Dual-specific adapters and their use with versatile CAR-T cells in tumor treatment and inhibition of cancer-associated fibroblasts.

Abstract

Chimeric antigen receptor (CAR)-T cells, bispecific adapters for linking CAR-T cells to tumor cells and / or cancer-associated fibroblasts (CAFs), and methods for treating cancer using them.

Description

[Technical Field]

[0001] Priority This patent application claims priority rights with respect to U.S. Provisional Application No. 63 / 451,448 filed on 10 March 2023, U.S. Provisional Application No. 63 / 595,456 filed on 2 November 2023, U.S. Provisional Application No. 63 / 451,453 filed on 10 March 2023, U.S. Provisional Application No. 63 / 600,612 filed on 17 November 2023, U.S. Provisional Application No. 63 / 451,457 filed on 10 March 2023, U.S. Provisional Application No. 63 / 600,616 filed on 17 November 2023, and U.S. Provisional Application No. 63 / 451,462 filed on 10 March 2023. The contents of each of the aforementioned applications are thus incorporated into this disclosure by reference in their entirety.

[0002] This disclosure relates to chimeric antigen receptor (CAR) T cells, a bispecific adapter capable of linking CAR-T cells to tumor cells expressing prostate-specific membrane antigen (PSMA) and / or folate receptor (FR) or cancer-associated fibroblasts (CAFs) expressing fibroblast-activating protein (FAP), a combination of the bispecific adapter linking CAR-T cells to PSMA or FR-expressing tumor cells and optionally to FAP-expressing CAFs, and a method for treating cancer using these. [Background technology]

[0003] Chimeric antigen receptor (CAR)-T cell therapy has proven effective in hematological malignancies, but in solid tumors, it has limited efficacy, at least partially, due to limited CAR-T cell infiltration. This CAR-T cell infiltration may be attributable to cancer-associated fibroblasts (CAFs), which are found in most solid tumors and have been shown to remodel the extracellular matrix, secrete immunosuppressive cytokines, promote tumor cell proliferation through growth factor secretion, facilitate tumor infiltration, inhibit the immune response, and form physical barriers that hinder T cell infiltration.

[0004] Fibroblast-activating protein (FAP) is expressed on the surface of CAFs and has been shown to correlate with poor patient prognosis in several solid tumors. In addition, virtually all human solid tumors overexpress FAP.

[0005] Traditionally, T cells expressing a single CAR have been used to target cell surface receptors on tumor cells. When a CAR in a T cell binds to a cell surface receptor on a tumor cell, the T cell can kill the tumor cell to which it is bound. This approach, while effective, can be very expensive, considering that CAR-T cells must be created for various cancers that express different cell surface receptors.

[0006] Despite the clear need for cancer prevention and treatment, cancer remains a major cause of death and suffering worldwide because there are currently no effective treatment options available to cure the condition. Furthermore, even when drugs or other therapies are available, they lack selectivity for the target cancer cells, and such treatments typically involve extremely potent drugs that pose a risk of systemic toxicity in subjects with underlying conditions.

[0007] What is needed is a bispecific adapter that can improve the immune response induced by CAR-T cells bound to tumor cells and inhibit the action of CAFs. This and other objectives and advantages, as well as the features of the present invention, will become apparent from the modes for carrying out the invention provided herein. [Overview of the project]

[0008] A dual-specific adapter is provided for use with anti-fluorescein chimeric antigen receptor (CAR)-T cells in the treatment of fibroblast-activating protein (FAP) expression (e.g., FAPα expression) and / or prostate-specific membrane antigen (PSMA) cancer, and for use in combination with cancer-associated fibroblasts (CAFs) expressing fibroblast-activating protein (FAP). In a particular embodiment, the dual-specific adapter has the following structure: FL-TL [wherein F comprises fluorescein, L comprises a linker, and TL comprises a targeting ligand]. Fluorescein may comprise fluorescein, fluorescein isothiocyanate (FITC), or N-hydroxysuccinimide (NHS)-fluorescein. The targeting ligand is

[0009] [ka] [In the formula,

[0010] [ka] [This is the linking point to the linker.] It may contain radicals of FAP ligands that include the structure shown.

[0011] A dual-specific adapter is also provided for use with anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) chimeric antigen receptor (CAR)-T cells in the treatment of fibroblast-activating protein (FAP)-expressing (e.g., FAPα-expressing) cancers and / or prostate-specific membrane antigen (PSMA) tumors.

[0012] In a particular embodiment, the adapter may include a fluorescein-linker-FAP ligand (e.g., fluorescein=fluorescein, FITC, or NHS-fluorescein), where the FAP ligand is

[0013] [ka] and the linker comprises (or consists essentially of or consists of) polyethylene glycol (PEG), and the adapter can be its pharmaceutically acceptable salt or hydrate. The linker can comprise (or consist essentially of or consist of) PEG4 to PEG , 1~6 , 1~6 , , 11 , 4 , , 9 , 2 , 7 , 5 , , 1~6 , 1~6 , 3 , , 1~6 , 1 , , 10 , 8 , 6 , and can comprise (or consist essentially of or consist of) PEG6. The linker can comprise (or consist essentially of or consist of) PEG 16 and can comprise (or consist essentially of or consist of) it. The FAP ligand is of formula I-B:

[0014] [Chemical formula] [T is substituted or unsubstituted methylene (-CH2-), substituted or unsubstituted amino (-NH-), -O-, or -S-, R 1 and R 2 are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl, R 3 and R 4 are each independently selected from the group consisting of -H, -OH, F, Cl, Br, I, -C 1~6 alkyl, -O-C 1~6 alkyl, and -S-C 1~6 alkyl, R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of H, alkyl, and halo, R 9 , R 10 , and R 11 are each independently selected from the group consisting of H, -C 1~6 alkyl, -O-C 1~6Alkyl, -SC 1~6 [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] It may have a structure represented by [this].

[0015] FAP ligand is formula IC:

[0016] [ka] [T is a substituted or unsubstituted methylene group (-CH2-), a substituted or unsubstituted amino group (-NH-), -O-, or -S-, R 1 and R 2 Each of these is independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl. R 3 and R 4 These are -H, -OH, F, Cl, Br, I, and -C, respectively. 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 5 , R 6 , R 7 , and R 8 Each is independently selected from the group consisting of H, alkyl, and halo. R 9 , R 10 , and R 11 These are H and -C respectively. 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 The structure may be represented by [independently selected from the group consisting of alkyl, F, Cl, Br, and I]. The dual specificity adapter may have one of the structures shown in Figure 25.

[0017] Another bispecific adapter for use in the treatment of FAP-expressing (e.g., FAPα-expressing) cancer in combination with anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) CAR-T cells, wherein the adapter comprises a fluorescein-linker-FAP ligand (e.g., fluorescein = fluorescein, FITC, or NHS-fluorescein), the FAP ligand is

[0018] [Chemical formula] is or comprises the same, the linker comprises PEG (or consists essentially of or consists of the same), and the adapter may be its pharmaceutically acceptable salt or hydrate. A bispecific adapter is provided. The linker may comprise (or consist essentially of or consist of) PEG3 to PEG 15 and may comprise (or consist essentially of or consist of) PEG 15 and may comprise (or consist essentially of or consist of) PEG 16 The FAP8 ligand has the structure:

[0019] [Chemical formula] [wherein,

[0020] [Chemical formula] represents a functionalized 5- to 10-membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocyclic ring, optionally further comprising 1 to 3 heteroatoms selected from O, N, and S, R1 and R2 are independently selected from the group consisting of -H, -D, -OH, -F, -Cl, -Br, -I, -C 1~6 alkyl, -O-C 1~6 alkyl, and -S-C 1~6 alkyl, R3 and R4 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R5 and R6 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R7 is selected from the group consisting of -H, -D, OH, CH2=, -CH3, CH3CH2-, (CH3)2CH-, (CH3)3C-, -CH2Ph, and substituted -CH2Ph. R8~R 10 is -H, -OH, -F, -Cl, -Br, -I, -NO2, -SO3H, -SO2NH2, -NH2, -N3, -NH=NH, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 11 -H, -D, C1~C 10 Alkyl, C3~C 10 Cycloalkyl, adamantyl,

[0021] [ka] , substituted or unsubstituted aryl, substituted or unsubstituted C7~C 20 Selected from the group consisting of alkylaryls, the aryl is

[0022] [ka] (In the formula, R 12 and R 16 These include -H, -D, halogens, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23It is selected from the group consisting of H, D, halogens, C1-C4 alkyls, and C1-C3 alkoxys. R 13 , R 14 and R 15 These include -H, -D, halogens, -OMe, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 (These are selected from the group consisting of -H, -D, halogens, C1-C4 alkyls, and C1-C3 alkoxys.) And, R 17 , R 18 , R 20 , and R 21 It is selected independently of -H and -CH3, R 19 and R 22 [These are independently selected from the group consisting of phenyl, dimethoxyphenyl, and aryl compounds.] It may have.

[0023] The dual-specific adapter has the following structure: FL-TL or a pharmaceutically acceptable salt or hydrate thereof [in the formula, F contains fluorescein, FITC, or NHS-fluorescein. L includes the linker, TL includes targeted ligands containing radicals of FAP ligand or PSMA ligand. It may include.

[0024] Targeted ligands are,

[0025] [ka] [In the formula,

[0026] [ka] [This is the linking point to the linker.] It may contain a radical of a FAP ligand including the structure of

[0027] The targeting ligand is of formula I-B:

[0028]

Chemical formula

[0029]

Chemical formula

[0030] The targeting ligand of the bispecific adapter is of formula I-C:

[0031] [Chemical formula] [wherein,

[0032] [Chemical formula] is a bonding point to the linker, T is substituted or unsubstituted methylene (-CH2-), substituted or unsubstituted amino (-NH-), -O-, or -S-, R 1 and R 2 are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl,<00,01006>R 3 and R 4 are each independently selected from the group consisting of -H, -OH, F, Cl, Br, I, -C 1~6 alkyl, -O-C 1~6 alkyl, and -S-C<00,00095>alkyl, R 5 R 6 R 7 and R 8 are each independently selected from the group consisting of H, alkyl, and halo, R 9 R 10 and R 11 are each independently selected from the group consisting of H, -C 1~6 alkyl, -O-C 1~6 alkyl, -S-C 1~6 alkyl, F, Cl, Br, and I] may include a radical of a FAP5 ligand having a structure represented by

[0033] The targeted ligand has the following structure:

[0034] [ka] [In the formula,

[0035] [ka] This represents a functionalized 5-10 member nitrogen-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, optionally further containing 1-3 heteroatoms selected from O, N, and S. R1 and R2 are -H, -D, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R3 and R4 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R5 and R6 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R7 is selected from the group consisting of -H, -D, OH, CH2=, -CH3, CH3CH2-, (CH3)2CH-, (CH3)3C-, -CH2Ph, and substituted -CH2Ph. R8~R 10 is -H, -OH, -F, -Cl, -Br, -I, -NO2, -SO3H, -SO2NH2, -NH2, -N3, -NH=NH, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 11 -H, -D, C1~C 10Alkyl, C3~C 10 Cycloalkyl, adamantyl,

[0036] [ka] , substituted or unsubstituted aryl, substituted or unsubstituted C7~C 20 Selected from the group consisting of alkylaryls, the aryl is

[0037] [ka] (In the formula, R 12 and R 16 These include -H, -D, halogens, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 It is selected from the group consisting of H, D, halogens, C1-C4 alkyls, and C1-C3 alkoxys. R 13 , R 14 and R 15 These include -H, -D, halogens, -OMe, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 (These are selected from the group consisting of -H, -D, halogens, C1-C4 alkyls, and C1-C3 alkoxys.) And, R 17 , R 18 , R 20 , and R 21 It is selected independently of -H and -CH3, R 19 and R 22 [These are independently selected from the group consisting of phenyl, dimethoxyphenyl, and aryl compounds.] It may contain radicals of FAP8 ligands, including

[0038] In certain embodiments, the targeted ligand comprises a radical of a PSMA ligand, and is either PSMAL1 or DUPA.

[0039] The linker is polyethylene glycol (PEG) or a PEG derivative, for example, optionally PEG3 to PEG 16 , and optionally PEG4~PEG 15 Or PEG3~PEG 12 PEG 12 PEG 15 PEG 16 , or PEG 18 PEG4~PEG 16 PEG 16 PEG3~PEG 15 PEG 15 PEG3~PEG 12 PEG6; PEG3-PEG8; or may include or be essentially derived from PEG6.

[0040] The bispecific adapter may be intended for use with anti-fluorescein CAR-T cells in the treatment of cancer.

[0041] The bispecific adapter may be used with anti-fluorescein CAR-T cells in the treatment of FAP-expressing cancers, and optionally, the linker may be PEG3-PEG. 15 , and optionally PEG 15 It includes or is essentially derived from it.

[0042] The dual-specificity adapter may be used with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancer, and optionally, the linker may be PEG3-PEG. 12 , and optionally PEG6 or PEG3-PEG8, and optionally including or essentially being PEG6.

[0043] In a particular embodiment, a bispecific adapter for use with anti-fluorescein CAR-T cells in the treatment of FAP-expressing cancer, comprising the following structure:

[0044] [ka]

[0045] [ka] Having or including one of the following, A pharmaceutically acceptable salt or hydrate of any of the aforementioned structures, A dual-specificity adapter is provided.

[0046] In a particular embodiment, a bispecific adapter for use with anti-fluorescein CAR-T cells in the treatment of FAP-expressing cancer, comprising the following structure:

[0047] [ka] A bispecific adapter is provided that has or contains one of the above structures, or contains a pharmaceutically acceptable salt or hydrate of any of the above structures.

[0048] In a particular embodiment, a bispecific adapter for use with anti-fluorescein CAR-T cells in the treatment of PSMA cancer, comprising the following structure:

[0049] [ka]

[0050] [ka] A bispecific adapter is provided that has or contains one of the aforementioned, or contains a pharmaceutically acceptable salt or hydrate of any of the aforementioned.

[0051] In one particular embodiment, a dual-specific adapter for use with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancer, comprising:

[0052] [ka] A dual-specificity adapter is provided which has or is a pharmaceutically acceptable salt or hydrate thereof.

[0053] In one particular embodiment, a dual-specific adapter for use with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancer, comprising:

[0054] [ka] A dual-specificity adapter is provided, which has or is a pharmaceutically acceptable salt or hydrate thereof.

[0055] Pharmaceutical compositions for the treatment of FAP-expressing (e.g., FAPα-expressing) cancers are also provided, comprising the bispecific adapter described above and a pharmaceutically acceptable carrier or excipient.

[0056] In certain embodiments, a pharmaceutical composition for the treatment of cancer comprises one of the dual-specific adapters described herein and a pharmaceutically acceptable carrier or excipient.

[0057] A combination of dual-specific adapters for use with anti-fluorescein CAR-T cells in cancer treatment is also provided. In a particular embodiment, the combination is: (i) A first bispecific adapter comprising any bispecific adapter described herein or a pharmaceutically acceptable salt or hydrate thereof, wherein the targeting ligand of the first bispecific adapter is

[0058] [ka] [In the formula,

[0059] [ka] [This is the linking point to the linker.] A first bispecific adapter comprising a radical of an FAP ligand having the formula, (ii) The following structure: FL-TL or a pharmaceutically acceptable salt or hydrate thereof [in the formula, F contains fluorescein, FITC, or NHS-fluorescein. L includes the linker, TL includes targeted ligands containing radicals of FR ligands or PSMA ligands. A second bispecific adapter including Includes.

[0060] The radical of the FAP ligand of the first bispecific adapter in the combination is given by formula IB:

[0061] [ka] [In the formula, T is a substituted or unsubstituted methylene (-CH2-), a substituted or unsubstituted amino (-NH-), -O- or -S-, R 1 and R 2 Each of these is independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl. R 3 and R 4 These are -H, -OH, F, Cl, Br, I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6Independently selected from the group consisting of alkyls, R 5 , R 6 , R 7 , and R 8 Each is independently selected from the group consisting of H, alkyl, and halo. R 9 , R 10 , and R 11 These are H and -C respectively. 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] The structure represented by, Formula IC:

[0062] [ka] [In the formula,

[0063] [ka] This is the linking point to the linker, T is a substituted or unsubstituted methylene group (-CH2-), a substituted or unsubstituted amino group (-NH-), -O-, or -S-. R 1 and R 2 Each of these is independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl. R 3 and R 4 These are -H, -OH, F, Cl, Br, I, and -C, respectively. 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 5 , R 6 , R 7 , and R8 Each is independently selected from the group consisting of H, alkyl, and halo. R 9 , R 10 , and R 11 These are H and -C respectively. 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] The structure represented by, The following formula:

[0064] [ka] [In the formula,

[0065] [ka] This represents a functionalized 5-10 member nitrogen-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, optionally further containing 1-3 heteroatoms selected from O, N, and S. R1 and R2 are -H, -D, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R3 and R4 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R5 and R6 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R7 is selected from the group consisting of -H, -D, OH, CH2=, -CH3, CH3CH2-, (CH3)2CH-, (CH3)3C-, -CH2Ph, and substituted -CH2Ph. R8~R 10 is -H, -OH, -F, -Cl, -Br, -I, -NO2, -SO3H, -SO2NH2, -NH2, -N3, -NH=NH, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 11 -H, -D, C1~C 10 Alkyl, C3~C 10 Cycloalkyl, adamantyl,

[0066] [ka] , substituted or unsubstituted aryl, substituted or unsubstituted C7~C 20 Selected from the group consisting of alkylaryls, the aryl is

[0067] [ka] (In the formula, R 12 and R 16 These include -H, -D, halogens, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 It is selected from the group consisting of H, D, halogens, C1-C4 alkyls, and C1-C3 alkoxys. R 13 , R 14 and R 15 These include -H, -D, halogens, -OMe, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 (These are selected from the group consisting of -H, -D, halogens, C1-C4 alkyls, and C1-C3 alkoxys.) And, R 17 , R 18 , R 20 , and R 21It is selected independently of -H and -CH3, R 19 and R 22 [These are independently selected from the group consisting of phenyl, dimethoxyphenyl, and aryl compounds.] It may have a structure represented by [this].

[0068] The targeting ligand of the second bispecific adapter of the combination, or a pharmaceutically acceptable salt or hydrate thereof, may contain a radical of a PSMA ligand. The linker of the first bispecific adapter of the combination may contain or be essentially derived from PEG.

[0069] The targeting ligand may be or may include PSMAL1 or DUPA.

[0070] The targeting ligand for the second bispecific adapter of the combination is a radical of folic acid or a functional fragment or analogue thereof.

[0071] Folic acid may be folic acid, dihydrofolic acid, tetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid (5,10-MTHF), 5-methyltetrahydrofolic acid (5-MTHF), or larcitrexed.

[0072] The PSMA ligand can be DUPA, and the linker can be PEG or a PEG derivative, for example, optionally PEG3-PEG. 12 , and optionally PEG6;PEG3~PEG 16 , and optionally PEG4~PEG 15 Or PEG3~PEG 12 PEG 12 PEG 15 PEG 16 , or PEG 18 PEG4~PEG 16 , and optionally PEG 16 PEG3~PEG 15 , and optionally PEG 15 PEG3 to PEG8, and optionally including or essentially including PEG6.

[0073] The combination can be used with anti-fluorescein CAR-T cells in the treatment of cancer. The combination can be used with anti-fluorescein CAR-T cells in the treatment of FAP-expressing cancer. The combination can be used with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancer. The combination can be used with anti-fluorescein CAR-T cells in the treatment of folate-expressing cancer. The first and second bispecific adapters of the combination can be formulated into separate pharmaceutical compositions.

[0074] A method for treating FAP-expressing (e.g., FAPα-expressing) cancer in a subject is further provided, comprising administering to the subject a cancer-treatment-effective amount of (i) an effective pharmaceutical composition comprising anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) CAR-T cells, or the same with a pharmaceutically acceptable carrier or excipient, and (ii) a dual-specific adapter as described herein, or the same with a pharmaceutically acceptable carrier or excipient, thereby treating the subject for cancer.

[0075] CARs may have a recognition region, which is a single-chain fragment variable (scFv) region of an anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) antibody. In certain embodiments, CARs may have a costimulatory domain, which is CD28, CD137(4-1BB), CD134(OX40), or CD278(ICOS). CARs may have an activation signaling domain, which is a T cell CD3ζ chain or Fc receptor γ.

[0076] In the methods described herein, (i) and (ii) may be administered simultaneously or sequentially by the same or different routes. Furthermore, optionally, (i) and (ii) of the method may be administered intravenously.

[0077] In a particular embodiment, a method is provided for treating cancer in a subject, comprising administering to the subject a cancer-treatment-effective amount of (i) anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, and (ii) any dual-specificity adapter described herein, a pharmaceutical composition described herein, or any combination described herein, thereby treating the subject for cancer.

[0078] The CAR of the method may include a recognition region containing a single-stranded fragment variable (scFv) region of an anti-fluorescein antibody, a costimulatory domain which is CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS), and / or an activation signaling domain which is a T cell CD3ζ chain or Fc receptor γ.

[0079] The fluorescein of the bispecific adapter of the method, upon exposure to anti-fluorescein CAR-T cells, can bind to the anti-fluorescein CAR-T cells with affinity, and when the targeted ligand of the bispecific adapter binds with affinity to the receptor of targeted cancer cells or cancer-associated fibroblasts (CAFs), the targeted ligand of the bispecific adapter ligates the bound anti-fluorescein CAR-T cells to such targeted cancer cells or CAFs. The receptors of the targeted cancer cells or targeted CAFs may be overexpressed FAP, overexpressed PSMA, and / or FR.

[0080] In certain embodiments, steps (i) and (ii) of the method are administered simultaneously or sequentially by the same or different routes. Step (ii) of the method may include any of the combinations described herein, where the first and second dual-specific adapters can be administered simultaneously to the subject by the same or different routes.

[0081] In a particular embodiment, step (ii) of the method comprises any of the combinations described herein, wherein the first dual-specific adapter and the second dual-specific adapter are administered to the subject sequentially in any order via the same or different routes.

[0082] Steps (i) and (ii) of the method may, for example, be administered intravenously, respectively.

[0083] The cancer may be an FAP-expressing cancer, and at least one of the bispecific adapters in step (ii) may contain a radical of the FAP ligand.

[0084] In certain embodiments of the method, the cancer is a PSMA-expressing cancer, and at least one bispecific adapter of step (ii) of the method contains a radical of a PSMA ligand.

[0085] The cancer may be an FR-expressing cancer, and step (ii) of the method may include any combination described herein.

[0086] Methods for treating FAP-expressing cancer in a subject are also provided. Such methods may include administering to the subject a cancer-treating amount of (i) anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, and (ii) any of the bispecific adapters or pharmaceutical compositions described herein, thereby treating the subject for cancer.

[0087] CARs may have a recognition region, which is the scFv region of an anti-fluorescein antibody. CARs may include a costimulatory domain that is CD28, CD137(4-1BB), CD134(OX40), or CD278(ICOS), and / or an activation signaling domain that is a T cell CD3ζ chain or Fc receptor γ.

[0088] Methods for treating cancer in a subject are also provided, such methods comprising administering to the subject a cancer-treating amount of (i) anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, and (ii) any combination described herein, thereby treating the subject for cancer. The CAR may comprise a recognition region including the scFv region of an anti-fluorescein antibody, a costimulatory domain which is CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS), and / or an activation signaling domain which is a T cell CD3ζ chain or Fc receptor γ.

[0089] In certain embodiments of the method, steps (i) and (ii) are administered simultaneously or sequentially by the same or different routes. The first and second dual-specific adapters of the combination can be administered to the subject simultaneously by the same or different routes. The first and second dual-specific adapters of the combination can be administered to the subject sequentially by the same or different routes. In certain embodiments of the method, steps (i) and (ii) are administered intravenously, respectively.

[0090] The methods described herein may further include imaging cancer in a subject. Imaging cancer may include imaging by optical imaging, positron emission tomography (PET), or single-photon emission computed tomography (SPECT).

[0091] The cancer may be ovarian cancer, endometrial cancer, breast cancer, glioma, for example, stage 3-4 glioma, or clear cell renal cell carcinoma, for example, stage 3-4 clear cell renal cell carcinoma,

[0092] Methods for enhancing CAR-T cell activation are also provided. In certain embodiments, such methods include preparing a bispecific adapter, a pharmaceutical composition, or a combination thereof, and exposing anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, to the bispecific adapter, the pharmaceutical composition, or the combination, wherein the CAR-T cells undergo enhanced activation after exposure compared to CAR-T cells not exposed to the bispecific adapter. When exposed to the bispecific adapter, the anti-fluorescein CAR-T cells may be systemically circulating in the subject.

[0093] Further kits are provided. The kits may include (i) a pharmaceutical composition comprising the bispecific adapter described above, or the same with a pharmaceutically acceptable carrier or excipient, and (ii) an anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) CAR-T cells, or the same with a pharmaceutically acceptable carrier or excipient.

[0094] In a particular embodiment, the kit comprises (i) at least one dose unit of any of the dual-specific adapters described herein, any of the pharmaceutical compositions described herein, or any combination described herein, and (ii) at least one dose unit of a pharmaceutical composition comprising anti-fluorescein CAR-T cells, or anti-fluorescein CAR-T cells with a pharmaceutically acceptable carrier or excipient, wherein (i) and (ii) are optionally in separate containers.

[0095] The above and other objects, features, and advantages of the present invention will become more apparent when considered in conjunction with the following description and drawings. [Brief explanation of the drawing]

