one skilled in the art will recognize, other orders of steps are possible. Thus, the specific order of steps disclosed herein should not be concluded as a limitation on the scope of the claims. Furthermore, claims directed to methods and / or processes should not be limited to performing those steps in the order described, and one skilled in the art can easily understand that the order can be changed and still be within the spirit and scope of the present disclosure.

[0192] It is therefore intended that the specification and appended claims cover all modifications and variations that would be apparent to one of ordinary skill in the art based on this disclosure. EXAMPLES

[0193] The following examples are intended to illustrate the present disclosure and are not intended in any way to limit the scope of the invention as claimed.

[0194] [Example 1] Generation of cancer cell lines and human anti-fluorescein CAR T cells MDA-MB-231 and KB cells were obtained from American Type Culture Collection (ATCC). Both cell lines were cultured using folate-free RPMI1640 (Gibco; Thermo Fisher Scientific, Waltham, MA) containing heat-inactivated 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. To obtain stable mCherry-expressing MDA-MB-231 cells, MDA-MB-231 cells were first transduced with a lentiviral vector (pLv-NLS-mCherry-puro; Vector Builder, Chicago, IL), and then positive clones were selected in puromycin-containing medium.

[0195] For the generation of human anti-fluorescein chimeric antigen receptor (CAR) T cells, peripheral blood mononuclear cells (PBMCs) were isolated from fresh human peripheral blood samples by Ficoll (GE Healthcare Life Sciences, Piscataway, NJ) density gradient centrifugation after informed consent. CD3 + T cells were harvested and enriched using the EasySep Human T-Cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada), and human anti-fluorescein CAR T cells were generated according to published lentiviral vector protocols. All cells were maintained at 37°C under 5% CO2 and regularly tested for mycoplasma contamination.

[0196] [Example 2] Analysis of TLR7 expression in human T cells To determine whether Toll-like receptor 7 (TLR7) is expressed in primary human T cells, freshly isolated CD3 + T cells were fixed, permeabilized, and stained with Alexa Fluor 488 anti-TLR7 (IC5875G, R&D Systems, Minneapolis, MN) according to the manufacturer's instructions (In(TRacellular Flow Cytome (TRy Staining Protocol, BioLegend, San Diego, CA). Cells were then washed twice with In(TRacellular Staining Permeabilization Wash Buffer (BioLegend, San Diego, CA) and resuspended in 1× phosphate-buffered saline (PBS) prior to analysis by flow cytometry.

[0197] [Example 3] Analysis of fluorescein-NIR dye binding to anti-fluorescein CAR T cells in vivo and in vitro Anti-fluorescein CAR T cells (along with MDA-MB-231 or KB cells as negative controls) were incubated with fluorescein near-infrared (NIR) dye (10 nM) for 1 h at room temperature prior to incubation in the presence or absence of a 1000-fold excess of sodium fluorescein (10 μM). Cells were then washed three times with PBS and NIR dye fluorescence was measured by flow cytometry.

[0198] Flow cytometry of anti-fluorescein CAR T cells in the absence (labeled A in Figure 3A) or presence (labeled B in Figure 3A) of fluorescein-NIR dye conjugate (10 nM), or in the presence of both fluorescein-NIR dye conjugate plus 1000-fold excess fluorescein (labeled C in Figure 3A), demonstrated that the fluorescein-NIR dye conjugate bound to the anti-fluorescein CAR T cells in a manner that could be quantitatively blocked by the addition of a 1000-fold excess of fluorescein.

[0199] Additionally, NSG mice were implanted with 1 million KB cells in one flank, and KB tumor volumes were ~50 mm 3 Upon reaching the target concentration, culture the anti-fluorescein CAR T cells (8 × 10 containing ~50% anti-fluorescein CAR T cells) 6 1000 cells) were injected into the mice. Fluorescein-folate (500 nmol / kg) was injected 4 and 24 hours later, and weekly thereafter. 15 days after CAR T cell infusion, mice were tail vein injected with 500 nmol / kg fluorescein-NIR dye. After 4 hours, tumors were dissociated and analyzed by flow cytometry for fluorescein-NIR dye uptake. CD3+ T cells were detected by anti-human CD3 in the APC-Cy7 channel, and green fluorescent protein (GFP)-transfected anti-fluorescein CAR T cells were detected using the GFP channel.

