ADAPTADORES BIESPECÍFICOS E SEU USO COM CÉLULAS CAR-T UNIVERSAIS NO TRATAMENTO DE TUMORES QUE EXPRESSAM CAIX

Abstract

Chimeric antigen receptor (CAR) T-cells, bispecific adapters that link the CAR-T cells to carbonic anhydrase IX (CAIX)-expressing tumor cells, and methods of treating CAIX-expressing cancer using same.

Description

1 / 94 “BI-SPECIFIC ADAPTERS AND ITS USE WITH UNIVERSAL CAR-T CELLS IN THE TREATMENT OF TUMORS THAT EXPRESS A BOX DESCRIPTIVE REPORT PRIORITY

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

[0002] This disclosure relates to chimeric antigen receptor (CAR) T cells, carbonic anhydrase-expressing (CAIX) tumors, bispecific adapters that link CAR T cells to CAIX-expressing tumor cells, and methods of cancer treatment using the same. BACKGROUND

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

[0004] Traditionally, a T cell expressing a single chimeric antigen receptor (CAR) has been used to target a cell surface receptor on a tumor cell. When the CAR on Petition 870250087052, dated 09 / 25 / 2025, page 5 / 121 2 / 94 T cells bind to a cell surface receptor on a tumor cell; the T cell can then kill the tumor cell to which it is bound. While effective, this approach can be quite costly, as CAR-T cells must be produced for different types of cancer that express different cell surface receptors.

[0005] Despite the clear need for cancer prevention and treatment, it remains a significant cause of death and suffering worldwide, as there are currently no effective therapeutic options that can cure the disease. Furthermore, when medications or other therapies are available, such treatments typically employ highly potent drugs that pose a risk of systemic toxicity to the underlying individual, as they are poorly selective for the cancer cells of interest.

[0006] What is needed are bispecific adapters that can facilitate the economical use of CAR-T cells that can bind to different types of cancer that express different cell surface receptors. This and other objects and advantages, as well as inventive features, will be evident from the detailed description provided herein. SUMMARY

[0007] A bispecific adapter is provided for use with cells. T anti-chimeric fluorescein antigen receptor (CAR) in the treatment of cancer expressing carbonic anhydrase IX (CAIX). The bispecific adapter may comprise the following structure: F — L — CAIX, or a pharmaceutically acceptable salt or hydrate thereof, wherein F comprises a fluorescein, L comprises a ligand, and CAIX comprises a CAIX ligand (for example, a radical of a CAIX ligand). The fluorescein may comprise fluorescein, isothiocyanate of Petition 870250087052, dated 09 / 25 / 2025, page 6 / 121 3 / 94 fluorescein (FITC) or N-hydroxysuccinimide (NHS)-fluorescein. The CAIX ligand may be or comprise a 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL) radical. The ligand may comprise, consist essentially of, or consist of polyethylene glycol (PEG).

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

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

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

[0011] The binder may comprise, consist essentially of, or consist of PEG1 to PEG9. The CAIX binder may be or comprise ortho-CAL or a derivative or analogue thereof, and the binder may comprise, consist essentially of, or consist of PEG1 to PEG9. In certain embodiments, the binder comprises, consists essentially of, or consists of PEG3 to PEG9.

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

[0013] The CAIX binder may be or comprise Aza or a derivative or analogue thereof. In certain embodiments, the binder comprises, consists essentially of, or consists of PEG6. In certain embodiments, the Petition 870250087052, dated 09 / 25 / 2025, page 7 / 121 4 / 94 binder comprises, consists essentially of, or consists of PEG9. In certain embodiments, the binder comprises, consists essentially of, or consists of PEG6.

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

[0015] In certain embodiments, the bispecific adapter comprises a structure of one of the following formulas: HO is either a pharmaceutically acceptable salt or hydrate of either of the above.

[0016] The bispecific adapter may comprise a structure with the following formulas: Petition 870250087052, dated 09 / 25 / 2025, page 8 / 121 5 / 94 OH or a pharmaceutically acceptable salt or carbohydrate thereof.

[0017] The bispecific adapter may comprise a structure of the following formulas: nh2 Aza-PEG3-FITC SR-402 Chemical Formula: C40H47N7O-12S3 compound code Chemical Formula: SR-380 C46H59N7O15S3 1046.19 N 'W H H Molecular weight: or eD'92 Chemical Formula: C52H71N7O18S3 Molecular Weight: 1178.35 O. 's. Petition 870250087052, dated 09 / 25 / 2025, p. 9 / 121 6 / 94 or be a pharmaceutically acceptable salt or carbohydrate of any of the above.