[0096] [Figure 1]This figure shows graphs of fibroblast-activating protein (FAP)-fluorescein isothiocyanate (FITC), concentration (nmol / L), and mean FITC fluorescence intensity (MFI) related to the KD of FAP5 in human FAP (left) and mouse FAP (right). [Figure 2] Figure 2A is a graph showing ligand and MFI (allophycocyanin (APC)) indicative of FITC exposure. FAP-FITC coated cells were stained with an anti-FITC antibody possessing an APC signal, and APC MFI representing FITC exposure was measured. Figure 2B is a graph showing ligand and FITC (MFI) indicative of Bmax for MDA-MB231-hFAP. [Figure 3] This graph shows the results of a binding affinity assay, comparing FAP5-FITC concentration (nmol / L) with FITC (MFI). [Figure 4] Figure 4A is a graph showing the relationship between FAP5-FITC concentration (nM) and solubility percentage. Figure 4B is a graph showing the relationship between FAP5-FITC concentration (nM) and interferon-gamma (IFNγ) (pg / ml). [Figure 5-1] Figure 5A is a graph showing the relationship between days after CAR-T injection and tumor volume (mm3) for each group of tumor size. Figure 5B is a graph showing the relationship between days after CAR-T injection and tumor volume (mm3) for single-tumor disease controls. [Figure 5-2] Figure 5C is a graph showing the relationship between days after CAR-T injection and tumor volume (mm3) for single tumor CAR-T only. Figure 5D is a graph showing the relationship between days after CAR-T injection and tumor volume (mm3) for single tumor FAP5-PEG16-FITC. [Figure 5-3] Figure 5E is a graph showing the relationship between days after CAR-T injection and tumor volume (mm3) for a single tumor FAP8-PEG15-FITC. Figure 5F is a bar graph showing the relationship between hCD3+ cells / total viable cells % for the control and treatment groups. [Figure 6-1] Figure 6A is a graph showing the binding affinity of FAP8-FITC with PEG linkers of different lengths to hFAP, between FAP-FITC concentration (nM) and MFI(FITC). [Figure 6-2] Figure 6B is a graph showing the binding affinity of FAP8-FITC with different PEG linker lengths to mFAP, comparing FAP-FITC concentration (nM) with MFI(FITC). Figure 6C is a graph of FITC showing the results normalized to the mode (unstained vs. FAP8-PEG8-FITC, FAP8-PEG12-FITC, and FAP8-PEG15-FITC). [Figure 7-1] Figure 7A shows graphs of anti-FITC APCs normalized to the mode (unstained vs. aFITC-APC, FAP8-PEG8-FITC, FAP8-PEG12-FITC, and -PEG15-FITC). [Figure 7-2] Figure 7B is a graph showing the geometric mean FITC exposure of FAP8-FITC with PEG linkers of different lengths, as measured by aFITC-APC antibody. Figure 7C is a graph showing the relationship between days after CAR-T injection and tumor volume (mm3). [Figure 7-3] Figure 7D is a bar graph showing the control and treatment groups and the percentage of hCD3+ cells / total viable cells. [Figure 8-1] Figure 8A is a graph showing the killing of MDA-hFAP by FAP8-FITC adapters with PEG linkers of different lengths under adapter co-culture conditions, comparing time with mCh+ surface area (μm2). [Figure 8-2] Figure 8A is a graph showing the killing of MDA-hFAP by FAP8-FITC adapters with PEG linkers of different lengths under adapter co-culture conditions, comparing time with mCh+ surface area (μm2). [Figure 8-3] Figure 8A is a graph showing the killing of MDA-hFAP by FAP8-FITC adapters with PEG linkers of different lengths under adapter co-culture conditions, comparing time with mCh+ surface area (μm2). [Figure 8-4]Figure 8B is a graph of FAP-FITC concentration (nM) and total lysis % at 68 hours of co-culture, showing MDA-hFAP sterilization by 4M5.3 with FAP8-FITC adapters having PEG linkers of different lengths. Figure 8C is a graph of FAP-FITC concentration (nM) and IFNγ (pg / ml) at 68 hours, showing cytokine release. [Figure 8-5] Figure 8D is a graph showing the sterilization of MDA-hFAP by FAP8-FITC adapters with PEG linkers of different lengths under adapter cleaning conditions, comparing time with mCh+ surface area (μm2). [Figure 8-6] Figure 8D is a graph showing the sterilization of MDA-hFAP by FAP8-FITC adapters with PEG linkers of different lengths under adapter cleaning conditions, comparing time with mCh+ surface area (μm2). [Figure 8-7] Figure 8E is a graph of FAP-FITC concentration (nM) and total lysis % showing MDA-hFAP killing by 4M5.3 CAR-T cells under washing conditions and FAP8-FITC adapters with PEG linkers of different lengths at 68 hours. Figure 8F is a graph of FAP-FITC concentration (nM) and IFNγ (pg / ml) showing cytokine release at 68 hours. [Figure 9] Figure 9A is a graph comparing FAP-FITC concentration (nmole / L) and MFI(FITC) to show a comparison of the binding of FAP8-PEG15-FITC and FAP5-PEG16-FITC to MDA-MB-231 cells overexpressing hFAP. Figure 9B is a graph comparing FAP-FITC concentration (nmole / L) and MFI(FITC) to show a comparison of the binding of FAP8-PEG15-FITC and FAP5-PEG16-FITC to MDA-MB-231 cells overexpressing mFAP. [Figure 10]Figure 10A is a graph of anti-FITC APC showing results normalized to the mode (unstained, aFITC-APC only, FAP8-PEG15-FITC, and FAP5-PEG16-FITC). Figure 10B is a graph showing the FITC exposure (geometric mean) of FAP8-FITC with PEG linkers of different lengths by aFITC-APC antibody. [Figure 11] This graph compares the dissociation of FAP8-PEG16-FITC, FAP8-PEG12-FITC, FAP8-PEG15-FITC, and FAP5-PEG16-FITC, showing the relationship between time after incubation (hours) and MFI (APC). [Figure 12-1] Figure 12A is a graph showing the relationship between time and mCh+ surface area (μm2) in which MDA-hFAP is killed by FAP8-PEG15-FITC and FAP5-PEG16-FITC adapters under adapter co-culture and adapter washing conditions. [Figure 12-2] Figure 12A is a graph showing the relationship between time and mCh+ surface area (μm2) in which MDA-hFAP is killed by FAP8-PEG15-FITC and FAP5-PEG16-FITC adapters under adapter co-culture and adapter washing conditions. [Figure 12-3] Figure 12B is a graph showing the relationship between FAP-FITC concentration (nM) and total solubility % (left) or IFNγ (pg / ml) (right) for FAP8-PEG15-FITC and FAP5-PEG16-FITC under adapter co-culture and adapter washing conditions. [Figure 12-4] Figure 12B is a graph showing the relationship between FAP-FITC concentration (nM) and total solubility % (left) or IFNγ (pg / ml) (right) for FAP8-PEG15-FITC and FAP5-PEG16-FITC under adapter co-culture and adapter washing conditions. [Figure 13-1] Figure 13A is a bar graph showing the control and treatment groups and the percentage of hCD3+ cells / total viable cells. [Figure 13-2] Figure 13B is a bar graph showing IFNγ (pg / ml) levels for the control and treatment groups. [Figure 14] This graph shows the relationship between the number of days after CAR T cell injection and the percentage change in body weight. [Figure 15] Figure 15A is a bar graph comparing an FAP-FITC adapter with an increased PEG linker length to MFI (anti-FITC APC). Figure 15B is a bar graph comparing an FAP-FITC adapter with an increased PEG linker length to FITC (MFI). [Figure 16] Figure 16A is a graph showing the relationship between FAP-FITC concentration (nmol / L) and total dissolved %. Figure 16B is a graph showing the relationship between FAP-FITC concentration (nmol / L) and IFNγ (pg / ml). [Figure 17] Figure 17A is a graph showing the relationship between FAP-FITC concentration (nmol / L) and total dissolved %. Figure 17B is a graph showing the relationship between FAP-FITC concentration (nmol / L) and IFNγ (pg / ml). [Figure 18] Figure 18A is a graph showing the relationship between FAP-FITC concentration (nmol / L) and total dissolved %. Figure 18B is a graph showing the relationship between FAP-FITC concentration (nmol / L) and IFNγ (pg / ml). [Figure 19-1] Figure 19A is a graph showing the relationship between the number of days after CAR T cell injection and tumor volume (mm3). Figure 19B is a graph showing the relationship between the number of days after CAR T cell injection and tumor volume (mm3). [Figure 19-2] Figure 19C is a graph showing the relationship between the number of days after CAR T cell injection and tumor volume (mm3). Figure 19D is a graph showing the relationship between the number of days after CAR T cell injection and tumor volume (mm3). [Figure 19-3] Figure 19E is a graph showing the relationship between the number of days after CAR T cell injection and tumor volume (mm3). Figure 19F is a graph showing the relationship between the number of days after CAR T cell injection and tumor volume (mm3). [Figure 20] Figure 20A shows graphs of IFNγ (pg / ml) for the control and treatment groups. Figure 20B shows graphs of CAR+ count / μl blood for the control and treatment groups. [Figure 21] Figure 21A shows graphs of IFNγ (pg / ml) for the control and treatment groups. Figure 21B shows graphs of CAR+ count / μl blood for the control and treatment groups. [Figure 22] Figure 22A shows graphs of IFNγ (pg / ml) for the control and treatment groups. Figure 22B shows graphs of CAR+ count / μl blood for the control and treatment groups. [Figure 23] Figure 23A shows graphs of IFNγ (pg / ml) for the control and treatment groups. Figure 23B shows graphs of CAR+ count / μl blood for the control and treatment groups. [Figure 24] This graph shows the control group, the treatment group, and the ratio of CAR+ cells to 50,000 live cells. [Figure 25-1] This is a diagram showing a dual specificity adapter structure. [Figure 25-2] This is a diagram showing a dual specificity adapter structure. [Figure 25-3] This is a diagram showing a dual specificity adapter structure. [Figure 25-4] This is a diagram showing a dual specificity adapter structure. [Figure 25-5] This is a diagram showing a dual specificity adapter structure. [Figure 25-6] This is a diagram showing a dual specificity adapter structure. [Figure 25-7] This is a diagram showing a dual specificity adapter structure. [Figure 26-1] Figure 26A is a graph comparing FAP5-PEG16-FITC and FAP8-PEG15-FITC, showing the relationship between days after CAR T injection and tumor volume (mm3). [Figure 26-2] Figure 26B is a graph showing the relationship between days after CAR T injection and tumor volume (mm3) for single-tumor disease controls. Figure 26C is a graph showing the relationship between days after CAR T injection and tumor volume (mm3) for single-tumor CAR T only. [Figure 26-3] Figure 26D is a graph showing the relationship between days after CAR T injection and tumor volume (mm3) for a single tumor FAP5-PEG16-FITC. Figure 26E is a graph showing the relationship between days after CAR T injection and tumor volume (mm3) for a single tumor FAP8-PEG15-FITC. [Figure 27] This graph shows the treatment group and the percentage of hCD3+ cells / total viable cells. [Figure 28-1] This figure shows the chemical structures of DUPA-FITC (2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) linked to fluorescein isothiocyanate (FITC)) and (((S)-5-amino-1-carboxypentyl)carbamoyl)-L-glutamic acid (PSMAL1), which have different PEG linkers. [Figure 28-2] This figure shows the chemical structures of DUPA-FITC (2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) linked to fluorescein isothiocyanate (FITC)) and (((S)-5-amino-1-carboxypentyl)carbamoyl)-L-glutamic acid (PSMAL1), which have different PEG linkers. [Figure 28-3] This figure shows the chemical structures of DUPA-FITC (2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) linked to fluorescein isothiocyanate (FITC)) and (((S)-5-amino-1-carboxypentyl)carbamoyl)-L-glutamic acid (PSMAL1), which have different PEG linkers. [Figure 29] This graph shows the effect of linker length on binding affinity to PSMA, comparing the average fluorescence intensity (MFI) of [FL-DUPA](nM) and FITC. [Figure 30] This is a graph showing the relationship between cell lines and MFI of APC-anti-PSMA. [Figure 31] Figure 31A is a graph of MFI between DUPA-FITC and FITC with different PEG linkers in MDA-PSMA cells. Figure 31B is a graph of MFI between DUPA-FITC and APC-anti-FITC with different PEG linkers in MDA-PSMA cells. [Figure 32-1]Figure 32A is a graph showing the cytotoxicity of anti-FITC CAR-T cells against HOS-143b-PSMA cells mediated by FITC-PEG-DUPA, between [DUPA-FITC] (nm) and lysis (%). Figure 32B is a graph showing the IFNγ release from anti-FITC CAR-T cells mediated by FITC-PEG-DUPA when co-cultured with HOS-PSMA cells, between [DUPA-FITC] (nm) and IFNγ (pg / ml). Figure 32C is a graph showing the cytotoxicity of anti-FITC CAR-T cells against LNCap cells mediated by FITC-PEG-DUPA, between [DUPA-FITC] (nm) and lysis (%). Figure 32D is a graph showing the IFNγ release from anti-FITC CAR-T cells mediated by FITC-PEG-DUPA when co-cultured with LNCap cells, between [DUPA-FITC] (nm) and IFNγ (pg / ml). [Figure 32-2] Figure 32E is a graph showing the cytotoxicity of anti-FITC CAR-T cells against 22Rv1 cells mediated by FITC-PEG-DUPA, between [DUPA-FITC] (nm) and lysis (%). Figure 32F is a graph showing the IFNγ release from anti-FITC CAR-T cells mediated by FITC-PEG-DUPA when co-cultured with 22Rv1 cells, between [DUPA-FITC] (nm) and IFNγ (pg / ml). [Figure 33-1] Figure 33A shows the protocol used in Example 33. [Figure 33-2] Figure 33B is a graph showing the relationship between the time after injection and the average radiance (photons / s / cm2 / sr). [Figure 34-1] Figure 34A shows the timeline and dosing schedule for the in vivo study of Example 8, which tested DUPA-FITC with different PEG linkers. [Figure 34-2] Figure 34B is a graph showing the relationship between the number of days after CAR-T injection and tumor volume (mm3). Figure 34C is a graph showing the relationship between the number of days after CAR-T injection and weight change (%). [Figure 35]This is a graph comparing [PSMA ligand-FITC](nm) and DUPA-PEG6-FITC. [Figure 36-1] Figure 36A shows a confocal image. [Figure 36-2] Figure 36B shows flow cytometry data. [Figure 36-3] Figure 36C shows flow cytometry data. [Figure 37] Figure 37A shows [PSMA ligand-FITC](nM) and its dissolution (%). Figure 37B shows [PSMA ligand-FITC](nM) and IFNγ (pg / ml). [Figure 38] This figure shows the protocol used in Example 38 and the fluorescence images at the indicated time points of MDA-PSMA tumor-bearing mice that were intravenously injected with the indicated adapter at 500 nmol / kg. [Figure 39] This figure shows the protocol used in Example 39, as well as the total FITC and surface FITC of the MDA-MB-231 PSMA tumor with respect to the indicated adapter 48 hours after injection. The results 144 hours after injection with PSMAL1-PEG6-FITC are also shown. [Figure 40-1] Figure 40A shows the protocol used in Example 14. Figure 40B shows a graph of the relationship between the number of days after CAR-T injection and tumor volume (mm3). [Figure 40-2] Figure 40C is a graph showing the relationship between the number of days after CAR-T injection and the change in body weight (%). [Figure 41-1] Figure 41A shows the protocol used in Example 15. [Figure 41-2] Figure 41B is a graph showing the relationship between the number of days after CAR-T injection and tumor volume (mm3). Figure 41C is a graph showing the relationship between the number of days after CAR-T injection and weight change (%). [Figure 42-1]Figure 42A illustrates a TagCAR lentivirus particle containing a multidomain fusion protein consisting of a coca glycoprotein and anti-CD3 scFV sandwiched between a coca glycoprotein and a costimulatory protein. [Figure 42-2] Figure 42B is a representative flow plot 1 hour after incubation of peripheral blood mononuclear cells with TagCAR lentiviral particles, where an anti-coca antibody is used to detect TagCAR lentiviral particles bound to cells. Figure 42C is a graph showing the mean (+ / -SEM) percent of coca and gMFI for a circulating immune cell subset after incubation of peripheral blood mononuclear cells (PBMCs) with TagCAR lentiviral particles. [Figure 42-3] Figure 42D is a graph showing the mean (+ / - SEM) percentages of CD25+ (activated) and TagCAR+ (transduced) CD3+ T cells on days 3 and 7, respectively, after transduction of TagCAR lentiviral particles into peripheral blood mononuclear cells. [Figure 43-1] Figure 43A illustrates a competitive assay defining the FITC ligand / TagCAR interaction, in which TagCAR T cells are incubated with saturation levels of FL-AF647 and then competitively replaced with gradually increasing concentrations of FITC ligand. [Figure 43-2] Figure 43B is a graph showing the mean FL-AF647 fluorescence intensity (MFI) for TagCAR+ T cells in the presence of gradually increasing concentrations of PSMAL1-PEG6-FITC or fluorescein sodium (NaFL) as an antigen-only control. Figure 43C is a graph showing the mean calculated inhibition constant (Ki) for NaFL and PSMAL1-PEG6-FITC (3 rows = TagCAR T cells from different donors). [Figure 44]Figure 44A illustrates the detection of surface fluorescein antigen (SurfaceTag) on ​​MDA-MB-231 tumor cells using an anti-fluorescein antibody. Figure 44B is a graph showing SurfaceTag levels (APC MFI) after incubation of PSMA-overexpressing (PSMA+) or wild-type (PSMA-) MDA-MB-231 tumor cells with PSMAL1-PEG6-FITC. [Figure 45-1] Figure 45A is a graph showing the mean (+ / -SEM) normalized tumor cell growth rate over time in the presence of TagCAR T cells containing PSMAL1-PEG6-FITC. Figure 45B is a graph showing the mean (+ / -SEM) cytokine (left, IFNg; right, IL-2) levels in the culture supernatant 24 hours after the addition of TagCAR T cells and different concentrations of PSMAL1-PEG6-FITC to PSMA-overexpressing MDA-MB-231 tumor cells. [Figure 45-2] Figure 45C is a graph showing the mean (+ / -SEM) percentage of TagCAR+ cells after 88 hours of co-culture with PSMA-overexpressing MDA-MB-231 tumor cells and different concentrations of PSMAL1-PEG6-FITC. Figure 45D is a graph showing the CD25+ percentage of TagCAR+ and TagCAR-CD3+ T cells after 88 hours of co-culture with PSMA-overexpressing MDA-MB-231 tumor cells and different concentrations of PSMAL1-PEG6-FITC. [Figure 46-1] Figure 46A shows a series of graphs comparing days and tumor volume (mm3) for grouped tumor size (4M5.3 group), single tumor disease control, single tumor 4M5.3 CAR-T cells only, and single tumor 4M5.3 and FAP5-PEG8-FITC. [Figure 46-2]Figure 46A shows a series of graphs showing the relationship between days and tumor volume (mm3) for grouped tumor size (4M5.3 group), single tumor disease control, single tumor 4M5.3 CAR-T cells only, and single tumor 4M5.3 and FAP5-PEG8-FITC. Figure 46B shows a series of graphs showing the relationship between days and tumor volume (mm3) for group tumor size (E2 group), single tumor disease control, single tumor E2 CAR-T cells only, and single tumor E2 and FAP5-PEG8-FITC. [Figure 46-3] Figure 46B shows a series of graphs comparing days and tumor volume (mm3) for group tumor size (E2 group), single tumor disease control, single tumor E2 CAR-T cells only, and single tumor E2 and FAP5-PEG8-FITC. [Figure 46-4] Figure 46C is a graph showing the mouse body weight over the course of the treatment, comparing the number of days with the percentage change in body weight. [Figure 47-1] Figure 47A is a graph showing the flow cytometry results of in vitro elimination (E:T=1:1) of MDA-MB-231-hFAP cells by 4M5.3 CAR-T cells after 24 hours of co-culture, comparing FAP5-PEG16-FITC concentration with total lysis percentage. [Figure 47-2] Figure 47B is a graph of time and AU showing the Incucyte results of an in vitro killing assay (E:T=1:1) of MDA-MB-231-hFAP cells by 4M5.3 CAR-T cells over 68 hours of co-culture. [Figure 48-1] Figure 48A is a graph showing the effect of treatment on tumor size, relating the number of days to tumor volume (mm3). [Figure 48-2] Figure 48B is a graph showing the effect of treatment on the body weight of mice, comparing the number of days with the percentage change in body weight. [Figure 48-3] Figure 48C shows graphs relating treatment to hCD3+ count / μL blood, and treatment to hIFNγ (pg / ml). [Figure 48-4] Figure 48D shows a graph of the relationship between treatment and hCD3+ / live cells (%). [Figure 48-5]Figure 48E shows the experimental design, a graph of the relationship between the number of days after CAR-T injection and tumor volume (mm3), a graph of the relationship between the treatment and hCD3+ T cells / total tumor cells (%), and a graph of the relationship between the treatment and hIFNγ (pg / mL). [Figure 48-6] Figure 48E shows the experimental design, a graph of the relationship between the number of days after CAR-T injection and tumor volume (mm3), a graph of the relationship between the treatment and hCD3+ T cells / total tumor cells (%), and a graph of the relationship between the treatment and hIFNγ (pg / mL). [Figure 48-7] Figure 48F shows immunohistochemistry (IHC) slides stained with anti-mouse alpha smooth muscle actin (a marker for CAF), and immunohistochemistry (IHC) slides stained with anti-mouse Ki67 as a cancer cell proliferation marker. [Figure 48-8] Figure 48F shows immunohistochemistry (IHC) slides stained with anti-mouse alpha smooth muscle actin (a marker for CAF), and immunohistochemistry (IHC) slides stained with anti-mouse Ki67 as a cancer cell proliferation marker. [Figure 49-1] Figure 49A shows the drug administration schedule for combination therapy with FAP5-PEG8-FITC and DUPA-PEG6-FITC. Figure 49B is a graph showing the relationship between the number of days after CAR-T injection and tumor volume (mm3), illustrating HOS-PSMA4+ tumor growth. [Figure 49-2] Figure 49C is a graph showing the number of human T cells in the blood, with respect to treatment and human T cell count / μl. Figure 49D is a graph showing the number of human T cells in HOS-PSMA tumors (left) and h-IFNγ derived from mouse blood (right), with respect to treatment and human CD3+ T cells / live cells (%). [Figure 50-1] This figure shows the administration schedule for combination therapy with FAP5-PEG8-FITC and DUPA-PEG6-FITC, a graph of the relationship between days after CAR-T injection and tumor size (mm3) for DUPA-PEG6-FITC alone and DUPA-PEG6-FITC in combination with FAP5-FITC, and a graph of the relationship between treatment and human T cell count / μL blood, showing the number of human T cells in the blood on day 29. [Figure 50-2] This figure shows the administration schedule for combination therapy with FAP5-PEG8-FITC and DUPA-PEG6-FITC, a graph of the relationship between days after CAR-T injection and tumor size (mm3) for DUPA-PEG6-FITC alone and DUPA-PEG6-FITC in combination with FAP5-FITC, and a graph of the relationship between treatment and human T cell count / μL blood, showing the number of human T cells in the blood on day 29. [Figure 51-1] Figure 51A shows the timeline and dosing schedule for in vivo studies testing Aza-PEG6-FITC and ortho-CAL-PEG6-FITC in combination with EC17. [Figure 51-2] Figure 51B is a graph showing tumor growth curves for different treatment groups, comparing days after CAR-T cell injection with tumor volume (mm3). Aza-PEG6-FITC combined with EC17 significantly inhibited KB tumor growth. Ortho-CAL-PEG6-FITC combined with EC17 also showed slightly better inhibition of KB tumor growth. Figure 51C is a graph showing weight change in mice in different treatment groups, comparing days after CAR-T cell injection with weight change (%). Aza-PEG6-FITC and EC17 induced weight loss in mice. [Figure 52-1] Figure 52A shows the timeline and dosing schedule for in vivo studies testing different CAIX bispecificity adapters. [Figure 52-2]Figure 52B is a graph showing tumor growth curves for different treatment groups, comparing days after CAR-T cell injection with tumor volume (mm3). Aza-PEG6-FITC combined with FAP8-PEG18-FITC slightly inhibited KB tumor growth. Its efficacy was comparable to that of the EC17 and FAP8-PEG18-FITC combination. Ortho-CAL-PEG6-FITC combined with FAP8-PEG18-FITC showed better efficacy in inhibiting KB tumor growth. Figure 52C is a graph showing weight change (%) in mice across different treatment groups, comparing days after CAR-T cell injection with weight change (%). None of the combinations induced significant weight loss in treated mice. [Figure 53-1] Figure 53A shows the timeline and medication schedule for the in vivo study. [Figure 53-2] Figure 53B is a graph showing tumor growth curves for different treatment groups, comparing days after CAR-T cell injection with tumor volume (mm3). Aza-PEG6-FITC combined with FAP8-PEG18-FITC and EC17 showed comparable efficacy to ortho-CAR-PEG6-FITC combined with FAP8-PEG18-FITC and EC17. Both showed slightly better efficacy than the combination of EC17 and FAP8-PEG18-FITC in inhibiting KB tumor growth. Figure 53C is a graph showing weight change (%) in mice in different treatment groups, comparing days after CAR-T cell injection with weight change (%). Mice injected with Aza-PEG6-FITC and the combination of EC17 and FAP8-PEG18-FITC showed weight loss during treatment. Weight loss may have been due to cytokine release from increased CAR-T cells. Toxicity can be minimized by optimizing the dosage of the adapters. [Figure 54-1] Figure 54A is a graph of FAP-FITC concentration (nmol / L) and MFI(FITC). Figure 54B is a graph of FAP-FITC concentration (nmol / L) and MFI(FITC). [Figure 54-2]Figure 54C is an IHC image showing that KB tumors had less CAR-T cell infiltration than MDA-MB231 tumors (top panel), and that KB tumors contained more mFAP+CAF, which may be limiting CAR-T cell infiltration (bottom panel). [Figure 55-1] Figure 55A is a graph showing the relationship between FAP-FITC concentration (nmol / L) and total dissolution percentage. [Figure 55-2] Figure 55B is a graph showing the relationship between FAP-FITC concentration (nmol / L) and total dissolution percentage. [Figure 56-1] Figure 56A is a graph showing the relationship between the number of days after CAR T cell injection and tumor volume (mm3). [Figure 56-2] Figure 56B is a graph showing the relationship between the number of days after CAR T cell injection and tumor volume (mm3). Figure 56C is a graph showing the relationship between the number of days after CAR T cell injection and tumor volume (mm3). [Figure 56-3] Figure 56D is a graph showing the relationship between the number of days after CAR T cell injection and tumor volume (mm3). Figure 56E is a graph showing the relationship between the number of days after CAR T cell injection and tumor volume (mm3). [Figure 57] Figure 57A is a graph showing the relationship between the treatment group and IFNγ (pg / ml). Figure 57B is a graph showing the relationship between the treatment group and CAR+ count / μl blood. [Figure 58] Figure 58A is a graph showing the relationship between the treatment group and IFNγ (pg / ml). Figure 58B is a graph showing the relationship between the treatment group and CAR+ count / μl blood. [Figure 59] This graph shows the treatment group and the ratio of CAR+ cells to 50,000 live cells. [Figure 60] This figure shows the hCD3-stained IHC images of the treatment group, where an increase in T cell infiltration was observed after FAP-FITC treatment. [Modes for carrying out the invention]

[0097] This disclosure is based, at least in part, on the finding that cancer-associated fibroblasts (CAFs) may influence the efficacy of chimeric antigen receptor (CAR)-T cells in the treatment of solid tumors. For example, KB tumors (human epithelial carcinoma) and MDA-MB-231 tumors (human invasive ductal carcinoma) were transplanted into NOD scid gamma (NSG) mice. Mice from both groups were treated with anti-fluorescein isothiocyanate (FITC) CAR-T and EC17. Both tumors were then harvested from the mice for tumor fixation, fixed overnight with 10% formalin, and rinsed with 70% ethanol. The tumor cells were then subjected to immunohistochemical (IHC) staining with either an anti-human CD3 antibody to detect human CAR-T cells or an anti-mouse fibroblast-activating protein (FAP) antibody to detect mouse FAP+CAFs. Anti-human CD3 antibody staining showed that CAR-T cells invaded the MDA-MB-231 tumors but not the KB tumors. Anti-mouse FAP antibody staining showed significant invasion of FAP+CAF into KB tumors and substantially less invasion of FAP+CAF into MDA-MB-231 tumors.

[0098] In consideration of the foregoing, a bispecific adapter or a pharmaceutically acceptable salt or hydrate thereof is provided for use with anti-fluorescein (e.g., fluorescein, FITC, or N-hydroxysuccinimide (NHS)-fluorescein) CAR-T cells in the treatment of FAP-expressing (e.g., FAPα or FAPβ-expressing) cancer or prostate-specific membrane antigen (PSMA)-expressing cancer.

[0099] In a particular embodiment, the dual-specific adapter has the following structure: FL-TL containing or a pharmaceutically acceptable salt or hydrate thereof [in the formula, F includes a CAR-T cell targeting moiety, such as fluorescein, FITC, or NHS-fluorescein. L includes the linker, TLs include cancer-targeting or cancer-associated cell-targeting ligands containing FAP ligands, PSMA ligands, or any of the aforementioned radicals. That is the case.

[0100] The use of a bispecific adapter may enable the use of a single CAR-T cell that exhibits a molecule that binds to fluorescein on its surface, i.e., a "general-purpose" CAR-T cell. When a general-purpose CAR-T cell, e.g., one that exhibits a molecule that binds to fluorescein on its surface, is used with a bispecific adapter that binds to the cell surface receptors of CAFs and tumor cells, e.g., one containing FITCs attached to the molecule (e.g., by linkers and / or spacers), the T cell can kill the CAFs and tumor cells to which it is bound.

[0101] This method can reduce the cost of creating CAR-T cells that can bind to various cancers expressing diverse cell surface receptors. Instead, by altering a portion of the bispecific adapter that binds to tumor cell surface receptors, versatile CAR-T cells can bind to various types of cancer. Thus, the bispecific adapter can improve the immune response induced by CAR-T cells bound to tumor cells and inhibit the action of CAFs.

[0102] CAR T cell targeting moiety The CAR T cell targeting portion of the dual-specificity adapter may be fluorescein, FITC, NHS-fluorescein, or any other portion that can be manipulated to be recognized and specifically bound to CAR.

[0103] "Binding with specificity," "binding with high affinity," or "specifically" or "selectively" refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologics, when referring to ligand / receptor, recognition region / targeting region, antibody / antigen, or other binding pairs. Therefore, under given conditions, a specified ligand or recognition region will bind to a specific receptor (e.g., one present on cancer cells or CAR T cells) or targeting region, respectively, and not in significant amounts to other proteins present in the sample (e.g., those associated with normal, healthy cells). Specific binding or binding with high affinity may also mean, for example, that a binding compound, ligand, antibody, or binding composition derived from the antigen-binding site of an antibody binds to its target with an affinity that is often at least 25%, more often at least 50%, most often at least 100% (2x), generally at least 10x, more generally at least 20x, and most generally at least 100x greater than its affinity to any other binding compound. In a typical embodiment, the molecules that specifically bind to the target are determined, for example, by scatchard analysis, at least about 10 6 liters / mol(K) D =10 -6 M) Preferably, it may have an affinity of at least about 10 liters / mol.

[0104] Targeted ligand As described above, the bispecific adapter may contain cancer-targeting or cancer-associated cell-targeting ligands, including FAP ligands, PSMA ligands, folate receptors (FR), or radicals of any of the aforementioned. When administered, the targeting ligands target the bispecific adapter compound to the cancer or tumor of interest or to the cancer-associated cells of interest. In some embodiments, the targeting moieties (their free forms, i.e., in their radicals) do not bind to the uptake receptors of untargeted cells.

[0105] Generally, tumors may contain infiltrating immune and inflammatory cells that support tumor growth and development through growth factor secretion, immunosuppression, metastasis, resistance, etc., such as cancer-associated fibroblasts (CAFs), extracellular matrix (ECM) proteins, T cells, tumor-associated macrophages (TAMs), myeloid suppressor cells, and vascular and lymphatic systems. CAFs are one of the major types of cells present in the tumor stroma and play several crucial roles in promoting tumor growth. These functions include ECM production, remodeling, and cytokine secretion, which can lead to angiogenesis that promotes tumor growth, secretion of signaling factors that increase chemotherapy resistance, a denser tumor stroma resulting in physical blockage of immune cells, and enhanced cell motility leading to metastasis. In some cases, such processes are comparable to the behavior of pathogenic fibroblasts in fibrous diseases.

[0106] In some cases, a common marker for CAF is fibroblast-activated protein alpha (FAPα). FAPα is a serine protease found (primarily) on the cell surface of activated fibroblasts in affected cells and tissues in fibrous diseases, inflammatory diseases, and / or cancers (e.g., fibrosis, rheumatoid arthritis, wound healing, and cancer). FAP is expressed on the surface of CAF and has been shown to correlate with poor patient prognosis in several solid tumors. In addition, virtually all human solid tumors overexpress FAP.

[0107] For example, over 90% of epithelial carcinomas show FAPα expression in immunohistochemical (IHC) staining. Further FAPα expression has been observed in a subset of primary glioma cell cultures and TAMs. In recent years, FAPα expression has been detected in at least 28 different types of human cancer. However, FAPα expression is very low or absent in most healthy adult tissues. Therefore, because its expression is restricted to the surface of affected cells such as carcinomas, FAPα is given unique qualification as a receptor for the selective delivery of drug therapeutics to tumors via ligand targeting.

[0108] The dual-specificity adapters described herein may include a linker-bound FAP-targeting ligand (or its radical), where the linker is further bound to a CAR-targeting moiety. FAP is a type II membrane-bound serine protease that cleaves proline-amino acid peptide bonds and can be expressed in collagen-producing CAFs and myofibroblasts. In certain embodiments, the dual-specificity adapter can target CAR T cells coupled to the CAR-targeting moiety of the dual-specificity adapter to FAP-expressing cancers or fibrous or inflammatory diseases. In certain embodiments, this improved FAP ligand skeleton can be further used with an albumin-binding moiety to achieve targeted delivery of radiolabeled groups and other functional groups. In certain embodiments, the FAP ligand is a high-affinity FAP ligand comprising a triazole moiety (or its derivative) introduced into the ligand skeleton. In certain embodiments, the FAP ligand is a high-affinity FAP ligand comprising a triazole moiety (or its derivative) and a phenyl ring introduced into the ligand skeleton (e.g., an isoindoline ring skeleton). Unless otherwise specified, "high affinity" or "higher affinity" with respect to the target of a ligand means a ligand having a Schrödinger molecular docking score of at least approximately -8.0 kcal / mol. In certain embodiments, a high-affinity FAP ligand has improved affinity for FAP compared to a ligand without a triazole moiety.

[0109] The targeted portion may be a radical of an FAPα ligand having a molecular weight of, for example, less than approximately 10,000, less than 7,500, less than 5,000, less than 2,500, less than 1,000, less than 760, less than 500, approximately 500 to approximately 10,000 g / mol, approximately 1,000 to approximately 7,500 g / mol, approximately 750 g / mol to approximately 1,500 g / mol, approximately 1,000 to approximately 5,000 g / mol, or approximately 500 to approximately 2,500 g / mol.

[0110] The targeted ligand can bind to activated fibroblasts expressing FAP (e.g., FAPα or FAPβ), where such activated fibroblasts are involved in cancer. In certain embodiments, the targeted ligand may have a binding affinity for FAP (e.g., FAPα) between about 1 nM and about 25 nM, for example, in the range of 1 nM to about 25 nM or about 1 nM to 25 nM.

[0111] In a particular embodiment, the FAP ligand is

[0112] [ka] [In the formula,

[0113] [ka] This is the connection point of the adapter to the linker. FAP5 has or contains the following structure.

[0114] In a particular embodiment, the FAP ligand or its radical is

[0115] [ka] [In the formula,

[0116] [ka] This is the connection point of the adapter to the linker. FAP8 has or contains the following structure.

[0117] In a particular embodiment, the FAP ligand or its radical is given by formula IB:

[0118] [ka] [In the formula,

[0119] [ka] This is the connection point of the adapter to the linker, T is a substituted or unsubstituted methylene group (-CH2-), a substituted or unsubstituted amino group (-NH-), -O-, or -S-. R 1 and R 2 Each of these is independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl. R 3 and R 4 These are -H, -OH, F, Cl, Br, I, and -C, respectively. 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 5 , R 6 , R 7 , and R 8 Each is independently selected from the group consisting of H, alkyl, and halo. R 9 , R 10 , and R 11 These are H and -C respectively. 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] It may be or may contain an FAP5 ligand or radical having the structure represented by .

[0120] FAP ligand or its radical is given by formula IC:

[0121] [ka] [In the formula,

[0122] [ka] This is the linking point to the linker, T is a substituted or unsubstituted methylene group (-CH2-), a substituted or unsubstituted amino group (-NH-), -O-, or -S-. R 1 and R 2 Each of these is independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl. R 3 and R 4 These are -H, -OH, F, Cl, Br, I, and -C, respectively. 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 5 , R 6 , R 7 , and R 8 Each is independently selected from the group consisting of H, alkyl, and halo. R 9 , R 10 , and R 11 These are H and -C respectively. 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] It may be or may contain an FAP5 ligand or radical having the structure represented by .

[0123] In a particular embodiment, the FAP ligand or its radical has the following structure:

[0124] [ka] [In the formula,

[0125] [ka] This represents a functionalized 5-10 member N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, and optionally further contains 1-3 heteroatoms independently selected from O, N, and S. R1 and R2 are -H, -D, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R3 and R4 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R5 and R6 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R7 is selected from the group consisting of -H, -D, OH, CH2=, -CH3, CH3CH2-, (CH3)2CH-, (CH3)3C-, -CH2Ph, and substituted -CH2Ph. R8~R 10 is -H, -OH, -F, -Cl, -Br, -I, -NO2, -SO3H, -SO2NH2, -NH2, -N3, -NH=NH, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 11 -H, -D, C1~C 10 Alkyl, C3~C 10 Cycloalkyl, adamantyl,

[0126] [ka] , substituted or unsubstituted aryl, substituted or unsubstituted C7~C 20 Selected from the group consisting of alkylaryls, the aryl is

[0127] [ka] (In the formula, R 12 and R 16 These include -H, -D, halogens, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 It is selected from the group consisting of H, D, halogens, C1-C4 alkyls, and C1-C3 alkoxys. R 13 , R 14 and R 15 These include -H, -D, halogens, -OMe, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 (These are selected from the group consisting of -H, -D, halogens, C1-C4 alkyls, and C1-C3 alkoxys.) And, R 17 , R 18 , R 20 , and R 21 It is selected independently of -H and -CH3, R 19 and R 22 [These are independently selected from the group consisting of phenyl, dimethoxyphenyl, and aryl compounds.] It may have or contain an FAP8 ligand or radical.

[0128] In certain embodiments, the targeting ligand of the bispecific adapter includes a PSMA ligand or its radical. PSMA is expressed in the neovascular network of tumors in several cancers, including ovarian cancer (100%), endometrial cancer (100%), breast cancer (60%), stage 3-4 glioma (100%), and stage 3-4 clear cell renal cell carcinoma (100%). PSMA is also overexpressed in prostate cancer, but is hardly expressed in normal tissues. PSMA is expressed in the brain, but only minimally, and most of its ligands are polar and cannot cross the blood-brain barrier. Therefore, PSMA may be a useful targeting ligand in this context.

[0129] PSMA is an H-type cell surface membrane-bound glycoprotein with a molecular weight of approximately 110 kD, comprising an intracellular segment (amino acids 1-18), a transmembrane domain (amino acids 19-43), and a broad extracellular domain (amino acids 44-750). While the functions of the intracellular segment and transmembrane domain are currently considered unimportant, the extracellular domain is involved in several distinct activities. PSMA plays a role in the central nervous system, metabolizing N-acetyl-aspartylglutamate (NAAG) to glutamate and N-acetylaspartate. Therefore, it is sometimes referred to as N-acetyl-alpha-linked acidic dipeptidase (NAALADase). PSMA is also sometimes referred to as folate hydrolase 1 (FOLH I) or glutamate carboxypeptidase (GCP II) due to its role in the proximal small intestine, where it removes T-linked glutamate from poly-γ-glutamic folate and α-linked glutamate from peptides and small molecules.

