Bispecific adapters and their combination with universal CAR-T cells in the treatment of CAIX-expressing tumors

Abstract

Chimeric antigen receptor (CAR) T cells, bispecific adapters that link CAR-T cells to carbonic anhydrase IX (CAIX)-expressing tumor cells, and methods of using them to treat CAIX-expressing cancers.

Description

[Technical Field]

[0001] Priority This patent application is related to and claims the benefit of priority to U.S. Provisional Application No. 63 / 451,450, filed March 10, 2023, and U.S. Provisional Application No. 63 / 600,606, filed November 17, 2023. The contents of each of the foregoing applications are hereby incorporated by reference in their entirety into this disclosure.

[0002] The present disclosure relates to chimeric antigen receptor (CAR)-T cells, tumors expressing carbonic anhydrase (CAIX), bispecific adapters that link CAR-T cells to CAIX-expressing tumor cells, and methods of using them to treat cancer. [Background technology]

[0003] Carbonic anhydrase IX (CAIX) is upregulated in most solid tumors and is a marker of poor prognosis in at least ovarian, breast, lung, and bladder carcinomas. CAIX is also overexpressed in over 95% of clear cell renal cell carcinomas.

[0004] Traditionally, T cells expressing a single chimeric antigen receptor (CAR) have been used to target the cell surface receptor of tumor cells. When the CAR on a T cell binds to the cell surface receptor of a tumor cell, the T cell can kill the tumor cell to which it is bound. Although effective, this approach can be very expensive, considering that CAR-T cells must be generated for different cancers expressing different cell surface receptors.

[0005] Despite the clear need for cancer prevention and treatment, cancer remains a significant cause of death and suffering worldwide, as there are currently no effective therapeutic options that can cure the condition. Furthermore, even when drugs or other therapies are available, such treatments typically use highly potent drugs that carry the risk of systemic toxicity in subjects with underlying disease, as they have poor selectivity for the targeted cancer cells.

[0006] What is needed is a bispecific adapter that can facilitate the cost-effective use of CAR-T cells that can bind to a variety of cancers that express a variety of cell surface receptors. This and other objects and advantages, as well as features of the present invention, will be apparent from the detailed description provided herein. Summary of the Invention

[0007] A bispecific adapter is provided for use with anti-fluorescein chimeric antigen receptor (CAR)-T cells in the treatment of carbonic anhydrase IX (CAIX)-expressing cancers. The bispecific adapter has the following structure: FL-CAIX, or a pharmaceutically acceptable salt or hydrate thereof, wherein F comprises fluorescein, L comprises a linker, and CAIX comprises a CAIX ligand (e.g., a radical of a CAIX ligand). The fluorescein may comprise fluorescein, fluorescein isothiocyanate (FITC), or N-hydroxysuccinimide (NHS)-fluorescein. The CAIX ligand may be or comprise a radical of 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL). The linker may comprise, consist essentially of, or consist of polyethylene glycol (PEG).

[0008] The linker may comprise (or consist essentially of, or consist of) PEG3 to PEG9. The linker may comprise (or consist essentially of, or consist of) PEG6. The linker may comprise (CH2)4.

[0009] In certain embodiments, the bispecific adapter, or a pharmaceutically acceptable salt or hydrate thereof, comprises fluorescein, FITC, or NHS-fluorescein conjugated to a radical of a CAIX ligand via a linker, wherein the linker comprises, consists essentially of, or consists of PEG.

[0010] The CAIX ligand may be or may include 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL), 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (meta-CAL), acetazolamide (Aza), or a derivative or analog of any of the foregoing.

[0011] The linker can comprise, consist essentially of, or consist of PEG1 through PEG9. The CAIX ligand can be or comprise an ortho-CAL or a derivative or analog thereof, and the linker can comprise, consist essentially of, or consist of PEG1 through PEG9. In certain embodiments, the linker comprises, consists essentially of, or consists of PEG3 through PEG9.

[0012] In certain embodiments, the CAIX ligand is or comprises meta-CAL or a derivative or analog thereof, and the linker comprises, consists essentially of, or consists of PEG3-PEG9.

[0013] The CAIX ligand may be or may comprise Aza or a derivative or analog thereof. In certain embodiments, the linker comprises, consists essentially of, or consists of PEG6. In certain embodiments, the linker comprises, consists essentially of, or consists of PEG9. In certain embodiments, the linker comprises, consists essentially of, or consists of PEG6.

[0014] The linker may comprise, consist essentially of, or consist of alkyl. The linker may comprise, consist essentially of, or consist of (CH2)4.

[0015] In certain embodiments, the bispecific adapter has the following formula:

[0016] [ka] or a pharmaceutically acceptable salt or hydrate of any of the foregoing.

[0017] The bispecific adapter has the following formula:

[0018] [ka] or a pharmaceutically acceptable salt or hydrate thereof.

[0019] The bispecific adapter has the following formula:

[0020] [ka] or a pharmaceutically acceptable salt or hydrate of any of the foregoing.

[0021] The bispecific adapters can be used with anti-fluorescein chimeric antigen receptor (CAR)-T cells in the treatment of cancer. The bispecific adapters can be used with anti-

[0022] In certain embodiments, the bispecific adapter is for use with anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) CAR-T cells in the treatment of CAIX-expressing cancers. The bispecific adapter may comprise a fluorescein-linker-CAIX ligand (e.g., fluorescein = fluorescein, FITC, or NHS-fluorescein) or a pharmaceutically acceptable salt or hydrate thereof, where the CAIX ligand is or comprises 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (meta-CAL), and the linker comprises (or consists essentially of, or consists of) PEG. The linker may comprise (or consist essentially of, or consist of) PEG3 to PEG9. The linker may comprise (or consist essentially of, or consist of) PEG6. The linker may comprise (or consist essentially of, or consist of) PEG9. The bispecific adapter may have the structure:

[0023] [ka] or a pharmaceutically acceptable salt or hydrate thereof.

[0024] Yet another bispecific adapter is provided for use with anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) CAR-T cells in the treatment of CAIX-expressing cancers. The adapter can comprise a fluorescein-linker-CAIX ligand (e.g., fluorescein = fluorescein, FITC, or NHS-fluorescein) or a pharmaceutically acceptable salt or hydrate thereof, where the CAIX ligand is acetazolamide (Aza) and the linker comprises (or consists essentially of, or consists of) PEG. The linker can comprise (or consist essentially of, or consist of) PEG1 through PEG9.

[0025] Also provided is a pharmaceutical composition for the treatment of a CAIX-expressing cancer comprising any of the bispecific adapters described herein and a pharmaceutically acceptable carrier or excipient.

[0026] Further provided are methods for treating cancer (e.g., CAIX-expressing cancer) in a subject. The method can include administering to the subject a cancer-treating effective amount of (i) anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) 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 described herein, or a pharmaceutical composition comprising a bispecific adapter described herein and a pharmaceutically acceptable carrier or excipient, thereby treating the subject for cancer. The CAR can have a recognition region, which can be a single-chain fragment variable (scFv) region of an anti-fluorescein antibody. The CAR can have a costimulatory domain. The costimulatory domain of the CAR can be CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS). The CAR can have an activation signaling domain, such as the T cell CD3 zeta chain or Fc receptor gamma.

[0027] The fluorescein of the bispecific adapter of the method is capable of affinity binding to the anti-fluorescein CAR-T cells upon exposure to the anti-fluorescein CAR-T cells, and the CAIX ligand of the bispecific adapter is capable of ligating the bound anti-fluorescein CAR-T cells to the CAIX-expressing cancer cells upon affinity binding of the bispecific adapter to a receptor on such CAIX-expressing cancer cells.

[0028] In the method, steps (i) and (ii) can be administered by the same or different routes, simultaneously or sequentially in any order. In the method, steps (i) and (ii) can each be administered intravenously.

[0029] In certain embodiments, the method can further include imaging the cancer in the subject. Imaging the cancer can include imaging by optical imaging, positron emission tomography (PET), or single photon emission computed tomography (SPECT).

[0030] The cancer may be ovarian cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer, or clear cell renal cell carcinoma, for example, optionally clear cell renal cell carcinoma at stage 3 to 4. The cancer may be a CAIX-expressing cancer.

[0031] Also provided are methods for enhancing CAR-T cell activation. In certain embodiments, the method for enhancing CAR-T cell activation comprises providing a bispecific adapter described herein or a pharmaceutical composition described herein, 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 or pharmaceutical composition, wherein after exposure, the CAR-T cells undergo enhanced activation against cancer cells compared to CAR-T cells not exposed to the bispecific adapter.

[0032] When exposed to the bispecific adapter, the CAR-T cells may be circulating systemically in the subject. The CAR-T cells may be exposed to the bispecific adapter in vitro (e.g., before administration to the subject). The CAR-T cells may be CAIX-expressing cancer cells.

[0033] Kits are also provided. The kits may include (i) at least one dosage unit of a pharmaceutical composition comprising a bispecific adapter described herein, or a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier or excipient, and (ii) at least one dosage unit of an anti-fluorescein CAR-T cell, or a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier or excipient, optionally with (i) and (ii) in separate containers. The CAIX ligand of the bispecific adapter may be or may comprise an ortho-CAL or a derivative or analog thereof. The CAIX ligand of the bispecific adapter may be or may comprise a meta-CAL or a derivative or analog thereof. The CAIX of the bispecific adapter may be or may comprise an Aza or a derivative or analog thereof.

[0034] 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 drawings]

[0035] [Figure 1-1] FIG. 1 shows the chemical structure of a carbonic anhydrase IX (CAIX)-targeted bispecific adapter. [Figure 1-2] FIG. 1 shows the chemical structure of a carbonic anhydrase IX (CAIX)-targeted bispecific adapter. [Figure 1-3] FIG. 1 shows the chemical structure of a carbonic anhydrase IX (CAIX)-targeted bispecific adapter. [Figure 2] Figure 2A is a graph of mean fluorescence intensity (MFI) versus concentration (nM) of the indicated bispecific adapters. Figure 2B is a graph of mean fluorescence intensity (MFI) versus concentration (nM) of the indicated bispecific adapters. [Figure 3-1] Figure 3A is a graph of the MFI of the bispecific adapter versus fluorescein isothiocyanate (FITC) showing total binding of the CAIX bispecific adapter to HT29 cells, and Figure 3B is a graph of the MFI of the bispecific adapter versus APC-anti-FITC showing surface exposure of the FITC moiety on the CAIX bispecific adapter after binding to HT29 cells. [Figure 3-2] FIG. 3C is a graph of the MFI of the bispecific adapter versus FITC showing total binding of the CAIX bispecific adapter to MDA-CAIX cells. [Figure 3-3] FIG. 3D is a graph of the MFI of the bispecific adapter versus APC-anti-FITC showing the surface exposure of the FITC moiety on the CAIX bispecific adapter after binding to MDA-CAIX cells. [Figure 4] Figure 4A is a graph of concentration (nM) versus lysis (%) showing the cytotoxicity of anti-FITC CAR-T cells against MDA-CAIX cells mediated by a CAIX-targeting bispecific adapter. Figure 4B is a graph of concentration (nM) versus IFNγ (pg / ml) showing the IFNγ release from anti-FITC CAR-T cells mediated by a bispecific adapter. [Figure 5] Figure 5A is a graph of concentration (nM) versus lysis (%) showing that the meta-CAL-FITC bispecific adapter mediated cytotoxicity of anti-FITC CAR-T cells against MDA-CAIX cells. Figure 5B is a graph of concentration (nM) versus IFNγ (pg / ml) showing that meta-CAL-PEG6-FITC and meta-CAL-PEG9-FITC mediated higher levels of IFNγ release from anti-FITC CAR-T cells than meta-CAL-PEG3-FITC at low concentrations (between 0.001 nM and 1 nM). [Figure 6]Figure 6A is a graph of concentration (nM) versus lysis (%) showing that ortho-CAL-FITC bispecific adapters with different linkers mediated cytotoxicity of anti-FITC CAR-T cells against MDA-CAIX cells. Figure 6B is a graph of concentration (nM) versus IFNγ (pg / ml) showing that ortho-CAL-FITC bispecific adapters mediated IFNγ release from anti-FITC CAR-T cells. [Figure 7-1] Figure 7A shows the timeline and dosing schedule of an in vivo study testing different CAIX bispecific adapters. Figure 7B is a graph of tumor volume (mm) versus days after CAR-T injection showing tumor growth curves for different treatment groups. [Figure 7-2] Figure 7C is a graph of % weight change versus days after CAR-T injection showing the weight change of mice in different treatment groups over the course of treatment. [Figure 8-1] FIG. 8A is a schematic diagram of the experimental design. [Figure 8-2] Figure 8B is a graph of the MFI of bispecific adapters versus FITC showing the total FITC signal in HT29 tumor cells from mice injected with different bispecific adapters, and Figure 8C is a graph of the MFI of bispecific adapters versus APC-anti-FITC showing the surface exposure of FITC moieties in HT29 tumors from mice injected with the CAIX bispecific adapter. [Figure 9] Figure 9A is a graph of ligand vs. T cells / μL blood, showing the number of T cells in the blood. Figure 9B is a graph of ligand vs. CD3+ T cells / viable cells (%), showing tumor-infiltrating T cells. [Figure 10-1] Figure 10A shows the timeline and dosing schedule of an in vivo study testing different CAIX bispecific adapters. Figure 10B is a graph of tumor volume (mm) versus days after CAR-T injection showing tumor growth curves for different treatment groups. [Figure 10-2] Figure 10C is a graph of weight change (%) versus days after CAR-T injection showing the weight change of mice in different groups during treatment. [Figure 11-1] FIG. 11A is a schematic diagram of the experimental design. [Figure 11-2] Figure 11B is a graph of the MFI of bispecific adapters versus FITC, showing the total FITC signal in MDA-CAIX tumor cells from mice injected with different bispecific adapters. Figure 11C is a graph of the MFI of bispecific adapters versus FITC after in vitro staining with 100 nM meta-CAL-PEG6-FITC, showing the total FITC signal in tumor cells. In vitro staining of cells with 100 nM meta-CAL-PEG6-FITC saturates all surface CAIX protein and may reflect the CAIX protein level in tumor cells. The results show that ortho-CAL-PEG6-FITC mediated complete eradication of CAIX+ tumor cells by CAR T cells. [Figure 11-3] Figure 11D is a graph of the MFI of the bispecific adapter versus APC-anti-FITC, showing the surface exposure of FITC moieties in MDA-CAIX tumors from mice treated with the CAIX bispecific adapter. Figure 11E is a graph of the MFI of the bispecific adapter versus APC-anti-FITC after in vitro staining with 100 nM meta-CAL-PEG6-FITC, showing the total FITC signal in tumor cells. In vitro staining of cells with 100 nM meta-CAL-PEG6-FITC saturates all surface CAIX protein and may reflect CAIX protein levels in tumor cells. [Figure 12] FIG. 1 is a graph of concentration (nM) versus normalized MFI showing that the PEG spacer has no significant effect on the binding affinity of the acetazolamide (Aza)-FITC bispecific adapter. [Figure 13-1] Figure 13A is a graph of the MFI of the bispecific adapter versus FL showing the total binding of the Aza-FITC bispecific adapter to MDA-CAIX cells. Linker length did not significantly affect total binding. [Figure 13-2]Figure 13B is a graph of the MFI of bispecific adapters and APC-anti-FITC showing the surface exposure of the FITC moiety in Aza-FITC bispecific adapters with different PEG linkers after binding to MDA-CAIX cells. Increasing the PEG spacer length enhanced the surface exposure of FITC after binding to MDA-CAIX cells. [Figure 14] Figure 1 is a graph of concentration (nM) versus IFNγ (pg / ml) showing the effect of linker length on IFNγ release from anti-FITC CAR-T cells mediated by Aza-FITC bispecific adapters when co-cultured with HT29 cells. Aza-PEG3-FITC and Aza-PEG9-FITC mediated higher levels of IFNγ released from anti-FITC CAR-T cells than Aza-PEG0-FITC. Aza-PEG6-FITC mediated the highest level of IFNγ released from anti-FITC CAR-T cells. [Figure 15] Figure 1 is a graph of CA9 Ligand-PEG(n)-FITC concentration (nM) versus IFNγ (pg / ml) showing that CAIX-targeting bispecific adapters with optimal linkers mediated IFNγ release from anti-FITC CAR-T cells. Meta-CAL-PEG9-FITC mediated the highest levels of IFNγ released from anti-FITC CAR-T cells at low concentrations (<0.1 nM). Aza-PEG6-FITC mediated comparable levels of IFNγ release from anti-FITC CAR T cells at concentrations greater than 0.1 nM. Ortho-CAL-PEG6-FITC mediated lower levels of IFNγ released from anti-FITC CAR-T cells. [Figure 16-1] FIG. 16A shows the timeline and dosing schedule of the in vivo study testing Aza-PEG6-FITC and OrthoCAL-PEG6-FITC. [Figure 16-2]Figure 16B is a graph of tumor volume (mm3) versus days after CAR-T cell injection, showing tumor growth curves for different treatment groups. Both Aza-PEG6-FITC and ortho-CAL-PEG6-FITC slightly inhibited the growth of KB tumors. The efficacy of Aza-PEG6-FITC is slightly better than that of ortho-CAL-PEG6-FITC. Figure 16C is a graph of body weight change (%) versus days after CAR-T cell injection, showing the weight change of mice in different treatment groups. Neither Aza-PEG6-FITC nor ortho-CAL-PEG6-FITC induced significant weight loss. DETAILED DESCRIPTION OF THE INVENTION