[0018] Bispecific adapters can be used with an anti-fluorescein chimeric antigen receptor (CAR) CAR T cell in cancer treatment. Bispecific adapters can be used with an anti-

[0019] CAR T cell. In certain embodiments, the bispecific adapter is for use with an anti-fluorescein CAR T cell (e.g., fluorescein, FITC, or NHS-fluorescein) in the treatment of CAIX-expressing cancer. The bispecific adapter may comprise a fluorescein-ligand CAIX ligand (e.g., fluorescein = fluorescein, FITC or NHS-fluorescein) or a pharmaceutically acceptable salt or hydrate thereof, wherein the CAIX ligand is or comprises 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (metaCAL) and the ligand comprises (or consists essentially of) PEG. The ligand may comprise (or consist essentially of) PEG3 to PEG9. The ligand may comprise (or consist essentially of) PEG6.The binder may comprise (or consist essentially of) PEG9. The bispecific adapter may have the following structure: OH H2N° or be a pharmaceutically acceptable salt or hydrate thereof.

[0020] A further bispecific adapter is provided for use with Petition 870250087052, dated 09 / 25 / 2025, page 10 / 121 7 / 94 a CAR-T cell antifluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) in the treatment of CAIX-expressing cancer. The adapter may comprise a fluorescein-CAIX ligand (e.g., fluorescein = fluorescein, FITC, or NHS-fluorescein) or a pharmaceutically acceptable salt or hydrate thereof, wherein the CAIX ligand is acetazolamide (Aza) and the ligand comprises (or consists essentially of) PEG. The ligand may comprise (or consist essentially of) PEG1 to PEG9. The ligand may comprise (or consist of)

[0021] A pharmaceutical composition for the treatment of cancer expressed as CAIX is also provided, comprising any bispecific adaptor described herein and a pharmaceutically acceptable carrier or excipient.

[0022] In addition, a method is provided for treating a cancer (e.g., a cancer that expresses CAIX) in an individual. The method may comprise administering to the individual cancer-treatment-effective amounts of (i) antifluorescein CAR-T cells (e.g., fluorescein, FITC, or NHS-fluorescein) or a pharmaceutical composition comprising antifluorescein 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, whereupon the individual is treated for cancer. The CAR may have a recognition region, and the recognition region may be a single-strand fragment variable (scFv) region of an antifluorescein antibody. The CAR may have a co-stimulation domain.The co-stimulatory domain of the CAR may be CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS). The CAR may have an activation signaling domain, such as, for example, a CD3α chain of T cells or an Fcγ receptor.

[0023] The fluorescein from the bispecific adapter of the method can bind to the antifluorescein CAR-T cell with affinity after exposure to it, Petition 870250087052, dated 09 / 25 / 2025, page 11 / 121 8 / 94 and the bispecific adapter CAIX ligand can bind the antifluorescein-linked CAR-T cell to a CAIX-expressing cancer cell after the bispecific adapter binds to a receptor on that CAIX-expressing cancer cell with affinity.

[0024] In the method, steps (i) and (ii) can be administered simultaneously or sequentially, in any order, by the same route or by different routes. In the method, steps (i) and (ii) can each be administered intravenously.

[0025] In certain modalities, the method may also include obtaining images of the cancer in the individual. Obtaining images of the cancer may include optical imaging, positron emission tomography (PET), or single-photon emission computed tomography (SPECT).

[0026] The cancer may be ovarian cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer, or clear cell renal cell carcinoma, as optionally stage 3-4 clear cell renal cell carcinoma. The cancer may be a CAIX-expressing cancer.

[0027] Methods for increasing CAR-T cell activation are also provided. In certain embodiments, a method for increasing CAR-T cell activation comprises: providing a bispecific adapter described herein or a pharmaceutical composition described herein; and exposing CAR-T cells to antifluorescein or a pharmaceutical composition comprising CAR-T cells and a pharmaceutically acceptable carrier or excipient to the bispecific adapter or pharmaceutical composition; wherein the CAR-T cell undergoes increased activation against cancer cells after exposure, compared with a CAR-T cell not exposed to the bispecific adapter.