[0130] Unlike many other membrane-bound proteins, PSMA undergoes rapid internalization into cells, similar to cell surface-bound receptors such as vitamin receptors. PSMA internalizes via clathrin-coated pits and can then be recycled to the cell surface or translocated to lysosomes. The dimeric and monomeric forms of PSMA are suggested to be interconvertible, although direct evidence of interconversion remains debated. Even if so, only the dimer of PSMA possesses enzymatic activity, while the monomer does not.

[0131] While the activity of PSMA on the cell surface of prostate cells is still under investigation, PSMA is a feasible target for selective and / or specific delivery of CAR T cells to PSMA-expressing cells.

[0132] In certain embodiments, the targeting ligand of the bispecific adapter comprises a PSMA ligand or its radical. PSMA may be or may comprise (((S)-5-amino-1-carboxypentyl)carbamoyl)-L-glutamic acid (PSMAL1). PSMA may be or may comprise 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) or its derivatives (see, for example, International Publication 2015 / 057852, which describes a DUPA derivative and incorporates teachings relating thereto by reference herein). When administered, the bispecific adapter compound comprising the PSMA ligand or its radical can target PSMA-expressing cancers in a subject.

[0133] Dual-specific adapters can be specifically designed and synthesized to achieve specific binding affinities for PSMA. For example, when PSMAL1-PEG6-FITC was compared with DUPA-PEG6-FITC in vitro and in vivo, PSMAL1-PEG6-FITC showed higher binding affinity for PSMA and longer retention times in PSMA-positive cells. Furthermore, PSMAL1-PEG6-FITC showed higher efficacy at low concentrations in mediating the function of anti-FITC CAR-T cells. PSMAL1-PEG6-FITC also demonstrated efficacy in mediating the eradication of tumors expressing low levels of PSMA by anti-FITC CAR-T cells.

[0134] In certain embodiments, the targeting ligand of the bispecific adapter comprises folic acid or its radical. "Folic acid" refers to FR-binding molecules (e.g., FRα or FRβ) including, for example, synthetic folic acid and its analogues and derivatives, such as, but not limited to, folinic acid, pteroylpolyglutamic acid, pteroyl-D-glutamic acid, and FR-binding pteridines, such as tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid, and their deaza and dideza analogues. The folic acid in the adapter containing fluorescein-linker-folic acid may be folic acid, dihydrofolic acid, tetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid (5,10-MTHF), 5-methyltetrahydrofolic acid (5-MTHF), or larcitrexed (which binds to FRα but not to FRβ).

[0135] The terms “deaza” and “dideaza” analogs refer to analogs recognized in the art in which one or two nitrogen atoms in a naturally occurring synthetic folic acid structure, or its analogues or derivatives, are substituted with carbon atoms. For example, deaza analogs include folic acid, folic acid, pteroylpolyglutamic acid, and folic acid receptor-binding pteridines, such as tetrahydropterin, dihydrofolic acid, and 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs of tetrahydrofolic acid. For example, dideaza analogs include 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs of folic acid. Other folic acid molecules useful as complex-forming ligands include folic acid receptor-binding analogs such as pemetrexed, proguanil, pyrimethamine, trimethoprim, pralatrexate, larcitrexed, aminopterin, ametopterin (also known as methotrexate), and N 10 -Methylfolate, 2-deamino-dydroxyfolate, deaza analogs, e.g., 1-deazametopterin or 3-deazametopterin, and 3',5'-dichloro-4-amino-4-deoxy-N 10 - It is methylpteroylglutamic acid (dichloromethotrexate).

[0136] Synthetic folic acid, as well as the aforementioned analogues and / or derivatives, are also referred to as "a folate," "the folate," or "folates," reflecting their ability to bind to FR such as FRα or FRβ. When conjugated with exogenous molecules, such molecules are effective in enhancing transmembrane transport via folate-mediated endocytosis and other mechanisms. The aforementioned can be used in fluorescein-linker-folate bispecific adapters.

[0137] FR alpha (FRα) is overexpressed in approximately 90% of ovarian cancers, 70% of endometrial cancers, 80% of triple-negative breast cancers, 80% of non-small cell lung cancers, and 65% of renal cell carcinomas. FR beta (FRβ) is overexpressed in immunosuppressive myeloid suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in the tumor microenvironment (TME).

[0138] Immunohistochemical (IHC) staining of solid tumors demonstrates that the limited efficacy of CAR-T cells in solid tumors may be attributable to CAF. KB tumor cells and MDA-MB-231 tumor cells were transplanted into NOD scid gamma (NSG) mice. Both mice were treated with anti-FITC CAR-T and EC17 (folate-FITC conjugate). Both tumors were then harvested from the mice for tumor fixation, fixed overnight with 10% formalin, and rinsed with 70% ethanol. The tumor cells were then subjected to IHC staining with either an anti-human CD3 antibody to detect human CAR-T cells or an anti-mouse FAP antibody to detect mouse FAP+CAF.

[0139] Anti-human CD3 antibody staining showed that CAR-T cells infiltrated MDA-MB-231 tumors but not KB tumors. Anti-mouse FAP antibody staining showed significant infiltration of FAP+CAF into KB tumors and substantially less infiltration of FAP+CAF into MDA-MB-231 tumors.

[0140] KB tumor cells (i.e., cell lines that form immunologically "cold" FR-expressing solid tumors) were administered to NSG mice, and CAR T cell toxicity was quantified in the presence of one or two bispecific adapters. Administration of general-purpose CAR-T cells followed by intravenous injection of FR-targeted bispecific adapters achieved significant antitumor efficacy, but co-injection of FAP-targeted bispecific adapters enhanced this efficacy to a level where it could be measured without apparent toxicity. Analysis of tumor masses throughout the course of therapy further revealed that co-administration of FAP-targeted bispecific adapters not only promoted CAF disappearance but also enhanced CAR-T cell infiltration and activation.

[0141] Therefore, in certain embodiments, the bispecificity adapter may comprise fluorescein conjugated (e.g., via a linker) to a radical of an FR ligand. Such a fluorescein-linker-folate bispecificity adapter can be used in combination with other bispecificity adapters herein for the treatment of cancer in a subject (e.g., fluorescein-L-PSMA and / or fluorescein-L-FAP).

[0142] The fluorescein-linker-folate bispecific adapter is given by formula V:

[0143] [ka] [In the formula, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently nitrogen (N), NH, CH, CH2, oxygen (O), or sulfur (S). Y is C, CH, CH2, N, NH, O, or S. Z is glutamate, valine, or a substrate. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is H or alkyl. m and n are independently 0, 1, or between 0 and 1.

[0144] [ka] This represents either a single or double bond in CC. It may include ligands (or radicals thereof) having the structure or functional fragments or analogs thereof.

[0145] In a further embodiment, as a non-limiting example, the ligand (or radical) of formula V is VI:

[0146] [ka] [In the formula, X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamate, valine, or a substrate. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is H or alkyl. m and n are independently 0, 1, or between 0 and 1.

[0147] [ka] This represents either a single or double bond in CC. It has the structure (or a functional fragment or analogue thereof).

[0148] Another specific ligand (or radical) of formula V (or its functional fragment or analogue) is formula VII:

[0149] [ka] [In the formula, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamate, valine, or a substrate. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is H or alkyl. m and n are independently 0, 1, or between 0 and 1.

[0150] [ka] This represents either a single or double bond in CC. It may have a structure.

[0151] In a particular embodiment, the ligand (or radical) of formula VI is formula VIII:

[0152] [ka] [In the formula, X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamate, valine, or a substrate. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is H or alkyl. m is 0, 1, or between 0 and 1.

[0153] [ka] This represents either a single or double bond in CC. It may have the structure or functional fragments or analogues thereof.

[0154] The ligand (or radical) of formula VI is formula IX:

[0155] [ka] [In the formula, X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamate, valine, or a substrate. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is H or alkyl. m is 0, 1, or between 0 and 1.

[0156] [ka] This represents either a single or double bond in CC. It may have the structure (or a functional fragment or analogue thereof).

[0157] The ligand (or radical) of formula VII is formula X or XI:

[0158] [ka] [In the formula, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamate, valine, or a substrate. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is H or alkyl. m is 0, 1, or between 0 and 1.

[0159] [ka] This represents either a single or double bond in CC. or

[0160] [ka] [In the formula, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamate, valine, or a substrate. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is H or alkyl. m is 0, 1, or between 0 and 1.

[0161] [ka] This represents either a single or double bond in CC. It may have the structure (or any functional fragment or analogue) of the above.

[0162] Table 1 provides non-limiting examples of additional embodiments of targeted ligands, including FR-targeted ligands (e.g., or radicals thereof) having the structure of formula VIII.

[0163] [Table 1-1]

[0164] [Table 1-2]

[0165] [Table 1-3]

[0166] Table 2 provides non-limiting examples of additional embodiments of targeting ligands for a bispecific adapter, including FR-targeting ligands (e.g., or radicals thereof) having the structure of formula IX.

[0167] [Table 2-1]

[0168] [Table 2-2]

[0169] Table 3 provides non-limiting examples of additional embodiments of the targeting ligands of the dual-specificity adapter herein, including FR-targeting ligands (or their radicals) having the structure of formula X'.

[0170] [Table 3]

[0171] Instead of folic acid, the targeting ligand (e.g., its radical) may be one or more non-classical folate antimetabolite analogs, such as pyrido[2,3-d]pyrimidine, or similar analogs (or their radicals) having the formula (or its analogue or functional fragment) listed in Table 4 below (e.g., the radical of the formula).

[0172] [Table 4-1]

[0173] [Table 4-2]

[0174] [Table 4-3]

[0175] [Table 4-4]

[0176] Linker The linker of the dual-specificity adapter described herein is positioned between the targeting ligand (e.g., its radical) and the CAR T cell targeting moiety (e.g., including fluorescein, FITC, or NHS-fluorescein). The linker may be any suitable linker.

[0177] As used herein, the term "linker" includes a chain of atoms that is biofunctionally configured to form a chemical bond, connecting a CAR T cell targeting moiety to a cancer or CAF targeting ligand to form a conjugate. Illustratively, an atomic chain may consist of carbon, nitrogen, oxygen, sulfur, silicon (Si), and phosphorus (P), e.g., C,N,O,S, and P, or C,N,O, and S.

[0178] The linker may include a wide variety of links in the range of approximately 2 to 100 atoms in a continuous skeleton, for example. The linker may include a triblock copolymer containing a releaseable form of PEG, a non-releaseable form of PEG, polyproline, hydrophilic amino acids, sugars, unnatural peptidoglycans, polyvinylpyrrolidone, or a central hydrophobic block of polypropylene glycol flanked by hydrophilic blocks of PEG on both sides.

[0179] The linker may contain PEG or a PEG derivative. The linker may be (PEG)3.

[0180] A linker can be non-release, i.e., not unstable. However, in some embodiments, it may be desirable for the linker in a dual-specific adapter to be releaseable, i.e., unstable, e.g., photocleavable, acid-unstable, base-unstable, or enzymatically cleavable. The term “releaseable” in the context of a linker means a linker containing at least one bond that is readily ruptured (e.g., chemically or enzymatically hydrolyzed) under physiological conditions, such as reducing agent instability, pH instability, acid instability, base instability, oxidative instability, metabolic instability, biochemical instability, enzymatic instability, or a multivalent releaseable bond based on p-aminobenzyl. It is understood that the physiological conditions resulting in the rupture of the bond do not necessarily involve biological or metabolic processes, but may instead include standard chemical reactions, such as hydrolysis reactions resulting from compartmentalization into organelles such as endosomes at physiological pH or at pH lower than cytoplasmic matrix pH. The cleavable bond can connect two adjacent atoms within the releaseable linker, and / or, for example, at one or both ends of the releaseable linker, to another linker portion or a targeting portion and / or CAR T cell targeting portion as described herein. In some examples, the releaseable linker is broken into two or more fragments. In some examples, the releaseable linker is separated from the CAR T cell targeting portion.

[0181] In some embodiments, the linker is configured such that the CAR T cell targeting ligand is cleaved from the cancer or CAF targeting moiety only after sufficient time has elapsed since administration for the bispecific adapter to circulate throughout the subject's systemic circulation (e.g., to give time for capture and internalization by targeting cells and / or receptors). In some embodiments, the period for release may vary (e.g., between subjects (e.g., based on various factors)). In some embodiments, the releaseable linker can be engineered to not be cleaved / released for at least 24 hours, or even up to a week, after administration. In some embodiments, the bispecific adapter can safely pass through the subject's system, and any amount not captured by targeting cells (e.g., those expressing FRα, FRβ, or PSMA, etc.) may be effluxed.

[0182] In contrast, the term “non-released” in the context of linkers means a linker containing at least one bond that is neither easily nor rapidly destroyed under physiological conditions. In some embodiments, the non-released linker contains a physiologically stable backbone (e.g., this backbone is resistant to hydrolysis (e.g., hydrolysis by water or enzymatic hydrolysis)). In some embodiments, the compositions provided herein containing a non-released linker do not release any components of the bispecific adapter (e.g., cancer or CAF-targeting ligand or CAR T cell-targeting ligand). In some embodiments, the non-released linker lacks disulfide bonds (e.g., SS) or esters in its backbone. In some embodiments, the composition contains a cancer or CAF-targeting ligand or CAR T cell-targeting ligand linked by a substantially stable backbone throughout the entire circulation period of the bispecific adapter (e.g., during endocytosis to target cell endosomes). The non-released linker may contain amides, esters, ethers, amines, and / or thioethers (e.g., thiomaleimides). Specific examples are provided herein, but it will be understood that any molecule can be used in a non-release linker, provided that at least one bond is formed that is neither easily broken nor rapidly broken under physiological conditions.

[0183] Perhaps more specifically, the non-release linker includes a linker that, at neutral pH, for example, less than 10 percent (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, or less than 0.001%) hydrolyzes in water (e.g., a buffered (e.g., phosphate-buffered) solution) within a certain period (e.g., 24 hours). In some embodiments, when a non-release linker is used, less than about 10 percent (10%), preferably less than 5 percent (5%) of the administered bispecific adapter releases the connected portion (e.g., in systemic circulation before uptake by targeted cells / tissues), or none of the administered bispecific adapters release the connected portion (e.g., in systemic circulation before uptake by targeted cells / tissues).

[0184] In some embodiments, the cancer or CAF-targeting ligand is not cleaved in vivo from the CAR T cell-targeting ligand or any CAR T cell-targeting ligand of the bispecific adapter. In some embodiments, this is advantageous because it allows the bispecific adapter to bind to CAR T cells and deliver them to targeted cancer cells or CAFs.

[0185] The linker length can be selected to optimize the separation of molecules on the target cell surface provided by the linker, which can facilitate the uptake of bound CAR T cells into the target cells (e.g., when a bispecific adapter is administered). The linker may have a chain length of at least about 5 nm. In certain embodiments, each linker is approximately 5 nm to 15 nm long. In some embodiments, the linker is at least about 7 nm long. In certain embodiments, each linker is approximately 7 nm long and flexible. In certain embodiments, each linker is approximately 7 to 10 nm long. In some embodiments, the linker is at least about 14 nm long. In some embodiments, the linker is approximately 15 nm long. In some embodiments, the linker is between approximately 7 nm and approximately 31 nm in length (e.g., approximately 7 to 31, 7 to approximately 31, or 7 to 31), between approximately 7 nm and approximately 24 nm in length (e.g., approximately 7 to 24, 7 to approximately 24, or 7 to 24), or between approximately 7 nm and approximately 20 nm in length (e.g., approximately 7 to 20, 7 to approximately 20, or 7 to 20). In some embodiments, the linker is between approximately 14 nm and approximately 31 nm in length (e.g., approximately 14 to 31, 14 to approximately 31, or 14 to 31), between approximately 14 nm and approximately 24 nm in length (e.g., approximately 14 to 24, 14 to approximately 24, or 14 to 24), or between approximately 14 nm and approximately 20 nm in length (e.g., approximately 14 to 20, 14 to approximately 20, or 14 to 20). In some embodiments, the linkers have chain lengths of at least 7 nm, at least 14 nm, at least 20 nm, at least 25 nm, at least 30 nm, or at least 40 nm, or 5 nm to 15 nm, 5 nm to 10 nm, 7 nm to 10 nm, 5 nm to 20 nm, 10 nm to 40 nm, or 25 nm to 100 nm. In certain embodiments, the length of each linker is selected to promote microclustering of bound molecules on the cell surface, resulting in spacings of 7 to 10 nm or about 7 to 10 nm between them (e.g., about 7 nm to about 10 nm, 7 nm to about 10 nm, about 7 nm to 10 nm, or 7 nm to 10 nm). The ranges specified in this paragraph include the stated endpoints, and all values ​​in 1 nm increments are included within the stated ranges.

[0186] The linker may contain at least one carbon-carbon bond and / or at least one amide bond. The linker may contain one or more L or D configurations, natural or unnatural amino acids, or any combination thereof.

[0187] In a particular embodiment, the linker is a group comprising one or more covalently connected structural units.

[0188] The linker can be further manipulated to optimize in vivo distribution, bioavailability, and PK / PD (e.g., of the bispecific adapter), and / or to increase previously described uptake (e.g., of the bispecific adapter) into target tissue, such as via PEGylation, according to methods commonly known or hereafter developed in the art.

[0189] In some embodiments, the linker may include one or more spacers (for example, to facilitate a specific release time, to facilitate increased uptake into targeted tissue, and / or to optimize the in vivo distribution, bioavailability, and / or PK / PD of the dual-specific adapter provided herein). The spacers may include one or more alkyl chains, PEGs, peptides, sugars, peptidoglycans, clickable linkers (e.g., triazoles), rigid linkers (e.g., polyprolines and polypiperidines), and the like.

[0190] In some embodiments, the linker of the dual-specificity adapter includes PEG, a PEG derivative, or any other linker known or to be developed in the art that can achieve the purposes described herein. In some embodiments, the linker is repeated n times, where n is a positive integer. For example, but not limited to, n may be any integer selected from the range of 1 to 16, 1 to 32, 1 to 64, or 1 to 96. The number of repetitions in the linker (i.e., n) can be selected to achieve the desired functionality, size, and / or potency of the compound, and / or to take into consideration the desired application. In some embodiments, the linker includes one or more spacers (for example, these may also be used to specifically design the properties of the dual-specificity adapter).

[0191] In a particular embodiment, the linker is PEG3~PEG 18 It includes, consists of, or is essentially derived from. In a particular embodiment, the linker is PEG3~PEG 12 It includes, consists of, or is essentially composed of. In certain embodiments, the linker includes, consists of, or is essentially composed of PEG3-PEG8. In certain embodiments, the linker includes PEG5-PEG 15 It includes, consists of, or is essentially derived from. In a particular embodiment, the linker is PEG6~PEG 14 It includes, consists of, or is essentially derived from. In a particular embodiment, the linker is PEG7~PEG 13 It includes, consists of, or is essentially derived from. In a particular embodiment, the linker is PEG8~PEG 12 It includes, consists of, or is essentially derived from. In a particular embodiment, the linker is PEG9~PEG 12 It includes, consists of, or is essentially derived from. In a particular embodiment, the linker is PEG4~PEG 15It includes, consists of, or is essentially derived from. In a particular embodiment, the linker is PEG4~PEG 16 It includes, consists of, or is essentially derived from. In a particular embodiment, the linker is PEG3~PEG 11 It includes, consists of, or is essentially derived from. In a particular embodiment, the linker is PEG3~PEG 12 It includes, consists of, or is essentially derived from. In a particular embodiment, the linker is PEG3~PEG 15 It includes, consists of, or is essentially derived from. The linker is PEG4~PEG 16 For example, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG 10 PEG 11 PEG 12 PEG 13 PEG 14 PEG 15 , or PEG 16 It may include (or may essentially be or may be) the endpoints described in this paragraph.

[0192] The linker may be PEG6 or may contain it (or may be essentially it or may be). The linker is PEG 10 It may be or may include it. The linker is PEG 12 It may be or may include it. The linker is PEG 15 It may be or may include it. The linker is PEG 16 It may be possible or may include it.

[0193] In some embodiments, the linker is a hydrolyzable linker. In some embodiments, the linker is a non-hydrolyzable linker. In some embodiments, the linker is an optionally substituted heteroalkyl. In some embodiments, the linker is a substituted heteroalkyl comprising at least one substituent selected from the group consisting of alkyl, hydroxyl, oxo, PEG, carboxylate, and halo. In some embodiments, the linker comprises a spacer (e.g., as described elsewhere in this specification).

[0194] In some embodiments, the linker is a substituted heteroalkyl having at least one disulfide bond in its backbone. In some embodiments, the linker is a peptide having at least one disulfide bond in its backbone.

[0195] In some embodiments, the linker is -CONH-CH(COOH)-CH2-SS-CH2-CR a R b -O-CO-, -CONH-CH(COOH)CR a R b -O-CO-, -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)CR a R b -O-CO-, or -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)-CH2-SS-CH2-CR a R b -O-CO-[wherein, R a and R b [The elements independently include H, alkyl, or heteroalkyl (e.g., PEG)].

[0196] In some embodiments, the linker is

[0197] [ka] [In the formula, n or m (if applicable) is between 0 and 10.] It includes the structure.

[0198] In some embodiments, the linker is

[0199] [ka] [In the formula, n and m are independently between 0 and 10.] It includes the structure.

[0200] In some embodiments, the linker is

[0201] [ka] The structure includes [wherein n is 1 to 32]. In at least one exemplary embodiment, n is 1 to 30 and w is 0 to 5 (if applicable).

[0202] In some embodiments, the linker is

[0203] [ka] [In the formula, n is between 1 and 16] It includes the structure.

[0204] In a particular embodiment, the linker is

[0205] [ka] [In the formula, n is between 1 and 30, and w is between 0 and 5.] It may include the structure of

[0206] For further discussion of linkers relating to this disclosure, particularly folic acid and fluorescein-linker-folate adapters, see, for example, International Publication No. 2020 / 033129, whose teachings thereto are incorporated herein by reference.

[0207] Dual-specificity adapter A bispecific adapter or a pharmaceutically acceptable salt or hydrate thereof may be used in conjunction with anti-fluorescein CAR-T cells in the treatment of FAP-expressing (e.g., FAPα-expressing) cancers and may contain a fluorescein-linker-FAP ligand. The adapter or a pharmaceutically acceptable salt or hydrate thereof may contain a fluorescein-linker-FAP ligand (e.g., fluorescein=fluorescein, FITC, or NHS-fluorescein), where the FAP ligand is

[0208] [ka] The linker contains (or is essentially made from or consists of) PEG. The linker is PEG4~PEG 16 For example, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG 10 PEG 11 PEG 12 PEG 13 PEG 14 PEG 15 , or PEG 16 The linker may include (or be essentially or be essentially) PEG6. The linker may include (or be essentially or be essentially) PEG 16 It may contain (or be essentially or be) FAP ligands, formula IB:

[0209] [ka] [In the formula,

[0210] [ka] This is the connection point of the adapter to the linker, T is a substituted or unsubstituted methylene group (-CH2-), a substituted or unsubstituted amino group (-NH-), -O-, or -S-. R 1 and R 2 Each of these is independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl. R 3 and R 4 These are -H, -OH, F, Cl, Br, I, and -C, respectively. 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 5 , R 6 , R 7 , and R 8 Each is independently selected from the group consisting of H, alkyl, and halo. R 9 , R 10 , R 11 These are H and -C respectively. 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] It may have a structure represented by [this].

[0211] In a particular embodiment, the FAP ligand (or its radical) of the bispecific adapter is of formula IC:

[0212] [ka] [In the formula,

[0213] [ka] This is the connection point of the adapter to the linker, T is a substituted or unsubstituted methylene group (-CH2-), a substituted or unsubstituted amino group (-NH-), -O-, or -S-. R 1 and R 2 Each of these is independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl. R 3 and R 4 These are -H, -OH, F, Cl, Br, I, and -C, respectively. 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 5 , R 6 , R 7 , and R 8 Each is independently selected from the group consisting of H, alkyl, and halo. R 9 , R 10 , and R 11 These are H and -C respectively. 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] It may have a structure represented by [this].

[0214] In a particular embodiment, a bispecific adapter or a pharmaceutically acceptable salt or hydrate thereof is intended for use with anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) CAR-T cells in the treatment of FAP-expressing (e.g., FAPα-expressing) cancer, wherein the adapter comprises a fluorescein-linker-FAP ligand (e.g., fluorescein=fluorescein, FITC, or NHS-fluorescein), where the FAP ligand (or its radical) is

[0215] [ka] The FAP8 ligand has the following structure, and the linker contains (or is essentially composed of or consists of) PEG. The linker is PEG3-PEG 15 For example, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG 10 PEG 11 PEG 12 PEG 13 PEG 14 , or PEG 15 It may include (or may essentially be or may be) PEG. The linker is PEG 15 It may include (or may essentially be or may be) PEG. The linker is PEG 16 It may include (or may essentially be or could be) a part of it.

[0216] FAP ligand (or its radical) has the following structure:

[0217] [ka] [In the formula,

[0218] [ka] This represents a functionalized 5-10 member nitrogen-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, optionally further containing 1-3 heteroatoms selected from O, N, and S. R1 and R2 are -H, -D, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R3 and R4 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6Independently selected from the group consisting of alkyls, R5 and R6 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R7 is selected from the group consisting of -H, -D, OH, CH2=, -CH3, CH3CH2-, (CH3)2CH-, (CH3)3C-, -CH2Ph, and substituted -CH2Ph. R8~R 10 is -H, -OH, -F, -Cl, -Br, -I, -NO2, -SO3H, -SO2NH2, -NH2, -N3, -NH=NH, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 11 -H, -D, C1~C 10 Alkyl, C3~C 10 Cycloalkyl, adamantyl,

[0219] [ka] , substituted or unsubstituted aryl, substituted or unsubstituted C7~C 20 Selected from the group consisting of alkylaryls, the aryl is

[0220] [ka] (In the formula, R 12 and R 16 These include -H, -D, halogens, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 It is selected from the group consisting of H, D, halogens, C1-C4 alkyls, and C1-C3 alkoxys. R 13 , R 14 and R 15These include -H, -D, halogens, -OMe, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 (These are selected from the group consisting of -H, -D, halogens, C1-C4 alkyls, and C1-C3 alkoxys.) And, R 17 , R 18 , R 20 , and R 21 It is selected independently of -H and -CH3, R 19 and R 22 [These are independently selected from the group consisting of phenyl, dimethoxyphenyl, and aryl compounds.] It may have.

[0221] The dual-specificity adapter may have the structure shown in Figure 25.

[0222] The bispecific adapter may contain a fluorescein-linker-PSMA ligand. In certain embodiments, the bispecific adapter is intended for use with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancer. In certain embodiments, the PSMA ligand (or its radical) is or contains DUPA. In certain embodiments, the fluorescein in the adapter contains FITC and the PSMA ligand (or its radical) is or contains DUPA, and as a result, the bispecific adapter contains a PEG linker, e.g., PEG3, PEG6, PEG8 or PEG 12 The adapter includes FITC-DUPA conjugated with FITC. Such adapters are designed and synthesized to control the distance between FITC-bound CAR-T cells (chimeric antigen receptor T cells) and PSMA-expressing tumor cells. Compared to DUPA-FITC conjugates without any linker, linker incorporation and increased linker length can improve the function of anti-FITC CAR-T cells. In certain embodiments, the optimal linker length can be obtained using PEG6.

[0223] A bispecific adapter or a pharmaceutically acceptable salt or hydrate thereof may contain a fluorescein-linker-PSMA ligand (e.g., fluorescein=fluorescein, FITC, or NHS-fluorescein), where the PSMA ligand is PSMAL1 (i.e., its radical) and the linker contains (or is essentially derived from or consists of) PEG. The linker is PEG3~PEG 12 For example, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG 10 PEG 11 , or PEG 12 The linker may contain (or be essentially composed of) PEG6. The dual-specific adapter has the following structure:

[0224] [ka] It may have.

[0225] The dual-specificity adapter may be for use with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancers and may contain a fluorescein-linker-PSMA ligand, where the PSMA ligand (or its radical) is DUPA or a DUPA derivative or contains thereof, and the linker is PEG3-PEG 11 The adapter may contain (or be essentially or be derived from) a PEG-3 to PEG-3 12 For example, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG 10 PEG 11 , or PEG 12The linker may contain (or be essentially composed of) PEG6. The dual-specificity adapter may have the structure shown in Figure 28.

[0226] A bispecific adapter may contain one or more chiral centers, or may otherwise exist as multiple stereoisomers. Therefore, various embodiments of a bispecific adapter may include pure stereoisomers, as well as mixtures of stereoisomers, e.g., enantiomers, diastereomers, and mixtures of enantiomers or diastereomers. A bispecific adapter may also exist as geometric isomers, e.g., pure geometric isomers or mixtures of geometric isomers.

[0227] Bispecific adapter compounds can be synthesized according to methods known in the art. Various synthesis methods are exemplified herein.

[0228] Salts and hydrates The dual-specificity adapters described herein may be presented as pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” of a dual-specificity adapter means a salt whose counterion is usable in pharmaceuticals. Such salts include (i) acid addition salts that can be obtained by the reaction of the free base of the parent compound with an inorganic acid, such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid, etc., or with an organic acid, 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, malonic acid, etc., and (ii) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, an alkaline earth ion, or an aluminum ion, or coordinates with an organic base, such as ethanolamine, diethanolamine, triethanolamine, trimamine, N-methylglucamine, etc. Pharmaceutically acceptable salts are well known to those skilled in the art, and all such pharmaceutically acceptable salts are intended herein.

[0229] In various embodiments, suitable basic 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. Half-salts of acid-bases, such as half-sulfates and half-calcium salts, can also be formed.

[0230] pharmaceutically acceptable salts can be synthesized by conventional chemical methods from parental bispecific adapter compounds containing basic or acidic moieties. In some examples, such salts can be prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture thereof, generally in non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, whose disclosure is incorporated herein by reference.

[0231] The dual-specificity adapter or its pharmaceutically acceptable salts may exist in solvated forms, including unsolvated and hydrated forms. The solvated forms may be equivalent to the unsolvated forms. In each embodiment herein, it will be understood that the formula includes and represents not only all pharmaceutically acceptable salts of the dual-specificity adapter, but also all hydrates and / or solvates of the formula of the compound or any salt thereof. The term “solvate” means a compound or a salt thereof that further contains stoichiometric or nonstoichiometric amounts of solvent bonded by non-covalent intermolecular forces. If the solvent is water, the solvate is a hydrate.

[0232] Certain functional groups, such as hydroxyl and amino, can form complexes and / or coordination bonds with water and / or various solvents. Therefore, the formula should be understood to include and represent their various hydrates and / or solvates. The nonhydrates and / or non-solvates of the bispecific adapters are also included.

[0233] Pharmaceutical composition Taking the above into consideration, compositions for the treatment of cancer (e.g., pharmaceutical compositions) are also provided, comprising at least one dual-specificity adapter and a pharmaceutically acceptable carrier or excipient. “pharmaceutically acceptable carrier” includes any of the standard pharmaceutically acceptable carriers, for example, but not limited to, buffers, preservatives, anesthetics, solubilizers, isotonic agents, wetting agents, and stabilizers. The term also encompasses any drugs approved for use in animals (e.g., mammals, e.g., humans) by a regulatory authority such as the U.S. Food and Drug Administration or listed in the United States Pharmacopeia. The carrier may be phosphate-buffered saline, water, or an emulsion such as an oil / water or water / oil emulsion.

[0234] In certain embodiments, the pharmaceutical composition comprises a combination of two or more of the dual-specificity adapters described herein. For example, but not limited to, the pharmaceutical composition may comprise (i) a combination of a first set of dual-specificity adapters comprising or consisting of fluorescein-linker-FAP and a second set of dual-specificity adapters comprising or consisting of fluorescein-linker-FR, and (ii) a pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition may comprise (i) a combination of a first set of dual-specificity adapters comprising or consisting of fluorescein-linker-FAP and a second set of dual-specificity adapters comprising or consisting of fluorescein-linker-PSMA, and (ii) a pharmaceutically acceptable carrier or excipient.

[0235] Further combinations of distinct pharmaceutical compositions for the treatment of cancer are provided. These combinations may include (i) a pharmaceutical composition comprising a bispecific adapter containing fluorescein-linker-folic acid and a pharmaceutically acceptable carrier or excipient, and (ii) a pharmaceutical composition comprising a bispecific adapter containing fluorescein-linker-FAP ligand and a pharmaceutically acceptable carrier or excipient.

[0236] Further provided are combinations of separate pharmaceutical compositions for the treatment of cancer, comprising (i) a pharmaceutical composition comprising a bispecific adapter containing a fluorescein-linker-PSMA ligand and a pharmaceutically acceptable carrier or excipient, and (ii) a pharmaceutical composition comprising a bispecific adapter containing a fluorescein-linker-FAP ligand and a pharmaceutically acceptable carrier or excipient.