[0200] As shown in Figure 3B, CAR-negative cells (e.g., MDA-MD-231 cells and KB cells) do not express binding sites for the fluorescein dye conjugate (10 nM). In contrast, confocal microscopy evaluation shows that the fluorescein-Alexafluor 647 conjugate is endocytosed by CAR T cells that have anti-fluorescein cell surface receptors (Figure 3C shows endocytosis of Alexafluor 647 after 1 h of incubation at 4 ° C, while Figure 3D shows endocytosis of Alexafluor 647 after transfer to CAR T cells for 4 h at 37 ° C). Bars = 10 μm.

[0201] [Example 4] Stimulation of CAR T cell exhaustion and reversal of exhaustion in vivo To induce CAR T cell exhaustion, anti-fluorescein CAR T cells (10 4 folate receptor-expressing mCherry+MDA-MB-231 cells (10 4 Anti-fluorescein CAR T cells were then added to mCherry+MDA-MB-231 cells (10 cells / well) and CAR T cell-mediated killing of mCherry+MDA-MB-231 cells was then initiated by the addition of 10 nM fluorescein folate. Anti-fluorescein CAR T cells were then added to mCherry+MDA-MB-231 cells (10 cells / well) every 12 hours thereafter. 4The CAR T cells were transferred to new flasks (1000 cells / well) to ensure continuous exposure to tumor antigens as shown in Figure 5A. A fraction of anti-fluorescein CAR T cells was collected after 12 hours (first round), 24 hours (second round), and 36 hours (third round) and analyzed for the expression of exhaustion markers PD-1, TIM3, and LAG3 by flow cytometry. CAR T cells were considered exhausted when they simultaneously expressed PD-1, TIM3, and LAG3. As shown in Figure 5C, the T cell exhaustion markers PD-1, TIM3, and LAG3 were increased. The number of live mCherry+ (MDA-MB-231) cells was counted by Incucyte S3 every 4 hours and used to calculate the cancer cell killing efficiency. As shown in Figure 5B, the ability of anti-fluorescein CAR T cells to kill MDA-MB-231 cells was decreased.

[0202] For evaluation of the recovery of exhausted CAR T cells, at the start of the third round of exhaustion, combined CAR T cell and MDA-MB-231 cell cultures were incubated overnight with the desired recovery compound at concentrations ranging from 0.01 to 100 nM. CAR T cells were then assessed for recovery by quantifying their cancer cell killing efficiency and analyzing their expression of PD-1, TIM3, and LAG3.

[0203] Activity restoration of exhausted anti-fluorescein CAR T cells was demonstrated by incubation with either targeted or non-targeted TLR7-1a. As shown in Figure 5E, T cell exhaustion markers (programmed cell death protein 1 (PD-1+), T cell immunoglobulin and mucin domain-containing-3 (TIM3)) were not significantly increased. + ), and lymphocyte activation 3 (LAG3 + ) was decreased. Furthermore, as shown in Figure 5D, the ability of anti-fluorescein CAR T cells to kill MDA-MB-231 cells was increased. Data shown show the change in markers above baseline levels (i.e., vehicle (dimethyl sulfoxide (DMSO) treated)). Bar graphs represent the mean ± SD, n = 3.