[0028] CAR-T cells can be in an individual's systemic circulation when exposed to the bispecific adapter. CAR-T cells can be exposed to the bispecific adapter in vitro (e.g., before Petition 870250087052, dated 09 / 25 / 2025, page 12 / 121 9 / 94 of administration to an individual). CAR-T cells may be cancerous cells that express CAIX.

[0029] A kit is also provided. The kit may comprise (i) at least one dosage unit of 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 antifluorescein CAR-T cells or a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier or excipient, wherein (i) and (ii) are optionally in separate containers. The CAIX binder of the bispecific adapter may be or comprise ortho-CAL or a derivative or analogue thereof. The CAIX binder of the bispecific adapter may be or comprise meta-CAL or a derivative or analogue thereof. The CAIX binder of the bispecific adapter may be or comprise Aza or a derivative or analogue thereof. DESCRIPTION OF THE DRAWINGS

[0030] The above objectives, features and advantages of the present invention and others will become more apparent when considered in conjunction with the following description and drawings, in which:

[0031] FIG. 1 shows the chemical structures of bispecific adapters directed to carbonic anhydrase IX (CAIX).

[0032] FIG. 2A is a graph of the concentration (nM) of the indicated bispecific adapters in relation to the mean fluorescence intensity (MFI).

[0033] FIG. 2B is a graph of the concentration (nM) of the indicated bispecific adaptors in relation to the MFI.

[0034] FIG. 3A is a graph of the bispecific adapter in relation to the MFI of fluorescein isothiocyanate (FITC), which shows the total binding of CAIX bispecific adapters to HT29 cells.

[0035] FIG. 3B is a graph of the bispecific adapter vs. MFI of Petition 870250087052, dated 09 / 25 / 2025, page 13 / 121 10 / 94 APC-anti-FITC, which shows the surface exposure of FITC fractions in CAIX bispecific adapters after connection to HT29 cells.

[0036] FIG. 3C is a graph of the bispecific adapter vs. MFI of FITC, which shows the complete connection of the CAIX bispecific adapters to the MDA-CAIX cells.

[0037] FIG. 3D is a graph of the bispecific adapter vs. MFI of APC-anti-FITC, which shows the surface exposure of FITC fractions in bispecific CAIX adapters after connection to MDA-CAIX cells.

[0038] FIG. 4A is a graph of concentration (nM) vs. lysis (%), which shows the cytotoxicity of anti-FITC CAR-T cells mediated by the bispecific adapter directed to CAIX for MDA-CAIX cells.

[0039] FIG. 4B is a graph of concentration (nM) vs. IFNγ (pg / ml), which shows the release of IFNγ mediated by a bispecific adapter from anti-FITC CAR-T cells.

[0040] FIG. 5A is a graph of concentration (nM) vs. lysis (%), which shows the cytotoxicity of anti-FITC CAR-T cells mediated by bispecific meta-CAL-FITC adaptors to MDA-CAIX cells.

[0041] FIG. 5B is a graph of concentration (nM) vs. IFNy (pg / ml), which shows higher levels of IFNy release mediated by meta-CAL-PEG6-FITC and meta-CAL-PEG9-FITC from antiFITC CAR-T cells than by meta-CAL-PEG3-FITC at low concentrations (between 0.001 nM and 1 nM).

[0042] FIG. 6A is a graph of concentration (nM) vs. lysis (%), which shows the cytotoxicity of anti-FITC CAR-T cells mediated by ortho-CAL-FITC adapters with different ligands to MDACAIX cells.

[0043] FIG. 6B is a graph of concentration (nM) vs. IFNγ (pg / ml), which shows the release of IFNγ mediated by orthoCAL-FITC adapters from anti-FITC CAR-T cells.

[0044] FIG. 7A shows the timeline and dosing scheme of an in vivo study to test different bispecific CAIX adaptors.

[0045] FIG. 7B is a graph of days after CAR-T injection vs. Petition 870250087052, dated 09 / 25 / 2025, page 14 / 121 11 / 94 tumor volume (mm3), which shows the tumor growth curves of different treatment groups.

[0046] FIG. 7C is a graph of days after CAR-T injection versus change in body weight (%), showing the changes in body weight of mice in different treatment groups throughout the treatment.

[0047] FIG. 8A is a schematic diagram of an experimental design.

[0048] FIG. 8B is a graph of the bispecific adapter versus FITC MFI, showing the total FITC signal in HT29 tumor cells from mice injected with different bispecific adapters.