[0036] This disclosure is based, at least in part, on the design of bispecific adapters with varying antigen binding affinities and spacer lengths to optimize the efficacy of universal chimeric antigen receptor (CAR)-T cells in treating carbonic anhydrase IX (CAIX)-expressing tumors. Two new high-affinity CAIX ligands, 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (meta-CAL) and 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL), were tested with three or four spacer linkers, respectively, in the bispecific adapters. Studies supported the finding that PEG6 and PEG9 (PEG = polyethylene glycol) spacers may be optimal for targeting CAIX-positive tumors in vivo and in vitro. When used in bispecific adapters, high affinity CAIX ligands were more potent than adapters with lower affinity, such as 5-acetamido-1,3,4-thiadiazole-2-sulfonamide (Aza).

[0037] In view of the above, there is provided a bispecific adapter, or a pharmaceutically acceptable salt or hydrate thereof, for use with anti-fluorescein (e.g., fluorescein, FITC, or N-hydroxysuccinimide (NHS)-fluorescein) CAR-T cells in the treatment of CAIX-expressing cancers.

[0038] In certain embodiments, the bispecific adapter has the following structure: FL-CAIX or a pharmaceutically acceptable salt or hydrate thereof, wherein F comprises a CAR-T cell targeting moiety, such as fluorescein, FITC, or NHS-fluorescein; L comprises a linker; CAIX contains a radical of a CAIX ligand] is.

[0039] In certain embodiments, the CAIX ligand is or comprises 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL), and the linker comprises (or consists essentially of or consists of) PEG.

[0040] The use of a bispecific adapter can enable the use of a single CAR-T cell, i.e., a "universal" CAR-T cell, that displays, for example, a molecule that binds fluorescein on its surface. When a universal CAR-T cell, e.g., one that displays a molecule that binds fluorescein on its surface, is used with a bispecific adapter that binds to a cell surface receptor on a tumor cell, e.g., one that includes FITC attached to the molecule (e.g., by a linker and / or spacer), the T cell can kill the tumor cell to which it is bound.

[0041] This approach can reduce the cost of generating CAR-T cells that can bind to various cancers expressing different cell surface receptors. Instead, by changing the portion of the bispecific adapter that binds to the cell surface receptor of tumor cells, universal CAR-T cells can bind to various types of cancer. Therefore, the bispecific adapter can improve the immune response induced by CAR-T cells bound to tumor cells.

[0042] CAR T cell targeting moiety The CAR T cell targeting portion of the bispecific adapter can be fluorescein, FITC, NHS-fluorescein, or any other moiety that the CAR can be engineered to recognize and bind with specificity.

[0043] "Binding with specificity," "binding with high affinity," or "specifically" or "selectively" binding, when referring to a ligand / receptor, recognition region / targeting moiety, nucleic acid / complementary nucleic acid, antibody / antigen, or other binding pair, refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologics. Thus, under given conditions, a designated ligand or recognition region binds to a particular receptor (e.g., one present on cancer cells or CAR T cells) or targeting moiety, respectively, and does not bind 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 can also mean, for example, that a binding compound, ligand, antibody, or binding composition derived from an antigen-binding portion of an antibody binds to its target with an affinity that is often at least 25% greater, more often at least 50% greater, most often at least 100% (2-fold) greater, typically at least 10-fold greater, more typically at least 20-fold greater, and most typically at least 100-fold greater than the affinity of any other binding compound. In typical embodiments, molecules that specifically bind to a target have a binding affinity of at least about 10, as determined, for example, by Scatchard analysis. 6liters / mol (K D =10 -6 M), preferably at least about 10 liters / mol.

[0044] Targeting Ligands As noted above, the bispecific adapter can comprise a radical of a CAIX ligand. When administered, the targeting ligand targets the bispecific adapter conjugate to a cancer or tumor of interest that expresses the associated receptor. In some embodiments, the targeting moieties (in their free form, i.e., their radicals) do not bind to uptake receptors on non-targeted cells.

[0045] CAIX is a small molecule ligand that binds with specificity to receptors that are overexpressed in certain cancer cell types (i.e., the receptors for each of these ligands are overexpressed in cancer compared to the expression of such receptors in normal tissues, or potentially in diseased tissues that do not suffer from the targeted cancer type). Receptors for CAIX ligands have been found in, for example, kidney, ovarian, vulvar, and breast cancers, as well as cancers of the colon and pancreas. Its expression may also be associated with renal cell carcinoma, lung cancer, etc. Thus, upregulated CAIX expression may be a useful target for therapy.

[0046] The bispecific adapter can comprise a CAIX ligand (or a radical thereof) linked to a linker, wherein the linker is further linked to a CAR targeting moiety. In certain embodiments, the CAIX ligand is a high-affinity CAIX ligand. Unless otherwise specified, "high affinity" or "higher affinity" with respect to the affinity of a ligand for a target refers to a ligand having a Schrodinger molecular docking score of at least about -8.0 kcal / mol. In certain embodiments, a high-affinity CAIX ligand has improved affinity for the CAIX receptor compared to other ligands.

[0047] The targeting moiety can be, for example, a radical of a CAIX ligand having a molecular weight of less than about 10,000, less than 7,500, less than 5,000, less than 2,500, less than 1,000, less than 750, less than 500, about 500 to about 10,000 g / mol, about 1,000 to about 7,500 g / mol, about 750 g / mol to about 1,500 g / mol, about 1,000 to about 5,000 g / mol, or about 500 to about 2,500 g / mol.

[0048] The targeting ligand can bind to activated tumor or other cancer cells overexpressing the CAIX receptor. In certain embodiments, the targeting ligand can have a binding affinity to the CAIX receptor between about 1 nM and about 25 nM, e.g., between 1 nM and about 25 nM, or between about 1 nM and 25 nM. In certain embodiments, the targeting ligand can have a binding affinity to the CAIX receptor between about 0.002 nM and about 25 nM, e.g., between 0.002 nM and about 1 nM, or between about 0.002 nM and 1 nM. In certain embodiments, the targeting ligand can have a binding affinity to the CAIX receptor between about 0.01 nM and about 0.9 nM, e.g., between 0.01 nM and about 0.9 nM, or between about 0.01 nM and 0.9 nM. In certain embodiments, the targeting ligand may have a binding affinity for the CAIX receptor between about 0.02 nM and about 0.8 nM, e.g., between 0.02 nM and about 0.8 nM, or between about 0.02 nM and 0.8 nM. In certain embodiments, the targeting ligand may have a binding affinity for the CAIX receptor between about 0.03 nM and about 0.7 nM, e.g., between 0.03 nM and about 0.7 nM, or between about 0.03 nM and 0.7 nM. In certain embodiments, the targeting ligand may have a binding affinity for the CAIX receptor between about 0.04 nM and about 0.6 nM, e.g., between 0.04 nM and about 0.6 nM, or between about 0.04 nM and 0.6 nM. In certain embodiments, the targeting ligand may have a binding affinity for the CAIX receptor between about 0.05 nM and about 0.5 nM, e.g., between 0.05 nM and about 0.5 nM or between about 0.05 nM and 0.5 nM. In certain embodiments, the targeting ligand may have a binding affinity for the CAIX receptor between about 0.06 nM and about 0.4 nM, e.g., between 0.06 nM and about 0.4 nM or between about 0.06 nM and 0.4 nM. In certain embodiments, the targeting ligand may have a binding affinity for the CAIX receptor between about 0.07 nM and about 0.3 nM, e.g., between 0.07 nM and about 0.3 nM or between about 0.07 nM and 0.3 nM.In certain embodiments, the targeting ligand may have a binding affinity for the CAIX receptor between about 0.08 nM and about 0.2 nM, e.g., 0.08 nM to about 0.2 nM or in the range of about 0.08 nM to 0.2 nM. In certain embodiments, the targeting ligand may have a binding affinity for the CAIX receptor between about 0.09 nM and about 0.1 nM, e.g., 0.09 nM to about 0.1 nM or in the range of about 0.09 nM to 0.1 nM. The ranges set forth in this section are inclusive of the recited endpoints, and all values ​​in increments of 0.001 nM are encompassed thereby.

[0049] The CAIX ligand may be or may comprise 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL) or a derivative or analog thereof. The CAIX ligand may be or may comprise 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (meta-CAL) or a derivative or analog thereof. The CAIX ligand may be or may comprise acetazolamide (Aza) or a derivative or analog thereof. In certain embodiments, the CAIX ligand is selected from the group consisting of meta-CAL, ortho-CAL, and Aza. When administered, a bispecific adapter conjugate comprising a CAIX ligand or a radical thereof can target CAIX-expressing cancers in a subject.

[0050] Bispecific adapters can be specifically designed and synthesized to achieve particular binding affinities for CAIX.

[0051] Linker The linker of the bispecific adapter herein is disposed between the targeting ligand (e.g., a radical thereof) and the CAR T cell targeting moiety (e.g., comprising fluorescein, FITC, or NHS-fluorescein). The linker can be any suitable linker.

[0052] The term "linker" includes a chain of atoms that is biofunctionally configured to form a chemical bond and connects the CAR T cell targeting moiety and the cancer targeting ligand to form a conjugate. Illustratively, the chain of atoms can include carbon, nitrogen, oxygen, sulfur, silicon (Si), and phosphorus (P), e.g., C, N, O, S, and P, or C, N, O, and S.

[0053] Linkers can include a wide variety of linkages, for example, ranging from about 2 to about 100 atoms in the contiguous backbone. Linkers can include releasable forms of PEG, non-releasable forms of PEG, polyproline, hydrophilic amino acids, sugars, non-natural peptidoglycans, polyvinylpyrrolidone, or triblock copolymers containing a central hydrophobic block of polypropylene glycol flanked on either side by hydrophilic blocks of PEG.

[0054] The linker may comprise PEG or a PEG derivative. The linker may be (PEG)3.

[0055] The linker may be non-releasable, i.e., not labile. However, in some embodiments, it may be desirable for the linker in the bispecific adapter to be releasable, i.e., labile, e.g., photocleavable, acid-labile, base-labile, or enzymatically cleavable. The term "releasable" in the context of a linker refers to a linker that includes at least one bond that can be easily broken (e.g., chemically or enzymatically hydrolyzed) under physiological conditions, e.g., by reducing agent labile, pH labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile, enzymatically labile, or a p-aminobenzyl-based polyvalent releasable bond. It is understood that physiological conditions that result in bond breakdown do not necessarily include biological or metabolic processes, but may instead include standard chemical reactions, such as hydrolysis reactions at physiological pH or as a result of compartmentalization into organelles, such as endosomes, that have a pH lower than the cytosolic pH. The cleavable bond can connect two adjacent atoms in the releasable linker, and / or can connect to another linker moiety or to the targeting moiety and / or CAR T cell targeting moiety described herein, for example, at one or both ends of the releasable linker. In some instances, the releasable linker is broken into two or more fragments. In some instances, the releasable linker is separated from the CAR T cell targeting moiety.

[0056] In some embodiments, the linker is formed such that the CAR T cell targeting ligand (i.e., fluorescein) is cleaved from the cancer targeting moiety (i.e., CAIX ligand) only after sufficient time has passed for the bispecific adapter to circulate in the subject's systemic circulation following administration (e.g., to allow time for capture and internalization by targeted cells and / or receptors). In some embodiments, the period for release can vary (e.g., from subject to subject (e.g., based on various factors)). In some embodiments, the releasable linker can be engineered to not be cleaved / released for up to at least 24 hours, or even a week, following administration. In some embodiments, the bispecific adapter can safely pass through the subject's system, and any amount not captured by targeted cells (e.g., those expressing CAIX, etc.) can be excreted.

[0057] In contrast, the term "non-releasable" in the context of a linker refers to a linker that includes at least one bond that is not easily or rapidly broken down under physiological conditions. In some embodiments, a non-releasable linker includes a backbone that is stable under physiological conditions (e.g., the backbone is not susceptible to hydrolysis (e.g., hydrolysis by water or enzymes)). In some embodiments, a bispecific adapter including a non-releasable linker does not release any component of the bispecific adapter (e.g., a cancer-targeting ligand or a CAR T cell-targeting ligand). In some embodiments, a non-releasable linker lacks a disulfide bond (e.g., S-S) or an ester in the backbone. In some embodiments, a bispecific adapter includes a cancer-targeting ligand or a CAR T cell-targeting ligand connected by a backbone that is substantially stable throughout the circulation of the bispecific adapter (e.g., during endocytosis into a target cell endosome). A non-releasable linker may include an amide, an ester, an ether, an amine, and / or a thioether (e.g., a thiomaleimide). Although specific examples are provided, it will be understood that any molecule can be used in a non-releasable linker, provided that it forms at least one bond that is not easily or rapidly broken under physiological conditions.