[0204] Human CD3+ T cells were activated upon administration of different concentrations of either TLR7-54 or TLR7-1a agonists, as shown in Figure 4. Figure 4A shows the percentage increase in CD69+ cells after stimulation of isolated human peripheral blood CD3+ T cells with anti-CD3+ monoclonal antibodies (mAbs) in the absence or presence of increasing concentrations of TLR-54 or TLR7-1a, as measured by flow cytometry. Figure 4B shows the percentage increase in CD25+ cells after stimulation of isolated human peripheral blood CD3+ T cells with anti-CD3+ mAbs in the absence or presence of increasing concentrations of TLR-54 or TLR7-1a, as measured by flow cytometry. Figure 4C shows the levels of interferon gamma (INF-γ) in cell-free supernatants of stimulated T cells at 24 hours, as measured by enzyme-linked immunosorbent assay (ELISA). Figure 4D shows the levels of tumor necrosis factor alpha (TNF-α) in cell-free supernatants of stimulated T cells at 24 hours as measured by ELISA. The data shown support an increase in these parameters over baseline levels (i.e., vehicle (DMSO treatment)). Bar graphs represent the mean ± SD, n=3.

[0205] [Example 5] Restoration of anti-fluorescein CAR T cell activity in vivo following intravenous injection of a fluorescein-TLR7-1a conjugate The mouse is 10 6 KB cells were injected subcutaneously on day -7, followed by 8 × 10 6 The mice were divided into four groups, including a treatment group that was infused with 10 anti-fluorescein CAR T cells on day 1; a control group did not receive CAR T cells. After 6 h, 24 h, and 9 days, mice in the two treatment groups were intravenously injected with fluorescein-folate to induce binding of folate receptor-positive KB cancer cells to CAR T cells. The mice in the treatment groups were then divided into two cohorts, and on days 4–7 and 11–14, mice in the second cohort of treatment groups were intravenously injected with fluorescein-TLR7-1a. The timeline of the in vivo study is shown in Figure 8A.

[0206] Tumor volume (Figure 8B) and animal weight change (Figure 8C) were measured every 3 days. The cohort treated with CAR T + fluorescein-folate + fluorescein-TLR7-1a showed delayed tumor growth compared to all other groups (both control and other cohorts of treatment groups). The cohort treated with CAR T + fluorescein-folate + fluorescein-TLR7-1a showed negligible percent weight change compared to the cohort treated with CAR T + fluorescein-folate.

[0207] On day 16, tumors were excised and dissociated into component cells, and human CD3+ T cells were determined as a percentage of total tumor cells (Figure 8D). PD-1+TIM3+ cells were also determined as a percentage of total human CD3+ T cells (Figure 8E), and the ratio of mouse CD86+ cells to CD206+ cells, which also express myeloid markers F4 / 80 and CD11b, in the anti-fluorescein CAR T cell treatment group is also shown (Figure 8F). Our targeted delivery of TLR7-1a successfully restored the activity of exhausted CAR T cells without affecting myeloid cells. All data were plotted as mean ± SEM. Data shown are representative of at least two independent experiments. Data were analyzed by two-way analysis of variance (ANOVA) ( ** p<0.01; ns=not significant).

[0208] [Example 6] Comparison of CAR T cell eradication of solid KB and MDA-MB-231 cell tumors in the presence and absence of fluorescein-TLR7-1a conjugates Eight- to 10-week-old NSG mice (Jackson Lab strain no. 005557; The Jackson Laboratory, Bar Harbor, ME) were placed on a folate-deficient diet (TD.95247, Envigo, Indianapolis, IN) upon arrival to reduce their serum folate concentrations to levels similar to those in wild humans and mice. One week later, mice were placed on a folate-deficient diet (TD.95247, Envigo, Indianapolis, IN) with ∼10 0.001 mg / kg of folate in separate flanks of the same mouse. 6KB cells and 4 million MDA-MB-231 cells were implanted subcutaneously. KB tumors were 80 mm 3 and MDA-MD-231 tumors grew to ~160 mm 3 The tumors were allowed to grow for approximately 2 weeks (i.e., to adjust for their different growth rates) until they reached 8×10 6 anti-fluorescein CAR T cells were injected intravenously, followed by 500 nmol / kg fluorescein folate injections both 4 and 24 hours after CAR T cell infusion.