[0049] FIG. 8C is a graph of the bispecific adapter versus MFI of the APC-anti-FITC, showing the surface exposure of the FITC I fraction in nHT29 tumors from mice injected with CAIX bispecific adapters.

[0050] FIG. 9A is a graph of ligand versus T cell / μL of blood, showing the T cell counts in the blood.

[0051] FIG. 9B is a graph of ligand vs. CD3+ T cells / live cells (%), which shows T cells infiltrated in the tumor.

[0052] FIG. 10A shows the timetable and dosing scheme of an in vivo study to test different bispecific CAIX adaptors.

[0053] FIG. 10B is a graph of days after CAR-T injection vs. Tumor volume (mm3), which shows the tumor growth curves of different treatment groups.

[0054] FIG. 10C is a graph of days after CAR-T injection vs. Change in body weight (%), which shows the changes in body weight of mice in different groups during treatments.

[0055] FIG. 11A is a schematic diagram of the experimental design.

[0056] FIG. 11B is a graph of bispecific adapter vs. MFI of FITC, showing the total FITC signal in MDA tumor cells. Petition 870250087052, dated 09 / 25 / 2025, page 15 / 121 12 / 94 CAIX of mice injected with different bispecific adapters.

[0057] FIG. 11C is a graph of the bispecific adapter vs. MFI of 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 would saturate all surface CAIX proteins and reflect the CAIX protein level in tumor cells. The results indicate that orthoCAL-PEG6-FITC mediated the complete eradication of CAIX+ tumor cells by CAR T cells.

[0058] FIG. 11D is a graph of the bispecific adapter vs. MFI of APC-anti-FITC, which shows the surface exposure of the FITC fraction in MDA-CAIX tumors from mice treated with bispecific CAIX adapters.

[0059] FIG. 11E is a graph of the bispecific adapter vs. MFI of anti-FITC APCs 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 would saturate all surface CAIX proteins and reflect the CAIX protein level in tumor cells.

[0060] FIG. 12 is a graph of concentration (nM) vs. normalized MFI, which shows that PEG spacers have no significant effects on the binding affinity of acetazolamide (Aza)-FITC bispecific adapters.

[0061] FIG. 13A is a graph of bispecific adapter vs. FL MFI, showing the total bonding of AzaFITC bispecific adapters to MDA-CAIX cells. The length of the linker did not significantly affect the total bonding.

[0062] FIG. 13B is a graph of the bispecific adapter vs. MFI of APC-anti-FITC, showing the surface exposure of FITC fractions in Aza-FITC bispecific adapters with different PEG binders after bonding to MDA-CAIX cells. Increasing the spacer length Petition 870250087052, dated 09 / 25 / 2025, page 16 / 121 13 / 94 PEG increased the surface exposure of FITC after binding to MDA-CAIX cells.

[0063] FIG. 14 is a graph of concentration (nM) vs. IFNγ (pg / ml), showing the effect of ligand length on IFNγ release from anti-FITC CAR-T cells mediated by the bispecific adapter Aza-FITC when co-cultured with HT 29 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.

[0064] FIG. 15 is a graph of the concentration of the CA9PEG(n)-FITC ligand (nM) vs. IFNγ (pg / ml), showing bispecific adapters targeting CAIX with IFNγ-mediated release by anti-FITC CAR-T cells by optimal ligands. MetaCAL-PEG9-FITC mediated the highest level of IFNγ released by anti-FITC CAR-T cells at low concentrations (< 0.1 nM). Aza-PEG6-FITC mediated similar levels of IFNγ release by anti-FITC CAR-T cells to metaCAL-PEG9-FITC at concentrations above 0.1 nM. OrthoCAL-PEG6-FITC mediated the lowest levels of IFNγ released by anti-FITC CAR-T cells.

[0065] FIG. Figure 16A shows the timeline and dosing scheme of an in vivo study to test Aza-PEG6-FITC and orthoCAL-PEG6-FITC.

[0066] FIG. 16B is a graph of days after cell injection. CAR-T versus tumor volume (mm3), which shows the tumor growth curves of different treatment groups. Aza-PEG6-FITC and orthoCAL-PEG6-FITC slightly inhibited the growth of KB tumors. The efficacy of Aza-PEG6-FITC is slightly superior to that of orthoCAL-PEG6-FITC.