[0058] Perhaps more specifically, a non-releasable linker can include, for example, a linker that hydrolyzes less than ten percent (10%) (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%) in water (e.g., a buffered (e.g., phosphate buffered) solution) at neutral pH within a period of time (e.g., 24 hours). In some embodiments, when a non-releasable linker is used, less than about ten percent (10%), preferably less than five percent (5%), of the administered bispecific adapters release their attached moieties (e.g., in the systemic circulation prior to uptake by targeted cells / tissues), or none of the administered bispecific adapters release their attached moieties (e.g., in the systemic circulation prior to uptake by targeted cells / tissues).

[0059] In some embodiments, the cancer-targeting ligand is not cleaved from the CAR-T cell-targeting ligand of the bispecific adapter in vivo, which can be advantageous because it allows the bispecific adapter to bind to and deliver the CAR-T cell to the targeted cancer cell.

[0060] The length of the linker can be selected to optimize the separation of molecules on the surface of the targeted cell provided by the linker, which can facilitate uptake of the bound CAR T cells into the targeted cell (e.g., when a bispecific adapter is administered). The linker can have a chain length of at least about 5 nm. In certain embodiments, each linker is approximately 5 nm to 15 nm in length. In some embodiments, the linker is at least about 7 nm in length. In certain embodiments, each linker is approximately 7 nm in length and flexible. In certain embodiments, each linker is approximately 7 to 10 nm in length. In some embodiments, the linker is at least about 14 nm in length. In some embodiments, the linker is about 15 nm in length. In some embodiments, the linker is between about 7 nm and about 31 nm in length (e.g., about 7 to 31, 7 to about 31, or 7 to 31), between about 7 nm and about 24 nm in length (e.g., about 7 to 24, 7 to about 24, or 7 to 24), or between about 7 nm and about 20 nm in length (e.g., about 7 to 20, 7 to about 20, or 7 to 20). In some embodiments, the linker is between about 14 nm and about 31 nm in length (e.g., about 14 to 31, 14 to about 31, or 14 to 31), between about 14 nm and about 24 nm in length (e.g., about 14 to 24, 14 to about 24, or 14 to 24), or between about 14 nm and about 20 nm in length (e.g., about 14 to 20, 14 to about 20, or 14 to 20). In some embodiments, the linkers have a chain length 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 the bound molecules at the cell surface, resulting in 7 to 10 nm or about 7 to 10 nm spacing 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 are inclusive of the recited endpoints, with all values ​​in 1 nm increments being encompassed within the recited range.

[0061] The linker may comprise at least one carbon-carbon bond and / or at least one amide bond. The linker may comprise one or more L- or D-configuration, natural or unnatural amino acids, or any combination of the foregoing.

[0062] In certain embodiments, a linker is a group that includes one or more covalently connected structural units.

[0063] The linker can be engineered to optimize the biodistribution, bioavailability, and PK / PD (e.g., of the bispecific adapter) and / or to increase previously described uptake (e.g., of the CAR-T cells attached thereto, and / or of the bispecific adapter itself) into targeted tissues, such as via, for example, PEGylation, according to methods generally known in the art or hereafter developed.

[0064] In some embodiments, the linker may include one or more spacers (e.g., to facilitate a specific release time, to facilitate increased uptake into targeted tissues, and / or to optimize the biodistribution, bioavailability, and / or PK / PD of the bispecific adapters provided herein). The spacer may include one or more alkyl chains, PEG, peptides, sugars, peptidoglycans, clickable linkers (e.g., triazoles), rigid linkers such as polyproline and polypiperidine, and the like.

[0065] In some embodiments, the linker of the bispecific adapter comprises PEG, a PEG derivative, or any other linker known in the art or later developed that can achieve the objectives described herein. In some embodiments, the linker is repeated n times, where n is a positive integer. For example, without limitation, n can be any integer selected from the ranges of 1 to 16, 1 to 32, 1 to 64, or 1 to 96. The number of repeats in the linker (i.e., n) can be selected to achieve the desired functionality, size, and / or efficacy of the conjugate and / or taking into account the desired application. In some embodiments, the linker comprises one or more spacers (e.g., which may also be used to specifically design the properties of the bispecific adapter).

[0066] In certain embodiments, the linker comprises, consists of, or consists essentially of PEG1 through PEG9. In certain embodiments, the linker comprises, consists of, or consists essentially of PEG2 through PEG8. In certain embodiments, the linker comprises, consists of, or consists essentially of PEG3 through PEG9. In certain embodiments, the linker comprises, consists of, or consists essentially of PEG4 through PEG8. In certain embodiments, the linker comprises, consists of, or consists essentially of PEG5 through PEG7. In certain embodiments, the linker comprises, consists of, or consists essentially of PEG3 through PEG 18 In certain embodiments, the linker comprises, consists of, or consists essentially of PEG4 to PEG 17 In certain embodiments, the linker comprises, consists of, or consists essentially of PEG5 to PEG 16 In certain embodiments, the linker comprises, consists of, or consists essentially of PEG6 to PEG 15 In certain embodiments, the linker comprises, consists of, or consists essentially of PEG7 to PEG14 In certain embodiments, the linker comprises, consists of, or consists essentially of PEG. 13 In certain embodiments, the linker comprises, consists of, or consists essentially of PEG. 12 In certain embodiments, the linker comprises, consists of, or consists essentially of PEG. 10 ~PEG 11 The linker may comprise, consist of, or consist essentially of PEG3-PEG9, e.g., PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, or PEG9. The linker may comprise PEG6 (or may consist essentially of or consist of). The linker may comprise an alkyl. The linker may be, comprise, or consist essentially of (CH2)4. All ranges described in this paragraph include the recited endpoints.

[0067] The linker may be or may comprise (or may consist essentially of or may consist of) PEG1. The linker may be or may comprise (or may consist essentially of or may consist of) PEG6. The linker may be or may comprise PEG3. The linker may be or may comprise PEG4. The linker may be PEG 12 The linker may be or comprise PEG. 16 It may be or may include.

[0068] 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 containing at least one substituent selected from the group consisting of alkyl, hydroxyl, oxo, PEG, carboxylate, and halo. In some embodiments, the linker includes a spacer (e.g., as described elsewhere herein).

[0069] In some embodiments, the linker is a substituted heteroalkyl having at least one disulfide bond in its backbone, hi some embodiments, the linker is a peptide having at least one disulfide bond in its backbone.

[0070] In some embodiments, the linker is -CONH-CH(COOH)-CH-SS-CH-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 are independently H, alkyl, or heteroalkyl (e.g., PEG).

[0071] In some embodiments, the linker is

[0072] [ka] wherein n or m (if applicable) is 0 to 10. Includes the structure of

[0073] In some embodiments, the linker is

[0074] [ka] [In the formula, n and m each independently represent an integer of 0 to 10.] Includes the structure of

[0075] In some embodiments, the linker is

[0076] [ka] wherein n is 1 to 32. In at least one exemplary embodiment, n is 1 to 30, and w is 0 to 5 (where applicable).

[0077] In some embodiments, the linker is

[0078] [ka] [wherein n is 1 to 16] Includes the structure of

[0079] In certain embodiments, the linker is

[0080] [ka] [In the formula, n is 1 to 30, and w is 0 to 5] The structure may include:

[0081] Bispecific adapters The bispecific adapter, or a pharmaceutically acceptable salt or hydrate thereof, can be for use with anti-fluorescein CAR-T cells in the treatment of CAIX-expressing cancers and can include a fluorescein-linker-CAIX ligand. The bispecific adapter can have a structure according to the formula shown in Figure 1.

[0082] The bispecific adapter has the following formula:

[0083] [ka] or a pharmaceutically acceptable salt or hydrate of any of the foregoing structures.

[0084] The bispecific adapter has the following formula:

[0085] [ka] or a pharmaceutically acceptable salt or hydrate thereof.

[0086] In certain embodiments, the bispecific adapter is for use with anti-fluorescein CAR-T cells in the treatment of CAIX-expressing cancers, wherein the fluorescein comprises FITC, the radical of the CAIX ligand is ortho-CAL, and the linker comprises (or consists essentially of or consists of) PEG, or the adapter is a pharmaceutically acceptable salt or hydrate of the foregoing.

[0087] In certain embodiments, the bispecific adapter is for use with anti-fluorescein CAR-T cells in the treatment of CAIX-expressing cancers, wherein the fluorescein comprises FITC, the radical of the CAIX ligand is ortho-CAL, and the linker comprises (or consists essentially of or consists of) PEG1, PEG6, or PEG9, or the adapter is a pharmaceutically acceptable salt or hydrate of the foregoing.

[0088] In certain embodiments, the bispecific adapter is for use with anti-fluorescein CAR-T cells in the treatment of CAIX-expressing cancers, wherein the fluorescein comprises FITC, the radical of the CAIX ligand is ortho-CAL, and the linker comprises (or consists essentially of or consists of) (CH2)4, or the adapter is a pharmaceutically acceptable salt or hydrate of the foregoing.

[0089] In certain embodiments, the bispecific adapter is for use with anti-fluorescein CAR-T cells in the treatment of CAIX-expressing cancers, wherein the fluorescein comprises FITC, the radical of the CAIX ligand is meta-CAL, and the linker comprises (or consists essentially of or consists of) PEG3, PEG6, or PEG9, or the adapter is a pharmaceutically acceptable salt or hydrate of the foregoing.

[0090] Also provided is a bispecific adapter for use with anti-fluorescein CAR-T cells in treating CAIX-expressing cancers, wherein the fluorescein comprises fluorescein, FITC, or NHS-fluorescein, the radical of the CAIX ligand is Aza, and the linker comprises (or consists essentially of, or consists of) PEG, or the adapter is a pharmaceutically acceptable salt or hydrate of the foregoing. The linker may comprise (or consist essentially of, or consist of) PEG1 through PEG9. The linker may comprise (or consist essentially of, or consist of) PEG6. The linker may comprise (or consist essentially of, or consist of) PEG9.

[0091] The bispecific adapter has the following formula:

[0092] [ka] or a pharmaceutically acceptable salt or hydrate thereof.

[0093] The bispecific adapter has the following formula:

[0094] [ka] or may include a pharmaceutically acceptable salt or hydrate thereof.

[0095] Bispecific adapters may contain one or more chiral centers or may otherwise be capable of existing as multiple stereoisomers. Thus, various embodiments of bispecific adapters may include pure stereoisomers as well as mixtures of stereoisomers, e.g., enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures. Bispecific adapters may be capable of existing as geometric isomers, e.g., pure geometric isomers or mixtures of geometric isomers.

[0096] Bispecific adapter conjugates can be synthesized according to methods known in the art. Various synthesis methods are illustrated in the Examples.

[0097] salt The bispecific adapters herein may be presented as pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" of a bispecific adapter refers to a salt whose counterion is acceptable for pharmaceutical use. Such salts include (i) acid addition salts that can be obtained by reacting the free base of the parent conjugate with an inorganic acid, such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid, or 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, and the like; and (ii) salts formed when an acidic proton present in the parent conjugate is replaced with a metal ion, such as an alkali metal ion, alkaline earth ion, or aluminum ion, or coordinates with an organic base, such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucamine, and the like. Pharmaceutically acceptable salts are well known to those of skill in the art, and all such pharmaceutically acceptable salts are contemplated herein.

[0098] In various embodiments, suitable base salts are formed from bases that form non-toxic salts. Illustrative examples include arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases, such as hemisulfate and hemicalcium salts, can also be formed.

[0099] Pharmaceutically acceptable salts can be synthesized from parent bispecific adapter conjugates containing basic or acidic moieties by conventional chemical methods. In some instances, such salts can be prepared by reacting the free acid or base form of these conjugates with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of both, with non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile generally being preferred. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is incorporated herein by reference.

[0100] The bispecific adapter, or its pharmaceutically acceptable salts or hydrates, can exist in solvated forms, including unsolvated and hydrated forms. A solvated form may be equivalent to an unsolvated form. In each embodiment herein, the formula will be understood to include and represent not only all pharmaceutically acceptable salts of the bispecific adapter, but also any hydrates and / or solvates of the conjugate formula or its salts. The term "solvate" refers to a compound, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0101] Certain functional groups, such as hydroxy, amino, etc., can form complexes and / or coordinate bonds with water and / or various solvents. Thus, the formulas should be understood to include and represent various hydrates and / or solvates thereof. Non-hydrated and / or non-solvated forms of the bispecific adapters are also included.

[0102] Pharmaceutical Composition In view of the above, compositions (e.g., pharmaceutical compositions) for treating cancer (e.g., CAIX-expressing cancer) are also provided, comprising at least one bispecific adapter and a pharmaceutically acceptable carrier or excipient. A "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as, but not limited to, buffers, preservatives, anesthetics, solubilizers, isotonicity agents, humectants, and stabilizers. The term also encompasses any agent approved by a regulatory agency, such as the U.S. Food and Drug Administration, or listed in the U.S. Pharmacopeia, for use in animals (e.g., mammals, e.g., humans). The carrier can be a phosphate-buffered saline solution, water, or an emulsion, such as an oil-in-water or water-in-oil emulsion.

[0103] Also provided are pharmaceutical compositions for use in treating CAIX-expressing cancers comprising any of the bispecific adapters herein (eg, fluorescein-linker-CAIX) and a pharmaceutically acceptable carrier or excipient.

[0104] Bispecific adapters can be formulated as pharmaceutical compositions and administered to a mammalian host, e.g., a human patient, in a variety of forms compatible with the selected route of administration. For example, pharmaceutical compositions can be formulated for and administered via oral or parenteral, intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumor, intramuscular, topical, inhalation, and / or subcutaneous routes. Indeed, in at least one embodiment, the bispecific adapters and / or compositions described herein can be administered directly into the bloodstream, muscle, or an internal organ.

[0105] For example, in at least one embodiment, the bispecific adapters can be administered systemically (e.g., orally) in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral therapeutic administration, the bispecific adapters can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the compositions and preparations may vary and may be between about 1 and about 99% by weight of the active ingredient, as well as binders, excipients, disintegrants, lubricants, and / or sweeteners (as known in the art). The amount of active conjugate in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0106] The bispecific adapters and pharmaceutical compositions herein can be formulated as parenteral formulations. Parenteral formulations are typically aqueous solutions that may contain carriers or excipients such as salts, carbohydrates, and buffers (preferably at a pH of 3 to 9), but they can more preferably be formulated as sterile nonaqueous solutions or as dry forms for use with a suitable vehicle such as sterile pyrogen-free water or sterile saline. Preparation under sterile conditions by lyophilization to produce a sterile, lyophilized powder for parenteral formulations can be achieved using methods well known in the art. The solubility of the bispecific adapter or its pharmaceutically acceptable salt or hydrate for parenteral formulations can be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.