[0209] Four days after CAR T cell infusion, CAR T cell-treated mice were divided into an activity recovery group (fluorescein-TLR7-treated) and a control group (saline-treated). The fluorescein-TLR7-treated cohort received 500 nmol / kg fluorescein-TLR7 4× / week, while the control group received an equal volume of saline on the same schedule. Tumor volume was calculated according to the formula (l×w 2 ) / 2, where "l" is the longest width of the tumor and "w" is the dimension perpendicular to the longest transept.

[0210] On day 18, mice were sacrificed and tumor fragments were dissociated using a human tumor dissociation kit (130-095-929, MeMiltenyi Biotec, Bergisch Gladbach, Germany). The resulting single cell suspensions were stained for human CD3 (anti-CD3-APC-Cy7, BioLegend, Sand Diego, CA), PD-1 (anti-PD-1-PE, BioLegend, Sand Diego, CA), TIM3 (anti-TIM3-PE-Cy7, BioLegend, Sand Diego, CA), mouse CD11b (anti-CD11b-PE, BioLegend, Sand Diego, CA), F4 / 80 (anti-F4 / 80-APC-Cy7, BioLegend, Sand Diego, CA), M2 macrophage marker CD206 (anti-CD206-APC, BioLegend, Sand Diego, CA), and M1 macrophage marker CD86 (anti-CD86-PE-Cy7, BioLegend, Sand Diego, CA). All samples were then analyzed by flow cytometry.

[0211] FIG. 6 shows the effect of anti-fluorescein CAR T cell therapy on the growth and immune characteristics of MDA-MB-231 and KB tumors. FIG. 6A shows the tumor volumes of cohorts that were either left untreated (dotted line) or treated with both anti-fluorescein CAR T cells and fluorescein-folate bispecific adapters (solid line). FIG. 6B shows the percentage of CD3+ T cells (as a percentage of total cells in the tumor) on day 18 when tumors were excised and both cancer and stromal cells were released using a tumor dissociation kit before analysis by flow cytometry. FIG. 6C shows the exhaustion markers PD-1+ and TIM3+ (as a percentage of total human CD3+ T cells) on day 18 when tumors were excised and both cancer and stromal cells were released using a tumor dissociation kit before analysis by flow cytometry. FIG. 6D shows the ratio of CD86+F4 / 80+CD11b+ to CD206+F4 / 80+CD11b+ myeloid cells on day 18 when tumors were excised and both cancer and stromal cells were released using a tumor dissociation kit prior to analysis by flow cytometry. n=5 mice / group. All data were analyzed by two-way ANOVA and plotted as mean±SEM ( * p<0.05, ** p<0.01, *** p<0.001). Data shown are representative of two independent experiments. FIG. 6E shows representative images showing human CD3+ T cell infiltration into MDA-MB231 and KB cell solid tumors. Bars=200 μm. Chemical Examples

[0212] Example A Synthesis of FITC-TCR7-1a

[0213] [ka]

[0214] 3-Amino-2,2-dimethylpropan-1-ol (2,1.5 equiv.) and triethylamine (2 equiv.) were added to a stirred solution of 4-chloro-3-nitroquinoline (1,1.2 equiv.) in N,N-dimethylformamide (DMF) (10 mL) (i) of Scheme 1. The reaction mixture was heated at 70° C. for 60 min and monitored by liquid chromatography-mass spectrometry (LCMS). It was then cooled, diluted with water and stirred for another 15 min. The precipitated solid was filtered and washed with water. The solid was dried under vacuum to give substance 3 as a yellow solid. Yield—80%.