[0237] Pharmaceutical compositions for use in the treatment of FAP-expressing cancers are also provided, comprising a bispecific adapter containing fluorescein-linker-FAP and a pharmaceutically acceptable carrier or excipient. In certain embodiments, pharmaceutical compositions for use in the treatment of PSMA-expressing cancers are provided, where the pharmaceutical composition may comprise a bispecific adapter containing fluorescein-linker-PSMA and a pharmaceutically acceptable carrier or excipient. In certain embodiments, pharmaceutical compositions for use in the treatment of FR-expressing cancers (e.g., FRα or FRβ) are provided, such compositions comprising a bispecific adapter containing fluorescein-linker-FR and a pharmaceutically acceptable carrier or excipient.

[0238] The bispecific adapter can be formulated as a pharmaceutical composition and administered to a mammalian host, such as a human patient, in various forms suitable for a selected route of administration. For example, the pharmaceutical composition can be formulated for and administered via oral or parenteral, intravenous, intra-arterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, topical, inhalation, and / or subcutaneous routes. In fact, in at least one embodiment, the bispecific adapter and / or composition described herein can be administered directly into the bloodstream, muscle, or viscera.

[0239] For example, in at least one embodiment, the dual-specificity adapter can be administered systemically (e.g., orally) in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilated food carrier. For oral therapeutic administration, the dual-specificity adapter can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, cachets, etc. The percentages of compositions and preparations may vary, and may consist of an active ingredient between about 1 and about 99% by weight, as well as binders, excipients, disintegrants, lubricants, and / or sweeteners (as known in the art). The amount of the active compound in such therapeutically useful compositions is such that an effective dose level is obtained.

[0240] The dual-specificity adapters and pharmaceutical compositions described herein can be formulated as parenteral formulations. Parenteral formulations are typically aqueous solutions that may contain salts, carbohydrates, and carriers or excipients such as buffers (preferably pH 3-9), but they can more preferably be formulated as sterile non-aqueous solutions or as dry forms used with a suitable vehicle such as sterile pyrogen-free water or sterile saline. Preparation under sterile conditions by lyophilization to produce sterile lyophilized powders for parenteral formulations can be achieved using methods well known in the art. The solubility of the dual-specificity adapters or their pharmaceutically acceptable salts for parenteral formulations can be enhanced by the use of appropriate formulation techniques, such as the incorporation of solubility enhancers.

[0241] The dual-specificity adapters / compositions can also be administered by injection or by nebulizer (e.g., using needled (including microneedle) syringes and / or needleless syringes). Solutions of the compositions may be aqueous and optionally mixed with non-toxic surfactants, and / or contain carriers or excipients such as salts, carbohydrates, and buffers (preferably pH 3–9). For some applications, they may be formulated more preferably as sterile non-aqueous solutions or as dry forms used with a suitable vehicle such as sterile pyrogen-free water or phosphate-buffered saline (PBS). For example, dispersants can be prepared in glycerol, liquid PEG, triacetin, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations may further contain preservatives to prevent microbial growth.

[0242] Suitable pharmaceutical dosage forms for injection or infusion include sterile aqueous solutions or dispersants, or sterile powders containing active ingredients configured for immediate preparation of sterile injection or infusion solutions or dispersants, optionally encapsulated in liposomes. In all cases, the final dosage form should be sterile, fluid, and stable under manufacturing and storage conditions. The liquid carrier or vehicle may be a solvent or liquid dispersion medium containing, for example, but not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid PEG, etc.), vegetable oils, non-toxic glyceryl esters, and / or suitable mixtures thereof. In at least one embodiment, adequate fluidity can be maintained by liposome formation, by maintaining the required particle size in the case of dispersants, or by the use of surfactants. Microbial activity can be prevented by the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In certain cases, it is desirable to include one or more isotonic agents such as sugars, buffers, or sodium chloride. Long-term absorption of injectable compositions can be achieved by incorporating formulations designed to delay absorption, such as aluminum monostearate and gelatin.

[0243] Sterile injectable solutions can be prepared by incorporating the dual-specificity adapter and / or composition, along with one or more of the other components described above as needed, into the required amount of a suitable solvent, followed by filtration sterilization. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredients already present in the filtration sterilized solution and any additional desired components.

[0244] For topical administration, it may be desirable to administer the dual-specificity adapter to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which may be solid or liquid. For example, in certain embodiments, the solid carrier may be a finely divided solid, such as talc, clay, microcrystalline cellulose, silica, or alumina. Similarly, useful liquid carriers may include water, alcohol or glycol, or a water-alcohol / glycol blend, in which the compound can be dissolved or dispersed at an effective level using optionally non-toxic surfactants. Additionally or alternatively, adjuvants such as fragrances and antimicrobial agents may be added to optimize the properties for a given use. The resulting liquid composition may be applied from an absorbent pad, used to impregnate bandages and / or other bandaging materials, sprayed onto the target site using a pump-type or / or aerosol sprayer, or simply applied directly to the desired site of application.

[0245] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, aliphatic alcohols, modified cellulose, or modified mineral materials can also be used for direct application to the target skin, together with liquid carriers for forming spreadable pastes, gels, ointments, soaps, and the like.

[0246] The amount of bispecific adapter (or its pharmaceutically acceptable salt) administered to a subject can vary considerably depending on the cancer being treated, the route of administration, and the tissue distribution. As used herein, the terms “therapeutically effective,” “therapeutic effective dose,” “therapeutic effective amount,” “prophylactic effective dose,” or “prophylactic effective dose” mean (unless otherwise specifically stated) the amount of bispecific adapter administered once or over the course of a treatment cycle that affects the subject’s health, well-being, or mortality (e.g., delaying the onset of one or more cancer-associated symptoms and / or reducing their severity). Useful doses of bispecific adapters can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to human subjects are known in the art. In fact, the dosage of a bispecific adapter can vary considerably depending on the host subject's condition, the type of cancer being treated, the stage of the disease, the route and tissue distribution of the bispecific adapter, and whether it can be used in combination with other therapeutic procedures (e.g., radiotherapy, or combination therapy, such as additional drugs in CAR T-cell therapy). The amount of composition required for use in the procedure (e.g., therapeutic or prophylactic effective dose or dosage) will vary not only with respect to the specific application but also with respect to the selected salt (if applicable) and the properties of the subject (e.g., age, condition, sex, body surface area and / or mass of the subject, drug tolerance, etc.), and will ultimately be left to the discretion of the attending physician or clinician.

[0247] The amount administered to the target individual, for example, approximately 0.05 mg to 30 mg, approximately 0.05 mg to 25 mg, approximately 0.05 mg to 20 mg, approximately 0.05 mg to 15 mg, approximately 0.05 mg to 10 mg, approximately 0.05 mg to 9 mg, approximately 0.05 mg to 8 mg, approximately 0.05 mg to 7 mg, approximately 0.05 mg to 6 mg, approximately 0.05 mg to 5 mg, approximately 0.05 mg to 4 mg, approximately 0.05 mg The dosage may range from approximately 3 mg, 0.05 mg to 2 mg, 0.05 mg to 1 mg, 0.05 mg to 0.5 mg, 0.05 mg to 0.4 mg, 0.05 mg to 0.3 mg, 0.05 mg to 0.2 mg, 0.05 mg to 0.1 mg, 0.01 mg to 20 mg, 0.3 mg to 10 mg, 0.1 mg to 20 mg, or 0.8 mg to 3 mg. Those skilled in the art will readily understand that the dosage may vary within the various ranges provided above, based on the factors mentioned above, and may be left to the discretion of the physician administering the treatment.

[0248] The therapeutically effective or prophylactic effective dose or dosage may be, for example, about 0.05 mg / kg patient body weight to about 30.0 mg / kg patient body weight, or in the range of about 0.01 mg / kg patient body weight to about 5.0 mg / kg patient body weight, including but not limited to 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, and 5.0 mg / kg, all of which are in kg relative to patient body weight. The total therapeutic or prophylactic effective dose of the dual-specific adapter may be administered in single or divided doses, and may, at the discretion of the practitioner, deviate from the typical range shown herein.

[0249] In another embodiment, the dual-specificity adapter is approximately 0.5 mg / m² 2 ~about 500mg / m 2 , about 0.5mg / m 2 ~about 300mg / m 2, or approximately 100 mg / m² 2 ~about 200mg / m 2 It can be administered in a therapeutic or prophylactic effective dose. In other embodiments, the dose is approximately 0.5 mg / m². 2 ~about 500mg / m 2 , about 0.5mg / m 2 ~about 300mg / m 2 , about 0.5mg / m 2 ~about 200mg / m 2 , about 0.5mg / m 2 ~about 100mg / m 2 , about 0.5mg / m 2 ~about 50mg / m 2 , about 0.5mg / m 2 ~about 600mg / m 2 , about 0.5mg / m 2 ~about 6.0mg / m 2 , about 0.5mg / m 2 ~about 4.0mg / m 2 , or approximately 0.5 mg / m² 2 ~about 2.0mg / m 2 This may be the case. The total amount may be administered as a single dose or in divided doses and may, at the physician's discretion, deviate from the typical ranges shown herein. These amounts are based on meters relative to body surface area. All ranges specified in this paragraph include the stated endpoints and encompass 0.5 mg / m². 2 Includes all values ​​in the step size.

[0250] In other embodiments, the amount of the bispecific adapter (or its pharmaceutically acceptable salt or hydrate) administered to the subject is, for example, about 50 nmol / kg to about 3,000 nmol / kg of subject body weight, about 50 nmol / kg to about 2,000 nmol / kg, about 50 nmol / kg to about 1,000 nmol / kg, about 50 nmol / kg to about 900 nmol / kg, about 50 nmol / kg to about 800 nmol / kg, about 50 nmol / kg to about 700 nmol / kg, and about 50 nmol / kg to about 600 nmol / kg. The range of mol / kg may be approximately 50 nmol / kg to 500 nmol / kg, approximately 50 nmol / kg to 400 nmol / kg, approximately 50 nmol / kg to 300 nmol / kg, approximately 50 nmol / kg to 200 nmol / kg, approximately 50 nmol / kg to 100 nmol / kg, approximately 100 nmol / kg to 300 nmol / kg, approximately 100 nmol / kg to 500 nmol / kg, approximately 100 nmol / kg to 1,000 nmol / kg, or approximately 100 nmol / kg to 2,000 nmol / kg within the range of the target body weight. In other embodiments, the dose may be about 100 nmol / kg, about 150 nmol / kg, about 200 nmol / kg, about 250 nmol / kg, about 300 nmol / kg, about 350 nmol / kg, about 400 nmol / kg, about 450 nmol / kg, about 500 nmol / kg, about 600 nmol / kg, about 700 nmol / kg, about 800 nmol / kg, about 900 nmol / kg, about 1,000 nmol / kg, about 2,000 nmol / kg, or about 3,000 nmol / kg of target body weight. In other embodiments, doses of about 20 μg / kg to about 3 mg / kg of target body weight may be administered. The amount may be between about 0.2 mg / kg and about 0.4 mg / kg of target body weight, or about 50 μg / kg of target body weight. All ranges specified in this paragraph include the stated endpoints and, where applicable, all values ​​in 1 nmol / kg or 10 μg / kg increments that are included within each specified range.

[0251] Usage and Method A method for treating cancer in a subject is further provided, comprising administering to the subject a cancer-treatment-effective amount of (i) an effective amount of anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) CAR-T cells, or a pharmaceutical composition comprising the same with a pharmaceutically acceptable carrier or excipient, and (ii) a bispecific adapter, or a pharmaceutical composition comprising the same with a pharmaceutically acceptable carrier or excipient, wherein the subject is treated for cancer.

[0252] The terms “to treat,” “to treat,” “to be treated,” and “treatment” refer to therapeutic treatment. Such treatment may have a preventive effect. Cancer is treated when there is improvement in the symptoms or signs of cancer, such as a reduction in tumor size, complete or partial disappearance of a tumor, stabilization of cancer by inhibiting cancer progression (e.g., an increase in tumor size or number due to metastasis), or any other effect on cancer that a physician may consider to constitute a therapeutic (or preventive) treatment.

[0253] The term “subject” as used herein means an animal, such as a mammal, and in particular a human. In veterinary use, the subject may be a laboratory animal, an agricultural animal, a domestic animal, or a wild animal. Examples of such animals, but not limited to, include rodents, rabbits, monkeys, chimpanzees, dogs, cats, cattle, horses, pigs, sheep, goats, bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, or whales.

[0254] Anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) CAR-T cells are T cells (or NK cells may be used) that have been engineered to express a CAR that recognizes and binds to fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) in a bispecific adapter.

[0255] A CAR is a fusion protein comprising at least three domains, including (i) a recognition region (e.g., a variable single-strand fragment (scFv) region of an antibody) that specifically recognizes and binds to fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein), (ii) a costimulatory domain that enhances T lymphocyte proliferation and survival, and (iii) an activation signaling domain that generates a cytotoxic T lymphocyte activation signal.

[0256] The scFv region of an antibody that binds to fluorescein (e.g., in FITC) can be used and prepared from (i) an antibody known in the art that binds to fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein), (ii) a newly prepared anti-fluorescein antibody, or (iii) a sequence variant derived from the scFv region of such an antibody, e.g., an scFv region having at least about 80%, at least about 90%, at least about 95%, 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. The CAR binding portion may be, for example, the scFv, Fab, Fv, Fc, or (Fab')2 fragment of the antibody.

[0257] The “sequence identity percentage (%)” with respect to references to polypeptide sequences is defined as the percentage of each amino acid or nucleic acid residue in a candidate sequence that is identical to a residue in the reference sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage, and no conservative substitutions are considered as part of the sequence identity. Alignment aimed at determining the sequence identity percentage can be achieved by various means within the scope of the art, for example, using publicly available computer software. For example, the determination of the identity or similarity percentage between sequences can be done, for example, by using the GAP program (software by Genetics Computer Group, currently available online via Accelrys), and alignment can be done, for example, using the ClustalW algorithm (VNTI software, InforMax Inc.). Furthermore, sequence databases are searchable using the nucleic acid or amino acid sequence of interest. The algorithm for database searching is typically based on BLAST software (Altschul et al., 1990), but those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared. In some embodiments, the identity percentage can be determined over the entire length of the nucleic acid or amino acid sequence.

[0258] In various embodiments, the CAR has a recognition region, which is the scFv region of an anti-fluorescein antibody capable of binding to fluorescein, FITC, or NHS-fluorescein (see, for example, the E2 anti-fluorescein antibody described in Vaughan et al., Nature Biotechnol 14(3): 309-314 (1996), and the exemplary CAR construct expressing a CAR containing the E2 anti-fluorescein antibody, shown in Figure 1 of International Publication No. 2019 / 144091, and described on pages 66, line 16 to 69, line 12. Both of these are incorporated herein by reference with their teachings). CARs possess a co-stimulatory domain, which may be CD28 (differentiation antigen group 28), CD2 (differentiation antigen group 2), CD137 (differentiation antigen group 137, 4-1BB), a member of the tumor necrosis factor (TNF) family, CD134 (differentiation antigen group 134, OX40), a member of the TNF receptor (TNFR) superfamily receptors, CD27 (differentiation antigen group 27), CD30 (differentiation antigen group 30), CD150 (differentiation antigen group 150), DAP10, NKG2D, CD278 (differentiation antigen group 278, ICOS), CD28 superfamily co-stimulatory molecules expressed on activated T cells, signaling lymphocyte activator (SLAM) related receptor family (e.g., 2B4), or any combination thereof. Sequence variants of the above-mentioned co-stimulatory domains having the same or similar activity as the underlying domain of the modeling can also be used without adversely affecting the method. CAR possesses an activation signaling domain, which may be a T cell CD3ζ chain, CD3 delta receptor protein, mbl receptor protein, B29 receptor protein, or Fc receptor γ. Sequence variants of the aforementioned activation signaling domains having the same or similar activity as the underlying domain for modeling can also be used without adversely affecting the method.Such co-stimulatory domains and variants of such co-stimulatory domains and activation signaling domains may have sequence identity of at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% with respect to the amino acid sequence of the domain from which they originate.

[0259] In embodiments of the CAR containing an E2 anti-fluorescein antibody fragment, the CAR includes an IgG4 hinge domain and a CD28 transmembrane domain. The co-stimulatory domain is CD137(4-1BB), and the activation signaling domain is CD3ζ.

[0260] In a particular embodiment, the CAR comprises an anti-fluorescein antibody scFv as a recognition region, a CD137(4-1BB) costimulatory domain, and a CD3ζ as an activation signaling domain.

[0261] Constructs encoding CARs are prepared using genetic engineering techniques. Such techniques are incorporated herein by reference, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rdThis is described in ed., Cold Spring Harbor Laboratory Press (2001). For example, plasmids or viral expression vectors (e.g., lentiviral vectors, retroviral vectors, sleeping beauty, and piggyback (transposon / transposase systems including nonviral-mediated CAR gene delivery systems)) can be prepared to encode a fusion protein comprising a recognition region, one or more co-stimulatory domains, and an activation signaling domain, linked in-frame and in the 5' to 3' direction. Other arrangements may be acceptable and include the recognition region, activation signaling domain, and one or more co-stimulatory domains. The position of the recognition region in the fusion protein is generally such that presentation of that region on the extracellular surface is achieved. The CAR may also include additional elements, such as a signal peptide to ensure proper transport of the fusion protein to the cell surface, a transmembrane domain to ensure the fusion protein is maintained as an endogenous membrane protein, and a hinge domain to provide flexibility to the recognition region and enable strong binding to the CAR targeting region.

[0262] T lymphocytes (e.g., cytotoxic T lymphocytes) can be genetically engineered to express a CAR construct by transfecting a population of T lymphocytes with an expression vector encoding the CAR construct. Suitable methods for preparing a transduced population of T lymphocytes expressing a selected CAR construct are well known to those skilled in the art and are described in Sambrook et al. (2001), supra.

[0263] While T lymphocytes can be autologous, heterogeneous cells may be used, for example, if the patient being treated is receiving high doses of chemotherapy or radiation therapy to disrupt the patient's immune system. In various embodiments, allogeneic cells may be used.

[0264] T lymphocytes can be obtained from patients by means known in the art. For example, T cells can be obtained by collecting peripheral blood from a patient, subjecting the blood to Ficol density gradient centrifugation, and then isolating a population of cytotoxic T cells from the peripheral blood using a negative T cell isolation kit (e.g., EasySep® T cell isolation kit). In various embodiments, the population of cytotoxic T lymphocytes does not need to be pure and may contain other cells, such as other T cells, monocytes, macrophages, natural killer cells, and B cells. The population of cells collected may contain at least about 90% selected cell types, for example, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% selected cell types.

[0265] After T lymphocytes are obtained, the cells can be cultured under conditions that promote cell activation. The culture conditions may be such that the cells can be administered to a patient without concern regarding their reactivity to the components of the culture medium. For example, the culture conditions may not include bovine serum preparations, such as bovine serum albumin (BSA). Activation can be achieved by introducing known activators, such as anti-CD3 antibodies in the case of cytotoxic T cells, into the culture medium. Another suitable activator is anti-CD28 antibodies. The lymphocyte population can be cultured for about 1 to 4 days under conditions that promote activation. An appropriate level of activation can be determined by cell size, proliferation rate, or activation markers, as determined by flow cytometry.

[0266] After a population of cytotoxic T lymphocytes has been cultured under conditions that promote activation, the cells may be transfected with an expression vector encoding a CAR. Following transfection, the cells may be administered to the patient immediately, or they may be cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 days or more, or for about 5 to 12 days, about 6 to 13 days, about 7 to 14 days, or about 8 to 15 days, for example, to give the cells time to recover from transfection. Preferred culture conditions may be similar to those under which the cells were cultured for activation, with or without the drug used to promote activation.

[0267] Once the cells are transfected and activated, compositions containing CAR-T cells can be prepared and administered to the subject. Culture media lacking any animal preparations such as BSA may be used. Tissue culture conditions typically used in the art can be used to avoid contamination by bacteria, fungi, and mycoplasmas. The cells can be pelleted, washed, and resuspended in pharmaceutically acceptable carriers, diluents, or excipients.

[0268] Exemplary compositions containing CAR-T cells include compositions containing cells in 290 mOsm sterile saline, injectable cryopreservation medium in 0.9% NaCl containing 2% human serum albumin (HSA) (containing Plasma-Lyte A, dextrose, sodium chloride, HSA, and dimethyl sulfoxide (DMSO)), or any other injectable 290 mOsm sterile material. Depending on the properties of the culture medium, CAR-T cells may be administered as a composition in the culture medium, or they may be concentrated and resuspended in the culture medium before administration.

[0269] The CAR-T cell composition can be administered to the subject by any suitable means, for example, parenteral administration, such as intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, or intrathecally.

[0270] The total number and concentration of CAR-T cells in the composition administered to a subject will vary depending on several factors, including the type of CAR-T cells used, the binding specificity of the CAR, the nature of the CAR targeting moiety (FITC in the example herein), and the nature of the bispecific adapter's low molecular weight ligand / targeting ligand (e.g., PSMAL1, DUPA, FR ligand, and / or FAP ligand), the nature of the cancer, its location in the cancerous subject, the means used to administer the composition to the subject, and the health status, age, and weight of the subject being treated. A suitable composition containing transduced CAR-T cells is approximately 1 × 10⁶ 5 ~Approx. 1×10 15 Examples include compositions containing transduced CAR-T cells and having a volume between approximately 5 ml and approximately 200 ml. Typical compositions contain a volume between approximately 10 ml and approximately 125 ml and have approximately 1 × 10⁶ cells. 7 ~Approx. 1×10 10 It contains approximately 1 × 10¹ CAR-T cells. An exemplary composition contains approximately 1 × 10¹ CAR-T cells in a volume of approximately 100 ml. 9 It contains CAR-T cells. A single or multiple dose of CAR-T cells may be administered to the subject. The composition may contain, for example, about 1 million (1M), 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M, 10M, 11M, 12M, 12.5M, 13M, 14M, or 15M CAR-T cells per kg of the patient's body weight. When a CAR-T cell composition is administered into the bloodstream of a subject by injection, the CAR-T cells in the bloodstream of the subject will constitute at least 5%, 7%, 10%, 11%, 12%, 13%, 14%, or 15% of the total T cells of the subject in the bloodstream by approximately 4 weeks after injection, or at least 20%, 25%, 30%, 35%, 40%, or 50% of the total T cells of the subject in the bloodstream by approximately 2 weeks after injection, or at least 85%, 90%, or 95% of the total T cells of the subject by approximately 1 week after injection.

[0271] A bispecific adapter (or a pharmaceutically acceptable salt or hydrate thereof) or a pharmaceutical composition containing the same, or a combination thereof with anti-fluorescein CAR-T cells or a pharmaceutical composition containing anti-fluorescein CAR-T cells, can be administered to a patient using any preferred method known in the art. The terms “administer,” “to administer,” “to be administered,” and “administer” refer to a method of introducing a bispecific adapter (or a pharmaceutically acceptable salt or hydrate thereof) or a pharmaceutical composition containing a bispecific adapter (or a pharmaceutically acceptable salt or hydrate thereof), and a method of introducing a pharmaceutical composition containing anti-fluorescein CAR-T cells or a pharmaceutical composition containing anti-fluorescein CAR-T cells. Examples of preferred routes of administration include, but are not limited to, oral, intravenous, intramuscular, subcutaneous, and transdermal. The components can be administered directly into the bloodstream, muscle, or viscera. Suitable routes for parenteral administration include, but are not limited to, intravenous, intra-arterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous. Needle-type syringes, needle-free syringes, and injections can be used. The components described above can be administered in unit dosage forms and / or formulations containing conventional non-toxic, pharmaceutically acceptable carriers or excipients (or vehicles or adjuvants).

[0272] In the method, anti-fluorescein CAR-T cells (or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient), and the bispecific adapter, pharmaceutical composition, or combination may be administered simultaneously or sequentially by the same or different routes. When administered simultaneously by the same route, the formulations may be the same or different. In various embodiments, the bispecific adapter may be administered to the subject after the CAR-T cells. The timing between the administration of CAR-T cells and the administration of the bispecific adapter can vary considerably depending on factors including the type of CAR-T cells used, the binding specificity of the CAR, the nature of the CAR targeting moiety (fluorescein in the examples herein), and the low molecular weight ligand / targeting moiety of the bispecific adapter (PSMA ligand, FAP ligand, FR ligand, or a combination of PSMA and FAP ligands, or a combination of FAP ligand and FR ligand in the examples herein), the nature of the cancer, the location of the cancer in the subject, the means used to administer the CAR-T cells and bispecific adapter to the subject, and the patient's health status, age, and weight.

[0273] The bispecific adapter may be administered before or after CAR-T cell therapy, for example, within approximately 3, 6, 9, 12, 15, 18, 21, or 24 hours, or within approximately 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 days, or longer. The rate of tumor lysis can be adjusted, for example, by adjusting the administration rate of the bispecific adapter (depending on a dosing schedule such as continuous, once daily, twice daily, three times daily, once weekly, twice weekly, or three times weekly). "Continuous" means a schedule of administration for at least 1 hour, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, or at least 24 hours, or daily or weekly, for example, once / day, twice / day, three times / day, every other day, once / week, twice / week, three times / week, or any other suitable schedule. In the method, (i) and (ii) may be administered intravenously. The cancer may be ovarian cancer, endometrial cancer, breast cancer, glioma (e.g., stage 3-4 glioma), or clear cell renal cell carcinoma (e.g., stage 3-4 clear cell renal cell carcinoma).

[0274] Such combination therapy methods can be carried out using any engineered cells suitable for treating cancer and may involve using two or more of these types of agents. In certain embodiments, the engineered cells used in this combination therapy are CAR T cells and may further (or alternatively) include engineered stem cells and other cells.

[0275] The engineered cells used in combination with the bispecific adapter or composition of the present invention may be any CAR T cells, stem cells, or other engineered cells, or a combination thereof. Various adoptive cell therapies (also known as cellular immunotherapy) are well known in the art for their use in the treatment of cancer, with T cell immunotherapy in particular attracting considerable attention. Some non-exclusive examples of such therapies include engineered T cell receptor (TCR) therapy, CAR T cell therapy, and natural killer (NK) cell therapy.

[0276] In certain approaches, administering both a bispecific adapter compound and a modified cell therapy results in greater inhibition of cancer growth than additive inhibition.

[0277] When multiple therapeutic agents and / or therapies are administered concurrently, the dosages may be adjusted accordingly, as is recognized in the relevant art. "Concurrent administration" and combination therapy are not limited to administrations made simultaneously, but also include treatment plans in which a targeted bispecific adapter is administered at least once during the course of a treatment involving administration of cell therapy.

[0278] Cytokine release syndrome (CRS) can be controlled by varying the dose of the bispecific adapter. See, for example, International Publication 2017 / 177149.

[0279] A method for treating cancer as described herein may include administering one of the bispecific adapters to a patient and administering one of the manipulated cell compositions or manipulated cell therapies described above to a patient.

[0280] In a particular embodiment, a method for treating cancer in a subject is provided. The method comprises administering to the subject a cancer-treatment-effective dose of (i) anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, and (ii) any bispecific adapter, any pharmaceutical composition comprising the same, or any combination of bispecific adapters. Steps (i) and (ii) may be administered simultaneously or sequentially by the same or different routes.

[0281] Anti-fluorescein CAR-T cells may include any CAR T cells described herein or suitable for the described use. In certain embodiments, anti-fluorescein CAR-T cells include a recognition region containing the scFv region of an anti-fluorescein antibody, a costimulatory domain which is CD28, CD137(4-1BB), CD134(OX40), or CD278(ICOS), and / or an activation signaling domain which is a T cell CD3ζ chain or Fc receptor γ.

[0282] When the method administers a combination, the combination may include a first and a second bispecific adapter that can be administered simultaneously to the subject via the same or different routes. Alternatively, the first and second bispecific adapters may be administered sequentially to the subject via the same or different routes in either order.

[0283] In a particular embodiment, both steps (i) and (ii) of the method are administered intravenously.

[0284] When the fluorescein of the bispecific adapter is exposed to anti-fluorescein CAR-T cells, it can bind to the anti-fluorescein CAR-T cells with affinity. When the targeting ligand of the bispecific adapter binds to the receptor of the targeted cancer cell or CAF with affinity, the targeting ligand of the bispecific adapter can link the bound anti-fluorescein CAR-T cells to the targeted cancer cell or CAF. Thus, the compounds, compositions, and combinations herein promote the enhancement of the efficacy of CAR-T cell therapy.

[0285] In certain embodiments, the receptor for targeted cancer cells or targeted CAFs is overexpressed FAP, overexpressed PSMA, and / or FR (e.g., overexpressed FR). The cancer may be an FAP-expressing cancer, and at least one of the bispecific adapters of (ii) may contain a radical of the FAP ligand. The cancer may be a PSMA-expressing cancer, and at least one of the bispecific adapters of (ii) may contain a radical of the PSMA ligand. In certain embodiments, for example, in which the method administers the combinations herein, the cancer is an FR-expressing cancer, and (ii) may contain the combinations herein, including folic acid.

[0286] Methods for treating FAP-expressing cancers in subjects are also provided. In certain embodiments, the method comprises administering to a subject a cancer-treatment-effective amount of (i) anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, and (ii) any bispecificity adapter, or a pharmaceutical composition comprising a bispecificity adapter and a pharmaceutically acceptable carrier or excipient. As stated above, the CAR may have a recognition region, which may be the scFv region of an anti-fluorescein antibody. In certain embodiments, the CAR includes a costimulatory domain which may be CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS), and / or an activation signaling domain which may be a T cell CD3ζ chain or an Fc receptor γ.

[0287] In a particular embodiment, a method for treating cancer in a subject comprises administering to the subject a cancer-treatment-effective dose of any combination of (i) anti-fluorescein chimeric antigen receptor (CAR)-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, and (ii) a bispecific adapter, where the CAR may comprise a recognition region including the scFv region of an anti-fluorescein antibody, a costimulatory domain which may be CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS), and / or an activation signaling domain which is a T cell CD3ζ chain or Fc receptor γ. Steps (i) and (ii) may be administered simultaneously or sequentially by the same or different routes. In a particular embodiment, the first and second bispecific adapters of the combination are administered to the subject simultaneously by the same or different routes. In a particular embodiment, the first and second dual-specific adapters of the combination are administered to the subject sequentially by the same or different routes in either order. (i) and (ii) can each be administered intravenously.

[0288] The methods described herein may further include imaging cancer in a subject. Imaging cancer may include, for example, imaging by optical imaging, positron emission tomography (PET), or single-photon emission computed tomography (SPECT).

[0289] In the method, cancer may be further imaged before administration of a bispecific adapter to the target, or a pharmaceutically acceptable salt or hydrate thereof, or a manipulated cell composition (e.g., a CAR-expressing cytotoxic lymphocyte composition or a CAR-NK cell composition). Cancer may be additionally or alternatively imaged during or after administration, for example, to evaluate metastasis and the efficacy of the treatment. For example, imaging may be performed by PET imaging, magnetic resonance imaging (MRI), or SPECT / computed tomography (CT) imaging. The imaging method may be any suitable imaging method known in the art.

[0290] Cancer can be any type of cancer. "Cancer," when read in light of this specification, has its obvious and ordinary meaning and may include, but is not limited to, a group of diseases involving abnormal cell growth that have the potential to invade or spread (i.e., metastasize) to other parts of the body. Examples include, but are not limited to, cancers of the brain, thyroid, lung, pancreas, kidney, stomach, gastrointestinal stroma, endometrium, breast, cervix, ovaries, colon, prostate, leukemia, lymphoma, other hematological cancers, or head and neck cancers. In certain embodiments, the cancer being treated is a tumor. In certain embodiments, cancer is malignant. In certain embodiments, cancer is ovarian cancer, endometrial cancer, breast cancer, glioma, e.g., optionally, a glioma of stage 3-4, or clear cell renal cell carcinoma, e.g., optionally, a clear cell renal cell carcinoma of stage 3-4.

[0291] In some embodiments of these embodiments, the cancer is a folate receptor-expressing cancer, for example, a FRα-expressing cancer, though not limited to these embodiments. In other embodiments, the cancer is a FRβ-expressing cancer. In certain embodiments, the cancer is an FAP-expressing cancer. In certain embodiments, the cancer is a PSMA-expressing cancer.

[0292] In some embodiments, the cancer is imaged before administration to subjects (i) and (ii). Imaging can be performed by PET, MRI, or SPECT / CT.

[0293] In certain embodiments, the use of a bispecificity adapter, a pharmaceutically acceptable salt, hydrate, or solvate of a bispecificity adapter, or a composition thereof, in the manufacture of a pharmaceutical for the treatment of cancer in a subject is provided. The bispecificity adapter may be any compound or conjugate herein. The pharmaceutical may be used in combination with administration to a subject such as a manipulated cell therapy, for example, CAR T cell therapy in which CAR T cells express anti-fluorescein.

[0294] Furthermore, methods for enhancing CAR-T cell activation are provided. These methods may include preparing the bispecificity adapter, pharmaceutical composition, or combination described herein (e.g., a therapeutically effective amount of any of the foregoing) and exposing anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, to the bispecificity adapter, pharmaceutical composition, or combination, wherein the CAR-T cells exhibit enhanced activation after exposure compared to CAR-T cells not exposed to the bispecificity adapter. For example, when exposed to the bispecificity adapter, pharmaceutical composition, or combination, the anti-fluorescein CAR-T cells may be systemically circulating in the subject. Alternatively, the anti-fluorescein CAR-T cells may be exposed to the bispecificity adapter, pharmaceutical composition, or combination in vitro.