[0067] FIG. 16C is a graph of days after cell injection. CAR-T versus change in body weight (%), which shows the changes in body weight of mice in different treatment groups. Neither Aza-PEG6-FITC nor orthoCAL-PEG6-FITC induced significant body weight loss. Petition 870250087052, dated 09 / 25 / 2025, page 17 / 121 14 / 94 DETAILED DESCRIPTION

[0068] This disclosure is based, at least in part, on the design of bispecific adapters with diverse antigen-binding affinities and spacer lengths to optimize the efficacy of universal chimeric antigen receptor (CAR) T cells in the treatment of carbonic anhydrase IX (CAIX)-expressing tumors. Two novel high-affinity CAIX ligands, namely 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (metaCAL) and 3-((3-(cyclo-octylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL), were tested with three or four spacer ligands, respectively, in bispecific adapters. Studies have shown that PEG6 and PEG9 spacers (PEG = polyethylene glycol) can be excellent for targeting CAIX-positive tumors in vivo and in vitro.When used in bispecific adapters, high-affinity CAIX ligands were more potent than low-affinity adapters, such as 5-acetamido-1,3,4-thiadiazol-2-sulfonamide (Aza).

[0069] In view of the foregoing, a bispecific adapter or a pharmaceutically acceptable salt or hydrate thereof is provided for use with antifluorescein CAR-T cells (e.g., fluorescein, FITC or N-hydroxysuccinimide (NHS)-fluorescein) in the treatment of CAIX-expressing cancer.

[0070] In certain embodiments, the bispecific adapter comprises the following structure: F — L — CAIX, or is a pharmaceutically acceptable salt or hydrate thereof, wherein: F comprises a CAR-T cell targeting fraction, such as fluorescein, FITC or NHS-fluorescein. Petition 870250087052, dated 09 / 25 / 2025, page 18 / 121 15 / 94 L comprises a ligand, and CAIX comprises a radical of a CAIX ligand.

[0071] In certain embodiments, the CAIX ligand is or comprises 3-((3-(cyclo-octylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL) and the ligand comprises (or consists essentially of) PEG.

[0072] The use of bispecific adapters may allow the use of a single CAR-T cell, i.e., a “universal” CAR-T cell, which has, for example, a molecule on its surface that binds to fluorescein. When the universal CAR-T cell, such as one that has a molecule on its surface that binds to fluorescein, is used in conjunction with a bispecific adapter, such as one comprising FITC connected (e.g., by a linker and / or spacer) to a molecule that binds to a cell surface receptor of tumor cells, the T cell can kill the tumor cells to which it is bound.

[0073] This approach can reduce the cost of cell production. CAR-T cells can bind to different types of cancer that express different cell surface receptors. Conversely, the universal CAR-T cell can bind to different types of cancer by altering the part of the bispecific adapter that binds to a cell surface receptor on a tumor cell. Consequently, bispecific adapters can enhance the immune response caused by CAR-T cells bound to tumor cells.

[0074] CAR T-Cell Targeting Fragment

[0075] The bispecific adapter CAR T cell targeting fragment can be fluorescein, FITC, NHS-fluorescein, or any other fragment that a CAR can be designed to recognize and bind to specifically.

[0076] “Binds specifically”, “binds with high affinity” or “specifically” or “selectively” binds, when referring to a Petition 870250087052, dated 09 / 25 / 2025, page 19 / 121 16 / 94 ligand / receptor, a recognition region / targeting fragment, a nucleic acid / complementary nucleic acid, an antibody / antigen, or other binding pair, indicating a binding reaction that determines the presence of the protein in a heterogeneous population of proteins and other biological compounds. Thus, under designated conditions, a specified ligand or recognition region binds to a specific receptor (e.g., one present on a cancer cell or CAR T cell) or to the targeting fragment, respectively, and does not bind in significant quantity to other proteins present in the sample (e.g., those associated with normal, healthy cells).Specific binding or high-affinity binding can also mean, for example, that the binding compound, ligand, antibody, or binding composition derived from the antigen-binding site of an antibody binds to its target with an affinity that is often at least 25% higher, more often at least 50% higher, most often at least 100% (twice) higher, typically at least ten times higher, more typically at least 20 times higher, and most typically at least 100 times higher than the affinity for any other binding compound. In a typical embodiment, a molecule that binds specifically to a target will have an affinity of at least about 10⁶ L / mol (KD = 10⁻⁶ M) and preferably at least about 10 L / mol, as determined, for example, by Scatchard analysis.