[0215] Then, 2,2-dimethyl-3-(3-nitroquinolin-4-ylamino)propan-1-ol (1 g) was dissolved in methanol (15 mL) and reduced with Pd / C (100 mg, 10 mol%) as a catalyst under hydrogen balloon conditions for 4 hours (Scheme 1 (ii)). The solution was then filtered and evaporated under reduced pressure to give 3-(3-aminoquinolin-4-ylamino)-2,2-dimethylpropan-1-ol (4).

[0216] Triethylamine (2 equiv.) and valeryl chloride (5, 1.5 equiv.) were then added to a stirred solution of substance 4 (1 g) in anhydrous tetrahydrofuran (THF) (10 mL) (scheme 1 (iii)). The reaction mixture was then stirred for 4 h, followed by removal of the solvent under reduced pressure. The crude residue was then dissolved in ethyl acetate (EtOAc), washed with water, brine and dried over sodium sulfate. The combined organic layers were evaporated under vacuum and dried to give intermediate amide compound 6. Overall yield - 70%.

[0217] To a stirred solution of amide compound 6 (1 g) in methanol (MeOH) (15 mL), excess calcium oxide (10 equiv.) was added and the solution was heated at 110° C. for 96 h (scheme 1 (iv)). The solvent was then removed under vacuum and the residue was purified using flash column chromatography (MeOH / dichloromethane mobile phase) to give compound 7. Yield—60%.

[0218] Then, NaH (2 eq.) and N-Boc-PEG3-bromide (8, 2 eq.) were added to a stirred solution of intermediate hydroxyl compound 7 (500 mg) in THF (5 mL) (Scheme 1 (v)). It was stirred for about 5 h under nitrogen atmosphere, then the solvent was evaporated to dryness using a rotary evaporator. It was quenched with water and diluted with DMSO. The crude reaction mixture was purified by high performance liquid chromatography (HPLC) using ammonium acetate and acetonitrile as the mobile phase to give the product (9) as a colorless liquid. Yield-60%.

[0219] To a stirred solution of 9 (100 mg) in anhydrous dichloromethane (1 mL), 3-chloroperbenzoic acid (10, 1.5 equiv.) was added and the solution was refluxed at 45° C. for 30 min (Scheme 1(vi)). When the stirred material was completely consumed, the solvent was evaporated under vacuum and dried. The residue was then redissolved in anhydrous dichloromethane (1 mL) followed by the addition of trichloroacetyl isocyanate (11, 2.0 equiv.) and the reaction mixture was heated at 45° C. for 30 min (Scheme 1(vi)). After completion of the reaction, the solvent was removed under vacuum and the residue was redissolved in anhydrous MeOH (1 mL) followed by the addition of 25% sodium methoxide in methanol (0.2 mL). This was then heated at 75° C. for 1 h and cooled (Scheme 1(vi)). The solvent was removed under vacuum and the residue was purified using column chromatography (MeOH / dichloromethane) to give compound 12 as a colorless liquid. Overall yield - 40%.

[0220] To a stirred solution of intermediate compound 12 (100 mg) in dichloromethane (1 mL), trifluoroacetic acid (10 eq.) was added (scheme 1 (vii)). It was stirred under nitrogen atmosphere for about 1 h and the solvent was evaporated to dryness using a rotary evaporator. After complete removal of residual acid, it was dissolved in DMSO (1 mL).

[0221] To this DMSO solution, fluorescein isothiocyanate (FITC) (catalog number F7250; Sigma Aldrich, St. Louis, MO) (13, 1.2 eq.) and DIPEA (2 eq.) were added and the solution was stirred for 10 min (Scheme 1 (vii)). The reaction was monitored by LCMS. After complete consumption of the starting material, purification by HPLC using ammonium acetate and acetonitrile as the mobile phase gave the product (14) (FITC-TLR7-1a) as a yellow solid. Yield-90%. Example B Synthesis of fluorescein-drug conjugates

[0222] [ka]

[0223] Reagents and conditions: (a) Amino-PEG3-amine, N,N-diisopropylethylamine (DIPEA), dimethylsulfoxide (DMSO), 1 hour. (b) Alexa Fluor™ 647 NHS Ester (structure not disclosed by supplier), DIPEA, DMSO, 1 hour.