[0295] kit Further kits are provided. The kits may include (i) a pharmaceutical composition comprising a bispecific adapter, or a pharmaceutically acceptable carrier or excipient thereof, or a combination thereof, and (ii) a pharmaceutical composition comprising anti-fluorescein CAR-T cells (e.g., anti-FITC CAR-T cells), or a pharmaceutically acceptable carrier or excipient thereof.

[0296] In certain embodiments, the bispecificity adapter, pharmaceutical composition, or combination and CAR-T cells are stored in separate containers. In certain embodiments where the kit includes the combination described herein, the first bispecificity adapter and the second bispecificity adapter are stored in separate containers.

[0297] General Matters Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. Implementations should not be limited to the specific limitations described. Other implementations may be possible.

[0298] While the dual-specificity adapters and pharmaceutical compositions are illustrated and described in detail in the foregoing description, it should be understood that this is illustrative and not restrictive with respect to their features, and that only certain embodiments are shown and described, and that it is desirable that all changes and modifications that fall within the spirit of the invention be protected.

[0299] The scope of this dual-specificity adapter, composition, and method is intended to be defined by the following claims. However, this disclosure can be implemented in ways different from those specifically described and illustrated without departing from its spirit or scope. Those skilled in the art will understand that various alternative forms of the embodiments described herein can be used when implementing the claims without departing from the spirit and scope defined in the following claims.

[0300] The use of any section heading is intended to aid in the reading of the document and should not be interpreted as limiting. Furthermore, the information related to a section heading may be located within or outside that particular section.

[0301] All publications, patents, patent application publications, academic journal articles, textbooks, and other publications referenced herein represent the level of skill of a person skilled in the art to which this disclosure pertains. All such publications are incorporated herein by reference to the same extent as each individual publication specifically and individually indicates that it is incorporated by reference. Where there is a discrepancy in usage between this document and those documents thus incorporated by reference, the usage in the incorporated references should be considered supplementary to the usage in this document, and in the event of an unresolved discrepancy, the usage in this document shall prevail.

[0302] The connection or linkage between two components can be described by various techniques and mechanisms. Words such as joined, linked, coupled, connected, and similar terms with their inflectional morphemes are used interchangeably unless differences are pointed out or otherwise evident from the context. These words and expressions do not necessarily indicate a direct connection, but include connections through intervening components. It should be noted that a connection between two components does not necessarily mean a direct, unhindered connection, as various other components may exist between the two components being referred to. Therefore, unless otherwise noted, connection does not necessarily mean a direct, unhindered connection.

[0303] A certain definition As used herein, the following terms and phrases shall have the meanings set forth below. Unless otherwise defined, all technical and scientific terms used herein shall have the same meanings as those commonly understood by those skilled in the art.

[0304] The terms “about” or “approximately” mean a range of tolerance for a particular value as determined by those skilled in the art, which in part depends on the method by which the value is measured or determined, e.g., the limits of the measuring system. For example, “about” could mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. As further examples, “about” or “approximately” could mean within 90%, 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999%, or a number greater than or equal to the limits of the stated value or range. Alternatively, particularly with respect to biological systems or processes, the term could mean within 10 times a certain value, preferably up to 5 times, and more preferably up to 2 times. Unless otherwise stated, the term “about” means a range of tolerance for a particular value, e.g., ±1 to 20%, preferably ±1 to 10%, and more preferably ±1 to 5%.

[0305] If a range of values ​​is provided, it is understood that each value between the upper and lower limits of that range, as well as any other values ​​listed or between that range, are included. The upper and lower limits of these narrower ranges may be independently included within the narrower ranges, and these are also included, but the exclusion of any limits within the listed range is followed. If the listed range includes one or both of the limits, the range excluding one or both of those limits is also included.

[0306] The phrase "at least one of" in the list of items refers to any combination of these items that includes a single component. For example, "at least one of a, b, or c" is intended to include a, b, c, ab, ac, bc, and abc.

[0307] The terms “a,” “an,” or “the” are used to include one or more unless the context clearly indicates otherwise. The term “or” is used to refer to a non-exclusive “or” unless the context clearly indicates otherwise. In addition, it should be understood that any expressions or technical terms used herein, unless otherwise defined, are for illustrative purposes only and not for limitation.

[0308] The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. Furthermore, it should be understood that any expressions or terms used herein, unless otherwise defined, are for illustrative purposes only and not for limitation.

[0309] The terms and expressions used are for illustrative purposes only, not limiting ones. Where a particular term is defined and separately explained or discussed elsewhere in the "Modes for Carrying Out the Invention," all such definitions, explanations, and discussions are intended to be attributed to that term. Furthermore, the use of such terms and expressions is not intended to exclude any equivalents of the exhibited or described features or any part thereof. Additionally, while subheadings may be used in the "Modes for Carrying Out the Invention," such use is solely for the purpose of facilitating reference and is not intended to limit any disclosure made in a particular section to that section alone. Rather, any disclosure made under a subheading is intended to constitute disclosures under any other subheading.

[0310] It is recognized that various modifications are possible within the scope of the claimed invention. Therefore, although the present invention is specifically disclosed in the context of preferred embodiments and optional features, those skilled in the art may modify and transform the concepts disclosed herein. Such modifications and transformations are considered to fall within the scope of the claimed invention. [Examples]

[0311] The following examples are helpful in illustrating the present disclosure. These examples are not intended in any way to limit the scope of the claimed invention.

[0312] [Example 1] Synthesis of FAP8-PEG3-FITC conjugate The FAP8-PEG3-FITC conjugate can be synthesized according to Scheme 1.

[0313] [ka]

[0314] [ka]

[0315] To a stirred solution of compound 1 (1.0 g, 5.1 mmol) in dry methylene chloride (DCM) (3.0 mL) at 0°C, Et3N (5.0 equivalents) was added, followed by POCl3 (1.5 equivalents). The reaction mixture was allowed to continue at the same temperature for 1 hour. The progress of the reaction was monitored by thin-layer chromatography (TLC). After completion of the starting materials shown on the TLC plate, the reaction mixture was further diluted with DCM, then adsorbed onto a silica gel cartridge, and purified using ethyl acetate + hexane as the mobile phase to obtain the desired compound 2 (750 mg, 81%) as a pale yellow liquid.

[0316] Synthesis of compounds (5 and 6): A mixture of N-Boc-L-prolinal (1 equivalent, 200 mg, 0.851 mmol), N-protected glycine (1 equivalent, 161 mg, 0.851 mmol), and isocyanide (1 equivalent, 162 mg, 0.851 mmol) was dissolved in anhydrous DCM (10 mL) and stirred for 4 hours. After complete conversion of the starting materials (monitored by liquid chromatography-mass spectrometry (LC-MS)), trifluoroacetic acid (3.0 mL) was added to the reaction mixture and stirring was continued at room temperature for 1 hour. Volatile substances were evaporated under reduced pressure. The oily residue was dissolved again in anhydrous DCM (10 mL) and cooled to 0°C using an ice bath. Triethylamine (5.0 mL) was added dropwise and stirring was continued until complete conversion (monitored by LC-MS, different retention times for the same mass), which typically takes less than 2 hours. The liquid was evaporated under reduced pressure, the mixture was redissolved in DCM, and washed three times with water. The organic phase was washed with brine, dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting crude residue was purified by a combiflush using hexane + ethyl acetate as the mobile phase to obtain the desired α-hydroxyamide 5 brown solid. The product was then used in the next step by dissolving it in MeOH + AcOH (10 mL, 1:1) and added 10% Pd-C (100 mg per 1 g of starting material), and then stirred under a hydrogen atmosphere for 6 hours. The reaction mixture was filtered through a Celite pad, and the filtrate was evaporated under reduced pressure to obtain the crude residue. The crude residue was an azeotropic mixture with EtOH, which was purified by a combiflush using methanol (MeOH) + DCM to obtain amine 6 as a white solid.

[0317] Synthesis of compound (8): Cs2CO3 (2.65 gm, 7.93 mmol), followed by tertiary butyl bromoacetate (7.93 mmol), was added to a solution of compound 7 (500 mg, 2.64 mmol) in DMF (10 mL). The reaction mixture was stirred at 55°C for 4 hours, then (3 equivalents of KOH relative to the dialkylation product) + H2O (5.0 mL) was added to the same reaction mixture, and stirring was continued for a further 12 hours. The progress of the reaction was monitored by LC-MS, and the reaction mixture was carefully neutralized with 1N HCl and extracted multiple times with pharmaceutically acceptable phosphate. The combined organic extracts were evaporated under reduced pressure to obtain a crude residue, which was purified by a combiflush using pharmaceutically acceptable phosphate + hexane to obtain the desired compound 8 as a white solid (650 mg 81%).

[0318] Synthesis of compound (9): PyBOP (411 mg, 0.792 mmol) and DIPEA (0.22 mL, 1.32 mmol) were added to a stirred solution of compound 8 (200 mg, 0.66 mmol) in anhydrous CH2Cl2 (10.0 mL), and the mixture was stirred for 10 minutes. Then, amine 6 (0.66 mmol) was added to the reaction mixture, and stirring was continued for a further 2 hours. The reaction mixture was diluted with water and then extracted into DCM (2 × 20 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to obtain the crude residue. The crude residue was purified by a combiflush using siRNA + hexane for 15 minutes, followed by MeOH + DCM as the mobile phase for 25 minutes, to obtain compound 9 as a white solid.

[0319] Synthesis of compound (10): Trifluoroacetic acid (TFA) (2.0 mL) was added to a solution of compound 10 (200.0 mg, 0.297 mmol) in DCM (5 mL), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was then evaporated under reduced pressure and dried under vacuum to obtain a crude residue, which was purified by a combiflush using siRNA + hexane for 15 minutes, followed by MeOH + DCM for 25 minutes, to obtain acidic compound 9a as a white solid. Acidic compound 9a (1.0 equivalent) was then dissolved in DCM, followed by the addition of PyBOP (1.2 equivalents) + N,N-diisopropylethylamine (DIPEA) (2.0 equivalents). After stirring for 10 minutes, BocNH(PEG)3NH2 (1.2 equivalents) was added to the reaction mixture, and stirring was continued for another 2 hours. Workup and purification were performed according to the same procedure as described above to obtain compound 10 as a white solid. Finally, compound 10 was dissolved again in DCM, followed by Dess-Martin periodinane (DMP) (3.0 equivalents), and then water (10 equivalents) was added to compound 10 (1.0 equivalent). The solution was stirred overnight at room temperature. The reaction mixture was further diluted with water and extracted in DCM (2 × 30 mL).

[0320] The combined organic extracts were dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure to obtain a crude residue, which was purified by combiflash using MeOH + DCM as the mobile phase to obtain the desired keto compound 11 as a white solid.

[0321] Synthesis of compound 12 (FAP8-PEG3-FITC): TFA (0.5 mL) was added to a stirred solution of compound 11 (100 mg, 0.1126 mmol) in DCM (1.0 mL). Stirring was continued for 30 minutes. The reaction mixture was then evaporated under reduced pressure to obtain amine 12 as a brown, sticky solid, which was used for further steps without purification.

[0322] To a stirred solution of amine 12 (20 mg, 0.0253 mmol) in DMF (500 μl), DIPEA (0.11 mmol), followed by fluorescein isothiocyanate isomer 1 (1.2 equivalents), was added, and the reaction mixture was continuously stirred at room temperature for a further 2 hours. The reaction mixture was diluted with water and prepared using the 5-35 method (A = ammonium acetate (10 mM, P). H The desired fractions were purified by ultra-high performance liquid chromatography (U-HPLC) using 7.5 (B = acetonitrile) for 60 minutes. These fractions were rapidly frozen using liquid nitrogen and freeze-dried for 48 hours to obtain the desired compound FAP8-PEG3-FITC(13) as a yellow solid.

[0323] [Example 2] Synthesis of FAP5-PEG8-FITC conjugate The FAP5-PEG8-FITC conjugate can be synthesized according to Scheme 2.

[0324] [ka]

[0325] [ka]

[0326] Fluorescein isothiocyanate (FITC) (13.65 mg, 0.035 mmol, 0.95 equivalents) and DIPEA (13.0 μL, 0.074 mmol, 2 equivalents) were added to a stirred solution of compound 3 (40 mg, 0.037 mmol, 1 equivalent) in dry DMF (1.0 mL), and the mixture was continuously stirred at room temperature under N2 for 1 hour. After the completion of the reaction (confirmed by LC-MS), the solvent was removed, and the crude product was supported on Celite. This was purified by CombiFlash (column: 15.5 g HP C18 Aq, flow rate: 40 ml / min, wavelength 254 nm, eluent: A = 20 mM ammonium acetate buffer (pH = 7), B = acetonitrile) with a solvent gradient of 0% B to 100% for 60 minutes to obtain FAP5-PEG8-FITC as a yellow solid. LC-MS (m / z): [M+H] Measured value: 1458.4.

[0327] 1 H NMR (500 MHz, DMSO) δ 10.18 (s, 1H), 8.69 (d, J = 9.5 Hz, 1H), 8.54 - 8.51 (m, 1H), 8.24 (s, 2H), 8.05 - 8.01 (m, 2H), 7.97 - 7.74 (m, 5H), 7.73 (d, J = 8.0 Hz, 1H), 7.36 - 7.31 (m, 2H), 7.23 - 7.11 (m, 2H), 6.60 (d, J = 9.0 Hz, 4H), 6.52 (d, J = 8.5 Hz, 2H), 5.66 (d, J = 7.0 Hz, 2H), 5.12 - 5.05 (m, 1H), 4.96 (s, 1H), 4.85 (d, J = 6.0 Hz, 1H), 4.65 (d, J = 8.0 Hz, 2H), 4.32 - 4.28 (m, 1H), 4.19 - 4.06 (m, 2H), 3.66 (s, 2H), 3.59 - 3.53 (m, 7H), 3.51 - 3.39 (m, 26H), 3.24 - 3.16 (m, 3H), 3.00 - 2.60 (m, 5H), 2.46 - 2.42 (m, 1H), 2.38 - 2.27 (m, 3H), 2.14 - 2.04 (m, 1H).

[0328] 13 C NMR (126 MHz, DMSO) δ 181.96, 179.73, 178.88, 171.87, 170.87, 169.85, 169.80, 169.60, 168.99, 166.34, 152.75, 146.48, 146.43, 141.76, 138.30, 137.55, 136.78, 136.48, 135.85, 134.08, 133.56, 131.19, 130.94, 129.63, 12.10, 128.80, 128.69, 128.44, 128.34, 127.95, 127.75, 127.11, 125.31, 123.64, 123.44, 122.83, 122.86, 118.10, 110.51, 102.73, 70.22, 70.10, 69.97, 68.89, 67.23, 52.32, 51.15, 44.79, 44.14, 38.70, 36.65, 36.45, 35.47, 35.40, 31.78, 31.73.

[0329] [Example 3] Synthesis of FAP5-PEG6-FITC conjugate The FAP5-PEG6-FITC conjugate can be synthesized according to Scheme 3.

[0330] [ka]

[0331] [ka]

[0332] FITC (5.96 mg, 0.014 mmol, 0.95 equivalents) and DIPEA (5.32 μL, 0.03 mmol, 2 equivalents) were added to a stirred solution of compound 3 (15 mg, 0.015 mmol, 1 equivalent) in dry DMF (1.0 mL), and stirring was continued at room temperature under N2 gas for 1 hour. After the completion of the reaction (confirmed by LC-MS), the solvent was removed, the crude product was supported on Celite, and purified by combiflash (column: 15.5 g HP C18 Aq, flow rate: 40 ml / min, wavelength: 254 nm, eluent: A = 20 mM ammonium acetate buffer (pH = 7), B = acetonitrile) with a solvent gradient of 0% B to 100% for 60 minutes to obtain FAP5-PEG6-FITC as a yellow solid (compound 4). LC-MS (m / z): [M + H] Measured value: 1370.4.

[0333] 1 H NMR (500 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.72 (d, J = 9.5 Hz, 1H), 8.55 - 8.51 (m, 1H), 8.26 (s, 2H), 8.06 - 8.02 (m, 2H), 7.94 - 7.90 (m, 5H), 7.76 (d, J = 8.0 Hz, 1H), 7.36 - 7.33 (m, 2H), 7.25 - 7.15 (m, 2H), 6.63 (d, J = 9.0 Hz, 4H), 6.55 (d, J = 8.5 Hz, 2H), 5.68 (d, J = 7.0 Hz, 2H), 5.15 - 5.07 (m, 1H), 4.98 (s, 1H), 4.88 (d, J = 6.0 Hz, 1H), 4.69 (d, J = 8.0 Hz, 2H), 4.34 - 4.30 (m, 1H), 4.21 - 4.08 (m, 2H), 3.68 (br-s, 2H), 3.60 - 3.54 (m, 7H), 3.50 - 3.46 (m, 18H), 3.245 - 3.19 (m, 3H), 2.89 - 2.71 (m, 5H), 2.47 - 2.41 (m, 1H), 2.37 - 2.27 (m, 3H), 2.15 - 2.07 (m, 1H).

[0334] 13 C NMR (126 MHz, DMSO-d6) δ180.99, 178.91, 178.85, 171.87, 170.86, 170.83, 169.85, 169.78, 168.98, 166.33, 152.72, 146.48, 146.41, 141.71, 138.30, 137.55, 136.49, 135.86, 134.08, 133.57, 133.15, 131.18, 130.93, 129.64, 128.79, 128.69, 128.58, 128.34, 128.25, 127.94, 127.75, 127.12, 125.34, 123.63, 123.43, 122.86, 122.62, 118.10, 110.48, 102.73, 70.22, 70.10, 69.96, 67.23, 52.31, 51.98, 51.42, 44.76, 44.14, 38.70, 36.84, 36.65, 36.45, 35.48, 35.31, 31.79, 31.67.

[0335] [Example 4] Synthesis of FAP5-PEG4-FITC conjugate The FAP5-PEG4-FITC conjugate can be synthesized according to Scheme 4.

[0336] [ka]

[0337] [ka]

[0338] [ka]

[0339] FITC (5.96 mg, 0.018 mmol, 0.95 equivalents) and DIPEA (6.6 μL, 0.038 mmol, 2 equivalents) were added to a stirred solution of compound 3 (17 mg, 0.019 mmol, 1 equivalent) in dry DMF (1.0 mL), and stirring was continued at room temperature under N2 gas for 1 hour. After the completion of the reaction (confirmed by LC-MS), the solvent was removed, the crude product was supported on Celite, and purified by combiflash (column: 15.5 g HP C18 Aq, flow rate: 40 ml / min, wavelength 254 nm, eluent: A = 20 mM ammonium acetate buffer (pH = 7), B = acetonitrile) with a solvent gradient of 0% B to 100% for 60 minutes to obtain FAP5-PEG4-FITC as a yellow solid (compound 4 above). LC-MS (m / z): [M+H] Measured value: 1282.4.

[0340] 1 H NMR (500 MHz, DMSO-d6) δ 10.18 9s, 1H), 8.69 (d, J = 9.5 Hz, 1H), 8.54 - 8.49 (m, 1H), 8.25 (s, 2H), 8.04 - 7.98 (m, 2H), 7.93 - 7.87 (m, 5H), 7.72 (d, J = 8.0 Hz, 1H), 7.34 - 7.30 (m, 2H), 7.22 - 7.13 (m, 2H), 6.62 - 6.59 (m, 4H), 6.53 (d, J = 8.5, 2H), 5.66 (d, J = 7.0 Hz, 2H), 5.10 - 5.07 (m, 1H), 4.97 (s, 1H), 4.85 (d, J = 6.5 Hz, 1H), 4.67 (d, J = 8.0 Hz, 2H), 4.32 - 4.29 (m, 1H), 4.20 - 4.04 (m, 2H), 3.66 (br-s, 2H), 3.59 - 3.51 (m, 8H), 3.49 - 3.42 (m, 9H), 3.23 - 3.17 (m, 3H), 2.88 - 2.70 (m, 5H), 2.46 - 2.38 (m, 1H), 2.31 - 2.24 (m, 1H), 2.11 - 2.07 (m, 1H).

[0341] 13 C NMR (126 MHz, DMSO-d6) δ181.01, 178.91, 178.87, 171.88, 170.86, 169.85, 169.80, 169.01, 166.34, 152.69, 146.48, 146.43, 141.80, 138.30, 137.56, 136.49, 135.86, 134.09, 133.56, 133.11, 131.20, 130.94, 129.62, 128.80, 128.69, 128.57, 128.34, 128.21, 127.95, 127.76, 127.15, 125.32, 123.64, 123.45, 122.84, 122.61, 118.11, 110.45, 102.73, 70.22, 70.12, 69.97, 67.23, 52.32, 51.98, 51.43, 50.95, 44.76, 44.12, 38.71, 36.86, 36.65, 36.45, 35.48, 35.31, 31.67.

[0342] [Example 5] The 5FAP-FITC conjugate has a binding affinity of less than 10 nM (K) to human FAP and mouse FAP. D ) has To evaluate binding affinity, MDA-MB-231 cells (approximately 100,000 cells / well) overexpressing human fibroblast-activating protein (hFAP) or mouse fibroblast-activating protein (mFAP) were suspended in complete RPMI medium in 96-well plates. Complete RPMI was prepared by supplementing RPMI1640 (Gibco #21870076) with 10% fetal bovine serum (FBS, bio-techne #S11150H), 1% streptomycin / penicillin (Corning #30002CI), and 1% L-glutamine (ATCC #302214).

[0343] FAP-FITC conjugates were diluted with phosphate-buffered saline (PBS, Sigma #D8537). Their concentrations were confirmed by measuring the absorbance at 495 nm using a nanodrop spectrophotometer. Next, FAP-FITC was added to MDA-MB-231 cells at different final concentrations (3 sets).

[0344] Cells were incubated with FAP5-FITC at room temperature for 40 minutes, washed three times with PBS containing 2% FBS, and subjected to flow cytometry to measure the mean fluorescence intensity in the FITC channel. D This was calculated via Prism8.

[0345] The results are shown in Figure 1. As shown in Figure 1, FAP5-FITC has a K content of less than 10 nM. D It binds to hFAP and mFAP.

[0346] [Example 6] 6FAP-FITC was retained on the cell surface for approximately 24 hours. MDA-MB231-hFAP cells (approximately 100,000 cells / well) were seeded overnight in complete RPMI in a confocal chamber (Thermo Scientific #155382). FAP5-PEG4-FITC (25 nM) was added to the cells, and the mixture was incubated in a cell incubator at 37°C and 5% CO2 for 1 hour. The cells were washed twice with PBS containing 2% FBS to remove free FAP5-PEG4-FITC, and incubation was continued in complete RPMI for 0 minutes, 1 hour, 2 hours, 4 hours, 24 hours, or 48 hours. The cells were then examined at different time points using confocal microscopy.

[0347] FAP5-FITC was retained on the cell surface for approximately 24 hours.

[0348] [Example 7] Optimization of polyethylene glycol length for FAP5-FITC conjugate The protein structure of the hFAP protein (PDB 1z68) was investigated using Pymol. The FAP5-PEG4-FITC with the hFAP protein was molecularly modeled to examine the depth of the FAP binding pocket for FAP5-PEG4-FITC. Molecular modeling revealed a deep binding pocket in hFAP, which typically requires a longer PEG length for better FITC exposure.

[0349] MDA-MB-231 cells (approximately 100,000 cells / well) overexpressing hFAP were suspended in complete RPMI medium in a 96-well plate. The cells were then treated with 100 nM FAP5-PEG4-FITC, FAP5-PEG6-FITC, FAP5-PEG8-FITC, and FAP5-PEG 12 -FITC, or FAP5-PEG 16 - The cells were incubated with FITC at room temperature for 40 minutes. Then, the cells were washed twice and incubated with anti-FITC antibody (APC) at 4°C for 30 minutes (for FITC exposure) before being subjected to flow cytometry, or immediately subjected to flow cytometry. The results are shown in Figures 2A and 2B. Figure 2A shows that increased PEG linker length in FAP5-FITC allowed for better FITC exposure, and Figure 2B shows that increased PEG linker length in FAP5-FITC allowed for better bispecific adapter B max This indicates that nothing has changed.

[0350] The effect of PEG linker length on binding affinity was also investigated. MDA-MB-231 cells (approximately 100,000 cells / well) overexpressing hFAP or mFAP were suspended in complete RPMI medium in 96-well plates. FAP5-PEG6-FITC, FAP5-PEG8-FITC, FAP5-PEG 12 -FITC, or FAP5-PEG 16The concentration of -FITC was confirmed by measuring the absorbance using a nanodrop spectrophotometer. Dual-specific adapters were added separately to MDA-MB-231 cells at different final concentrations (triple chain). The cells were incubated with the adapters at room temperature for 40 minutes, washed three times with PBS + 2% FBS, centrifuged at 300g for 5 minutes, and subjected to flow cytometry for average fluorescence intensity measurement. The results are shown in Figure 3. Figure 3 shows the K of FAP5-FITC with different PEG linker lengths relative to hFAP. D This indicates that nothing has changed.

[0351] The killing efficacy mediated by FAP5-FITC with different PEG linker lengths was also investigated. MDA-MB-231 cells (approximately 7,000 cells / well) were seeded overnight on 96-well plates. On day 18, 4M5.3 CAR-T cells were added to the target cells in an effector:target cell ratio of 1:4. FAP5-PEG8-FITC, FAP5-PEG 12 -FITC, or FAP5-PEG 16 -FITC was incubated with cells for 24 hours or aspirated after 1 hour of incubation at room temperature and washed with PBS (all in 3-cell systems, 0nM, 0.1nM, 1nM, 10nM, 100nM, or 1,000nM). Live cells were determined by mCherry-positive cells. Killing percentage was determined by (1 - (live cells) / (live cells in wells with tumor cells only)) × 100%. Results are shown in Figures 4A and 4B.

[0352] As shown in Figure 4A, longer linkers (e.g., PEG) 16 FAP5-FITC with a longer linker (e.g., PEG) was more effective at killing tumor cells. As shown in Figure 4B, 16 FAP5-FITC, which possesses ), was more effective in activating CAR-T cells.

[0353] [Example 8] FAP8-FITC is superior to FAP5-FITC, which has a similar PEG length, in terms of better serum stability. MDA-MB-231-hFAP cells were subcutaneously injected into NSG mice, and 150 mm 3 They were allowed to grow to this stage. At that point, E2 (anti-fluorescein antibody) CAR-T cells (8 million) were injected intravenously, and FAP8-PEG8-FITC and FAP8-PEG were administered. 12 -FITC, FAP8-PEG 15 -FITC, or FAP5-PEG 16 -FITC was injected at 500 nmol / kg three times per week. The following six groups of mice were used: Group 1 (n=6): Disease control group (tumor cells only), Group 2 (n=6): Group with only E2 CAR-T cells (PBS 3 times / week), Group 3 (n=6): FAP8-PEG8-FITC group, Group 4 (n=7): FAP8-PEG 12 -FITC group, Group 5 (n=7): FAP8-PEG 15 -FITC group, and Group 6 (n=6): FAP5-PEG 16 -FITC group.

[0354] As shown in Figures 5A-5E, FAP5-PEG 16 -FITC and FAP8-PEG 15 -FITC functions similarly in vitro, while in vivo, FAP8-PEG 15 -FITC has better serum stability and mediates better binding with CAR T cells. FAP8-PEG, as shown in Figure 5F. 15 -When treated with FITC, FAP5-PEG 16-More CAR T cells infiltrated the tumors than when treated with FITC. At the end of the study, tumors were collected from mice and digested using a human tumor dissociation kit (Miltenyi) containing 50% enzyme R to enhance lymphocyte recovery. The cells were then stained with Zombie violet and an Fc blocker on ice for 30 minutes, followed by two washes, stained with an anti-hCD3 antibody on ice for 30 minutes, washed three times, and performed flow cytometry.

[0355] [Example 9] FAP8-FITC with optimal PEG linker length MDA-MB-231 cells (approximately 100,000 cells / well) overexpressing hFAP or mFAP were suspended in complete RPMI medium in a 96-well plate. FAP8-FITC adapters with increased PEG linker length were diluted with PBS. Their concentrations were confirmed by measuring absorbance using a nanodrop spectrophotometer. Then, FAP-FITC bispecific adapters with different PEG linker lengths were separately added to MDA-MB-231 cells at different final concentrations (triple chains). The cells were incubated with the adapters at room temperature for 40 minutes, washed three times with PBS and 2% FBS, centrifuged at 300 g for 5 minutes, and subjected to flow cytometry for MFI. The results are shown in Figures 6A-6C. As shown in Figures 6A-6C, FAP8-FITC binds to FAP+ cells with high specificity. A decrease in Kd was observed with increasing linker length.

[0356] MDA-MB231-FAP cells were suspended in 1.5 mL Eppendorf tubes (approximately 100,000 cells / tube). The cells were treated with 100 nM FAP8-PEG8-FITC and FAP8-PEG8. 12 -FITC, or FAP8-PEG 15- The cells were incubated with FITC at room temperature for 40 minutes. Then, the cells were washed twice and incubated with anti-FITC antibody (APC) at 4°C for 30 minutes (for FITC exposure), and then subjected to flow cytometry. The results are shown in Figures 7A-7B. As shown in Figures 7A-7B, increased PEG linker length allowed for better exposure, and FAP8-PEG 15 -FITC showed the best FITC exposure. As shown in Figures 7C-7D, increased PEG linker length allows for better binding with CAR T cells, thereby enhancing tumor disappearance.

[0357] MDA-MB-231-FAP-mCh cells were seeded overnight in 96 wells (approximately 5,000 cells / well). FAP8-PEG3-FITC, FAP8-PEG8-FITC, FAP8-PEG 12 -FITC and FAP8-PEG 15 -FITC was added separately to target cells at different concentrations (all in triplicates: 0nM, 0.1nM, 1nM, 10nM, 100nM, or 1,000nM). FAP5-PEG8-FITC was either co-incubated with the cells or aspirated after 1 hour of incubation at room temperature and washed away with complete RPMI. On day 19, 4M5.3 CAR-T cells were added to the target cells in an effector:target cell ratio of 1:3, followed by co-incubation for 68 hours. Incucyte images were acquired every 2 hours to measure total mCherry+ surface area. The results are shown in Figures 8A-8F. As shown in Figures 8A-8F, increased PEG linker length in FAP8-FITC enhanced the killing efficacy of FAP8-PEG. 15 -FITC demonstrated the best in vitro efficacy in killing pathogens and CAR-T activation.

[0358] [Example 10] FAP8-PEG 15 -FITC has shown superior efficacy against FAP5-PEG in in vitro killing activity. 16 -Comparable to FITC, but shows significantly better FITC exposure. MDA-MB-231 cells overexpressing hFAP or mFAP were suspended in a complete RPMI 96-well plate (approximately 100,000 cells / well). FAP8-PEG 15 -FITC and FAP5-PEG 16 -FITC concentration was confirmed by measuring absorbance using a nanodrop spectrometer. Adapters were added to MDA-MB-231 cells at different final concentrations (3-pack). Cells were incubated with the compound at room temperature for 40 minutes, washed three times with PBS and 2% FBS, centrifuged at 300g for 5 minutes, and subjected to flow cytometry for MFI measurement. Results are shown in Figures 9A and 9B. FAP8-PEG as shown in Figures 9A and 9B. 15 -FITC binding affinity was observed in hFAP and mFAP overexpressing MDA-MB-231 cells, compared to FAP5-PEG. 16 - Its binding affinity is comparable to that of FITC, but not quite as good.

[0359] MDA-MB-231 cells overexpressing FAP were suspended in 1.5 mL Eppendorf tubes (approximately 100,000 cells / tube). The cells were then treated with 100 nM FAP5-PEG. 16 -FITC or FAP8-PEG 15 -The cells were incubated with FITC at room temperature for 40 minutes. Then, the cells were washed twice and incubated with anti-FITC antibody (APC) at 4°C for 30 minutes, followed by flow cytometry. The results are shown in Figures 10A and 10B. FAP8-PEG, as shown in Figures 10A and 10B, 15 -FITC is FAP5-PEG 16 - It mediates more FITC exposure than FITC.

[0360] MDA-MB-231 cells overexpressing FAP were suspended in 1.5 mL Eppendorf tubes (approximately 200,000 cells / tube). The cells were then treated with 100 nM FAP8-PEG8-FITC and FAP8-PEG8-FITC. 12 -FITC, FAP8-PEG 15 -FITC, or FAP5-PEG 16-The cells were incubated with FITC at room temperature for 40 minutes. Then, the cells were washed twice and incubated at 37°C for 0 minutes or 1 hour, then incubated with anti-FITC antibody (APC) at 4°C for 30 minutes and subjected to flow cytometry. The results are shown in Figure 11. FAP8-PEG, as shown in Figure 11, 15 -FITC adapter is FAP5-PEG 16 - Compared to FITC, there was less dissociation (i.e., internalization) from the FAP protein, resulting in better FITC exposure.