[0107] The bispecific adapters / compositions can also be administered via infusion or injection (e.g., using needle (including microneedle) and / or needleless injectors). Solutions of the compositions can be aqueous, optionally mixed with nontoxic surfactants, and / or contain carriers or excipients such as salts, carbohydrates, and buffers (preferably pH 3-9), although for some applications, they may be more suitably formulated as sterile nonaqueous solutions or as dry forms for use with a suitable vehicle such as sterile pyrogen-free water or phosphate-buffered saline (PBS). For example, dispersions can be prepared in glycerol, liquid PEG, triacetin, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations may further contain a preservative to prevent microbial growth.

[0108] Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions, or sterile powders containing the active ingredient, optionally encapsulated in liposomes, configured for extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. Liquid carriers or vehicles can be solvents or liquid dispersion media, including, 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, proper fluidity can be maintained by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. The action of microorganisms can be prevented by the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In certain cases, it may be desirable to include one or more isotonic agents, such as sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents in the formulation to delay absorption, for example, aluminum monostearate and gelatin.

[0109] Sterile injectable solutions can be prepared by incorporating the bispecific adapter and / or composition in the required amount of an appropriate solvent, along with one or more of the other ingredients described above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredient already present in the filtered sterile solution.

[0110] For topical administration, it may be desirable to administer the bispecific 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, solid carriers can include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Similarly, useful liquid carriers can include water, alcohol, or glycol, or water-alcohol / glycol blends, in which the conjugate can be dissolved or dispersed at effective levels, optionally utilizing non-toxic surfactants. Additionally or alternatively, adjuvants such as fragrances and antimicrobial agents can be added to optimize the properties for a given use. The resulting liquid composition can be applied from an absorbent pad, used to impregnate bandages and / or other dressings, sprayed onto the targeted site using a pump-type or aerosol sprayer, or simply applied directly to the desired area on the subject.

[0111] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified mineral materials can also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the subject's skin.

[0112] The amount of bispecific adapter (or its pharmaceutically acceptable salt or hydrate) administered to a subject can vary widely depending on the cancer being treated, the route of administration, and tissue distribution. As used herein, the terms "therapeutically effective," "therapeutically effective dose," "therapeutically effective amount," "prophylactically effective amount," or "prophylactically effective dose" (unless specifically stated otherwise) refer to an amount of bispecific adapter that, when administered once or over the course of a treatment cycle, affects the health, well-being, or mortality of a subject (e.g., delays the onset and / or reduces the severity of one or more symptoms associated with cancer). Useful dosages of bispecific adapters can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to human subjects are known in the art. Indeed, the dosage of the bispecific adapter can vary widely depending on the condition of the host subject, the cancer being treated, the progression of the condition, the route of administration and tissue distribution of the bispecific adapter, and whether other therapeutic treatments (e.g., radiation therapy, or additional drugs in combination therapies such as CAR T cell therapy) are used concomitantly. The amount of the composition required for use in treatment (e.g., a therapeutically or prophylactically effective amount or dose) will vary not only with the particular application, but also with the salt selected (if applicable) and the characteristics of the subject (e.g., age, condition, sex, subject's body surface area and / or mass, tolerance to drugs, etc.), and is ultimately at the discretion of the attending physician, clinician, etc.

[0113] The amount administered to a subject is, for example, about 0.05 mg to about 30 mg, about 0.05 mg to about 25 mg, about 0.05 mg to about 20 mg, about 0.05 mg to about 15 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 9 mg, about 0.05 mg to about 8 mg, about 0.05 mg to about 7 mg, about 0.05 mg to about 6 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 4 mg, about 0.05 mg The dosage may range from about 0.05 mg to about 3 mg, about 0.05 mg to about 2 mg, about 0.05 mg to about 1 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.4 mg, about 0.05 mg to about 0.3 mg, about 0.05 mg to about 0.2 mg, about 0.05 mg to about 0.1 mg, about 0.01 mg to about 20 mg, about 0.3 mg to about 10 mg, about 0.1 mg to about 20 mg, or about 0.8 mg to about 3 mg. Those skilled in the art will readily appreciate that dosages may vary within the various ranges provided above based on the factors noted above and may be left to the discretion of the treating physician.

[0114] A therapeutically or prophylactically effective amount or dose can be, for example, from 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 of patient body weight. The total therapeutically or prophylactically effective amount of the bispecific adapter may be administered in single or divided doses and, at the practitioner's discretion, may fall outside of the typical range given herein.

[0115] In another embodiment, the bispecific adapter is at about 0.5 mg / m 2 ~about 500mg / m 2 , about 0.5mg / m 2 ~about 300mg / m 2, or about 100 mg / m 2 ~about 200mg / m 2 In other embodiments, the amount can be administered in a therapeutically or prophylactically effective amount of about 0.5 mg / m 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 about 0.5 mg / m 2 ~about 2.0mg / m 2 The total amount can be administered in single or divided doses and, at the physician's discretion, may fall outside the typical ranges given herein. These amounts are based on meters of body surface area. All ranges specified in this paragraph are inclusive of the stated endpoints, with the ranges up to 0.5 mg / m2 subsumed within each specified range. 2 Includes all values ​​of the step.

[0116] In other embodiments, the amount of bispecific adapter (or a pharmaceutically acceptable salt or hydrate thereof) administered to a subject is, for example, about 50 nmol / kg to about 3,000 nmol / kg of the subject's 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, or about 50 nmol / kg to about 600 nmol / kg , about 50 nmol / kg to about 500 nmol / kg, about 50 nmol / kg to about 400 nmol / kg, about 50 nmol / kg to about 300 nmol / kg, about 50 nmol / kg to about 200 nmol / kg, about 50 nmol / kg to about 100 nmol / kg, about 100 nmol / kg to about 300 nmol / kg, about 100 nmol / kg to about 500 nmol / kg, about 100 nmol / kg to about 1,000 nmol / kg, or about 100 nmol / kg to about 2,000 nmol / kg of the subject's 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 the subject's body weight. In other embodiments, about 20 μg / kg to about 3 mg / kg of the subject's body weight may be administered. The amount may be between about 0.2 mg / kg to about 0.4 mg / kg of the subject's body weight, or about 50 μg / kg of the subject's body weight. All ranges specified in this paragraph include the recited endpoints and, where applicable, all values ​​in increments of 1 nmol / kg or 10 μg / kg subsumed within each specified range.

[0117] Uses and Methods Further provided is a method of treating cancer (e.g., a CAIX-expressing cancer) in a subject, comprising administering to the subject a cancer-treating effective amount of (i) anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) CAR-T cells, or a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier or excipient, and (ii) a bispecific adapter, or a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier or excipient.

[0118] The terms "treat," "treating," "treated," and "treatment" refer to therapeutic treatment. Such treatment may have a prophylactic effect. Cancer is treated if the symptoms or signs of the cancer are improved, such as a reduction in tumor size, complete or partial disappearance of the tumor, stabilization of the cancer, such as by inhibiting cancer progression (e.g., an increase in tumor size or number of tumors due to metastasis, etc.), or any other effect on the cancer that a physician may consider to constitute therapeutic (or prophylactic) treatment.

[0119] The term "subject" as used herein refers to an animal, such as a mammal, particularly a human. In veterinary applications, the subject may be a laboratory animal, an agricultural animal, a domestic animal, or a wild animal. Examples of such animals include, but are not limited to, rodents, rabbits, monkeys, chimpanzees, dogs, cats, cows, horses, pigs, sheep, goats, bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, or whales.

[0120] Anti-fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) CAR-T cells are T cells (alternatively, NK cells can 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.

[0121] A CAR is a fusion protein that contains at least three domains, including: (i) a recognition region (e.g., a single-chain fragment variable (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.

[0122] The scFv region of an antibody that binds fluorescein (e.g., in FITC) can be used and prepared from (i) antibodies known in the art that bind fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein), (ii) newly prepared anti-fluorescein antibodies, or (iii) sequence variants derived from the scFv region of such antibodies, e.g., scFv regions 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 binding portion of the CAR can be, for example, an scFv, Fab, Fv, Fc, or (Fab')2 fragment of an antibody.

[0123] "Percent sequence identity (%)" in reference to a polypeptide or nucleotide sequence is defined as the percentage of amino acid or nucleic acid residues in a candidate sequence that are identical to the residues in a reference sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, and does not consider any conservative substitutions as part of the sequence identity. Alignment for determining percent sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software. For example, the determination of percent identity or similarity between sequences can be performed, for example, by using the GAP program (software by Genetics Computer Group, currently available online through Accelrys), and alignment can be performed, for example, using the ClustalW algorithm (VNTI software, InforMax Inc.). Furthermore, sequence databases can be searched using the nucleic acid or amino acid sequence of interest. Algorithms for database searching are typically based on BLAST software (Altschul et al., 1990), although one of skill in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, percent identity can be determined over the full length of the nucleic acid or amino acid sequence.

[0124] In various embodiments, the CAR has a recognition region, which is an scFv region of an anti-fluorescein antibody that can bind to fluorescein, FITC, or NHS-fluorescein (see, e.g., 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 comprising an E2 anti-fluorescein antibody shown in Figure 1 of WO 2019 / 144091 and described from page 66, line 16 to page 69, line 12, both of which are incorporated herein by reference for their teachings). The CAR has a costimulatory domain, which can be CD28 (cluster of differentiation 28), CD2 (cluster of differentiation 2), CD137 (cluster of differentiation 137, 4-1BB), a member of the tumor necrosis factor (TNF) family, CD134 (cluster of differentiation 134, OX40), a member of the TNF receptor (TNFR) superfamily of receptors, CD27 (cluster of differentiation 27), CD30 (cluster of differentiation 30), CD150 (cluster of differentiation 150), DAP10, NKG2D, CD278 (cluster of differentiation 278, ICOS), a CD28 superfamily costimulatory molecule expressed on activated T cells, a member of the signaling lymphocyte activation molecule (SLAM)-related receptor family (e.g., 2B4), or any combination thereof. Sequence variants of costimulatory domains with the same or similar activity as the domains on which they are modeled can also be used without adversely affecting the method. The CAR has an activation signaling domain, which can be a T cell CD3 zeta chain, a CD3 delta receptor protein, an mbl receptor protein, a B29 receptor protein, or an Fc receptor gamma. Sequence variants of the activation signaling domain that have the same or similar activity as the domain on which the modeling is based can also be used without adversely affecting the method.Such costimulatory domains and variants of such costimulatory domains and activation signaling domains can have 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 domain from which they are derived.

[0125] In one embodiment of a CAR comprising an E2 anti-fluorescein antibody fragment, the CAR comprises an IgG4 hinge domain and a CD28 transmembrane domain, the costimulatory domain is CD137 (4-1BB), and the activation signaling domain is CD3ζ.

[0126] In certain embodiments, the CAR comprises an scFv of an anti-fluorescein antibody as the recognition region, a CD137 (4-1BB) costimulatory domain, and CD3ζ as the activation signaling domain.

[0127] Constructs encoding CARs are prepared using genetic engineering techniques, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3, 2003, incorporated herein by reference. rded., Cold Spring Harbor Laboratory Press (2001). By way of example, a plasmid or viral expression vector (e.g., a lentiviral vector, a retroviral vector, sleeping beauty, and piggyback (a transposon / transposase system including a non-viral-mediated CAR gene delivery system)) can be prepared to encode a fusion protein comprising a recognition region, one or more costimulatory domains, and an activation signaling domain linked in-frame and in a 5' to 3' direction. Other arrangements may be acceptable, including a recognition region, an activation signaling domain, and one or more costimulatory domains. The location of the recognition region in the fusion protein is generally such that presentation of the region on the outside of the cell is achieved. CARs can 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 that the fusion protein is maintained as an integral membrane protein, and a hinge domain to provide flexibility to the recognition region and enable strong binding to the CAR targeting moiety.

[0128] T lymphocytes (e.g., cytotoxic T lymphocytes) can be 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.

[0129] The T lymphocytes can be autologous, although xenogeneic cells can be used, such as when the patient being treated is undergoing high-dose chemotherapy or radiation treatment to destroy the patient's immune system. In various embodiments, allogeneic cells can be used.

[0130] T lymphocytes can be obtained from patients by means well known in the art. For example, T cells can be obtained by collecting peripheral blood from a patient, subjecting the blood to Ficoll 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 collected population of cells can contain at least about 90% of the selected cell type, for example, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the selected cell type.

[0131] After obtaining T lymphocytes, the cells can be cultured under conditions that promote cell activation. The culture conditions can be such that the cells can be administered to patients without concern for reactivity to components of the culture medium. For example, the culture conditions can be free of bovine serum preparations, such as bovine serum albumin (BSA). Activation can be achieved by introducing a known activating agent, such as an anti-CD3 antibody in the case of cytotoxic T cells, into the culture medium. Other suitable activating agents include anti-CD28 antibodies. The lymphocyte population can be cultured for about 1 to about 4 days under conditions that promote activation. The appropriate level of activation can be determined by cell size, proliferation rate, or activation markers determined by flow cytometry.

[0132] After the population of cytotoxic T lymphocytes has been cultured under conditions that promote activation, the cells can be transfected with an expression vector encoding a CAR. After transfection, the cells can be administered immediately to the patient, or the cells can 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 about 12 days, about 6 to about 13 days, about 7 to about 14 days, or about 8 to about 15 days, e.g., to allow the cells time to recover from the transfection. Suitable culture conditions can be similar to those under which the cells were cultured for activation, with or without agents used to promote activation.

[0133] After the cells are transfected and activated, the composition containing CAR-T cells can be prepared and administered to the subject.Culture medium that does not contain any animal preparations, such as BSA, can be used.Tissue culture conditions typically used in the art can be used to avoid contamination with bacteria, fungi, and mycoplasma.Cells can be pelleted, washed, and resuspended in pharmaceutically acceptable carriers, diluents, or excipients.

[0134] Exemplary compositions comprising CAR-T cells include those comprising cells in 290 mOsm sterile saline, injectable cryopreservation medium (containing Plasma-Lyte A, dextrose, sodium chloride, HSA, and dimethyl sulfoxide (DMSO)) in 0.9% NaCl containing 2% human serum albumin (HSA), or any other injectable 290 mOsm sterile material. Depending on the nature of the culture medium, the CAR-T cells may be administered as a composition in the culture medium or may be administered after being concentrated and resuspended in the culture medium.

[0135] The CAR-T cell composition can be administered to a subject by any suitable means, for example, parenteral administration, for example, intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, or intrathecally.