[0224] FITC was added dropwise to a solution of amino-PEG3-amine (3 eq.) and DIPEA (5 eq.) in DMSO. The solution was stirred at room temperature for 1 h. The resulting product was purified by preparative reversed-phase high performance liquid chromatography (HPLC) (92% yield). To obtain FITC-PEG3-Alexa Fluor647, Alexa Fluor 647 NHS ester (Thermo Fisher Scientific, Waltham, MA), FITC-PEG3-amine (2 eq.) and DIPEA (5 eq.) were dissolved in DMSO and the solution was stirred for 1 h. The product was then purified using HPLC to give the compound in 90% yield.

[0225] [ka]

[0226] Reagents and conditions: (c) HATU, DIPEA, DMSO, 12 hours.

[0227] Near infrared (NIR) dye, HATU (1 eq.), and DIPEA (5 eq.) were dissolved in DMSO and stirred for 25 min, after which FITC-PEG3-amine (1 eq.) was added. The reaction was stirred at room temperature for 12 h, and the product was purified using HPLC (71%).

Claims

1. 1. A chimeric antigen receptor (CAR) T cell activity restoring conjugate comprising an agonist of toll-like receptor 7, toll-like receptor 8, or toll-like receptor 7 and toll-like receptor 8 conjugated via a linker to a targeting moiety that binds with specificity to the CAR on a CAR T cell, the conjugate comprising the structure: 【Chemical 1】 [In the formula: R 1 is acyclic alkyl or cyclic alkyl optionally substituted with one or more substituents independently selected from alkyl, halo, heteroalkyl, alkoxy, and cycloalkyl; R 2 is -NR 2x R 2y , H, -OR z , -SO 2 N (R z ) 2 , or N 3 (In the formula: R 2x and R 2y are each independently hydrogen (H), —N(R z ) 2 , -CON(R z ) 2 , -C(R z ) 2 -N(R z ) 2 , -CS-N(R z ) 2 or alkyl optionally substituted by one or more substituents independently selected from oxo, halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; Each R Z is independently H or optionally substituted alkyl; or R 2x and R 2y taken together to form an optionally substituted 3-10 membered monocyclic or bicyclic heterocycloalkyl; Each R 3 are independently H, halo, -N 3 , -CN, -NO 2 , alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, or alkoxy is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halo, or cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally substituted; X 1 , X 2 , and X 3 are independently N or CR q (In the formula, each R q are independently H, halo, or optionally substituted alkyl; Z is GL-, where L is a linker and G is a targeting moiety; In formula (I), n is 1 to 6; m in formula (I) is 0 to 4. or a pharmaceutically acceptable salt thereof; structure: 【Chemistry 2】 [In the formula: R 1 , R 3 , R 4 , and R 5 are each independently H, alkyl, alkoxyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR 2x , 【Chemistry 3】 (In the formula, R 2x and R 2y are H, —OH, —CH 2 —OH, —NH 2 , -CH 2 -NH 2 , -COOMe, -COOH, -CONH 2 , -COCH 3 , alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl; Z is GL-, where L is a linker and G is a targeting moiety; X 1 , X 2 , and X 3 is an independent CR q or N (wherein each R q are independently H, halo, or optionally substituted alkyl; Y is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO 3 R z , -N 3 , -COR z , -COOR z , -CON(R z ) 2 , -COSR z , -SO 2 N (R z ) 2 , or -CON(R z ) 2 (In the formula: R 2x and R 2y are each independently hydrogen, —N(R z ) 2 , -CON(R z ) 2 , -C(R z ) 2 -N(R z ) 2 , -CS-N(R z ) 2 or optionally substituted alkyl; Each R z are independently hydrogen or optionally substituted alkyl; or R 2x and R 2y taken together to form an optionally substituted heterocycloalkyl; n in formula (II) is 0 to 30. or a pharmaceutically acceptable salt thereof; or structure: 【Chemistry 4】 [In the formula: R 1 is acyclic alkyl or cyclic alkyl optionally substituted with one or more substituents independently selected from halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; Y is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO 3 R z , -N 3 , -COR z , -COOR z , -CONR z 2 , -COSR z , -SO 2 N (R z ) 2 , or -CON(R z ) 2 (In the formula: Each R Z are independently H or optionally substituted alkyl; R 2x and R 2y are each independently H, —N(R z ) 2 , -CON(R z ) 2 , -C(R z ) 2 -N(R z ) 2 , -CS-N(R z ) 2 or alkyl optionally substituted by one or more substituents independently selected from oxo, halo, alkyl, heteroalkyl, alkoxy, and cycloalkyl; or R 2x and R 2y taken together to form an optionally substituted 3-10 membered monocyclic or bicyclic heterocycloalkyl; Each R 3 are independently halo, -N 3 , -CN, -NO 2 , alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein the alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halogen, or cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally substituted; X 1 , X 2 , and X 3 is an independent CR q or N (wherein each R q are independently hydrogen, halogen, or optionally substituted alkyl; Z is LG-, where L is a linker and G is a targeting moiety; In formula (III), n is 0 to 30; In formula (III), m is 0 to 4. or a pharmaceutically acceptable salt thereof, G in formula (I), formula (II) or formula (III) has the structure: 【Chemistry 5】 or a pharmaceutically acceptable salt thereof.