[0361] MDA-MB-23a-FAP-mCh cells were seeded overnight in 96-well plates (approximately 5,000 cells / well). FAP8-PEG 15 -FITC and FAP5-PEG 16 -FITC adapters were added to target cells at different concentrations (all in triplicates: 0nM, 0.1nM, 1nM, 10nM, 100nM, or 1,000nM). FAP5-PEG 16 -FITC and FAP8-PEG 15 -FITC was either co-incubated with cells or aspirated after 1 hour of incubation at room temperature and washed away with complete RPMI. On D19, 4M5.3 CAR-T was added to target cells in a 1:3 E:T ratio and then co-incubated for 68 hours. Incucyte images were acquired every 2 hours to measure the total mCherry+ surface area. The results are shown in Figures 12A-12B. FAP8-PEG as shown in Figures 12A-12B. 15 -FITC is more effective in vitro than FAP5-PEG under adapter co-culture and adapter washing conditions. 16 -It was equivalent to FITC.

[0362] [Example 11] CAR T cell proliferation and activation in the blood At the end of the procedure, blood samples were collected from mouse hearts. A portion of the blood was incubated with erythrocyte lysis buffer on ice for 30 minutes and washed three times. The remaining blood was centrifuged at 1,000 g for 10 minutes to collect serum for human IFNγ ELISA analysis. The cells were then stained with zombie violet and an Fc blocker on ice for 30 minutes, washed twice, stained with anti-hCD3 on ice for 30 minutes, washed three times, and subjected to flow cytometry analysis. The results are shown in Figures 13A-13B. As shown in Figures 13A-13B, the FAP8-FITC adapter with a longer PEG linker resulted in better CAR T cell proliferation and activation.

[0363] [Example 12] FAP8-PEG 15 -FITC treatment did not cause toxicity in mice. FAP8-PEG at 500 nmol / kg was administered to disease control mice. 15 -FITC or phosphate-buffered saline was injected, and imaging was performed 24 hours later using the Spectral Ami optical imaging system. The tumor and all major organs were resected for imaging of the FITC signal.

[0364] Mouse body weight was measured twice per week and compared to initial body weight before any treatment or tumor transplantation. Data were obtained using FAP8-PEG. 15 -FITC specifically localizes to tumor sites and does not bind to cells in major organs. As shown in Figure 14, body weight analysis showed that FAP8-PEG 15 - This demonstrates that FITC treatment did not cause toxicity in mice.

[0365] [Example 13] PEG8~PEG 23 Bmax and FITC exposure of FAP8-FITC adapter with linker length MDA-MB231-FAP cells were suspended in 1.5 mL Eppendorf tubes (approximately 100,000 cells / tube). The cells were treated with 100 nM PEG8-PEG. 23Cells were incubated at room temperature for 1 hour with a FAP8-FITC adapter having a linker length (n=3). The cells were then washed twice and incubated with anti-FITC antibody (APC) at 4°C for 30 minutes (for FITC exposure) or subjected to flow cytometry (for Bmax).

[0366] The results are shown in Figures 15A-15B. As shown in Figure 15A, FITC exposure from the FAP binding pocket increased with increasing PEG linker length. As shown in Figure 15B, Bmax gradually decreased with increasing PEG linker length.

[0367] [Example 14] 4M5.3 PEG8-PEG in in vitro co-culture kill assay using CAR-T 23 Comparison of FAP8-FITC with linker length MDA-MB231-FAP-mCh cells (approximately 7,000 cells / well) were seeded overnight on a 96-well plate. PEG8~PEG 23 FAP8-FITC with linker length was added to target cells at different concentrations (0 nM to 1,000 nM, n=3). The adapter was either co-incubated with the cells or aspirated after 1 hour of incubation at room temperature and replaced with complete RMI. On approximately day 20, 4M5.3 CAR-T was added to the target cells in an E:T ratio of 1:3, followed by co-incubation for 48 hours. Live cells were measured by mCherry+ cells via flow cytometry. The results are shown in Figures 16A-16B.

[0368] As shown in Figures 16A-16B, 4M5.3 CAR-T and FAP8-PEG 23 - Better killing and T cell activation were observed in FITC. The same results were observed under the adapter washing conditions of the in vitro killing assay, as shown in Figures 17A-17B.

[0369] [Example 15] PEG8-PEG in in vitro co-culture kill assay using E2 CAR-T 23 Comparison of FAP8-FITC with linker length MDA-hFAP cells or Hs894 CAF cells were seeded overnight on 96-well plates. PEG8-PEG 23 FAP8-FITC with linker length was added to target cells at different concentrations (0 nM to 1,000 nM, n=3). E2 CAR-T was added to the target cells in an E:T ratio of 1:3 for MDA-hFAP and an E:T ratio of 2:1 for CAF, and then co-incubated for 48 hours. Live cells were measured by CellTrace+ cells via flow cytometry. The results are shown in Figures 18A to 18B.

[0370] As shown in Figures 18A-18B, the effectiveness of in vitro killing increased with increasing PEG linker length, but the difference was slight.

[0371] [Example 16] PEG8~PEG in combination with EC17 23 In vivo effects of FAP8-FITC with linker length on KB tumors KB tumors are FAP- and FR-, while CAF is FAP-. KB tumors were transplanted into NSG mice, and the tumors were approximately 50mm in size. 3 They were allowed to grow to this stage. The mouse groups were as follows: Group 1 (n=5) (FAP-FITC adapter only): FAP8-FITC only (CAR-T not included) Group 2 (n=6) (CAR T only): E2 CAR-T and PBS Group 3 (n=6) (FAP-FITC only): E2 CAR-T and FAP-FITC Group 4 (n=6) (EC17 only): E2 CAR-T and EC17 Groups 5, 6, 7, and 8 (n=6) (FAP-FITC and EC17): E2 CAR-T, FAP8-PEG 8~23 -FITC and EC17.

[0372] 10x10 on the mouse 6 Individual E2 CAR-T (VPN404) cells were intravenously administered with FAP8-FITC and / or EC17. Disease control tumors were 1,500 mm². 3 Tumors were harvested when the tumor reached a certain level or when the tumor disappeared in the treatment group. The results are shown in Figures 19A to 19F.

[0373] As shown in Figures 19A to 19F, PEG 18 FAP8-FITC with a linker mediated better regression in KB tumors.

[0374] [Example 17] FAP8-PEG 15 -FITC and FAP8-PEG 18 -FITC showed comparable cytokine release and CAR T count at the interim stage of the study. At an intermediate stage of the study, blood was collected from the tail vein of mice. A portion of the blood was incubated with red blood cell (RBC) lysis buffer on ice for 30 minutes and washed three times. The remaining blood was centrifuged at 1,000 g for 10 minutes to collect serum for human IFNγ enzyme-linked immunosorbent assay (ELISA) analysis. The cells were then stained with zombie violet and an Fc blocker on ice for 30 minutes, washed twice, stained with anti-hCD3 on ice for 30 minutes, washed three times, and subjected to flow cytometry analysis. The results are shown in Figures 20A-20B.

[0375] As shown in Figures 20A-20B, at the interim stage of the study, FAP8-PEG 15 -FITC and FAP8-PEG 18 - Similar cytokine release and CAR T cell counts were observed for FITC.

[0376] [Example 18] FAP8-PEG 18 -FITC was FAP8-PEG at the end of the study. 15 - Showed slightly better cytokine release and CAR T count than FITC. At the end of the study, blood was collected from the tail vein of mice. A portion of the blood was incubated with RBC lysis buffer on ice for 30 minutes and washed three times. The remaining blood was centrifuged at 1,000 g for 10 minutes to collect serum for human IFNγ ELISA analysis. The cells were then stained with zombie violet and an Fc blocker on ice for 30 minutes, washed twice, stained with anti-hCD3 on ice for 30 minutes, washed three times, and subjected to flow cytometry analysis. The results are shown in Figures 21A-21B.

[0377] As shown in Figures 21A-21B, FAP8-PEG 18 -FITC was FAP8-PEG at the end of the study. 15 - It showed slightly better cytokine release and CAR T count compared to FITC.

[0378] [Example 19] Combination therapy resulted in better CAR T proliferation and activation at the interim stage of the study. At an intermediate stage of the study, blood was collected from the tail vein of mice. A portion of the blood was incubated with RBC lysis buffer on ice for 30 minutes and washed three times. The remaining blood was centrifuged at 1,000 g for 10 minutes to collect serum for human IFNγ ELISA analysis. The cells were then stained with zombie violet and an Fc blocker on ice for 30 minutes, washed twice, stained with anti-hCD3 on ice for 30 minutes, washed three times, and subjected to flow cytometry analysis. The results are shown in Figures 22A-22B.

[0379] As shown in Figures 22A-22B, FAP8-PEG 15 -FITC and FAP8-PEG 18 -FITC showed comparable cytokine release and CAR T count at the interim stage of the study.

[0380] [Example 20] Combination therapy resulted in better CAR T proliferation and activation at the end of the study. At the end of the study, blood was collected from the tail vein of mice. A portion of the blood was incubated with RBC lysis buffer on ice for 30 minutes and washed three times. The remaining blood was centrifuged at 1,000 g for 10 minutes to collect serum for human IFNγ ELISA analysis. The cells were then stained with zombie violet and an Fc blocker on ice for 30 minutes, washed twice, stained with anti-hCD3 on ice for 30 minutes, washed three times, and subjected to flow cytometry analysis. The results are shown in Figures 23A-23B.

[0381] As shown in Figures 23A-23B, FAP8-PEG with EC17 18 -FITC was FAP8-PEG at the end of the study. 18 - It showed better cytokine release and CAR T count compared to EC17 without FITC.

[0382] [Example 21] Combination therapy resulted in better CAR T invasion into KB tumors at the end of the study. At the end of the study, tumors were collected from the mice and digested using a human tumor dissociation kit (Miltenyi) containing 50% enzyme R to enhance lymphocyte recovery. The cells were then stained with Zombie violet and an Fc blocker on ice for 30 minutes, washed twice, stained with anti-E2 CAR antibody on ice for 30 minutes, washed three times, and subjected to flow cytometry analysis. The results are shown in Figure 24.

[0383] As shown in Figure 24, more CAR T cell infiltration was observed in combination therapy with FAP8-FITC and EC17.

[0384] When FAP8-FITC (500 nmol / kg) was injected into disease control mice (KB tumor) and imaged 24 hours later, FAP8-PEG 23 -FITC is FAP8-PEG 15 -FITC and FAP8-PEG 18 - Showed weaker retention than FITC.

[0385] [Example 22] Immunohistochemistry of KB tumors and MDA-MB231 tumors To characterize and compare invasive FAP+CAF in cold KB tumors and hot MDA-MB-231 tumors, KB tumors or MDA-MB-231 tumors were transplanted into NOD scid gamma (NSG) mice, and the mice were treated with general-purpose anti-FITC CAR-T and EC17 (folate-fluorescein). Both tumors were then harvested from the mice for tumor fixation, fixed overnight with 10% formalin, and rinsed with 70% ethanol. The tumor cells were then subjected to IHC staining with either an anti-human CD3 antibody to detect human CAR-T cells or an anti-mouse FAP antibody to detect mouse FAP+CAF.

[0386] Results from immunohistochemical (IHC) staining demonstrated a lack of CAR-T cell infiltration in immunologically cold KB tumors, but not in hot MDA-MB-231 tumors. Increased FAP+CAF infiltration was observed in cold KB tumors, forming a physical barrier around the tumor, while invasive CAFs were significantly less abundant in hot MDA-MB-231 tumors. We hypothesized that these invasive fibroblasts contribute to the immunologically cold solid tumor characteristics.

[0387] [Example 23] Confocal microscopy of FAP8-FITC specific binding To confirm the adapter specificity of FAP8-FITC in FAP-expressing cells, MDA-MB231-hFAP (human FAP), MDA-MB231-mFAP (mouse FAP), and parental MDA-MB231 cells (without FAP expression) were seeded overnight on a confocal chamber (approximately 100,000 cells / well). FAP8-PEG 18 FAP8-FITC (50 nM) was added to the cells, and the mixture was incubated at room temperature for 1 hour. The cells were washed twice with PBS + 2% FBS and immediately examined under a confocal microscope. FAP8-FITC specifically bound to hFAP and mFAP.

[0388] [Example 24] In vivo study to determine the optimal linker for FAP8-FITC in mice FAP8-FITC with different PEG linkers was tested in mice to determine the optimal linker. 8、12、15 -FITC and FAP5-PEG 16 -The effectiveness of FITC was also compared. MDA-MB231-hFAP (5 million cells) was subcutaneously injected into one NSG mouse. The tumor size was approximately 100 mm. 3 When this stage was reached, each mouse was injected with 8 million E2 CAR T cells. 500 nmol / kg of FAP8-FITC was injected via the tail vein three times per week. The mouse groups were as follows: Group 1 (n=6) (disease control): Only tumor cells were injected. Group 2 (n=6) (CAR T cells only); CAR T cells (8 × 10 6 The patient was injected with (individual cells) and FAP-FITC was injected three times per week. ·Group 3 (n=6) (FAP8-PEG8-FITC treatment); CAR T cells (8×10 6 The patient was injected with (individual cells) and FAP-FITC was injected three times per week. Group 4 (n=7) (FAP8-PEG) 12 -FITC treatment); CAR T cells (8 x 10 6 The patient was injected with (individual cells) and FAP-FITC was injected three times per week. Group 5 (n=7) (FAP8-PEG) 15 -FITC treatment); CAR T cells (8 x 10 6 The patient was injected with (individual cells) and FAP-FITC was injected three times per week. Group 6 (n=6) (FAP5-PEG) 16 -FITC treatment); CAR T cells (8 x 10 6 The patient was injected with (individual cells) and FAP-FITC was injected three times per week.

[0389] FAP5-FITC functions comparably to FAP8-FITC in vitro, but in vivo, FAP8-FITC, with its superior serum stability, appears to mediate better binding to CAR T cells. See Figures 26A-26E.

[0390] [Example 25] CAR T cell tumor infiltration At the end of the treatment cycle, tumors were collected from mice and digested using a human tumor dissociation kit (Miltenyi) containing 50% enzyme R to enhance lymphocyte recovery. The cells were then stained with Zombie violet and an Fc blocker on ice for 30 minutes, followed by two washes. Next, the cells were stained with an anti-hCD3 antibody on ice for 30 minutes, followed by three washes. The cells were then subjected to flow cytometry. More CAR T cell infiltration was observed with FAP8-FITC treatment than with FAP5-FITC treatment. See Figure 27.

[0391] [Example 26] FAP8-FITC tumor retention analysis via imaging FAP8-FITC (500 nmol / kg) was injected into disease control mice (KB tumor), and imaging was performed 24 hours later. PEG 23 FAP8-FITC with a linker showed weaker retention in tumors, but PEG 15 or PEG 18 FAP8-FITC with a linker showed good retention.

[0392] [Example 27] Synthesis of DUPA-PEG6-FITC 2-[3-(3-benzyloxycarbonyl-1-tert-butoxycarbonylpropyl)-ureido]pentanedioate ditert-butyl ester (2). TEA (1.0 mL, 8.19 mmol) was added at -65°C to a solution of L-glutamic acid ditert-butyl hydrochloride (1.0 g, 3.39 mmol) and triphosgene (329.8 mg, 1.12 mmol) in dichloromethane (DCM) (25.0 mL) (Scheme 5). The mixture was stirred under argon at -65°C for 2 hours, and then L-Glu(OBn)-OtBu (1.2 g, 3.72 mmol) and TEA (600 μL, 4.91 mmol) in DCM (5.0 mL) were added. The reaction mixture was brought to room temperature over a period of 1 hour and stirred overnight at room temperature.

[0393] The reaction was quenched with 1 M HCl, the organic layer was washed with brine, and dried over Na₂SO₄. The crude product was purified by flash chromatography (hexane:HCl, 1:1) to obtain 2 (1.76 g, 90.2%) as a colorless oil. LCMS (m / z): (M+H) + C 30 H 47 Calculated value of N2O9 = 579.33; measured value = 579.30.

[0394] [ka]

[0395] 2-[3-(1,3-bis-tert-butoxycarbonylpropyl)-ureido]pentanedioic acid 1-tert-butyl ester (3). 10% Pd / C was added to a solution of 2 (1.00 g, 432) in DCM. The reaction mixture was hydrogenated at 1 atm at room temperature for 24 hours. Pd / C was filtered through a Celite pad and washed with DCM. The crude product was purified by flash chromatography (hexane:siRNA, 40:60) to obtain 3 (80.2%) as a colorless oil. LCMS (m / z): (M+H) + C 23 H 41Calculated value of N2O9 = 489.28; measured value = 489.28.

[0396] Solid-phase synthesis of 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) conjugate. The DUPA conjugate was synthesized by solid-phase method as follows (see Scheme 5). 500 mg, 0.400 mM, 200-400 mesh 1,2-diaminoethanetrityl resin bonded to a polymer was swollen by bubbling argon through DCM (8 mL) for 10 minutes, draining the DCM, bubbling dimethylformamide (DMF, 8 mL) for 10 minutes, and then draining. The resin was washed with DMF (3 × 8 mL). The resin was swollen in DMF, and a solution of Fmoc-Phen-OH (2.0 equivalents), PyBOP (2.0 equivalents), and DIPEA (5.0 equivalents) in DMF (8.0 mL) was added (Scheme 6). Argon was blown over the resin for 5 hours, and the resin was washed with DMF (3 × 8 mL). A 20% piperidine solution in DMF (3 × 8 mL) was added to the resin, and argon was blown over it for 15 minutes after each addition, and the resin was washed with DMF (3 × 8 mL). The above procedure was repeated for three further coupling steps using Fmoc-Phen OH (2.0), 8-aminooctanoic acid (1.5 equivalents), and DUPA(OtBu)-OH 3 (1.5 equivalents), as shown in Scheme 7. The final compound was cleaved from the resin using a trifluoroacetic acid (TFA):H2O:triisopropylsilane cocktail (95:2.5:2.5) and concentrated under vacuum. The concentrated product was precipitated in diethyl ether and dried under vacuum. The crude product was dissolved in the minimum amount of DMF and purified using a C-18 column (reverse phase) [solvent gradient: 0%B to 80%B over a 55-minute run, A=A=milli Q water (TFA) pH=3, B=acetonitrile (ACN)]. The pure fraction was collected, frozen at -80°C for 3 hours, and lyophilized to obtain conjugate 4. HRMS(ESI)(m / z):(M+H) + C 39 H 56 N7O 11 Calculated value = 798.40, measured value = 798.40.

[0397] [ka]

[0398] [ka]

[0399] DUPA-PEG6NHFmoc(6). DUPA conjugate 4 (0.100 g, 1 equivalent) and PEG6NH ester 5 (0.092 g, 1.1 equivalents) were stirred in DMF (1.5 mL). DIPEA (0.044 mL, 2.0 equivalents) was added under argon at room temperature, and the solution was stirred for 3 hours as described in Scheme 7. The reaction was monitored by LC-MS and purified by C-18 column reversed phase (mobile phase: A=milli Q water (TFA) pH=3, B=ACN, method: 0%B to 90%B in 50 minutes). The pure fraction was pooled and freeze-dried to obtain DUPA-PEG6NHFmoc 6.

[0400] [ka]

[0401] DUPA-PEG6NH2(7). DUPA-PEG6NHFmoc(6) was dissolved in 20% piperidine in DMF (1.0 mL) at room temperature and stirred under argon for 2 hours (Scheme 7). The reaction was monitored by LC-MS and purified by C-18 column reversed phase (mobile phase: A=milli Q water (TFA) pH=3, B=ACN, method: 0%B to 90%B in 50 minutes). The pure fraction was pooled and freeze-dried to obtain DUPA-PEG6NH27.

[0402] DUPA-PEG6-FITC(8). DUPA-PEG6NH27 (20 mg, 1.0 equivalent) was dissolved in DMF (1.0 mL) under an argon atmosphere, and then FITC (1.2 equivalents) was added under dark conditions (Scheme 7). After stirring for 5 minutes, DIPEA (4.0 equivalents) was added to the solution, and the reaction was monitored using LC-MS (the reaction was completed within 2.0 hours). The crude product was purified by C-18 column reversed phase (mobile phase: A=milli Q water (TFA) pH=3, B=ACN, method: 0%B~90%B in 60 minutes). The pure fraction was collected, frozen at -80°C for 3 hours, and lyophilized to obtain DUPA-PEG6-FITC conjugate 8. LC-MS(ESI)(m / z):(M+H) + C 75 H 95 N9O 25 Calculated value of S+H = 1522.6 Measured value (m / 2): (M+H) + 762.4 and (m / 3):(M+H) + 508.4.

[0403] [Example 28] Synthesis of PSMAL1-PEG6-FITC The PSMAL1-PEG6-FITC conjugate was synthesized by solid-phase method as follows (see the scheme below):

[0404] DIPEA (1.3 mL, 10 equivalents, 7.5 mmol) was added at -65°C to a solution of L-glutamic acid di-tert-butyl hydrochloride (1.33 g, 6.00 equivalents, 4.50 mmol) and triphosgene (0.445 g, 2.00 equivalents, 1.5 mmol) in DCM (15.0 mL) (Scheme 8). After stirring under argon at -65°C for 1 hour, the solution was warmed to room temperature and stirred at room temperature for 1 hour to generate the corresponding isocyanate intermediate in situ.

[0405] HL-LYS(ALLOC)-2-Cl-trityl resin (1.0 g, 1.0 equivalent, 0.75 mM) was swollen in a peptide vessel with argon for 10 minutes using dry DCM (8 mL). The DCM was then drained, and the resin was washed twice with dry DCM and then drained. Immediately after swelling the resin in DCM, the isocyanate produced in situ was transferred to a peptide vessel under an argon atmosphere. Argon was blown in at room temperature overnight (16 hours), and the isocyanate was washed with DCM. Completion of the reaction was confirmed by the Kaiser test.

[0406] Subsequently, tetrakis(triphenylphosphine)palladium (0) (100 mg) and 0.800 ml of morpholine were dissolved in 8 mL of dry DCM in a round-bottom flask under an argon atmosphere. The mixture was then transferred to a peptide vessel, and argon was blown in at room temperature for 3 hours to remove the allyloxy protecting group. After deprotection, the resin was washed three times with dry DCM. The completion of the reaction was again confirmed by the Kaiser test. Furthermore, the resin was washed twice with DMF and three times with sodium diethyldithiocarbamate (0.03 M in DMF) to remove any residual Pd catalyst. Finally, the resin was washed three times with DMF and DCM.

[0407] [ka]

[0408] Immediately, the resin was swollen in DMF for further coupling, and a solution of Fmoc-3(2-naphthyl)-L-alanine (2.0 equivalents), PyBOP (2.0 equivalents), and DIPEA (5.0 equivalents) in DMF (8.0 mL) was added (Scheme 8). Argon was blown in for 5 hours, and the resin was washed with DMF (3 × 8 mL). A solution of 20% piperidine in DMF (3 × 8 mL) was added to the resin, and argon was blown in for 15 minutes after each addition, and the resin was washed with DMF (3 × 8 mL). The above procedure was repeated for two further coupling steps using N-Fmoc-tranexamic acid (2.0) and Fmoc-N-amide-PEG6-acid (2.0 equivalents), as shown in Scheme 9. The final compound was cleaved from the resin using a trifluoroacetic acid (TFA):H2O:triisopropylsilane cocktail (95:2.5:2.5) and concentrated under vacuum. The concentrated product was precipitated in diethyl ether and dried under vacuum. The crude product was dissolved in a minimum amount of DMF and purified using a C-18 column (reverse phase) [solvent gradient: 0%B to 80%B in a 55-minute run, A=milli Q water (TFA pH=3), B=acetonitrile (ACN)]. The pure fraction was collected, frozen at -80°C for 3 hours, and lyophilized to obtain the conjugate PSMAL1-PEG6-NH2 intermediate. HRMS(ESI)(m / z):(M+H) + C 48 H 75 N6O 16 The calculated value is 991.52, and the measured value is 991.5.

[0409] [ka]

[0410] Synthesis of PSMAL1-PEG6-FITC conjugate. PSMAL1-PEG6-NH2 (10 mg, 1.0 equivalent) was dissolved in DMF (1.0 mL) under an argon atmosphere, and then FITC (1.2 equivalents) was added under dark conditions (Scheme 10). After stirring for 5 minutes, DIPEA (4.0 equivalents) was added to the solution, and the reaction was monitored using LC-MS (the reaction was completed within 1.0 to 2 hours). The crude product was purified by C-18 column reversed phase (mobile phase: A = milli Q water (TFA, pH = 3), B = ACN, method: 0% B to 90% B in 60 minutes). The pure fraction was collected, frozen at -80°C for 3 hours, and lyophilized to obtain PSMAL1-PEG6-FITC conjugate 8. LC-MS (ESI) (m / z): (M + H) + C 69 H 86 N7O 21 Calculated value of S+H = 1380.56 Measured value (m / 2): (M+H) + 690.9 and (m / 3):(M+H) + 461.0

[0411] [ka]

[0412] [Example 29] Effect of linker length on binding affinity to PSMA MDA-PSMA cells (200,000) were incubated in complete RPMI medium (RPMI + 10% FBS) with adapters serially diluted 2-fold from 500 nM for 1 hour at room temperature. The cells were washed twice with 2% FBS in PBS and resuspended in the same buffer. The fluorescence intensity of fluorescein isothiocyanate (FITC) was analyzed by flow cytometry. The results are shown in Figure 29.

[0413] As shown in Figure 29, the coupling affinity and total coupling of the DUPA-FITC adapter decreased as the linker length between DUPA and FITC increased.

[0414] [Example 30] PSMA expression levels in various cell lines used Cells (200,000) were stained with APC-anti-PSMA antibody on ice for 30 minutes and washed twice with 2% FBS in PBS. The fluorescence intensity of APC-anti-PSMA was analyzed by flow cytometry. The results are shown in Figure 30, which illustrates the level of PSMA expression in the indicated cell lines.

[0415] [Example 31] The length of the PEG linker affects the binding and surface exposure of the FITC portion to target cells. MDA-PSMA cells (200,000) were incubated in complete RPMI medium (RPMI + 10% FBS) with 1 μM DUPA-FITC conjugates containing different PEG linkers at room temperature for 1 hour. Free compounds were washed away, and the fluorescence intensity of FITC was analyzed by flow cytometry. To analyze surface exposure of the FITC portion, stained cells were incubated with APC-anti-FITC on ice for 30 minutes. The cells were then washed, and the fluorescence intensity of APC was analyzed by flow cytometry. The results are shown in Figures 31A-31B.

[0416] As shown in Figure 31A, the total bond between DUPA and FITC decreases as the linker length increases. As shown in Figure 31B, the surface exposure of FITC increases as the linker length increases.

[0417] [Example 32] Increasing linker length to the maximum PEG6 enhances the activation of anti-FITC CAR-T cells. Anti-FITC CAR-T cells were incubated with equal numbers of target cells in the presence of different concentrations of FITC-PEG-DUPA. The number of target cells was determined at the end of co-culture, and cytotoxicity was calculated using the formula: [(number of untreated cells - number of treated cells) / number of untreated cells] × 100%. IFNγ secretion from CAR-T cells in the co-culture medium was analyzed by ELISA using the human IFNγ ELISA kit (Biolegend). The results are shown in Figures 32A-32F. As shown in the figures, increasing the linker length in the bispecific adapter enhanced the activation of anti-FITC CAR-T cells. When the linker length reached PEG6, CAR-T cells reached the highest activation level. Longer linker lengths did not dramatically improve CAR-T cell activation.

[0418] [Example 33] Time course of DUPA-FITC accumulation in KB-PSMA tumors In KB-PSMA tumor-carrying mice, the indicated doses of DUPA-PEG6-FITC or DUPA-PEG were administered. 12 -FITC was injected. The mice were euthanized at the desired time, and the tumors were excised. FITC fluorescence images of the tumors were acquired using the Spectral Ami imaging system, and the mean radiance of the images was analyzed using Aura software. The results are shown in Figure 33B. As shown in Figure 33B, DUPA-PEG6-FITC and DUPA-PEG 12 -The peak accumulation of FITC in KB-PSMA tumors occurred approximately 2 hours after injection, and almost complete elimination occurred at approximately 96 hours.

[0419] [Example 34] DUPA-FITC with different PEG linkers mediates the eradication of MDA-PSMA tumors in mice without causing weight loss. MDA-PSMA cells (high PSMA level, 5 million cells) were transplanted into each NSG mouse by subcutaneous injection. The tumor volume was approximately 100 mm². 3When this was reached, the treatment group received 10 million anti-FITC CAR T cells and a bispecific adapter (without linker, PEG3, PEG6, or PEG) as shown in Figure 34A. 12 The patient was injected with ). Tumor volume and body weight were monitored regularly. Tumor volume was calculated using the formula: (length × width 2 The calculation was performed using ) / 2. The results are shown in Figures 34B and 34C. As shown in Figure 34B, all DUPA-FITC adapters with different PEG linkers successfully mediated tumor eradication. As shown in Figure 34C, all DUPA-FITC adapters tested did not cause weight loss during the procedure.

[0420] [Example 35] PSMAL1-PEG6-FITC has higher binding affinity and higher total binding than DUPA-PEG6-FITC. MDA-PSMA cells (200,000) were incubated at room temperature for 1 hour in complete RPMI medium (RPMI + 10% FBS) with adapters serially diluted from 500 nM to 2-fold. The cells were washed twice with 2% FBS in PBS and resuspended in the same buffer. The fluorescence intensity of FITC was analyzed by flow cytometry. The results are shown in Figure 35.

[0421] As shown in Figure 35, the binding affinity of PSMAL1-PEG6-FITC to MDA-PSMA cells is five times higher than that of DUPA-PEG6-FITC.

[0422] [Example 36] PSMAL1-PEG6-FITC is PSMA + Cellular retention is superior to that of DUPA-PEG6-FITC. MDA-PSMA cells or 22Rv1 cells were incubated with 1 μM DUPA-PEG6-FITC or PSMAL1-PEG6-FITC at room temperature for 1 hour. Unbound compounds were washed away, and the cells were cultured in RPMI containing 10% FBS at 37°C and 5% CO2. At the indicated time points, cells were imaged under a confocal microscope, and the intensity and location of FITC were analyzed. To quantify the intensity of FITC and surface-bound FITC, cells stained with DUPA-PEG6-FITC and PSMAL1-PEG6-FITC were analyzed by flow cytometry at the indicated time points, and the FITC fluorescence intensity was analyzed. To analyze surface-bound FITC, cells were stained with APC-anti-FITC antibody on ice for 30 minutes at different time points. The fluorescence intensity of APC was analyzed by flow cytometry. The results are shown in Figures 36A-36C. As shown in Figure 36A, PSMAL1-PEG6-FITC persisted longer than DUPA-PEG6-FITC in MDA-MB-231-PSMA cells. Figure 36B shows that PSMAL1-PEG6-FITC persisted longer than DUPA-PEG6-FITC in MDA-PSMA cells expressing different levels of PSMA. As shown in Figure 36C, PSMAL1-PEG6-FITC persisted longer than DUPA-PEG6-FITC in 22Rv1 cells that spontaneously express low levels of PSMA.

[0423] [Example 37] The efficacy of anti-FITC CAR-T cells mediated by PSMAL1-PEG6-FITC is higher than that of DUPA-PEG6-FITC at low concentrations. Equal numbers of 22Rv1 and anti-FITC CAR T cells were co-cultured in 96-well plates with different concentrations of DUPA-PEG6-FITC or PSMAL1-PEG6-FITC for 48 hours. The number of 22Rv1 cells was counted at the end of co-culture. Cytotoxicity of anti-FITC CAR T cells was calculated using the formula: (Number of untreated 22Rv1 cells - Number of treated 22Rv1 cells) / Number of untreated 22Rv1 cells × 100%. The concentration of IFNγ in the co-culture medium was analyzed by ELISA using a human IFNγ ELISA kit (Biolegend). The results are shown in Figures 37A-37B. As shown in Figure 37A, lysis of 22Rv1 cells by anti-FITC CAR-T cells mediated by PSMAL1-PEG6-FITC was much higher than that by DUPA-PEG6-FITC at low concentrations. As shown in Figure 37B, when co-cultured with 22Rv1 cells in the presence of PSMAL1-PEG6-FITC, IFNγ secreted by anti-FITC CAR-T cells was higher than that secreted by DUPA-PEG6-FITC at low concentrations.

[0424] [Example 38] PSMAL1-PEG6-FITC shows superior retention in MDA-MB-231-PSMA tumors compared to DUPA-PEG6-FITC and DUPA-FITC. MDA-PSMA tumor-carrying mice were intravenously injected with the indicated adapter at 500 nmol / kg. Fluorescence images of the mice were acquired using the Spectral Ami imaging system at the indicated time after injection. The results are shown in Figure 38.