[0136] The total number of CAR-T cells and the concentration of 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 examples herein) and the nature of the small molecule ligand / targeting ligand (e.g., CAIX ligand) of the bispecific adapter, the nature of the cancer, the location of the cancer in the subject, the means used to administer the composition to the subject, and the health, age, and weight of the subject to be treated. Suitable compositions comprising transduced CAR-T cells include approximately 1 x 10 5 ~Approx. 1×10 15 Examples of compositions include compositions having a volume of between about 5 ml and about 200 ml containing transduced CAR-T cells. Typical compositions include compositions having a volume of between about 10 ml and about 125 ml, containing about 1 x 10 7 ~Approx. 1×10 10 An exemplary composition contains about 1 x 10 CAR-T cells in a volume of about 100 ml. 9 The composition comprises CAR-T cells. A single dose or multiple doses of CAR-T cells can be administered to a subject. The composition can include, 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 patient body weight. When the CAR-T cell composition is administered by injection into a subject's bloodstream, the CAR-T cells in the subject's bloodstream represent at least 5%, 7%, 10%, 11%, 12%, 13%, 14%, or 15% of the subject's total T cells in the subject's bloodstream by about 4 weeks after injection, or at least 20%, 25%, 30%, 35%, 40%, or 50% of the subject's total T cells in the subject's bloodstream by about 2 weeks after injection, or at least 85%, 90%, or 95% of the subject's total T cells by about 1 week after injection.

[0137] The bispecific adapter (or a pharmaceutically acceptable salt or hydrate thereof) or a pharmaceutical composition comprising it, or a combination thereof with anti-fluorescein CAR-T cells or a pharmaceutical composition comprising anti-fluorescein CAR-T cells, can be administered to a patient using any suitable method known in the art. The terms "administer," "administering," "administered," and "administration" refer to methods of introducing a bispecific adapter (or a pharmaceutically acceptable salt or hydrate thereof) or a pharmaceutical composition comprising a bispecific adapter (or a pharmaceutically acceptable salt or hydrate thereof), and methods of introducing anti-fluorescein CAR-T cells or a pharmaceutical composition comprising anti-fluorescein CAR-T cells. Examples of suitable routes of administration include, but are not limited to, oral, intravenous, intramuscular, subcutaneous, and transdermal. Components can be administered directly into the bloodstream, muscle, or an internal organ. Suitable routes for parenteral administration include but are not limited to intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular and subcutaneous.Needle syringes, including microneedles, needleless syringes and infusions can be used.Components can be administered in unit dosage forms and / or formulations containing conventional non-toxic pharmaceutically acceptable carriers or excipients (or vehicles or adjuvants).

[0138] In the methods, the anti-fluorescein CAR-T cells (or a pharmaceutical composition comprising the anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient) and the bispecific adapter can be administered simultaneously or sequentially in any order by the same or different routes. When administered simultaneously by the same route, the formulations can be the same or different. In various embodiments, the bispecific adapter can be administered to the subject after the CAR-T cells. The timing between the administration of the CAR-T cells and the bispecific adapter can vary widely 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 example herein) and the small molecule ligand / targeting moiety (i.e., CAIX ligand) of the bispecific adapter, the nature of the cancer, the location of the cancer in the subject, the means used to administer the CAR-T cells and the bispecific adapter to the subject, and the patient's health, age, and weight.

[0139] The bispecific adapter can be administered before or after the CAR-T cells, e.g., within about 3, 6, 9, 12, 15, 18, 21, or 24 hours, or within about 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, or more days. The rate of tumor lysis can be modulated, for example, by adjusting the rate of administration of the bispecific adapter (e.g., according to a dosing schedule such as continuous, once daily, twice daily, three times daily, once weekly, twice weekly, or three times weekly). By "continuously" is meant 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 a daily or weekly administration schedule, e.g., once daily, twice daily, three times daily, every other day, once weekly, twice weekly, three times weekly, or any other suitable schedule. In the method, the anti-fluorescein CAR-T cells (or a pharmaceutical composition comprising the anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient) and the bispecific adaptor can be administered intravenously. The cancer can be ovarian cancer, breast cancer, lung cancer, bladder cancer, or clear cell renal cell carcinoma (e.g., stage 3-4 clear cell renal cell carcinoma). The cancer can be endometrial cancer or glioma (e.g., stage 3-4 glioma).

[0140] Cytokine release syndrome (CRS) can be controlled by varying the dose of the bispecific adapter. See, e.g., WO 2017 / 177149.

[0141] Such combination therapy methods can be practiced using any engineered cells suitable for treating cancer and may include the use of 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 additionally (or alternatively) include engineered stem cells and other cells.

[0142] The engineered cell used in combination with bispecific adaptor or composition can be any CAR T cell, stem cell or other engineered cell, or their combination.A variety of adoptive cell therapy (also referred to as cellular immunotherapy) is known in the art for use in cancer treatment, and T cell immunotherapy in particular has attracted much attention.Some non-limiting examples of such therapy include engineered T cell receptor (TCR) therapy, CAR T cell therapy and natural killer (NK) cell therapy.

[0143] In certain approaches, administering both the bispecific adapter conjugate and the engineered cell therapy results in greater than additive inhibition of cancer growth.

[0144] When multiple therapeutic agents and / or therapies are co-administered, dosages may be adjusted accordingly, as recognized in the relevant art. "Co-administration" and combination therapy are not limited to simultaneous administration, but also include treatment regimens in which a targeting bispecific adapter is administered at least once during a course of treatment that involves administering a cell therapy to a subject.

[0145] The methods of treating cancer herein can include administering to a patient any of the bispecific adapters and administering to the patient either an engineered cell composition or an engineered cell therapy.

[0146] In certain embodiments, a method for treating cancer in a subject is provided. The method comprises administering to the subject a cancer-treating 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 bispecific adapter or any pharmaceutical composition comprising it and a pharmaceutically acceptable carrier or excipient. Steps (i) and (ii) can be administered by the same or different routes, simultaneously, or sequentially, in any order.

[0147] The anti-fluorescein CAR-T cells can include any CAR T cells suitable for the described uses. In certain embodiments, the anti-fluorescein CAR-T cells include a recognition region comprising an scFv region of an anti-fluorescein antibody, a costimulatory domain that is CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS), and / or an activation signaling domain that is T cell CD3 zeta chain or Fc receptor γ.

[0148] In certain embodiments, both steps (i) and (ii) of the method are administered intravenously.

[0149] The fluorescein of the bispecific adapter, when exposed to anti-fluorescein CAR-T cells, can bind with affinity to the anti-fluorescein CAR-T cells, and the targeted ligand of the bispecific adapter can link the bound anti-fluorescein CAR-T cells to the targeted cancer cells when the targeted ligand binds with affinity to a receptor on the targeted cancer cells. Thus, the conjugates and compositions herein facilitate enhanced efficacy of CAR-T cell therapy.

[0150] In certain embodiments, the receptor of the targeted cancer cells is overexpressed CAIX. The cancer may be a CAIX-expressing cancer, and the at least one bispecific adapter of (ii) may comprise a radical of a CAIX ligand.

[0151] Also provided are methods for treating CAIX-expressing cancer in a subject. In certain embodiments, the method comprises administering to the subject a cancer-treating 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 bispecific adaptor or a pharmaceutical composition comprising a bispecific adaptor and a pharmaceutically acceptable carrier or excipient. As described above, the CAR can have a recognition region, which is an scFv region of an anti-fluorescein antibody. In certain embodiments, the CAR comprises a costimulatory domain that is CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS), and / or an activation signaling domain that is T cell CD3 zeta chain or Fc receptor γ.

[0152] The methods herein can further include imaging the cancer in the subject. Imaging the cancer can include, for example, imaging by optical imaging, positron emission tomography (PET), or single photon emission computed tomography (SPECT).

[0153] In the methods described herein, the cancer can be further imaged before administering the bispecific adapter, or a pharmaceutically acceptable salt or hydrate thereof, or an engineered cell composition (e.g., a CAR-expressing cytotoxic lymphocyte composition or a CAR-NK cell composition) to the subject. Additionally or alternatively, the cancer can be imaged during or after administration, for example, to assess metastasis and treatment efficacy. For example, imaging can be performed by PET imaging, magnetic resonance imaging (MRI), or SPECT / computed tomography (CT) imaging. The imaging method can be any suitable imaging method known in the art.

[0154] The cancer may be any cancer. "Cancer," when read in light of this specification, has its plain and ordinary meaning and may include, but is not limited to, a group of diseases involving abnormal cell growth that has the potential to invade or spread (i.e., metastasize) to other parts of the body. Examples include, but are not limited to, cancer of the brain, thyroid, lung, pancreas, kidney, stomach, gastrointestinal stromal, endometrial, breast, cervical, ovarian, colon, or prostate, leukemia, lymphoma, other blood-related cancers, and head and neck cancer. In certain embodiments, the cancer to be treated is a tumor. In certain embodiments, the cancer is malignant. In certain embodiments, the cancer is ovarian cancer, endometrial cancer, breast cancer, glioma, e.g., optionally, stage 3-4 glioma, or clear cell renal cell carcinoma, e.g., optionally, stage 3-4 clear cell renal cell carcinoma.

[0155] In some aspects of these embodiments, the cancer is a CAIX-expressing cancer.

[0156] In some embodiments, the cancer is imaged prior to administering (i) and (ii) to the subject. Imaging can be performed, for example, by PET, MRI, or SPECT / CT.

[0157] In certain embodiments, the use of a bispecific adaptor, a pharmaceutically acceptable salt, hydrate, or solvate of a bispecific adaptor, or a composition is provided for the manufacture of a medicament for the treatment of cancer in a subject.The bispecific adaptor can be any conjugate.The medicament can be used in combination with the administration of engineered cell therapy, such as CAR T cell therapy, in which CAR T cells express anti-fluorescein, to a subject.

[0158] Further provided are methods for enhancing CAR-T cell activation. The methods may include providing a bispecific adapter described herein, a pharmaceutical composition comprising the same, or a combination thereof (e.g., a therapeutically effective amount of any of those described above), 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 exhibit enhanced activation after exposure compared to CAR-T cells not exposed to the bispecific adapter. For example, when exposed to the bispecific adapter, pharmaceutical composition, or combination, the anti-fluorescein CAR-T cells may be systemically circulating in a subject. Alternatively, the anti-fluorescein CAR-T cells may be exposed to the bispecific adapter, pharmaceutical composition, or combination in vitro (e.g., prior to administration to a subject).

[0159] kit Kits are also provided. The kits may include (i) a bispecific adapter, or a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier or excipient, and (ii) anti-fluorescein CAR-T cells (e.g., anti-FITC CAR-T cells), or a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the bispecific adapter (or a pharmaceutical composition comprising the same) and the CAR-T cells (or a pharmaceutical composition comprising the same) are stored in separate containers.

[0160] General Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above, and implementations should not be limited to the particular embodiments described. Other implementations may be possible.

[0161] While the bispecific adapters and pharmaceutical compositions have been illustrated and described in detail in the foregoing description, it is to be understood that the same is to be considered illustrative and not restrictive in character, that only certain embodiments have been shown and described, and that all changes and modifications that come within the spirit of the invention are desired to be protected.

[0162] It is intended that the scope of the present bispecific adapters, compositions, and methods be defined by the following claims. However, the present disclosure may be practiced otherwise than as specifically described and exemplified without departing from its spirit or scope. Those skilled in the art will recognize that various alternatives to the embodiments described herein may be employed in practicing the following claims without departing from the spirit and scope thereof as defined therein.

[0163] The use of any section headings is intended to aid in the reading and comprehension of the document and should not be construed as limiting. Furthermore, the information associated with a section heading may be found either within that particular section or outside of that section.

[0164] All publications, patents, patent application publications, journal articles, textbooks, and other publications mentioned in this document are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. In the event of inconsistent usage between this document and those documents so incorporated by reference, the usage in the incorporated references should be considered supplementary to the usage in this document, and in the event of any irresolvable inconsistency, the usage in this document will control.

[0165] A connection or coupling between two components may be described by various techniques and mechanisms. Words such as bonded, linked, coupled, and connected, and similar terms with their inflected morphemes, are used interchangeably unless a difference is noted or the context makes clear otherwise. 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 imply a direct, unobstructed connection because various other components may exist between the two components being referenced. Thus, unless otherwise noted, a connection does not necessarily imply a direct, unobstructed connection.

[0166] A specific 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 have the same meaning as commonly understood by one of ordinary skill in the art.

[0167] The term "about" or "approximately" refers to a range within which a particular value is within acceptable tolerance, as determined by one of ordinary skill in the art, which depends, in part, on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can refer to 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 a further example, "about" or "approximately" can mean within 90%, 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999%, or greater, of a stated value or a stated range limit. Alternatively, particularly with respect to biological systems or processes, the term can refer to within 10-fold, preferably within 5-fold, and more preferably within 2-fold, of a value. Unless otherwise specified, the term "about" refers to a range within acceptable error, e.g., ±1-20%, preferably ±1-10%, and more preferably ±1-5% of a particular value.

[0168] When a range of values ​​is provided, it is understood that each value between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is included. The upper and lower limits of these narrower ranges may independently be included in the narrower ranges, and are also included, subject to any explicitly excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding one or both of those limits are also included.

[0169] A phrase referring to "at least one of" a list of items refers to any combination of those items, including single members. As an example, "at least one of a, b, or c" is intended to encompass a, b, c, ab, ac, bc, and abc.

[0170] The terms "a," "an," or "the" are used to include one or more, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or," unless otherwise indicated. In addition, phraseology or terminology used herein and not otherwise defined should be understood to be for purposes of description only and not of limitation.

[0171] The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. In addition, phrases or terms used herein and not otherwise defined should be understood to be for purposes of description only and not of limitation.

[0172] The terms and phrases employed are used as terms of description and not of limitation. Where a particular term is defined and otherwise explained or discussed elsewhere in the Detailed Description, all such definitions, explanations, and discussions are intended to be attributed to such terms. Furthermore, the use of such terms and phrases is not intended to exclude any equivalents of the features shown and described or portions thereof. Furthermore, although subheadings may be used in the Detailed Description, such use is merely for ease of reference and is not intended to limit any disclosure made in a section to only that section. Rather, any disclosure made under a subheading is intended to constitute a disclosure under all other subheadings.

[0173] It is recognized that various modifications are possible within the scope of the claimed invention. Thus, while the present invention has been specifically disclosed in the context of preferred embodiments and optional features, those skilled in the art may make modifications and variations of the concepts disclosed herein. Such modifications and variations are considered to be within the scope of the invention as claimed herein. [Example]

[0174] The following examples serve to illustrate the present disclosure and are not intended to limit the scope of the claimed invention in any way.

[0175] [Example 1] General Procedure for the Synthesis of Ortho-CAIX-PEG-FITC and Ortho-(CH)-FITC Conjugates Ortho-CAIX-PEG1-FITC and ortho-(CH2)4-FITC conjugates can be synthesized according to Scheme 1.