2. X in formula (I), formula (II), and formula (III) 1 , X 2 , and X 3 2. The CAR T cell activity restoring conjugate of claim 1, wherein each of

3. structure: 【Chemistry 6】 or a pharmaceutically acceptable salt thereof. 【Request 4】 【Chemical 7】 [In the formula, n=0 to 200] or a pharmaceutically acceptable salt of any of the foregoing structures.

5. 【Chemical 8】 2. The CAR T cell activity restoring conjugate of claim 1, having a structure selected from:

6. 【Chemical 9】 or a pharmaceutically acceptable salt of any of the foregoing structures. 【Request 7】 【Chemical 10】 or a pharmaceutically acceptable salt of any of the foregoing structures.

8.

11. or a pharmaceutically acceptable salt of any of the foregoing structures.

9.

12. or a pharmaceutically acceptable salt of any of the foregoing structures.

10. 2. The CAR T cell activity restoring conjugate of claim 1, wherein the linker is releasable or non-releasable.

11. The linker is C 1 ~C 20 Alkyl, alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, carboxylate, carbonate, carbamate, urea, thiourea, -S-S, -CO-O-(CH 2 ) n -S-S- (n=2-6), -O-CO-O-(CH 2 ) n -S-S- (n=2-6), -S-CO-O-(CH 2 ) n -S-S- (n=2-6), -NH-CO-O-(CH 2 ) n 2. The CAR T cell activity restoring conjugate of claim 1, comprising -S-S-(n=2-6), PEG, polyproline, oligo-(4-piperidine)carboxylic acid, oligopiperidine, amino acid (e.g., hydrophilic amino acid), peptide, saccharopeptide, sugar, peptidoglycan, polyvinylpyrrolidone, Pluronic F-127, or any combination of two or more of the foregoing.

12. 11. The CAR T cell activity restoring conjugate of claim 1 or 10, wherein the linker comprises PEG.

13. A pharmaceutical composition comprising the CAR T cell activity restoring conjugate of claim 1 and a pharmaceutically acceptable carrier.