[0425] [Example 39] PSMAL1-PEG6-FITC shows longer retention on the surface of MDA-MB-231-PSMA tumor cells in vivo. MDA-PSMA tumor-bearing mice were injected with DUPA-PEG6-FITC, PSMAL1-PEG6-FITC, or folic acid-FITC as a control. Mice were euthanized at the indicated time after injection. Tumors were excised and dissociated into single cells. The fluorescence intensity of FITC, indicating total adapter retention in tumor cells, was analyzed by flow cytometry. To analyze adapter surface retention on the tumor cell surface, dissociated tumor cells were stained with APC-anti-FITC antibody, and the fluorescence intensity of APC was analyzed by flow cytometry. The results are shown in Figure 39. After injection (48 hours after injection), 77% of tumor cells still had PSMAL1-PEG6-FITC inside the cell, and 65% of cells retained PSMAL1-PEG6-FITC on the surface. In contrast, only 13% of tumor cells had DUPA-PEG6-FITC, and 9% of cells retained DUPA-PEG6-FITC on the surface. At 144 hours post-injection, 45% of tumor cells retained PSMAL1-PEG6-FITC, and 34% of cells retained PSMAL1-PEG6-FITC on their surface.

[0426] [Example 40] PSMAL1-PEG6-FITC and DUPA-PEG6-FITC can mediate the eradication of MDA-PSMA tumors expressing high levels of PSMA. MDA-PSMA cells (5 million) were transplanted into each NSG mouse by subcutaneous injection. The tumor volume was approximately 100 mm². 3 When the tumor reached a certain stage, 10 million anti-FITC CAR T cells and the corresponding adapter were injected into the mice. Tumor volume and mouse body weight were monitored regularly. Tumor volume was calculated using the formula: (length × width) 2 The calculation was performed using ) / 2. The results are shown in Figures 40A-40C. Both DUPA-PEG6-FITC and PSMAL1-PEG6-FITC showed good efficacy in clearing MDA-PSMA tumors expressing high levels of PSMA, and no weight loss was observed in mice during treatment.

[0427] [Example 41] PSMAL1-PEG6-FITC significantly inhibited the growth of 22Rv1 tumors. 22Rv1 cells (3 million) were transplanted into NSG mice by subcutaneous injection. The tumor volume was approximately 50 mm². 3 When the target was reached, 10 million anti-FITC CAR T cells and the indicated adapter were injected into the mice. Tumor volume and mouse body weight were monitored regularly. Tumor volume was calculated using the formula: (length × width) 2 The calculation was performed using ) / 2. The results are shown in Figures 41A-41C. PSMAL1-PEG6-FITC and anti-FITC CAR-T cells significantly inhibited the growth of 22Rv1 tumors, while DUPA-PEG6-FITC showed little efficacy. No significant weight loss was observed in any of the treatment groups during the treatment period.

[0428] [Example 42] Efficacy of PSMAL1-PEG6-FITC binding to TagCAR T cells generated using lentiviral vectors. This study was conducted to evaluate the efficacy of a combination of administered PSMAL1-PEG6-FITC and T cells engineered in vivo using a lentiviral vector encoding a general-purpose anti-fluorescein CAR (TagCAR). Such a method may be advantageous over other costly or toxic autologous methods.

[0429] Selective generation of versatile anti-fluorescein CAR (TagCAR) T cells using a lentiviral vector. An exemplary TagCAR lentiviral vector (Figure 42A) was used to promote TagCAR transduction and payload expression in T cells. TagCAR lentiviral particles were pseudotyped and surface-engineered using coca glycoprotein to express a multi-domain fusion protein (MDF) consisting of an anti-CD3 single-strand variable fragment (scFv) sandwiched between two T-cell costimulatory ligands. The vector also contained a TagCAR payload as a polynucleotide encoding TagCAR, having the following components from N-terminus to C-terminus: scFv (e.g., anti-FITC E2), a hinge (spacer), a transmembrane domain, and an internal domain containing a costimulatory signaling domain and a CD3 zeta signaling domain (Z).

[0430] To evaluate selective T cell binding, activation, and transduction by TagCAR lentiviral particles, peripheral blood mononuclear cells (PBMCs) were transduced with TagCAR lentiviral particles containing a TagCAR payload, or, as a negative control, with a lentiviral vector without TagCAR or a TagCAR lentiviral vector without MDF. A representative flow plot of PBMCs and the anti-coca antibody used to detect lentiviral vector binding is shown in Figure 42B. In addition, the percentage of circulating immune cell subsets depending on the amount of TagCAR lentiviral particles delivered is shown in Figure 42C. Furthermore, T cell activation and transduction were measured as the percentage of CD25+ 3 days after transduction and TagCAR+ 7 days after transfection, respectively (Figure 42D). In summary, these data suggest that TagCAR lentiviral particles selectively generate TagCAR T cells after treatment of PBMCs.

[0431] PSMAL1-PEG6-FITC strongly binds to TagCAR T cells generated using TagCAR lentiviral particles. To demonstrate that TagCAR T cells generated using TagCAR lentiviral particles can strongly bind to PSMAL1-PEG6-FITC, the interaction between TagCAR and PSMAL1-PEG6-FITC was defined using a competitive assay, which is shown as a graph in Figure 43A. Since fluorescein fluorescence is quenched when TagCAR is bound, TagCAR T cells were instead incubated with saturation levels of FL-AF647, and then competitively replaced with increasing concentrations of PSMAL1-PEG6-FITC. The results from the competitive assay are shown in Figures 43B-43C. The concentration of PSMAL1-PEG6-FITC required to compete with FL-AF647 was lower compared to the antigen-only control of sodium fluorescein (NaFL), as detected by the mean fluorescence intensity (MFI) of FL-AF647. In addition, the average calculated inhibition constant (Ki) or semi-maximal inhibitory concentration of a triplicate composed of TagCAR T cells from different donors was calculated for PSMAL1-PEG6-FITC and NaFL using the equation: Ki = IC50 / (1 + [S] / Km) [where [S] = [FL-AF647] = 100 nM, and the Km of FL-AF647 is 9.9 nM]. PSMAL1-PEG6-FITC had a lower Ki at 9.9 nM compared to NaFL, suggesting that PSMAL1-PEG6-FITC strongly bound to TagCAR T cells generated using TagCAR lentiviral particles.

[0432] [Example 43] In vitro efficacy of TagCAR T cells generated using PSMAL1-PEG6-FITC-mediated TagCAR lentiviral particles against MDA-PSMA tumors This study was conducted to evaluate whether the combination of administered PSMAL1-PEG6-FITC and TagCAR T cells generated as described in Example 42 is effective in treating PSMA-positive tumor cells in vitro.

[0433] PSMAL1-PEG6-FITC strongly localizes to the surface of PSMA-positive tumor cells. To characterize the cell binding affinity of PSMAL1-PEG6-FITC to PSMA-expressing tumor cells, different concentrations of PSMAL1-PEG6-FITC were used to overexpress PSMA or (PSMA) + MDA-MB-321 tumor cells were incubated with either PSMA- or wild-type (PSMA-) cells. The level of surface fluorescein antigen on MDA-MB-321 tumor cells was detected using an anti-fluorescein antibody (Figure 44A). As shown in Figure 44B, PSMAL1-PEG6-FITC, at escalating ligand concentrations, was superior to PSMA-controls. + It was detected on the surface of tumor cells. Its affinity, or Kd, was calculated to be 1.292 nM, and it was found to be PSMA. + The total antigen amount, or Bmax, in tumor cells is 2.6 × 10⁻⁶. 6 It was MFI. These data suggest that PSMAL1-PEG6-FITC was specifically localized to the surface of PSMA-expressing tumors.

[0434] PSMAL1-PEG6-FITC induces dose-dependent TagCAR T cell function in vitro. The efficacy of TagCAR T cells generated by PSMAL1-PEG6-FITC, as described in Example 42, against PSMA-positive tumor cells was investigated in vitro. PSMA overexpression (PSMA + )Normalized tumor cell growth rate of MDA-MB-231 tumor cells in the presence of TagCAR T cells generated as described in Example 42 in PSMA +Tumor size was measured every 4 hours during 88 hours of co-culture of MD-MB-231 tumor cells with different concentrations of PSMAL1-PEG6-FITC. As shown in Figure 45A, tumor size was reduced in the presence of PSMAL1-PEG6-FITC compared to negative controls in the absence of PSMAL1-PEG6-FITC.

[0435] In addition, cytokine levels were adjusted using different concentrations of PSMAL1-PEG6-FITC in the TagCAR T cells generated as described in Example 42 and PSMA. + The levels were measured in the culture supernatant 24 hours after addition to MD-MB-231 tumor cells. As shown in Figure 45B, levels of both interferon-gamma (IFNg) and interleukin-2 (IL-2) increased with increasing dose of PSMAL1-PEG6-FITC.

[0436] The presence of TagCAR+ cells (Figure 45C) and CD25+ cells (Figure 45D) in CD3+ T cells is also related to PSMA. + The measurements were taken after 88 hours of co-culture of MDA-MB-231 tumor cells with different concentrations of PSMAL1-PEG6-FITC. TagCAR-bearing CD3+ T cells had a higher percentage of CD25+ compared to TagCAR-less CD3+ T cells.

[0437] In summary, these data suggest that TagCAR T cells can inhibit the growth of PSMA-positive tumors by mediating dose-dependent cytolytic activity and cytokine release against PSMA-positive tumor cells.

[0438] [Example 44] FAP-FITC mediates the killing of hFAP+ tumor cells by anti-FITC CAR-T cells. MDA-MB-231-hFAP-mCh cells were seeded overnight on 96-well plates (approximately 7,000 cells / well). On day 18, 4M5.3 CAR-T cells were added to the target cells in a 1:1 effector:target cell ratio. FAP5-PEG 16-FITC was added to target cells and CAR-T cells at different concentrations (all in triplicates, 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, or 1,000 nM) and incubated for 24 hours. Viability was determined by flow cytometry by mCherry-positive cells or by mCherry-positive surface area measured by Incucyte, which acquired images every 2 hours. Killing percentage was determined by (1 - (viable cells) / (viable cells in wells with tumor cells only)) × 100%. Results are shown in Figures 47A and 47B.

[0439] As shown in Figures 47A-47B, FAP5-FITC mediated effective killing of human FAP+ cells. Killing was maximized at FAP5-FITC concentrations of 1 nM to 10 nM.

[0440] The above experiment was repeated using human fibrosarcoma cells that overexpress hFAP. Similar results were obtained.

[0441] [Example 45] FAP5-FITC mediates FAP+ tumor disappearance via 4M5.3 or E2 CAR-T cells without toxicity. MDA-MB-231-hFAP cells were subcutaneously injected into NSG mice, and 150 mm 3 The cells were allowed to grow to this stage. Then, 4M5.3 or E2 CAR-T cells (8 million) were intravenously injected, followed by injection of FAP5-PEG8-FITC at 400 nmol / kg three times per week. The following five groups of mice (n=6) were used: • Disease control group (tumor cells only), • Group consisting only of 4M5.3 CAR-T cells (4M5.3 CAR-T cells and PBS), • 4M5.3 CAR-T cells and FAP5-PEG8-FITC group (administered 3 times / week), • Group consisting only of E2 CAR-T cells (E2 CAR-T cells and PBS), and • E2 CAR-T cells and FAP5-PEG8-FITC group (administered 3 times / week).

[0442] E2 CAR-T cells target and attack cancer cells that express estrogen receptor alpha (ERα), also known as E2. They are designed to recognize and bind to cancer cells that overexpress ERα, which is often found in hormone receptor-positive breast cancer and other hormone-dependent cancers.

[0443] The results are shown in Figures 46A-46C. As shown in Figure 46A, FAP5-PEG8-FITC was able to mediate FAP+ tumor clearance via 4M5.3 CAR-T cells. As shown in Figure 46B, FAP5-PEG8-FITC was able to mediate FAP+ tumor clearance via E2 CAR-T cells. As shown in Figure 46C, treatment with FAP5-PEG8-FITC did not mediate any toxicity in mice treated with either 4M5.3 CAR-T cells or E2 CAR-T cells.

[0444] [Example 46] FAP-FITC treatment enhanced CAR-T cell activation and proliferation, and reduced tumor cell proliferation by eliminating FAP+CAF in TMEs. At the end of the in vivo study, mouse blood samples were collected via cardiac puncture. The mouse blood samples were then centrifuged at approximately 1,000 g for 10 minutes, and serum was subsequently obtained for detection of hIFNγ via ELISA assay (Figure 48C). The blood samples were then incubated and washed in erythrocyte (RBC) lysis buffer according to the manufacturer's protocol and stained for zombie violet (i.e., live / dead staining) and anti-human CD3 antibody (i.e., to detect human CAR-T cells) (Figure 48C). Combination therapy with FAP-FITC + EC17 suppressed KB tumor growth without significant toxicity (Figures 48A-48B).

[0445] After euthanasia of the mice, mouse tumors were collected at the end of the study. Small pieces from each tumor sample were cut and fixed in 10% formalin for the preparation of IHC slides. The remaining tumor was digested using a human tumor dissociation kit from Miltenyi Biotec according to the manufacturer's protocol (Milteny Biotec, Bergisch Galdbach, Germany). For the IHC slides, CAF was stained with anti-mouse alpha smooth muscle actin (a marker for CAF), and cancer cells were stained with anti-mouse Ki67 as a cancer cell proliferation marker. Subsequently, tumor cells derived from tumor dissociation were stained with anti-human CD3 antibody for the detection of human CAR-T cells. The results are shown in Figures 48A-49D.

[0446] As shown in Figures 48C-48D, mice treated with combination therapy had greater CAR-T cell proliferation and higher levels of hIFNγ in their blood. CAR-T infiltration into KB tumors was similar between the EC17-treated group and the EC17+FAP5-FITC-treated group, and these results may have been due to late-stage tumor harvesting and the majority of CAR-T cells being non-functional or no longer viable. IHC staining with alpha-smooth muscle actin showed a decrease in CAF in the peripheral and central parts of tumors after FAP-FITC treatment, as well as a decrease in tumor growth rate (Figure 48F). The decrease in tumor growth rate was attributed to CAF elimination, which eliminates the release of multiple CAF-derived growth factors that stimulate tumor cell growth. As shown in Figure 48E, combination therapy using 4M5.3 anti-FITC CAR-T cells showed similar results.

[0447] As shown in Figures 49A-49D, combination therapy with FAP5-PEG8-FITC and DUPA-PEG6-FITC also enhanced tumor resolution. Mice treated with the combination therapy showed higher CAR-T cell proliferation in the blood and tumors. Similar results were observed in different PSMAs. + This was obtained in a cold tumor model (KB-PSMA) (see Figure 50).

[0448] [Example 47] In vivo efficacy of Aza-PEG6-FITC and ortho-CAL-PEG6-FITC combined with EC17 (folate-FITC) KB cells (1 million) were transplanted into each NSG mouse by subcutaneous injection. The tumor volume was approximately 50 mm². 3 When the target was reached, the treatment group was injected with 10 million anti-FITC CAR T cells and the indicated bispecific adapter, as shown in Figure 51A. Tumor volume and body weight were monitored regularly. Tumor volume was calculated using the formula: (length × width) 2 The calculation was performed using ) / 2. The results are shown in Figures 51A to 51C.

[0449] Figure 51B shows tumor growth curves for different treatment groups, relating to days after CAR-T cell injection and tumor volume (mm²). 3 Figure 51C is a graph showing the relationship between Aza-PEG6-FITC in combination with EC17 and KB tumor growth. Aza-PEG6-FITC in combination with EC17 significantly inhibited KB tumor growth. Ortho-CAL-PEG6-FITC in combination with EC17 also showed slightly better inhibition of KB tumor growth. Figure 51C is a graph showing the relationship between days after CAR-T cell injection and weight change (%) in mice in different treatment groups. Aza-PEG6-FITC and EC17 induced weight loss in mice. Weight loss may have been due to cytokine release from increased CAR-T cells. Toxicity can be minimized by optimizing the dosage of the adapter.

[0450] [Example 48] FAP8-PEG 18 -In vivo efficacy of Aza-PEG6-FITC in combination with FITC KB cells (1 million) were transplanted into each NSG mouse by subcutaneous injection. The tumor volume was approximately 50 mm². 3 When the target was reached, the treatment group was injected with 10 million anti-FITC CAR T cells and the indicated bispecific adapter, as shown in Figure 52A. Tumor volume and body weight were monitored regularly. Tumor volume was calculated using the formula: (length × width) 2The calculation was performed using ) / 2. The results are shown in Figures 52B to 52C.

[0451] Figure 52B shows tumor growth curves for different treatment groups, relating to the number of days after CAR-T cell injection and tumor volume (mm²). 3 This is a graph of FAP8-PEG. 18 Aza-PEG6-FITC, combined with FITC, slightly inhibited the growth of KB tumors. Efficacy was observed with EC17 and FAP8-PEG. 18 -It was equivalent to the combination with FITC. FAP8-PEG 18 Ortho-CAL-PEG6-FITC, combined with -FITC, showed better efficacy in inhibiting KB tumor growth. Figure 52C is a graph showing the change in body weight (%) between days after CAR-T cell injection and body weight change in mice in different treatment groups. None of the combinations induced significant weight loss in the treated mice.

[0452] [Example 49] EC17 (folate-FITC) and FAP8-PEG 18 -In vivo efficacy of Aza-PEG6-FITC in combination with FITC KB cells (1 million) were transplanted into each NSG mouse by subcutaneous injection. The tumor volume was approximately 50 mm². 3 When the target was reached, the treatment group was injected with 10 million anti-FITC CAR T cells and the indicated bispecific adapter, as shown in Figure 53A. Tumor volume and body weight were monitored regularly. Tumor volume was calculated using the formula: (length × width) 2 The calculation was performed using ) / 2. The results are shown in Figures 53B to 53C.

[0453] Figure 53B shows the growth curves of different treatment groups, relating to the number of days after CAR-T cell injection and tumor volume (mm²). 3 This is a graph of FAP8-PEG. 18 - Aza-PEG6-FITC combined with FITC and EC17 is FAP8-PEG 18- It has comparable efficacy to ortho-CAL-PEG6-FITC in combination with FITC and EC17. Both have comparable efficacy to EC17 and FAP8-PEG in inhibiting KB tumor growth. 18 -It showed slightly better efficacy than the combination with FITC. Figure 53C is a graph showing the change in body weight (%) in mice in different treatment groups: Aza-PEG6-FITC, EC17, and FAP8-PEG. 18 Mice injected with the combination of -FITC showed weight loss during treatment. This weight loss may have been due to cytokine release from enlarged CAR-T cells. Toxicity can be minimized by optimizing the adapter dosage.

[0454] [Example 50] IHC of KB tumors and MDA-MB231 tumors To characterize and compare invasive FAP+CAF in cold KB tumors and hot MDA-MB-231 tumors, KB tumors or MDA-MB-231 tumors were transplanted into NOD scid gamma (NSG) mice, and the mice were treated with general-purpose anti-FITC CAR-T and EC17 (folate-fluorescein). Both tumors were then harvested from the mice for tumor fixation, fixed overnight with 10% formalin, and rinsed with 70% ethanol. The tumor cells were then subjected to IHC staining with either an anti-human CD3 antibody to detect human CAR-T cells or an anti-mouse FAP antibody to detect mouse FAP+CAF.

[0455] As shown in Figure 54C, results from IHC staining demonstrated a lack of CAR-T cell infiltration in immunologically cold KB tumors, but not in hot MDA-MB-231 tumors. Increased FAP+CAF infiltration was observed in cold KB tumors, forming a physical barrier around the tumor, while invasive CAFs were significantly less numerous in hot MDA-MB-231 tumors. We hypothesized that these invasive fibroblasts are one reason for the immunologically cold solid tumors.

[0456] [Example 51] FAP8-FITC binding affinity To determine the binding affinity of FAP8-FITC in human FAP (hFAP) and mouse FAP (mFAP), MDA-MB231-FAP cells (approximately 100,000 cells) were suspended and incubated with gradually increasing concentrations of FAP8-FITC at room temperature for 1 hour. The cells were washed twice and subjected to flow cytometry for Bmax.

[0457] As shown in Figures 54A-54B, FAP8-FITC binds to hFAP and mFAP with high affinity and specificity. A decrease in Kd was observed with increasing linker length.

[0458] [Example 52] In vitro efficacy of FAP8-FITC in killing FAP+ cells FAP + To determine the in vitro efficacy of FAP8-FITC in killing cells, MDA-hFAP cells (approximately 7,000 cells / well) and Hs894 CAF cells (approximately 4,000 cells / well) were separately seeded overnight on 96-well plates. FAP8-FITC was added to target cells at various concentrations (0nM, 0.001nM, 0.01nM, 0.1nM, 1nM, 10nM, 100nM, and 1,000nM, n=3). E2 CAR T was added to target cells at an E:T ratio of 1:3 for MDA-hFAP and an E:T ratio of 2:1 for CAF, and concurrently incubated for 48 hours. Live cells were measured by CellTrace+ cells via flow cytometry. FAP-FITC mediated the killing of FAP-expressing cells. See Figures 55A-55B.

[0459] [Example 53] The effects of FAP8-FITC and EC17 on KB tumors To compare FAP8-FITCs with different PEG lengths in vivo, KB (tumor FAP) - FR + CAF FAP+ The tumor was transplanted into NSG mice, and the size was approximately 50 mm. 3 It was grown to this size. For the mouse, 10x10 6 Individual E2 CAR T (VPN404) cells were intravenously administered with FAP8-FITC and / or EC17. Disease controls were 1500 mm. 3 Tumors were harvested when the tumor reached a certain stage or when it disappeared in the treatment group. The mouse groups were as follows: Group 1 (n=5) (FAP-FITC adapter only): FAP8-FITC only (CAR T not included) Group 2 (n=6) (CAR T only): E2 CAR T + PBS Group 3 (n=6) (FAP-FITC only): E2 CAR T + FAP-FITC Group 4 (n=6) (EC17 only): E2 CAR T + EC17 ·Group 5 (n=6) (FAP-FITC+EC17): E2 CAR T+FAP8-FITC+EC17

[0460] The results indicate that FAP8-FTC mediates the efficacy of combination therapy in KB tumors. See Figures 56A-56E.

[0461] [Example 54] Combination therapy resulted in better CAR T proliferation and activation at the interim stage of the study. At an intermediate stage of the study, blood was collected from the tail vein of mice. A portion of the blood was incubated with RBC lysis buffer on ice for 30 minutes and washed three times. The remaining blood was centrifuged at 1,000 g for 10 minutes to collect serum for human IFNγ ELISA analysis. The cells were then stained with zombie violet and an Fc blocker on ice for 30 minutes and washed twice. The cells were then stained with anti-hCD3 for 30 minutes, washed three times, and subjected to flow cytometry.

[0462] PEG 15 / PEG 18FAP8-FITC, possessing [specific characteristic], showed comparable cytokine release and CAR T count at the interim stage of the study. See Figures 57A-57B.

[0463] [Example 55] Combination therapy resulted in better CAR T proliferation and activation at the end of the study. At an intermediate stage of the study, blood was collected from the tail vein of mice. A portion of the blood was incubated with RBC lysis buffer on ice for 30 minutes and washed three times. The remaining blood was centrifuged at 1,000 g for 10 minutes to collect serum for human IFNγ ELISA analysis. The cells were then stained with zombie violet and an Fc blocker on ice for 30 minutes and washed twice. The cells were then stained with anti-hCD3 for 30 minutes, washed three times, and subjected to flow cytometry.

[0464] FAP8-PEG 18 -FITC showed better cytokine release and CAR T count at the end of the study. See Figures 58A-58B.

[0465] [Example 56] Combination therapy resulted in better CAR T invasion into KB tumors at the end of the study. At the end of the study, tumors were collected from the mice and digested using a human tumor dissociation kit (Miltenyi) containing 50% enzyme R to enhance lymphocyte recovery. The cells were then stained with Zombie violet and an Fc blocker on ice for 30 minutes and washed twice. The cells were then stained with an anti-E2 CAR antibody on ice for 30 minutes, washed three times, and subjected to flow cytometry.

[0466] More CAR T cell infiltration was observed in combination therapy with FAP8-FITC and EC17. See Figure 59.

[0467] [Example 57] IHC staining Tumor samples from mouse studies (CAR T+EC17, CAR T+FAP-FITC, and CAR T+EC17+FAP-FITC) were fixed with 10% formalin, paraffin-treated, and sectioned for IHC staining of the cancer growth marker Ki67. After FAP-FITC treatment, a decrease in cancer growth was observed, likely due to the disappearance of CAF, which is known to secrete various growth factors (Figure 60).

[0468] Staining for the human T cell marker CD3 showed increased T cell infiltration after FAP-FITC treatment, likely due to the disappearance of CAFs, which are known to form a physical barrier that blocks the function of immune cells (Figure 60).

[0469] List of embodiments of the claimed invention Clause 1. The following structure: FL-TL or a pharmaceutically acceptable salt or hydrate thereof [in the formula, F includes fluorescein, fluorescein isothiocyanate (FITC), or N-hydroxysuccinimide (NHS)-fluorescein. L includes the linker, TLs include targeted ligands, such as radicals of fibroblast-activating protein (FAP) ligands or radicals of prostate-specific membrane antigen (PSMA) ligands. A dual-specificity adapter including a bispecific adapter.

[0470] Clause 2. The targeted ligand is

[0471] [ka] [In the formula,

[0472] [ka] [This is the linking point to the linker.] A bispecific adapter according to Clause 1, comprising a radical of an FAP ligand containing the structure.

[0473] Clause 3. The targeted ligand is of formula IB:

[0474] [ka] [In the formula,

[0475] [ka] This is the linking point to the linker, T is a substituted or unsubstituted methylene group (-CH2-), a substituted or unsubstituted amino group (-NH-), -O-, or -S-. R 1 and R 2 Each of these is independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl. R 3 and R 4 These are -H, -OH, F, Cl, Br, I, and -C, respectively. 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 5 , R 6 , R 7 , and R 8 Each is independently selected from the group consisting of H, alkyl, and halo. R 9 , R 10 , and R 11 These are H and -C respectively. 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] A dual-specific adapter according to Clause 2, comprising a radical of an FAP5 ligand having a structure represented by [the specified structure].

[0476] Clause 4. The targeted ligand is of formula IC:

[0477] [ka] [In the formula,

[0478] [ka] This is the linking point to the linker, T is a substituted or unsubstituted methylene group (-CH2-), a substituted or unsubstituted amino group (-NH-), -O-, or -S-. R 1 and R 2 Each of these is independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl. R 3 and R 4 These are -H, -OH, F, Cl, Br, I, and -C, respectively. 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 5 , R 6 , R 7 , and R 8 Each is independently selected from the group consisting of H, alkyl, and halo. R 9 , R 10 , and R 11 These are H and -C respectively. 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] A dual-specific adapter according to Clause 2, comprising a radical of an FAP5 ligand having a structure represented by [the specified structure].

[0479] Clause 5. The target ligand has the following structure:

[0480] [ka] [In the formula,

[0481] [ka] This represents a functionalized 5-10 member nitrogen-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, optionally further containing 1-3 heteroatoms selected from O, N, and S. R1 and R2 are -H, -D, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R3 and R4 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R5 and R6 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R7 is selected from the group consisting of -H, -D, OH, CH2=, -CH3, CH3CH2-, (CH3)2CH-, (CH3)3C-, -CH2Ph, and substituted -CH2Ph. R8~R 10 is -H, -OH, -F, -Cl, -Br, -I, -NO2, -SO3H, -SO2NH2, -NH2, -N3, -NH=NH, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 11 -H, -D, C1~C 10 Alkyl, C3~C 10Cycloalkyl, adamantyl,

[0482] [ka] , substituted or unsubstituted aryl, substituted or unsubstituted C7~C 20 Selected from the group consisting of alkylaryls, the aryl is

[0483] [ka] (In the formula, R 12 and R 16 These include -H, -D, halogens, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 It is selected from the group consisting of H, D, halogens, C1-C4 alkyls, and C1-C3 alkoxys. R 13 , R 14 and R 15 These include -H, -D, halogens, -OMe, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 (These are selected from the group consisting of -H, -D, halogens, C1-C4 alkyls, and C1-C3 alkoxys.) And, R 17 , R 18 , R 20 , and R 21 It is selected independently of -H and -CH3, R 19 and R 22 [These are independently selected from the group consisting of phenyl, dimethoxyphenyl, and aryl compounds.] A bispecific adapter as described in Clause 2, comprising a radical of the FAP8 ligand.

[0484] Clause 6. The dual-specific adapter according to Clause 1, wherein the targeting ligand comprises a radical of a PSMA ligand and is (S)-5-amino-1-carboxypentyl)carbamoyl)-L-glutamic acid (PSMAL1) or 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA).

[0485] Clause 7. The linker may be polyethylene glycol (PEG) or a PEG derivative, for example, optionally PEG3 to PEG 16 , and optionally PEG4~PEG 15 Or PEG3~PEG 12 PEG 12 PEG 15 PEG 16 , or PEG 18 PEG4~PEG 16 PEG 16 PEG3~PEG 15 PEG 15 PEG3~PEG 12 A dual-specific adapter as described in any one of clauses 1 to 6, including or essentially comprising PEG6; PEG3-PEG8; or PEG6.

[0486] Clause 8. A dual-specific adapter as described in any one of Clauses 1 to 6, for use in conjunction with anti-fluorescein chimeric antigen receptor (CAR)-T cells in the treatment of cancer.

[0487] Clause 9. Optionally, the linker may be configured to use PEG3-PEG 15 , and optionally PEG 15 A dual-specific adapter as described in any one of Clauses 1 to 5, for use with anti-fluorescein CAR-T cells in the treatment of FAP-expressing cancer, comprising or essentially derived therefrom.

[0488] Clause 10. Optionally, the linker may be PEG3~PEG 12A dual-specific adapter as described in Clause 1 or 6, for use with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancer, and optionally comprising PEG6 or PEG3-PEG8, and optionally comprising or essentially comprising PEG6.

[0489] Clause 11. A pharmaceutical composition for the treatment of cancer comprising a dual-specific adapter as described in any one of Clauses 1 to 10 and a pharmaceutically acceptable carrier or excipient.

[0490] Clause 12. A combination of dual-specific adapters for use with anti-fluorescein chimeric antigen receptor (CAR)-T cells in the treatment of cancer, (i) A first bispecific adapter comprising the bispecific adapter described in item 1 or a pharmaceutically acceptable salt or hydrate thereof, wherein the targeting ligand of the first bispecific adapter is

[0491] [ka] [In the formula,

[0492] [ka] [This is the linking point to the linker.] A first bispecific adapter comprising a radical of an FAP ligand having the formula, (ii) The following structure: FL-TL or a pharmaceutically acceptable salt or hydrate thereof [in the formula, F contains fluorescein, FITC, or NHS-fluorescein. L includes the linker, TLs include targeted ligands containing radicals of folate receptor (FR) ligands or prostate-specific membrane antigen (PSMA) ligands. A second bispecific adapter including A combination that includes this.

[0493] Clause 13. The radical of the FAP ligand of the first bispecific adapter is of formula IB:

[0494] [ka] [T is a substituted or unsubstituted methylene (-CH2-), substituted or unsubstituted amino (-NH-), -O- or -S-, R 1 and R 2 These are -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, Independently selected from the group consisting of -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl, R 3 and R 4 These are -H, -OH, F, Cl, Br, I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 5 , R 6 , R 7 , and R 8 Each is independently selected from the group consisting of H, alkyl, and halo. R 9 , R 10 , and R 11 These are H and -C respectively. 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] The structure represented by, Formula IC:

[0495] [ka] [In the formula,

[0496] [ka] This is the linking point to the linker, T is a substituted or unsubstituted methylene group (-CH2-), a substituted or unsubstituted amino group (-NH-), -O-, or -S-. R 1 and R 2 Each of these is independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, and 5-tetrazolyl. R 3 and R 4 These are -H, -OH, F, Cl, Br, I, and -C, respectively. 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 5 , R 6 , R 7 , and R 8 Each is independently selected from the group consisting of H, alkyl, and halo. R 9 , R 10 , and R 11 These are H and -C respectively. 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] The structure represented by, The following formula:

[0497] [ka] [In the formula,

[0498] [ka] This represents a functionalized 5-10 member nitrogen-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, optionally further containing 1-3 heteroatoms selected from O, N, and S. R1 and R2 are -H, -D, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R3 and R4 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R5 and R6 are -H, -OH, -F, -Cl, -Br, -I, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R7 is selected from the group consisting of -H, -D, OH, CH2=, -CH3, CH3CH2-, (CH3)2CH-, (CH3)3C-, -CH2Ph, and substituted -CH2Ph. R8~R 10 is -H, -OH, -F, -Cl, -Br, -I, -NO2, -SO3H, -SO2NH2, -NH2, -N3, -NH=NH, -C 1~6 Alkyl, -OC 1~6 alkyl, and -SC 1~6 Independently selected from the group consisting of alkyls, R 11 -H, -D, C1~C 10 Alkyl, C3~C 10 Cycloalkyl, adamantyl,

[0499] [ka] , substituted or unsubstituted aryl, substituted or unsubstituted C7~C 20 Selected from the group consisting of alkylaryls, the aryl is

[0500] [ka] (In the formula, R 12 and R 16 These include -H, -D, halogens, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 It is selected from the group consisting of H, D, halogens, C1-C4 alkyls, and C1-C3 alkoxys. R 13 , R 14 and R 15 These include -H, -D, halogens, -OMe, C1-C3 alkyl, C1-C3 alkoxy, -CF3, and -C(=O)-OR 23 Independently selected from the group consisting of R 23 (These are selected from the group consisting of -H, -D, halogens, C1-C4 alkyls, and C1-C3 alkoxys.) And, R 17 , R 18 , R 20 , and R 21 It is selected independently of -H and -CH3, R 19 and R 22 [These are independently selected from the group consisting of phenyl, dimethoxyphenyl, and aryl compounds.] Structure represented by The combinations described in Clause 12, which include the following:

[0501] Clause 14. The combination described in Clause 12, wherein the targeting ligand of the second bispecific adapter or a pharmaceutically acceptable salt or hydrate thereof comprises a radical of a PSMA ligand.