[0176] [ka]

[0177] [ka]

[0178] PyBOP (1.2 equiv.) and DIPEA (2.0 equiv.) were added to a stirred solution of the acidic compound (1.0 equiv.) in dimethylformamide (DMF). After 10 min of stirring, BocNH-PEG1-NH2 or BocNH(CH2)4NH2 (1.2 equiv.) was added to the reaction mixture, and stirring was continued for an additional 2 h. After complete conversion of the starting material (as determined by liquid chromatography-mass spectrometry (LC-MS)), the reaction mixture was diluted with water and then extracted with dichloromethane (DCM) (2 × 20 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to give the crude residue, which was then purified using Method 5-95 (A = 10 mM ammonium acetate, P H The resulting desired fraction was kept on a freeze dryer for 48 hours to give the coupled product as a white solid.

[0179] To the lyophilized solid (0.0133 mmol) in DCM (3 mL) was added trifluoroacetic acid (TFA) (1 mL) and stirring was continued for 30 min. The reaction mixture was then evaporated under reduced pressure to give the free amine as a brown sticky solid. The resulting amine was used in the next step without purification. To a stirred solution of the amine (1 equiv) in DMF (500 μL) was added N,N-diisopropylethylamine (DIPEA) (10 equiv), followed by fluorescein isothiocyanate isomer 1 (1 equiv), and stirring was continued for an additional 2 h at room temperature. The reaction mixture was diluted with water and purified using the 5-35 method (A = 10 mM ammonium acetate, P H The resulting fraction was purified by ultra-high performance liquid chromatography (U-HPLC) using 1000 kJ / ml hexane (B = 7.5, B = acetonitrile) for 60 min, and the desired fraction was immediately placed in a freeze-dryer for 48 h to obtain the desired ortho-CAIX-FITC compound as a yellow solid.

[0180] [Example 2] Binding affinity of a bispecific adaptor targeting carbonic anhydrase IX MDA-carbonic anhydrase IX (CAIX) cells (200,000) were incubated for 1 hour at room temperature with bispecific adapters serially diluted 2-fold from 250 nM in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS). The cells were washed once to remove unbound adapters. The fluorescence intensity of the adapters bound to the cells was analyzed by flow cytometry. The results are shown in Figure 2A-2B.

[0181] For ortho-CALs, binding affinity to CAIX+ cells decreased with increasing linker length. For meta-CALs, binding affinity to CAIX+ cells decreased slightly with increasing linker length, but all meta-CAL bispecific adapters had higher binding.

[0182] [Example 3] Total binding and surface exposure of CAIX bispecific adapters HT29 or MDA-CAIX cells were incubated with different ligands for CAIX and 1 μM bispecific adapters with different polyethylene glycol (PEG) linkers in complete RPMI medium (RPMI + 10% FBS) for 1 h at room temperature. Free compounds were washed away, and fluorescein isothiocyanate (FITC) fluorescence intensity was analyzed by flow cytometry.

[0183] To analyze the surface exposure of the FITC moiety, stained cells were incubated with allophycocyanin (APC)-anti-FITC antibody 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 3A–3D. As shown in Figure 3A, meta-CAL-PEG3 and Aza-FITC had better total binding to HT29 cells than the other adapters. As shown in Figure 3B, all three meta-CAL-FITC adapters had better surface binding and FITC exposure in HT29 cells. As shown in Figure 3C, meta-CAL-FITC and Aza-FITC with different linkers had better total binding to MDA-CAIX cells. As shown in Figure 3D, all meta-CAL-FITC adapters had better surface binding and FITC exposure in MDA-CAIX cells than the other tested adapters.

[0184] [Example 4] Anti-FITC CAR-T cell cytotoxicity against MDA-CAIX cells and IFNγ release mediated by a CAIX-targeting bispecific adapter Anti-FITC CAR-T cells were incubated with equal numbers of MDA-CAIX cells in the presence of different concentrations of bispecific adapter. The number of MDA-CAIX cells was determined at the end of the coculture, and the percentage of cell lysis was calculated using the following formula: [(number of untreated cells - number of treated cells) / number of untreated cells] x 100%.

[0185] Interferon gamma (IFNγ) secretion from anti-FITC CAR-T cells was analyzed by enzyme-linked immunosorbent assay (ELISA) using the supernatant of the co-culture medium at the end of the co-culture. The results are shown in Figures 4A-4B.

[0186] As shown in Figure 4A, the efficacy of meta-CAL-PEG6-FITC was higher than that of ortho-CAL-PEG6-FITC and Aza-FITC at adapter concentrations between 0.001 nM and 10 nM. As shown in Figure 4B, meta-CAL-PEG6-FITC mediated higher levels of IFNγ release from anti-FITC CAR-T cells than ortho-CAL-PEG6-FITC and Aza-FITC when the adapter concentration was between 0.001 nM and 1 nM.

[0187] [Example 5] Effect of linker length on anti-FITC CAR-T cell cytotoxicity and IFNγ release mediated by meta-CAL-FITC bispecific adapter when co-cultured with MDA-CAIX cells Anti-FITC CAR-T cells were incubated with equal numbers of MDA-CAIX cells in the presence of different concentrations of bispecific adapter. The number of MDA-CAIX cells was determined at the end of the coculture, and the percentage of cell lysis was calculated using the following formula: [(number of untreated cells - number of treated cells) / number of untreated cells] x 100%.

[0188] IFNγ secretion from anti-FITC CAR T cells was analyzed by ELISA using the coculture medium supernatant at the end of the coculture. The results are shown in Figures 5A-5B. As shown in Figure 5A, meta-CAL-PEG6-FITC and meta-CAL-PEG9-FITC were more effective than meta-CAL-PEG3-FITC at low concentrations (between 0.001 nM and 0.1 nM). As shown in Figure 5B, meta-CAL-PEG6-FITC and meta-CAL-PEG9-FITC mediated higher levels of IFNγ release from anti-FITC CAR T cells than meta-CAL-PEG3-FITC at low concentrations (between 0.001 nM and 1 nM).

[0189] [Example 6] Effect of linker length on anti-FITC CAR-T cell cytotoxicity and IFNγ release mediated by ortho-CAL-FITC bispecific adapter when co-cultured with MDA-CAIX cells Anti-FITC CAR T cells were incubated with equal numbers of MDA-CAIX cells in the presence of different concentrations of bispecific adapter. The number of MDA-CAIX cells was determined at the end of coculture, and the percentage of cell lysis was calculated using the following formula: [(number of untreated cells - number of treated cells) / number of untreated cells] x 100%.

[0190] IFNγ secretion from anti-FITC CAR T cells was analyzed by ELISA using the supernatant of the co-culture medium at the end of the co-culture study, and the results are shown in Figures 6A-6B.

[0191] As shown in Figure 6A, the ortho-CAL-FITC bispecific adapter had comparable efficacy, but the longer PEG spacer was slightly better than the (CH2)4 spacer. As shown in Figure 6B, the ortho-CAL-FITC bispecific adapter showed comparable efficacy, but the longer PEG spacer was slightly better than the (CH2)4 spacer.

[0192] [Example 7] In vivo efficacy of CAIX-targeting bispecific adapters in the HT29 tumor model HT29 cells (1.5 million) were implanted subcutaneously into each NOD scid gamma (NSG) mouse. Tumor volumes were approximately 100 mm 3 When tumor size reached 10,000,000, the treatment group was injected with 10 million anti-FITC CAR-T cells and bispecific adapters as shown in Figure 7A. Tumor volume and body weight were monitored periodically. Tumor volume was calculated using the formula: (length x width) 2 ) / 2. The results are shown in Figures 7B-7C.

[0193] As shown in Figure 7B, Aza-FITC and meta-CAL-PEG3-FITC slightly inhibited HT29 tumor growth, whereas meta-CAL-PEG9-FITC significantly inhibited HT29 tumor growth. As shown in Figure 7C, only mice treated with meta-CAL-PEG9-FITC lost weight. The weight loss was likely due to increased cytokine release from CAR-T cells, an indicator of better functionality of meta-CAL-PEG9-FITC. Toxicity can be minimized by optimizing adapter dosing.

[0194] [Example 8] Retention and surface exposure of CAIX bispecific adaptors in HT29 tumor cells HT29 tumor-bearing mice were injected with 500 nmol / kg of CAIX bispecific adapter. After injection (24 h), tumors were excised and digested into single cells. Total retention of the bispecific adapter was determined by FITC fluorescence intensity analyzed by flow cytometry. To analyze FITC exposure of the bispecific adapter on the tumor cell surface, digested tumor cells were stained with APC-anti-FITC antibody for 30 min on ice. After washing away unbound antibody, the APC fluorescence intensity was analyzed by flow cytometry. The results are shown in Figures 8B-8C.

[0195] As shown in Figure 8B, all compounds had very low retention in HT29 tumors, which may be due to the low expression of CAIX in HT29 tumor cells. As shown in Figure 8C, meta-CAL-FITC with different linkers had better surface retention and FITC exposure than the other compounds. meta-CAL-PEG6-FITC and meta-CAL-PEG9-FITC had better FITC exposure in vivo than meta-CAL-PEG3-FITC.

[0196] [Example 9] T cell counts in blood and HT29 tumors from mice at the end of treatment At the end of the in vivo study in the HT29 solid tumor model described in Figures 7A-7C, blood was collected from the mice, and red blood cells in the blood were lysed using red blood cell lysis buffer (BioLegend, San Diego, CA). The remaining cells were stained with anti-human CD3 antibody on ice for 30 minutes. The number of T cells was analyzed by flow cytometry. To analyze tumor-infiltrating T cells, tumors were excised and digested into single cells. The digested cells were stained with Zombie Violet cell viability dye (BioLegend, San Diego, CA) and anti-human CD3 antibody. The percentage of CD3 T cells among total live cells was analyzed by flow cytometry. The results are shown in Figures 9A-9B.

[0197] As shown in Figure 9A, blood from mice treated with meta-CAL-PEG9-FITC had more T cells than blood from other treatment groups, and as shown in Figure 9B, tumors from mice treated with meta-CAL-PEG9-FITC had more tumor-infiltrating T cells.

[0198] [Example 10] In vivo efficacy of CAIX-targeting bispecific adapters in the MDA-CAIX tumor model MDA-CAIX cells (5 million) were implanted into each NSG mouse by subcutaneous injection. Tumor volumes were approximately 100 mm 3 When tumor volume reached 100 μg / mL, treatment groups were injected with 10 million anti-FITC CAR T cells and the indicated bispecific adapters, as shown in Figure 10A. Tumor volume and body weight were monitored periodically. Tumor volume was calculated using the formula: (length x width) 2 ) / 2. The results are shown in Figures 10B and 10C.

[0199] As shown in Figure 10B, meta-CAL-PEG3-FITC slightly inhibited the growth of MDA-CAIX tumors, whereas ortho-CAL-PEG6-FITC significantly inhibited the growth of MDA-CAIX tumors. As shown in Figure 10C, neither bispecific adapter was toxic to mice during treatment.

[0200] [Example 11] Retention and surface exposure of the CAIX bispecific adaptor in MDA-CAIX tumor cells Twenty-four hours after the final injection of 500 nmol / kg of CAIX bispecific adapter, MDA-CAIX tumors from mice in the different treatment groups were excised and digested to single cells. Total retention of the bispecific adapter was determined by FITC fluorescence intensity analyzed by flow cytometry. To estimate CAIX protein levels in tumor cells, cells were stained with 100 nM meta-CAL-PEG6-FITC for 1 hour at room temperature and analyzed by flow cytometry. FITC exposure of the bispecific adapter on the tumor cell surface was analyzed by flow cytometry after staining with APC-anti-FITC antibody for 30 minutes on ice. Surface exposure of FITC was also analyzed in cells stained with 100 nM meta-CAL-PEG6-FITC. The results are shown in Figures 11B-11E.

[0201] As shown in Figure 11B, tumors treated with Aza-FITC and meta-CAL-FITC with PEG3 or PEG6 linkers had comparable total FITC retention in the cells. As shown in Figure 11C, tumor cells treated with Aza-FITC had slightly higher CAIX levels than tumors treated with meta-CAL-FITC with PEG3 or PEG6. As shown in Figure 11D, meta-CAL-PEG6-FITC had slightly better surface retention and FITC exposure than the other compounds. As shown in Figure 11E, tumor cells from mice treated with ortho-CAL-PEG6-FITC had little to no anti-FITC antibody staining, indicating that CAIX-expressing tumor cells were killed by CAR-T cells.

[0202] [Example 12] General Procedure for the Synthesis of Acetazolamide (Aza)-PEG-FITC Conjugates

[0203] [ka]

[0204] [ka]

[0205] [Example 13] Effect of linker length on the binding affinity of Aza-FITC bispecific adapters MDA-CAIX cells (200,000) were incubated with 4-fold serial dilutions of the bispecific adapter starting from 1000 nM in PBS containing 2% FBS for 1 hour at room temperature. Cells were washed once to remove unbound adapter. The fluorescence intensity of the cell-bound adapter was analyzed by flow cytometry. The results are shown in Figure 12, a graph of concentration (nM) vs. normalized MFI, demonstrating that the PEG spacer has no significant effect on the binding affinity of the acetazolamide (Aza)-FITC bispecific adapter.

[0206] [Example 14] Effect of linker length on total binding and surface exposure of Aza-FITC bispecific adapters MDA-CAIX cells were incubated with 500 nM Aza-FITC bispecific adapters with different PEG spacers in complete RPMI medium (RPMI + 10% FBS) for 1 hour at room temperature. Free compounds were washed away, and the fluorescence intensity of FITC was analyzed by flow cytometry. To analyze the surface exposure of the FITC moiety, the stained cells were incubated with APC-anti-FITC antibody 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 13A-13B. Figure 13A is a graph of the MFI of the bispecific adapter versus FL, showing the total binding of the Aza-FITC bispecific adapter to MDA-CAIX cells. Linker length did not significantly affect total binding. Figure 13B is a graph of the MFI of the bispecific adapter versus APC-anti-FITC, showing the surface exposure of the FITC moiety in Aza-FITC bispecific adapters with different PEG linkers after binding to MDA-CAIX cells. Increasing the PEG spacer length enhanced the surface exposure of FITC after binding to MDA-CAIX cells.

[0207] [Example 15] Effect of linker length on IFNγ release of anti-FITC CAR-T cells mediated by Aza-FITC bispecific adapter when co-cultured with HT29 cells Anti-FITC CAR-T cells were incubated with HT29 cells at a 1:5 ratio in the presence of different concentrations of bispecific adapter for 40 hours. IFNγ secretion from anti-FITC CAR-T cells was analyzed by ELISA using the coculture medium supernatant at the end of the coculture study. The results are shown in Figure 14, which is a graph of concentration (nM) versus IFNγ (pg / ml) showing the effect of linker length on IFNγ release of anti-FITC CAR-T cells mediated by the Aza-FITC bispecific adapter when cocultured with HT29 cells.

[0208] Aza-PEG3-FITC and Aza-PEG9-FITC mediated higher levels of IFNγ released from anti-FITC CAR-T cells than Aza-PEG0-FITC. Aza-PEG6-FITC mediated the highest level of IFNγ released from anti-FITC CAR-T cells.