14. The chimeric antigen receptor (CAR) T cell activity restoring conjugate of claim 1 or the pharmaceutical composition of claim 13 for use in restoring the activity of exhausted CAR-T cells in a subject with cancer, comprising: The subject is (a) a vector comprising (i) a promoter operably linked to a nucleic acid sequence encoding a CAR, or (ii) a T cell expressing the CAR, wherein the CAR is bound to a first targeting moiety, a second targeting moiety, or a first targeting moiety and a second targeting moiety; and (b) a cancer-binding conjugate that binds with specificity to cancer cells and comprises a ligand conjugated to said first targeting moiety, wherein said cancer and said ligand bound by said first targeting moiety are optionally conjugated via a first linker. a subject being treated by the CAR T cell activity restoring conjugate comprises an agonist of toll-like receptor 7, toll-like receptor 8, or toll-like receptor 7 and toll-like receptor 8 (TLR7 / 8 agonist) conjugated to either the first targeting moiety or the second targeting moiety, wherein the agonist and either the first targeting moiety or the second targeting moiety are conjugated via a second linker; The CAR T cell activity restoring conjugate or the pharmaceutical composition.

15. The first targeting moiety, the second targeting moiety, or the first targeting moiety and the second targeting moiety have the structure: 【Chemistry 13】 or 【Chemistry 14】 is or comprises a group having the formula 15. The CAR T cell activity restoring conjugate of claim 14.

16. The CAR T cell activity restoring conjugate of claim 14, wherein the ligand that binds with specificity to cancer cells is selected from the group consisting of folate, 5-methyltetrahydrofolate, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) ligand, neurokinin 1 receptor (NK-1R) ligand, carbonic anhydrase IX (CAIX) ligand, gamma glutamyl transpeptidase ligand, luteinizing hormone releasing hormone (LHRHR) ligand, CD73 ligand, heat shock protein (HSP) ligand, glucose transporter 1 (glut-1) ligand, fibroblast activation protein ligand, natural killer group 2D receptor (NKG2D) ligand, and cholecystokinin B receptor (CCKBR or CCK2) ligand.

17. The CAR T cell activity restoring conjugate of claim 14, wherein the first targeting moiety and the second targeting moiety are independently selected from the group consisting of 2,4-dinitrophenyl (DNP), L-rhamnose, tacrolimus (FK506), 2,4,6-trinitrophenol (TNP), biotin, rapamycin, digoxigenin, folate, 5-methyltetrahydrofolate, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centirin, and DARPin.

18. The method of claim 17, wherein the first targeting moiety and the second targeting moiety are independently selected from the group consisting of DNP, FK506, TNP, biotin, rapamycin, digoxigenin, folate, 5-methyltetrahydrofolate, fluorescein, FITC, NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centirin, and DARPin; The ligand that binds to cancer cells with specificity is selected from the group consisting of folate, DUPA ligand, NK-1R ligand, CAIX ligand, gamma glutamyl transpeptidase ligand, LHRHR ligand, CD73 ligand, HSP ligand, glut-1 ligand, fibroblast activation protein ligand, NKG2D ligand, and CCKBR or CCK2 ligand.

15. The CAR T cell activity restoring conjugate of claim 14.

19. The method of claim 19, wherein the first linker and the second linker are selected from the group consisting of C 1 to C 20 alkyl, alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, -S-S, -CO-O-(CH 2 ) n -S-S-(n=2 to 6), -O-CO-O-(CH 2 ) n -S-S-(n=2 to 6), -S-CO-O-(CH 2 ) n -S-S-(n=2 to 6), -NH-CO-O-(CH 2 ) n 15. The CAR T cell activity restoring conjugate of claim 14, independently comprising -S-S-(n=2-6), carboxylate, carbonate, carbamate, urea, thiourea, polyethylene glycol (PEG), polyproline, oligo-(4-piperidine)carboxylic acid, oligopiperidine, amino acid, peptide, saccharopeptide, sugar, peptidoglycan, polyvinylpyrrolidone, Pluronic F-127, or any combination of two or more of the foregoing.

20. The CAR T cell activity restoration conjugate of claim 14, wherein the first linker, the second linker, or both the first linker and the second linker comprise PEG.

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