[0502] Clause 15. The linker of the first dual-specificity adapter is a combination of the components described in Clause 12, which includes or is essentially derived from a PEG.

[0503] Clause 16. The combination described in Clause 14, wherein the targeting ligand is PSMAL1 or DUPA or includes them.

[0504] Clause 17. The combination described in Clause 12, wherein the targeting ligand of the second bispecific adapter is a radical of folic acid or a functional fragment or analog thereof.

[0505] Clause 18. The combinations described in Clause 17, wherein folic acid is folic acid, dihydrofolic acid, tetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid (5,10-MTHF), 5-methyltetrahydrofolic acid (5-MTHF), or larcitrexed.

[0506] Clause 19. The PSMA ligand is DUPA, and the linker is PEG or a PEG derivative, for example, optionally PEG3-PEG. 12 , and optionally PEG6;PEG3~PEG 16 , and optionally PEG4~PEG 15 Or PEG3~PEG 12 PEG 12 PEG 15 PEG 16 , or PEG 18 PEG4~PEG 16 , and optionally PEG 16 PEG3~PEG 15 , and optionally PEG 15 ; The combinations described in Clause 16, including or essentially comprising PEG3 to PEG8, and optionally PEG6.

[0507] Clause 20. Any combination described in any one of Clauses 12 to 19 for use in conjunction with anti-fluorescein CAR-T cells in the treatment of cancer.

[0508] Clause 21. Any combination described in any one of Clauses 12 to 19 for use in conjunction with anti-fluorescein CAR-T cells in the treatment of FAP-expressing cancer.

[0509] Clause 22. Combinations described in any one of Clauses 12 to 16 and 19 for use in conjunction with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancer.

[0510] Clause 23. Combinations described in any one of Clauses 12 to 15, 17, and 18 for use in conjunction with anti-fluorescein CAR-T cells in the treatment of folate-expressing cancers.

[0511] Clause 24. The combination according to any one of Clauses 12 to 19, wherein the first dual-specificity adapter and the second dual-specificity adapter are formulated into separate pharmaceutical compositions.

[0512] Clause 25. A dual-specific adapter for use with anti-fluorescein CAR-T cells in the treatment of FAP-expressing cancer, comprising the following structure:

[0513] [ka]

[0514] [ka] A dual-specific adapter having or containing one of the above structures, or containing a pharmaceutically acceptable salt or hydrate of any of the aforementioned structures.

[0515] Clause 26. A dual-specific adapter for use with anti-fluorescein CAR-T cells in the treatment of FAP-expressing cancer, comprising the following structure:

[0516] [ka]

[0517] [ka] A dual-specific adapter having or containing one of the above structures, or containing a pharmaceutically acceptable salt or hydrate of any of the aforementioned structures.

[0518] Clause 27. A dual-specific adapter for use with anti-fluorescein CAR-T cells in the treatment of PSMA cancer, comprising the following structure:

[0519] [ka]

[0520] [ka] A dual-specific adapter having or containing one of the above, or containing a pharmaceutically acceptable salt or hydrate of any of the above.

[0521] Clause 28. A dual-specific adapter for use with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancer, comprising:

[0522] [ka] A dual-specific adapter having or a pharmaceutically acceptable salt or hydrate thereof.

[0523] Clause 29. A dual-specific adapter for use with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancer, comprising:

[0524] [ka] A dual-specific adapter having or a pharmaceutically acceptable salt or hydrate thereof.

[0525] Clause 30.(i) a kit comprising at least one dose unit of a bispecific adapter as described in any one of Clauses 1 to 10 or 25 to 29, a pharmaceutical composition comprising the bispecific adapter as described in any one of Clauses 1 to 10 or 25 to 29 and a pharmaceutically acceptable carrier or excipient, or a combination as described in any one of Clauses 12 to 24, and (ii) at least one dose unit of an anti-fluorescein CAR-T cell, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, wherein (i) and (ii) are optionally in separate containers.

[0526] Clause 31. A method for treating cancer in a subject, comprising administering to the subject a cancer-treatment-effective amount of (i) anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, and (ii) a combination of the bispecific adapter described in any one of Clauses 1 to 10 and 25 to 29, the pharmaceutical composition described in Clause 11, or any one of Clauses 12 to 24, thereby treating the subject for cancer.

[0527] Clause 32. The method according to Clause 31, wherein the CAR comprises a recognition region containing a single-stranded fragment variable (scFv) region of an anti-fluorescein antibody, a costimulatory domain which is CD28, CD137(4-1BB), CD134(OX40), or CD278(ICOS), and / or an activation signaling domain which is a T cell CD3ζ chain or Fc receptor γ.

[0528] Clause 33. The method according to Clause 31, wherein when the fluorescein of the bispecific adapter is exposed to anti-fluorescein CAR-T cells, it binds to the anti-fluorescein CAR-T cells with affinity, and when the targeting ligand of the bispecific adapter binds to the receptor of a targeted cancer cell or CAF with affinity, the targeting ligand of the bispecific adapter ligates the bound anti-fluorescein CAR-T cells to such targeted cancer cells or CAFs.

[0529] Clause 34. The method according to Clause 33, wherein the receptor for the targeted cancer cell or targeted CAF is overexpressed FAP, overexpressed PSMA, and / or FR.

[0530] The method according to Clause 31, wherein Clauses 35(i) and (ii) are administered simultaneously or sequentially by the same or different routes.

[0531] The method according to Clause 31, wherein Clause 36(ii) includes the combination described in any one of Clauses 25 to 29, and the first bispecific adapter and the second bispecific adapter are administered simultaneously to the target by the same or different routes.

[0532] The method according to Clause 31, wherein Clause 37(ii) includes the combination described in any one of Clauses 25 to 29, and the first bispecific adapter and the second bispecific adapter are administered to the subject sequentially in any order by the same or different routes.

[0533] The method according to any one of the provisions of 31 to 37, wherein provision 38(i) and (ii) are administered intravenously, respectively.

[0534] Clause 39. The method according to Clause 31, wherein the cancer is an FAP-expressing cancer and at least one of the bispecific adapters of (ii) comprises a radical of an FAP ligand.

[0535] Clause 40. The method according to Clause 31, wherein the cancer is a PSMA-expressing cancer and at least one of the bispecific adapters in (ii) comprises a radical of a PSMA ligand.

[0536] Clause 41. The method according to Clause 31, wherein the cancer is a folate receptor-expressing cancer, and (ii) comprises any combination of any one of Clauses 12 to 15, 17, and 18.

[0537] Clause 42. A method for treating FAP-expressing cancer in a subject, comprising administering to the subject a cancer-treating dose of (i) anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, and (ii) a dual-specific adapter as described in any one of Clauses 1 to 5, or a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier or excipient, thereby treating the subject for cancer.

[0538] Clause 43. The method according to Clause 42, wherein the CAR has a recognition region, and the recognition region is the scFv region of an anti-fluorescein antibody.

[0539] The method according to Clause 44. The method according to Clause 42 or 43, wherein the CAR comprises a costimulatory domain which is CD28, CD137(4-1BB), CD134(OX40), or CD278(ICOS), and / or an activation signaling domain which is a T cell CD3ζ chain or Fc receptor γ.

[0540] Clause 45. A method for treating cancer in a subject, comprising administering to the subject a cancer-treating dose of (i) an effective dose of a pharmaceutical composition comprising anti-fluorescein CAR-T cells, or anti-fluorescein CAR-T cells with a pharmaceutically acceptable carrier or excipient, and (ii) a combination described in any one of Clauses 12 to 24, thereby treating the subject for cancer.

[0541] The method according to Clause 46. The method according to Clause 45, wherein the CAR comprises a recognition region including the scFv region of an anti-fluorescein antibody, a costimulatory domain which is CD28, CD137(4-1BB), CD134(OX40), or CD278(ICOS), and / or an activation signaling domain which is a T cell CD3ζ chain or Fc receptor γ.

[0542] The method according to Clause 45, wherein Clauses 47(i) and (ii) are administered simultaneously or sequentially by the same or different routes.

[0543] Clause 48. The method according to Clause 45, wherein the first and second bispecific adapters of the combination are administered simultaneously to the target via the same or different routes.

[0544] Clause 49. The method according to Clause 45, wherein the first and second bispecific adapters of the combination are administered sequentially to the target via the same or different routes in any order.

[0545] The method according to any one of the provisions of 45 to 49, wherein provision 50(i) and (ii) are administered intravenously, respectively.

[0546] Clause 51. The method of any one of Clauses 31 to 49, further comprising imaging cancer in a subject.

[0547] Clause 52. The method according to Clause 51, wherein imaging of cancer includes imaging by optical imaging, positron emission tomography (PET), or single-photon emission computed tomography (SPECT).

[0548] Clause 53. The method according to any one of Clauses 31 to 49, wherein the cancer is ovarian cancer, endometrial cancer, breast cancer, glioma, e.g., optionally, a glioma of stage 3-4, or clear cell renal cell carcinoma, e.g., optionally, a clear cell renal cell carcinoma of stage 3-4.

[0549] Clause 54. A method for enhancing CAR-T cell activation, comprising providing a bispecific adapter as described in any one of Clauses 1 to 10 and 25 to 29, a pharmaceutical composition as described in Clause 11, or a combination as described in any one of Clauses 12 to 24, and exposing anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, to the bispecific adapter, pharmaceutical composition, or combination, wherein the CAR-T cells, after exposure, exhibit enhanced activation compared to CAR-T cells not exposed to the bispecific adapter.

[0550] Clause 55. The method according to Clause 54, wherein anti-fluorescein CAR-T cells are systemically circulating in the subject when exposed to a bispecific adapter.

Claims

1. The following structure: F-L-TL or a pharmaceutically acceptable salt or hydrate thereof [in the formula, F comprises fluorescein, fluorescein isothiocyanate (FITC), or N-hydroxysuccinimide (NHS)-fluorescein. L includes the linker, TL includes targeted ligands, which include radicals of fibroblast-activating protein (FAP) ligands or radicals of prostate-specific membrane antigen (PSMA) ligands. A dual-specificity adapter including a bispecific adapter.

2. The targeted ligand is 【Chemistry 1】 [In the formula, 【Chemistry 2】 [This is the connection point to the linker.] The dual-specific adapter according to claim 1, comprising a radical of a FAP ligand having the structure.

3. The aforementioned targeted ligand is of formula I-B: 【Transformation 3】 [In the formula, 【Chemistry 4】 This is the connection point to the linker, T is a substituted or unsubstituted methylene group (-CH ２ -), substituted or unsubstituted amino(-NH-), -O-, or -S-, R １ and R ２ are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH) ２ , -C(O)alkyl, -C(O)aryl-, -C═C-C(O)aryl, -C═C-S(O) ２ aryl, -CO ２ H, -SO ３ H, -SO ２ NH ２ , -PO ３ H ２ , -SO ２ F and 5-tetrazolyl R ３ and R ４ These are -H, -OH, F, Cl, Br, I, and -C, respectively. １～６ Alkyl, -O-C １～６ Alkyl and -S-C １～６ Independently selected from the group consisting of alkyls, R ５ , R ６ , R ７ , and R ８ Each is independently selected from the group consisting of H, alkyl, and halo. R ９ , R １０ , and R １１ H and -C, respectively. １～６ Alkyl, -O-C １～６ Alkyl, -S-C １～６ [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] The dual-specific adapter according to claim 2, comprising a radical of a FAP5 ligand having a structure represented by [the specified structure].

4. The aforementioned targeted ligand is of formula I-C: 【Transformation 5】 [In the formula, 【Transformation 6】 This is the connection point to the linker, T is a substituted or unsubstituted methylene group (-CH ２ -), substituted or unsubstituted amino(-NH-), -O-, or -S-, R １ and R ２ These are -H, -CN, -CHO, and -B(OH), respectively. ２ -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O) ２ Aryl, -CO ２ H, -SO ３ H, -SO ２ NH ２ , -PO ３ H ２ , -SO ２ Independently selected from the group consisting of F and 5-tetrazolyl, R ３ and R ４ These are -H, -OH, F, Cl, Br, I, and -C, respectively. １～６ Alkyl, -O-C １～６ Alkyl and -S-C １～６ Independently selected from the group consisting of alkyls, R ５ , R ６ , R ７ , and R ８ Each is independently selected from the group consisting of H, alkyl, and halo. R ９ , R １０ , and R １１ H and -C, respectively. １～６ Alkyl, -O-C １～６ Alkyl, -S-C １～６ [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] The dual-specific adapter according to claim 2, comprising a radical of a FAP5 ligand having a structure represented by [the specified structure].

5. The aforementioned targeted ligand has the following structure: 【Transformation 7】 [In the formula, 【Transformation 8】 This represents a functionalized 5-10 member N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, and optionally further contains 1-3 heteroatoms selected from O, N, and S. R １ and R ２ -H, -D, -OH, -F, -Cl, -Br, -I, -C １～６ Alkyl, -O-C １～６ Alkyl and -S-C １～６ Independently selected from the group consisting of alkyls, R ３ and R ４ -H, -OH, -F, -Cl, -Br, -I, -C １～６ Alkyl, -O-C １～６ Alkyl and -S-C １～６ Independently selected from the group consisting of alkyls, R ５ and R ６ -H, -OH, -F, -Cl, -Br, -I, -C １～６ Alkyl, -O-C １～６ Alkyl and -S-C １～６ Independently selected from the group consisting of alkyls, R ７ -H, -D, OH, CH ２ = IEEE - CH ３ ,CH ３ CH ２ -, (CH ３ ) ２ CH-, (CH ３ ) ３ C-, -CH ２ Ph, and substitution-CH ２ Selected from the group consisting of Ph, R ８ to R １０ is independently selected from the group consisting of -H, -OH, -F, -Cl, -Br, -I, -NO ２ , -SO ３ H, -SO ２ NH ２ , -NH ２ , -N ３ , -NH = NH, -C １～６ alkyl, -O-C １～６ alkyl, and -S-C １～６ alkyl, and is independently selected from the group R １１ is -H, -D, C １ ~C １０ Alkyl, C ３ ~C １０ Cycloalkyl, adamantyl, 【Chemistry 9】 , substituted or unsubstituted aryl, substituted or unsubstituted C ７ -C ２０ selected from the group consisting of alkylaryl, wherein said aryl is 【Chemistry 10】 (In the formula, R １２ and R １６ -H, -D, halogen, C １ ~C ３ Alkyl, C １ ~C ３ Alkoxy, -CF ３ , and -C(=O)-OR ２３ Independently selected from the group consisting of, R ２３ H, D, halogen, C １ ~C ４ Alkyl and C １ ~C ３ Selected from the group consisting of alkoxys, R １３ , R １４ and R １５ -H, -D, halogen, -OMe, C １ ~C ３ Alkyl, C １ ~C ３ Alkoxy, -CF ３ , and -C(=O)-OR ２３ Independently selected from the group consisting of, R ２３ -H, -D, halogen, C １ ~C ４ Alkyl and C １ ~C ３ (Selected from the group consisting of alkoxys) And, R １７ , R １８ , R ２０ , and R ２１ -H and -CH ３ Selected independently from, R １９ and R ２２ [The compound is independently selected from the group consisting of phenyl, dimethoxyphenyl, and aryl compounds.] The dual-specific adapter according to claim 2, comprising a radical of a FAP8 ligand.

6. The dual-specific adapter according to claim 1, wherein the targeted ligand comprises a radical of a PSMA ligand and is (S)-5-amino-1-carboxypentyl)carbamoyl)-L-glutamic acid (PSMAL1) or 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA).

7. The linker is polyethylene glycol (PEG) or a PEG derivative, for example, optionally, PEG ３ ~PEG １６ , and optionally, PEG ４ ~PEG １５ Or PEG ３ ~PEG １２ , PEG １２ PEG １５ PEG １６ , or PEG １８ , P.E. ４ ~PEG １６ 、 PEG １６ 、 P.E. ３ ~PEG １５ 、 PEG １５ 、 P.E. ３ ~PEG １２ 、 PEG ６ 、 PEG ３ ~PEG ８ ,or PEG ６ A dual-specific adapter according to any one of claims 1 to 6, comprising or essentially comprising.

8. A dual-specific adapter according to any one of claims 1 to 6, for use in conjunction with anti-fluorescein chimeric antigen receptor (CAR)-T cells in the treatment of cancer.

9. Optionally, the linker is PEG ３ ~PEG １５ , and optionally PEG １５ A dual-specific adapter according to any one of claims 1 to 5, for use in conjunction with anti-fluorescein CAR-T cells in the treatment of FAP-expressing cancer, comprising or essentially being therefor.

10. Optionally, the linker is PEG ３ ~PEG １２ , and optionally PEG ６ or PEG ３ ~PEG ８ , and optionally PEG ６ A dual-specific adapter according to claim 1 or 6, for use in conjunction with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancer, comprising or essentially being therefor.

11. A pharmaceutical composition for the treatment of cancer, comprising a dual-specific adapter according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier or excipient.

12. A combination of dual-specific adapters for use with anti-fluorescein chimeric antigen receptor (CAR)-T cells in cancer treatment, (i) A first bispecific adapter comprising the bispecific adapter according to claim 1 or a pharmaceutically acceptable salt or hydrate thereof, wherein the targeted ligand of the first bispecific adapter is 【Chemistry 11】 [In the formula, 【Chemistry 12】 [This is the connection point to the linker.] A first bispecific adapter comprising a radical of a FAP ligand having the formula, (ii) The following structure: F-L-TL or a pharmaceutically acceptable salt or hydrate thereof [in the formula, F contains fluorescein, FITC, or NHS-fluorescein. L includes the linker, TL includes targeted ligands containing radicals of folate receptor (FR) ligands or prostate-specific membrane antigen (PSMA) ligands. A second dual-specificity adapter including A combination that includes this.

13. The radical of the FAP ligand of the first bispecific adapter is of formula I-B: 【Chemistry 13】 [T is a substituted or unsubstituted methylene group (-CH ２ -), substituted or unsubstituted amino(-NH-), -O-, or -S-, R １ and R ２ These are -H, -CN, -CHO, and -B(OH), respectively. ２ -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O) ２ Aryl, -CO ２ H, -SO ３ H, -SO ２ NH ２ , -PO ３ H ２ , -SO ２ Independently selected from the group consisting of F and 5-tetrazolyl, R ３ and R ４ These are -H, -OH, F, Cl, Br, I, and -C, respectively. １～６ Alkyl, -O-C １～６ Alkyl and -S-C １～６ Independently selected from the group consisting of alkyls, R ５ , R ６ , R ７ , and R ８ Each is independently selected from the group consisting of H, alkyl, and halo. R ９ , R １０ , and R １１ H and -C, respectively. １～６ Alkyl, -O-C １～６ Alkyl, -S-C １～６ [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] The structure represented by, Formula IC: 【Chemistry 14】 [In the formula, 【Chemistry 15】 This is the connection point to the linker, T is a substituted or unsubstituted methylene group (-CH ２ -), substituted or unsubstituted amino(-NH-), -O-, or -S-, R １ and R ２ These are -H, -CN, -CHO, and -B(OH), respectively. ２ -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O) ２ Aryl, -CO ２ H, -SO ３ H, -SO ２ NH ２ , -PO ３ H ２ , -SO ２ Independently selected from the group consisting of F and 5-tetrazolyl, R ３ and R ４ These are -H, -OH, F, Cl, Br, I, and -C, respectively. １～６ Alkyl, -O-C １～６ Alkyl and -S-C １～６ Independently selected from the group consisting of alkyls, R ５ , R ６ , R ７ , and R ８ Each is independently selected from the group consisting of H, alkyl, and halo. R ９ , R １０ , and R １１ H and -C, respectively. １～６ Alkyl, -O-C １～６ Alkyl, -S-C １～６ [Independently selected from the group consisting of alkyl, F, Cl, Br, and I] The structure represented by, The following formula: 【Chemistry 16】 [In the formula, 【Chemistry 17】 This represents a functionalized 5-10 member N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, and optionally further contains 1-3 heteroatoms selected from O, N, and S. R １ and R ２ -H, -D, -OH, -F, -Cl, -Br, -I, -C １～６ Alkyl, -O-C １～６ Alkyl and -S-C １～６ Independently selected from the group consisting of alkyls, R ３ and R ４ -H, -OH, -F, -Cl, -Br, -I, -C １～６ Alkyl, -O-C １～６ Alkyl and -S-C １～６ Independently selected from the group consisting of alkyls, R ５ and R ６ -H, -OH, -F, -Cl, -Br, -I, -C １～６ Alkyl, -O-C １～６ Alkyl and -S-C １～６ Independently selected from the group consisting of alkyls, R ７ -H, -D, OH, CH ２ = IEEE - CH ３ ,CH ３ CH ２ -, (CH ３ ) ２ CH-, (CH ３ ) ３ C-, -CH ２ Ph, and substitution-CH ２ Selected from the group consisting of Ph, R ８ ~R １０ -H, -OH, -F, -Cl, -Br, -I, -NO ２ , -SO ３ H, -SO ２ NH ２ , -NH ２ , -N ３ , -NH=NH, -C １～６ Alkyl, -O-C １～６ Alkyl and -S-C １～６ Independently selected from the group consisting of alkyls, R １１ is -H, -D, C １ ~C １０ Alkyl, C ３ ~C １０ Cycloalkyl, adamantyl, [Chemistry 18] , substituted or unsubstituted aryl, substituted or unsubstituted C ７ ~C ２０ Selected from the group consisting of alkylaryls, the aryl is 【Chemistry 19】 (In the formula, R １２ and R １６ -H, -D, halogen, C １ ~C ３ Alkyl, C １ ~C ３ Alkoxy, -CF ３ , and -C(=O)-OR ２３ Independently selected from the group consisting of, R ２３ H, D, halogen, C １ ~C ４ Alkyl and C １ ~C ３ Selected from the group consisting of alkoxys, R １３ , R １４ and R １５ -H, -D, halogen, -OMe, C １ ~C ３ Alkyl, C １ ~C ３ Alkoxy, -CF ３ , and -C(=O)-OR ２３ Independently selected from the group consisting of, R ２３ -H, -D, halogen, C １ ~C ４ Alkyl and C １ ~C ３ (Selected from the group consisting of alkoxys) And, R １７ , R １８ , R ２０ , and R ２１ -H and -CH ３ Selected independently from, R １９ and R ２２ [The compound is independently selected from the group consisting of phenyl, dimethoxyphenyl, and aryl compounds.] Structure represented by The combination according to claim 12, having the following characteristics.

14. The combination according to claim 12, wherein the targeted ligand of the second bispecific adapter or a pharmaceutically acceptable salt or hydrate thereof comprises a radical of a PSMA ligand.

15. The combination according to claim 12, wherein the linker of the first dual-specific adapter contains or is essentially made of polyethylene glycol (PEG).

16. The combination according to claim 14, wherein the targeted ligand is (((S)-5-amino-1-carboxypentyl)carbamoyl)-L-glutamic acid (PSMAL1) or 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) or comprising the same.

17. The combination according to claim 12, wherein the targeting ligand of the second bispecific adapter is a radical of folic acid or a functional fragment or analog thereof.

18. The combination according to claim 17, wherein the folic acid is folic acid, dihydrofolic acid, tetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid (5,10-MTHF), 5-methyltetrahydrofolic acid (5-MTHF), or larcitrexed.

19. The PSMA ligand is DUPA, and the linker is PEG or a PEG derivative, for example, optionally PEG ３ ~PEG １２ , and optionally PEG ６ , PEG ３ ~PEG １６ , and optionally PEG ４ ~PEG １５ Or PEG ３ ~PEG １２ , PEG １２ PEG １５ PEG １６ , or PEG １８ , PEG ４ ~PEG １６ , and optionally PEG １６ , PEG ３ ~PEG １５ , and optionally PEG １５ , PEG ３ ~PEG ８ , and optionally PEG ６ The combination according to claim 16, which includes or is essentially derived from.

20. A combination according to any one of claims 12 to 19 for use in conjunction with anti-fluorescein chimeric antigen receptor (CAR)-T cells in the treatment of cancer.

21. A combination according to any one of claims 12 to 19 for use in conjunction with anti-fluorescein CAR-T cells in the treatment of FAP-expressing cancer.

22. A combination according to any one of claims 12 to 16 and 19 for use in conjunction with anti-fluorescein CAR-T cells in the treatment of PSMA-expressing cancer.

23. A combination according to any one of claims 12 to 15, 17, and 18 for use in conjunction with anti-fluorescein CAR-T cells in the treatment of folate-expressing cancer.

24. The combination according to any one of claims 12 to 19, wherein the first dual-specificity adapter and the second dual-specificity adapter are formulated into separate pharmaceutical compositions.

25. A dual-specific adapter for use with anti-fluorescein chimeric antigen receptor (CAR) T cells in the treatment of fibroblast-activating protein (FAP) expressing cancer, comprising the following structure: 【Chemistry 20-1】 【Chemistry 20-2】 A dual-specific adapter having or containing one of the above structures, or containing a pharmaceutically acceptable salt or hydrate of any of the aforementioned structures.

26. A dual-specific adapter for use with anti-fluorescein chimeric antigen receptor (CAR) T cells in the treatment of fibroblast-activating protein (FAP) expressing cancer, comprising the following structure: 【Chemistry 21-1】 【Chemistry 21-2】 A dual-specific adapter having or containing one of the above structures, or containing a pharmaceutically acceptable salt or hydrate of any of the aforementioned structures.

27. A bispecific adapter for use with anti-fluorescein chimeric antigen receptor (CAR) T cells in the treatment of prostate-specific membrane antigen (PSMA) cancer, comprising the following structure: 【Chemistry 22-1】 【Chemistry 22-2】 A dual-specific adapter having or containing one of the above, or containing a pharmaceutically acceptable salt or hydrate of any of the above.

28. A dual-specific adapter for use with anti-fluorescein chimeric antigen receptor (CAR) T cells in the treatment of prostate-specific membrane antigen (PSMA) expressing cancer, comprising: 【Chemistry 23】 A dual-specific adapter having or a pharmaceutically acceptable salt or hydrate thereof.

29. A dual-specific adapter for use with anti-fluorescein chimeric antigen receptor (CAR) T cells in the treatment of prostate-specific membrane antigen (PSMA) expressing cancer, comprising: 【Chemistry 24】 A dual-specific adapter having or a pharmaceutically acceptable salt or hydrate thereof.

30. (i) at least one dose unit of a bispecific adapter according to any one of claims 1 to 10 or 25 to 29, a pharmaceutical composition comprising a bispecific adapter according to any one of claims 1 to 10 or 25 to 29 and a pharmaceutically acceptable carrier or excipient, or a combination according to any one of claims 12 to 24, and (ii) A pharmaceutical composition comprising at least one dose unit of anti-fluorescein chimeric antigen receptor (CAR)-T cells, or anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient. A kit that includes, A kit in which (i) and (ii) are optionally in separate containers.

31. A method for treating cancer in a subject, wherein the subject is given an effective amount for treating cancer. (i) Anti-fluorescein chimeric antigen receptor (CAR)-T cells, or pharmaceutical compositions comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, and (ii) A dual specificity adapter according to any one of claims 1 to 10 and 25 to 29, a pharmaceutical composition according to claim 11, or a combination according to any one of claims 12 to 24 This includes administering, A method by which the subject is treated for cancer.

32. The aforementioned CAR, Recognition region containing a single-stranded fragment variable (scFv) region of anti-fluorescein antibody, Costimulatory domains that are CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS), and / or The activation signaling domain is either the T cell CD3ζ chain or the Fc receptor γ. The method according to claim 31, including the method described in claim 31.

33. The method according to claim 31, wherein when the fluorescein of the bispecific adapter is exposed to the anti-fluorescein CAR-T cells, it binds to the anti-fluorescein CAR-T cells with affinity, and when the targeting ligand of the bispecific adapter binds to the receptor of a targeted cancer cell or cancer-associated fibroblast (CAF) with affinity, the targeting ligand of the bispecific adapter links the bound anti-fluorescein CAR-T cells to such targeted cancer cells or CAFs.

34. The method according to claim 33, wherein the receptor of the targeted cancer cell or the targeted CAF is an overexpressed fibroblast-activating protein (FAP), an overexpressed prostate-specific membrane antigen (PSMA), and / or folate receptor (FR).

35. The method according to claim 31, wherein (i) and (ii) are administered simultaneously or sequentially by the same or different routes.

36. The method according to claim 31, wherein (ii) includes the combination described in any one of claims 25 to 29, and the first bispecific adapter and the second bispecific adapter are administered to the subject simultaneously by the same or different routes.

37. The method according to claim 31, wherein (ii) comprises the combination described in any one of claims 25 to 29, and the first bispecific adapter and the second bispecific adapter are administered to the subject sequentially in any order by the same or different routes.

38. The method according to any one of claims 31 to 37, wherein (i) and (ii) are administered intravenously, respectively.

39. The method according to claim 31, wherein the cancer is a FAP-expressing cancer, and at least one of the bispecific adapters of (ii) comprises a radical of a FAP ligand.

40. The method according to claim 31, wherein the cancer is a prostate-specific membrane antigen (PSMA) expressing cancer, and at least one of the bispecific adapters of (ii) comprises a radical of a PSMA ligand.

41. The method according to claim 31, wherein the cancer is a folate receptor-expressing cancer, and (ii) comprises a combination according to any one of claims 12 to 15, 17, and 18.

42. A method for treating cancers expressing fibroblast-activating protein (FAP) in a subject, wherein the subject is given an effective amount for cancer treatment. (i) Anti-fluorescein chimeric antigen receptor (CAR)-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, and (ii) A pharmaceutical composition comprising a dual specificity adapter according to any one of claims 1 to 5, or the same and a pharmaceutically acceptable carrier or excipient. This includes administering, A method by which the subject is treated for cancer.

43. The method according to claim 42, wherein the CAR has a recognition region, and the recognition region is a single-stranded fragment variable (scFv) region of an anti-fluorescein antibody.

44. The aforementioned CAR, Costimulatory domains that are CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS), and / or The activation signaling domain is either the T cell CD3ζ chain or the Fc receptor γ. The method according to claim 42 or 43, including the method described in claim 42 or 43.

45. A method for treating cancer in a subject, wherein the subject is given an effective amount for treating cancer. (i) Anti-fluorescein chimeric antigen receptor (CAR)-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, and (ii) The combination according to any one of claims 12 to 24 This includes administering, A method by which the subject is treated for cancer.

46. The aforementioned CAR, Recognition region containing a single-stranded fragment variable (scFv) region of anti-fluorescein antibody, Costimulatory domains that are CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS), and / or The activation signaling domain is either the T cell CD3ζ chain or the Fc receptor γ. The method according to claim 45, including the method described in claim 45.

47. The method according to claim 45, wherein (i) and (ii) are administered simultaneously or sequentially by the same or different routes.

48. The method according to claim 45, wherein the first and second dual-specific adapters of the combination are administered simultaneously to the subject by the same or different routes.

49. The method according to claim 45, wherein the first bispecific adapter and the second bispecific adapter of the combination are administered to the subject sequentially in either the same or different route.

50. The method according to any one of claims 45 to 49, wherein (i) and (ii) are administered intravenously, respectively.

51. The method according to any one of claims 31 to 49, further comprising imaging the cancer in the subject.

52. The method according to claim 51, wherein imaging the cancer includes imaging by optical imaging, positron emission tomography (PET), or single-photon emission computed tomography (SPECT).

53. The method according to any one of claims 31 to 49, wherein the cancer is ovarian cancer, endometrial cancer, breast cancer, glioma, for example optionally, a glioma of stage 3 to 4, or clear cell renal cell carcinoma, for example optionally, a clear cell renal cell carcinoma of stage 3 to 4.

54. A method for enhancing chimeric antigen receptor (CAR)-T cell activation, The dual specificity adapter according to any one of claims 1 to 10 and 25 to 29, the pharmaceutical composition according to claim 11, or the combination according to any one of claims 12 to 24, Exposing anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, to the bispecific adapter, the pharmaceutical composition, or the combination thereof. Includes, A method wherein the CAR-T cells exhibit enhanced activation after exposure compared to CAR-T cells that have not been exposed to the bispecific adapter.

55. The method according to claim 54, wherein the anti-fluorescein CAR-T cells are systemically circulating in the subject when exposed to the bispecific adapter.

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