[0209] [Example 16] IFNγ release of anti-FITC CAR-T cells mediated by CAIX-targeting bispecific adapter with optimal linker Anti-FITC CAR T cells were incubated with HT29 cells at a 1:5 ratio in the presence of different concentrations of bispecific adapter for 40 hours. IFNγ secretion from anti-FITC CAR T cells was analyzed by ELISA using the supernatant of the coculture medium at the end of the coculture study. The results are shown in Figure 15, which is a graph of CA9 ligand-PEG(n)-FITC concentration (nM) versus IFNγ (pg / ml), demonstrating that CAIX-targeted bispecific adapters with optimal linkers mediated IFNγ release from anti-FITC CAR-T cells. Meta-CAL-PEG9-FITC mediated the highest levels of IFNγ released from anti-FITC CAR-T cells at low concentrations (<0.1 nM). Aza-PEG6-FITC mediated comparable levels of IFNγ release from anti-FITC CAR T cells as meta-CAL-PEG9-FITC at concentrations higher than 0.1 nM. Ortho-CAL-PEG6-FITC mediated lower levels of IFNγ released from anti-FITC CAR-T cells.

[0210] [Example 17] In vivo efficacy of Aza-PEG6-FITC and ortho-CAL-PEG6-FITC KB cells (1 million) were implanted into each NSG mouse by subcutaneous injection. Tumor volumes were approximately 50 mm 3 When tumor volume reached 100 μg / mL, treatment groups were injected with 10 million anti-FITC CAR-T cells and the indicated bispecific adapters, as shown in FIG. 16A. Tumor volume and body weight were monitored periodically. Tumor volume was calculated using the formula: (length x width)2 ) / 2. The results are shown in Figures 16B-16C.

[0211] Figure 16B shows tumor growth curves for different treatment groups, showing tumor volume (mm ) versus days after CAR-T cell injection. 3 ) Both Aza-PEG6-FITC and ortho-CAL-PEG6-FITC slightly inhibited the growth of KB tumors. The efficacy of Aza-PEG6-FITC is slightly better than that of ortho-CAL-PEG6-FITC. Figure 16C is a graph of body weight change (%) versus days after CAR-T cell injection, showing the weight changes of mice in different treatment groups. Neither Aza-PEG6-FITC nor ortho-CAL-PEG6-FITC induced significant weight loss.

[0212] [Example 18] Synthesis of Aza-FITC Synthesis of 5-amino-2-sulfamoyl-1,3,4-thiadiazole monohydrochloride B: Hydrochloric acid (1 M aqueous solution) (70 mL, 70.00 mmol, 5.2 equiv.) was added to acetazolamide A (3 g, 13.34 mmol, 1.0 equiv.), and the mixture was stirred under reflux for 3 h (Scheme 3). Water was then evaporated using a rotary evaporator. The crude material (dissolved in a small amount of methanol with TEA for solid support) was purified by column chromatography (CHCl3 / MeOH: 100 / 0 to 70 / 30) to give product B (2.768 g, 96%).

[0213] [ka]

[0214] Synthesis of (9H-fluoren-9-yl)methyl (8-oxo-8-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)octyl)carbamate D: N-methylmorpholine (0.063 mL, 1.1 equiv.) was added to a stirred solution of N-Fmoc-8-aminooctanoic acid C (0.200 g, 1 equiv.) and ethyl chloroformate (0.055 mL, 1.1 equiv.) in fresh, dry DMF (1.5 mL) at room temperature under argon, followed by stirring for 30 minutes (Scheme 3). 5-Amino-2-sulfamoyl-1,3,4-thiadiazole B (0.568, 1.5 equiv.) was then added at room temperature, and stirring was continued overnight under argon. The reaction was monitored by liquid chromatography-mass spectrometry (LCMS) and purified by preparative high-performance liquid chromatography (HPLC) (mobile phase: A = 20 mM ammonium acetate pH = 7, B = acetonitrile (ACN), method: 0% B to 80% B in 40 min at 13 mL / min). Pure fractions were pooled and freeze-dried to give (9H-fluoren-9-yl)methyl (8-oxo-8-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)octyl)carbamate D.

[0215] Synthesis of 8-amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)octanamide E: (9H-Fluoren-9-yl)methyl (8-oxo-8-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)octyl)carbamate D (0.080 g) was dissolved in 20% piperidine in DMF (1.0 mL) at room temperature, and stirring was continued for 2 hours under argon (Scheme 3). The reaction was monitored by LCMS and purified by preparative HPLC (mobile phase: A = 20 mM ammonium acetate pH = 7, B = ACN, method: 0% B to 50% B in 30 minutes at 13 mL / min). Pure fractions were pooled and freeze-dried to give 8-amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)octanamide E.

[0216] Synthesis of Aza-FITC F: DIPEA (3.0 equiv.) was added at room temperature to a stirred solution of 8-amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)octanamide E (0.0160 g) and FITC (0.019 g, 1.0 equiv., 0.80 equiv. + 0.20 equiv. added in small portions over 1 h) in dimethyl sulfoxide (DMSO) (1 mL) and stirred continuously under argon for 3 h. The reaction was monitored by LCMS and purified by preparative HPLC (mobile phase: A = 20 mM ammonium acetate pH = 7, B = ACN, method: 0% B to 60% B in 40 min at 13 mL / min). Pure fractions were pooled and freeze-dried to give Aza-FITC F. LCMS (ESI) (m / z): (M+H) + C 31 H 30 Calculated value for N6O8S3+H = 711.13 Measured value (m / z): (M+H) + 710.80.

[0217] [Example 19] Synthesis of CAL-PEG-FITC conjugate

[0218] [ka]

[0219] Synthesis of meta-CAL-P3-FITC conjugate Meta-CAL-PEG3NHBoc (8). Meta-CA ligand 6 was synthesized by a known procedure described in the literature. More specifically, to a stirred solution of meta-CA ligand 6 (0.039 g, 1.0 equiv.), t-Boc-N-amido-PEG3amine 7 (0.027 g, 1.1 equiv.), and hexafluorophosphate azabenzotriazole tetramethyluronium (HATU) (0.036 g, 1.15 equiv.) in DMF (1.5 mL), DIPEA (0.0173 mL, 1.2 equiv.) was added at room temperature under argon, and the solution was stirred for 3-4 h as described in Scheme 5. The reaction was monitored by LCMS and purified by reversed-phase C-18 column (mobile phase: A = 20 mM ammonium acetate pH = 7, B = ACN, method: 5% B to 95% B in 50 min). Pure fractions were pooled and freeze-dried to give meta-CAL-PEG3NHBoc 8.

[0220] Meta-CAL-PEG3NH2 (9). Meta-CAL-PEG3NHBoc 8 (0.050 g) was dissolved in 30% TFA in wet DCM (1.5 mL) at room temperature and stirred for 1–2 h (Scheme 5). The reaction was monitored by LCMS, and volatiles were removed by rotary evaporation. The residue was dissolved in DCM and purified by column chromatography on silica gel (DCM:methanol:TFA = 89:10:1). Further purification of trace impurities by preparative thin-layer chromatography (TLC) (DCM:methanol:TFA = 89:10:1) gave meta-CAL-PEG3NH29.

[0221] Meta-CAL-P3-FITC (11). Meta-CAL-PEG3NH28 (0.041 g, 1.0 equiv.) was dissolved in DMF (1.0 mL) under an argon atmosphere, followed by the addition of FITC 10 (0.024 g, 1.0 equiv., 0.80 equiv. + 0.20 equiv. added in small portions over 2 h) and DIPEA (1.0 equiv.) at room temperature in the dark (Scheme 5). The reaction mixture was stirred for an additional 1 h. The reaction was monitored by LCMS and purified on a C-18 reverse-phase column (mobile phase: A = milli-Q water (TFA) pH = 3, B = ACN, method: 5% B to 95% B in 50 min). Pure fractions were collected, frozen at -80 °C for 3 h, and lyophilized to give meta-CAL-P3-FITC conjugate 11. The purity of compound meta-CAL-P3-FITC conjugate 11 was analyzed by LCMS.

[0222] [ka]

[0223] Synthesis of meta-CAL-P6-FITC conjugate Meta-CAL-PEG6NHBoc (13). DIPEA (0.0177 mL, 1.2 equiv.) was added to a stirred solution of meta-CAL ligand 6 (0.040 g, 1.0 equiv.), t-Boc-N-amido-PEG6amine 12 (0.039 g, 1.1 equiv.), and HATU (0.037 g, 1.15 equiv.) in DMF (1.5 mL) at room temperature under an argon atmosphere. The solution was stirred for 3–4 h as described in Scheme 6. The reaction was monitored by LCMS and purified on a C-18 reverse-phase column (mobile phase: A = 20 mM ammonium acetate pH = 7, B = ACN, method: 5% B to 95% B in 50 min). Pure fractions were pooled and freeze-dried to give meta-CAL-PEG6NHBoc 13.

[0224] Meta-CAL-PEG6NH2 (14). Meta-CAL-PEG6NHBoc 13 (0.056 g, 1.0 equiv.) was dissolved in 30% TFA in wet DCM (1.5 mL) at room temperature and stirred for 1–2 h (Scheme 6). The reaction was monitored by LCMS, and volatiles were removed by rotary evaporation. The residue was dissolved in DCM and purified by column chromatography on silica gel (DCM:methanol:TFA=94:5:1). Further purification of trace impurities by preparative TLC (DCM:methanol:TFA=94:5:1) gave meta-CAL-PEG6NH2 14.

[0225] Meta-CAL-P6-FITC (15). Meta-CAL-PEG6NH214 (0.057 g, 1.0 equiv.) was dissolved in DMF (1.0 mL) under an argon atmosphere, followed by the addition of FITC 10 (0.028 g, 1.0 equiv., 0.80 equiv. + 0.20 equiv. added in small portions over 2 h) and DIPEA (1.0 equiv.) at room temperature in the dark (Scheme 6). The reaction mixture was stirred for an additional 1 h. The reaction was monitored by LCMS and purified on a C-18 reverse-phase column (mobile phase: A = milli-Q water (TFA) pH = 3, B = ACN, method: 5% B to 95% B in 50 min). Pure fractions were collected, frozen at -80 °C for 3 h, and lyophilized to give meta-CAL-P6-FITC conjugate 15. The purity of compound meta-CAL-P6-FITC conjugate 15 was analyzed by LCMS.

[0226] [ka]

[0227] Synthesis of meta-CAL-P9-FITC conjugate Meta-CAL-PEG9NHBoc (17). To a stirred solution of meta-CA ligand 6 (0.031 g, 1.0 equiv.), t-Boc-N-amido-PEG9amine 16 (0.040 g, 1.1 equiv.), and HATU (0.028 g, 1.15 equiv.) in DMF (1.0 mL), DIPEA (0.014 mL, 1.2 equiv.) was added at room temperature under argon, and the solution was stirred for 3–4 h as described in Scheme 7. The reaction was monitored by LCMS and purified on a C-18 reverse-phase column (mobile phase: A = 20 mM ammonium acetate pH = 7, B = ACN, method: 5% B to 95% B in 50 min). Pure fractions were pooled and freeze-dried to give meta-CAL-PEG9NHBoc 17.

[0228] Meta-CAL-PEG9NH2 (18). Meta-CAL-PEG9NHBoc 17 (0.067 g, 1.0 equiv.) was dissolved in 30% TFA in wet DCM (1.5 mL) at room temperature and stirred for 1–2 h (Scheme 7). The reaction was monitored by LCMS, and volatiles were removed by rotary evaporation. The residue was dissolved in DCM and purified by column chromatography on silica gel (DCM:methanol:TFA=94:5:1). Further purification of trace impurities by preparative TLC (DCM:methanol:TFA=94:5:1) gave meta-CAL-PEG9NH2 18.

[0229] Meta-CAL-P9-FITC (19). Meta-CAL-PEG9NH2 (0.041 g, 1.0 equiv.) was dissolved in DMF (1.0 mL) under an argon atmosphere, followed by the addition of FITC 10 (0.017 g, 1.0 equiv., 0.80 equiv. + 0.20 equiv. added in small portions over 2 h) and DIPEA (1.0 equiv.) at room temperature in the dark (Scheme 7). The reaction mixture was stirred for an additional 1 h. The reaction was monitored by LCMS and purified on a C-18 reverse-phase column (mobile phase: A = milli-Q water (TFA) pH = 3, B = ACN, method: 5% B to 95% B in 50 min). Pure fractions were collected, frozen at -80 °C for 3 h, and lyophilized to obtain meta-CAL-P9-FITC conjugate 19. The purity of compound meta-CAL-P9-FITC 19 was analyzed by LCMS.

[0230] [ka]

[0231] Synthesis of ortho-CAL-P6-FITC conjugate Ortho-CAL-PEG6NHBoc (21). To a stirred solution of ortho-CA ligand 20 (0.033 g, 1.0 equiv.), t-Boc-N-amido-PEG6amine 12 (0.032 g, 1.1 equiv.), and HATU (0.033 g, 1.15 equiv.) in DMF (1.5 mL) was added DIPEA (0.015 mL, 1.2 equiv.) at room temperature under argon, and the solution was stirred for 3–4 h as described in Scheme 8. The reaction was monitored by LCMS and purified on a C-18 reverse-phase column (mobile phase: A = 20 mM ammonium acetate pH = 7, B = ACN, method: 5% B to 95% B in 50 min). Pure fractions were pooled and freeze-dried to give ortho-CAL-PEG6NHBoc 21.

[0232] Ortho-CAL-PEG6NH2 (22). Ortho-CAL-PEG6NHBoc 21 (0.110 g, 1.0 equiv.) was dissolved in 25% TFA in wet DCM (1.5 mL) at room temperature and stirred for 2 h (Scheme 8). The reaction was monitored by LCMS, and volatiles were removed by rotary evaporation. The residue was dissolved in DMF and purified on a C-18 reverse-phase column (mobile phase: A = 20 mM ammonium acetate pH = 7, B = ACN, method: 5% B to 95% B in 60 min). Pure fractions were pooled and freeze-dried to give ortho-CAL-PEG6NH2 22.

[0233] Ortho-CAL-P6-FITC (23). Ortho-CAL-PEG6NH222 (0.060 g, 1.0 equiv.) was dissolved in DMF (1.0 mL) under an argon atmosphere, followed by the addition of FITC 10 (0.028 g, 0.9 equiv., 0.70 equiv. + 0.20 equiv. added in small portions over 1 h) and DIPEA (1.25 equiv.) at room temperature in the dark (Scheme 8). The reaction mixture was stirred for an additional 1 h. The reaction was monitored by LCMS and purified on a C-18 reverse-phase column (mobile phase: A = milli-Q water (TFA) pH = 3, B = ACN, method: 5% B to 95% B in 50 min). Pure fractions were collected, frozen at -80 °C for 3 h, and lyophilized to give ortho-CAL-P6-FITC conjugate 23. The purity of compound ortho-CAL-P6-FITC 23 was analyzed by LCMS.

[0234] [ka]

[0235] [ka]

[0236] Synthesis of ortho-CAL-P9-FITC conjugate Ortho-CAL-PEG9NHBoc (24). Ortho-CA ligand 20 was synthesized by a known procedure described in the literature. Specifically, to a stirred solution of ortho-CA ligand 20 (0.022 g, 1.0 equiv.), t-Boc-N-amido-PEG9amine 16 (0.028 g, 1.0 equiv.), and HATU (0.022 g, 1.15 equiv.) in DMF (1.0 mL), DIPEA (0.0104 mL, 1.2 equiv.) was added at room temperature under an argon atmosphere, and the solution was stirred for 3-4 h as described in Scheme 9. The reaction was monitored by LCMS and purified by reversed-phase C-18 column (mobile phase: A = 20 mM ammonium acetate pH = 7, B = ACN, method: 5% B to 95% B in 50 min). Pure fractions were pooled and freeze-dried to give ortho-CAL-PEG9NHBoc 24.

[0237] Ortho-CAL-PEG9NH2 (25). Ortho-CAL-PEG9NHBoc 24 (0.098 g, 1.0 equiv.) was dissolved in 25% TFA in wet DCM (1.5 mL) at room temperature and stirred for 2 h (Scheme 9). The reaction was monitored by LCMS, and volatiles were removed by rotary evaporation. The residue was dissolved in DMF and purified on a C-18 reversed-phase column (mobile phase: A = 20 mM ammonium acetate pH = 7, B = ACN, method: 5% B to 95% B in 60 min). Pure fractions were pooled and freeze-dried to give ortho-CAL-PEG9NH2 25.

[0238] ortho-CAL-P9-FITC (26). Ortho-CAL-PEG9Nhour (0.071 g, 1.0 equiv.) was dissolved in DMF (1.0 mL) under an argon atmosphere, followed by the addition of FITC 10 (0.028 g, 0.9 equiv., 0.70 equiv. + 0.20 equiv. added in small portions over 1 h) and DIPEA (1.25 equiv.) in the dark at room temperature (Scheme 9). The reaction mixture was stirred for an additional 1 h.

[0239] The reaction was monitored by LCMS and purified by reversed-phase C-18 column (mobile phase: A = milli Q water (TFA) pH = 3, B = ACN, method: 5% B to 95% B in 50 min). Pure fractions were collected, frozen at -80 °C for 3 h, and lyophilized to obtain ortho-CAL-P9-FITC conjugate 26. The purity of compound ortho-CAL-P9-FITC 26 was analyzed by LCMS.

[0240] [ka]

[0241] [ka]

[0242] Enumeration of embodiments of the claimed invention Clause 1. A bispecific adapter, or a pharmaceutically acceptable salt or hydrate thereof, comprising fluorescein, fluorescein isothiocyanate (FITC), or N-hydroxysuccinimide (NHS)-fluorescein conjugated via a linker to a radical of a carbonic anhydrase IX (CAIX) ligand, wherein the linker comprises, consists essentially of, or consists of polyethylene glycol (PEG).

[0243] Clause 2. The bispecific adapter of clause 1, wherein the CAIX ligand is or comprises 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL), 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (meta-CAL), acetazolamide (Aza), or a derivative or analog of any of the foregoing.

[0244] Clause 3. The bispecific adapter of clause 1, wherein the linker comprises, consists essentially of, or consists of PEG1-PEG9.

[0245] Clause 4. The bispecific adapter of clause 1, wherein the CAIX ligand is or comprises an ortho-CAL or a derivative or analogue thereof and the linker comprises, consists essentially of, or consists of PEG1 to PEG9.

[0246] Clause 5. The bispecific adaptor of clauses 1 to 3, wherein the linker comprises, consists essentially of, or consists of PEG3 to PEG9.

[0247] Clause 6. The bispecific adapter of clauses 1 to 3, wherein the CAIX ligand is or comprises meta-CAL or a derivative or analogue thereof and the linker comprises, consists essentially of, or consists of PEG3 to PEG9.

[0248] Clause 7. The bispecific adaptor of clause 3, wherein the CAIX ligand is or comprises Aza or a derivative or analogue thereof.

[0249] Clause 8. The bispecific adapter of any one of clauses 4, 6, and 7, wherein the linker comprises, consists essentially of, or consists of PEG6.

[0250] Clause 9. The bispecific adaptor of clause 6 or 7, wherein the linker comprises, consists essentially of, or consists of PEG9.

[0251] Clause 10. The bispecific adaptor of clause 7, wherein the linker comprises, consists essentially of, or consists of PEG6.

[0252] Clause 11. The bispecific adapter of any of clauses 1 to 10, wherein the linker comprises, consists essentially of, or consists of alkyl.

[0253] Clause 12. The bispecific adaptor of any one of clauses 1 to 10, wherein the linker comprises, consists essentially of, or consists of (CH2)4.

[0254] Article 13. The following formula:

[0255] [ka] or a pharmaceutically acceptable salt or hydrate of any of the foregoing.

[0256] Article 14. The following formula:

[0257] [ka] or a pharmaceutically acceptable salt or hydrate thereof.

[0258] Article 15. The following formula:

[0259] [ka] or a pharmaceutically acceptable salt or hydrate of any of the foregoing.

[0260] Clause 16. The bispecific adapter of any one of clauses 1 to 15 for use with anti-fluorescein chimeric antigen receptor (CAR)-T cells in the treatment of cancer.

[0261] Clause 17. The bispecific adapter of any one of clauses 1 to 15 for use with anti-fluorescein CAR-T cells in the treatment of CAIX-expressing cancer.

[0262] Clause 18. A pharmaceutical composition for the treatment of a CAIX-expressing cancer comprising the bispecific adapter of any one of clauses 1 to 16 and a pharmaceutically acceptable carrier or excipient.

[0263] Article 19. (i) at least one dosage unit of the bispecific adapter of any one of clauses 1 to 17, or the pharmaceutical composition of clause 18, and (ii) a pharmaceutical composition comprising at least one dosage 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 comprising: Optionally, the kit, wherein (i) and (ii) are in separate containers.

[0264] Clause 20. The kit of clause 19, wherein the CAIX ligand of the bispecific adaptor is or comprises ortho-CAL or a derivative or analogue thereof.

[0265] Clause 21. The kit of clause 19, wherein the CAIX ligand of the bispecific adaptor is or comprises meta-CAL or a derivative or analogue thereof.

[0266] Clause 22. The kit of clause 19, wherein the bispecific adaptor CAIX is or comprises Aza or a derivative or analogue thereof.

[0267] Clause 23. A method of treating cancer in a subject, comprising administering to the subject a cancer-treating 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 bispecific adapter according to any one of clauses 1 to 17, or a pharmaceutical composition according to clause 18. administering The method thereby treating the subject for cancer.

[0268] Clause 24. The method of clause 23, wherein the CAR comprises a recognition region comprising a single-chain fragment variable (scFv) region of an anti-fluorescein antibody, a costimulatory domain that is CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS), and / or an activation signaling domain that is T cell CD3 zeta chain or Fc receptor gamma.

[0269] Clause 25. The method of clause 23, wherein the fluorescein of the bispecific adapter, upon exposure to the anti-fluorescein CAR-T cells, binds with affinity to the anti-fluorescein CAR-T cells, and wherein the CAIX ligand of the bispecific adapter ligates the bound anti-fluorescein CAR-T cells to such CAIX-expressing cancer cells upon affinity binding of the bispecific adapter to a receptor on such CAIX-expressing cancer cells.

[0270] Clause 26. The method of clause 23, wherein (i) and (ii) are administered by the same or different routes, simultaneously or sequentially in any order.

[0271] Clause 27. The method of any one of clauses 23 to 26, wherein (i) and (ii) are each administered intravenously.

[0272] Clause 28. The method of any one of clauses 23 to 27, further comprising imaging the cancer in the subject.

[0273] Clause 29. The method of clause 28, wherein imaging the cancer comprises imaging by optical imaging, positron emission tomography (PET), or single photon emission computed tomography (SPECT).

[0274] Clause 30. The method of any one of clauses 23 to 29, wherein the cancer is a CAIX-expressing cancer.

[0275] Clause 31. The method of any one of clauses 23 to 30, wherein the cancer is ovarian cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer, or clear cell renal cell carcinoma, for example optionally stage 3-4 clear cell renal cell carcinoma.

[0276] Clause 32. A method for enhancing chimeric antigen receptor (CAR)-T cell activation, comprising: (i) providing a bispecific adapter according to any one of clauses 1 to 17, or a pharmaceutical composition according to clause 18; (ii) 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 or pharmaceutical composition; Including, The method wherein, after exposure, the CAR-T cells undergo enhanced activation against the cancer cells compared to CAR-T cells that have not been exposed to the bispecific adapter.

[0277] Clause 33. The method of clause 32, wherein the anti-fluorescein CAR-T cells are in systemic circulation in the subject when exposed to the bispecific adaptor.

[0278] Clause 34. The method of Clause 32, wherein the cancer cells are CAIX-expressing cancer cells.

Claims

1. 1. A bispecific adapter, or a pharmaceutically acceptable salt or hydrate thereof, comprising fluorescein, fluorescein isothiocyanate (FITC), or N-hydroxysuccinimide (NHS)-fluorescein conjugated via a linker to a radical of a carbonic anhydrase IX (CAIX) ligand, wherein the linker comprises, consists essentially of, or consists of polyethylene glycol (PEG).

2. 2. The bispecific adapter of claim 1, wherein the CAIX ligand is or comprises 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL), 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (meta-CAL), acetazolamide (Aza), or a derivative or analog of any of the foregoing.

3. The linker is PEG １ ~PEG ９ 2. The bispecific adapter of claim 1, comprising, consisting essentially of, or consisting of:

4. The CAIX ligand is or comprises ortho-CAL or a derivative or analog thereof, and the linker is PEG １ ~PEG ９ 2. The bispecific adapter of claim 1, comprising, consisting essentially of, or consisting of:

5. The linker is PEG ３ ~PEG ９ 2. The bispecific adapter of claim 1, comprising, consisting essentially of, or consisting of:

6. The CAIX ligand is or comprises meta-CAL or a derivative or analog thereof, and the linker is PEG ３ ~PEG ９ 2. The bispecific adapter of claim 1, comprising, consisting essentially of, or consisting of:

7. 4. The bispecific adapter of claim 3, wherein the CAIX ligand is or comprises Aza or a derivative or analog thereof.

8. The linker is PEG ６ 8. The bispecific adapter of any one of claims 4, 6, and 7, comprising, consisting essentially of, or consisting of:

9. The linker is PEG ９ 8. The bispecific adapter of claim 6 or 7, comprising, consisting essentially of, or consisting of:

10. The linker is PEG ６ 8. The bispecific adapter of claim 7, comprising, consisting essentially of, or consisting of:

11. 2. The bispecific adapter of claim 1, wherein the linker comprises, consists essentially of, or consists of an alkyl.

12. The linker is (CH ２ ) ４ 2. The bispecific adapter of claim 1, comprising, consisting essentially of, or consisting of:

13. The following formula: 【Chemistry 1】 or a pharmaceutically acceptable salt or hydrate of any of the foregoing.

14. The following formula: 【Chemistry 2】 2. The bispecific adapter of claim 1, comprising the structure:

15. The following formula: 【Transformation 3】 or a pharmaceutically acceptable salt or hydrate of any of the foregoing.

16. 16. A bispecific adapter according to any one of claims 1 to 7 and 10 to 15 for use with anti-fluorescein chimeric antigen receptor (CAR)-T cells in the treatment of cancer.

17. 16. A bispecific adapter according to any one of claims 1 to 7 and 10 to 15 for use with anti-fluorescein CAR-T cells in the treatment of CAIX-expressing cancer.

18. 20. A pharmaceutical composition for the treatment of a CAIX-expressing cancer, comprising a bispecific adapter according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier or excipient.

19. (i) at least one dosage unit of the bispecific adapter of any one of claims 1 to 17 or the pharmaceutical composition of claim 18, and (ii) A pharmaceutical composition comprising at least one dosage 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 comprising: Optionally, the kit wherein (i) and (ii) are in separate containers.

20. 20. The kit of claim 19, wherein the CAIX ligand of the bispecific adapter is or comprises 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL) or a derivative or analogue thereof.

21. 20. The kit of claim 19, wherein the CAIX ligand of the bispecific adapter is or comprises 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (meta-CAL) or a derivative or analog thereof.

22. 20. The kit of claim 19, wherein the bispecific adapter CAIX is or comprises acetazolamide (Aza) or a derivative or analog thereof.

23. 1. A method of treating cancer in a subject, comprising administering to the subject a cancer-treating effective amount of: (i) a pharmaceutical composition comprising anti-fluorescein chimeric antigen receptor (CAR)-T cells or anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient; and (ii) a bispecific adapter according to any one of claims 1 to 17 or a pharmaceutical composition according to claim 18 administering whereby the subject is treated for cancer.

24. The CAR is a recognition region comprising a single-chain fragment variable (scFv) region of an anti-fluorescein antibody; 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 CD3 ζ chain or an Fc receptor γ 24. The method of claim 23, comprising:

25. 24. The method of claim 23, wherein the fluorescein of the bispecific adapter, upon exposure to the anti-fluorescein CAR-T cells, binds with affinity to the anti-fluorescein CAR-T cells, and the CAIX ligand of the bispecific adapter, upon affinity binding of the bispecific adapter to a receptor on a CAIX-expressing cancer cell, ligates the bound anti-fluorescein CAR-T cells to such CAIX-expressing cancer cells.

26. 24. The method of claim 23, wherein (i) and (ii) are administered by the same or different routes, simultaneously or sequentially in any order.

27. 27. The method of any one of claims 23 to 26, wherein (i) and (ii) are each administered intravenously.

28. 28. The method of any one of claims 23 to 27, further comprising imaging the cancer in the subject.

29. 29. The method of claim 28, wherein imaging the cancer comprises imaging by optical imaging, positron emission tomography (PET), or single photon emission computed tomography (SPECT).

30. 30. The method of any one of claims 23 to 29, wherein the cancer is a CAIX-expressing cancer.

31. 31. The method of any one of claims 23 to 30, wherein the cancer is ovarian cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer, or clear cell renal cell carcinoma, such as optionally stage 3-4 clear cell renal cell carcinoma.

32. 1. A method for enhancing chimeric antigen receptor (CAR)-T cell activation, comprising: Providing a bispecific adapter according to any one of claims 1 to 17 or a pharmaceutical composition according to claim 18; exposing anti-fluorescein CAR-T cells, or a pharmaceutical composition comprising anti-fluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient, to said bispecific adapter or said pharmaceutical composition; Including, wherein, after exposure, the CAR-T cells undergo enhanced activation against cancer cells compared to CAR-T cells that have not been exposed to the bispecific adapter.

33. 33. The method of claim 32, wherein the anti-fluorescein CAR-T cells are in systemic circulation in the subject when exposed to the bispecific adaptor.

34. 33. The method of claim 32, wherein the cancer cells are CAIX-expressing cancer cells.

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