[0077] Target Ligands

[0078] As noted above, bispecific adapters may 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, a radical thereof) do not bind to uptake receptors on non-target cells. Petition 870250087052, dated 09 / 25 / 2025, page 20 / 121 17 / 94

[0079] CAIX is a small molecule ligand that binds specifically to a receptor that is overexpressed in certain types of cancer cells (i.e., the receptor for each of these ligands is overexpressed in cancers compared to the expression of that receptor in normal tissues or, potentially, in diseased tissues that do not have the target cancer type). Receptors for the CAIX ligand are found, for example, in renal, ovarian, vulvar, and breast cancers, and in colon and pancreatic cancers. Its expression may also be associated with renal cell carcinoma, lung cancer, and others. Consequently, increased CAIX expression may be a useful target for therapy.

[0080] Bispecific adapters may comprise a CAIX ligand (or a radical thereof) linked to a ligand, wherein the ligand is additionally linked to a CAR targeting moiety. In certain embodiments, the CAIX ligand is a high-affinity CAIX ligand. Unless otherwise specified, high affinity or greater affinity relative to the affinity of a ligand for a target means a ligand possessing a Schrodinger molecular docking index of at least about -8.0 kcal / mol. In certain embodiments, the high-affinity CAIX ligand possesses an improved affinity for the CAIX receptor compared to other ligands.

[0081] The targeting fraction may be, for example, a CAIX ligand radical with a molecular weight 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; from about 500 to about 10,000 g / mol, from about 1,000 to about 7,500 g / mol, from about 750 g / mol to about 1,500 g / mol, from about 1,000 to about 5,000 g / mol or from about 500 to about 2,500 g / mol.

[0082] The targeting ligand can bind to an activated tumor or other cancer cell that is overexpressing the CAIX receptor. In certain embodiments, the targeting ligand may have a binding affinity to a CAIX receptor in the range of about 1 Petition 870250087052, dated 09 / 25 / 2025, p. 21 / 121 18 / 94 nM to about 25 nM, such as 1 nM to about 25 nM or about 1 nM to 25 nM. In certain embodiments, the targeting ligand may have a binding affinity to a CAIX receiver in the range of about 0.002 nM to about 25 nM, such as 0.002 nM to about 1 nM or about 0.002 nM to 1 nM. In certain embodiments, the targeting ligand may have a binding affinity to a CAIX receiver in the range of about 0.01 nM to about 0.9 nM, such as 0.01 nM to about 0.9 nM or about 0.01 nM to 0.9 nM. In certain embodiments, the targeting ligand may have a binding affinity to a CAIX receptor in the range of about 0.02 nM to about 0.8 nM, such as 0.02 nM to about 0.8 nM or about 0.02 nM to 0.8 nM. In certain embodiments, the targeting ligand may have a binding affinity to a CAIX receptor in the range of about 0.03 nM to about 0.7 nM, such as 0.03 nM to about 0.7 nM or about 0.03 nM to 0.7 nM.In certain embodiments, the targeting ligand may have a binding affinity to a CAIX receptor in the range of about 0.04 nM to about 0.6 nM, such as 0.04 nM to about 0.6 nM or about 0.04 nM to 0.6 nM. In certain embodiments, the targeting ligand may have a binding affinity to a CAIX receptor in the range of about 0.05 nM to about 0.5 nM, such as 0.05 nM to about 0.5 nM or about 0.05 nM to 0.5 nM. In certain embodiments, the targeting ligand may have a binding affinity to a CAIX receptor in the range of about 0.06 nM to about 0.4 nM, such as 0.06 nM to about 0.4 nM or about 0.06 nM to 0.4 nM. In certain embodiments, the targeting ligand may have a binding affinity to a CAIX receptor in the range of about 0.07 nM to about 0.3 nM, such as 0.07 nM to about 0.3 nM or about 0.07 nM to 0.3 nM.In certain embodiments, the targeting ligand may have a binding affinity to a CAIX receptor in the range of about 0.08 nM to about 0.2 nM, such as 0.08 nM to about 0.2 nM or about 0.08 nM to 0.2 nM. In certain embodiments, the targeting ligand may have a binding affinity to a CAIX receptor in the range of about 0.09 nM to about 0.1 nM, such as 0.09 nM to about 0.1 nM or about 0.09 nM to 0.1 nM. Petition 870250087052, dated 09 / 25 / 2025, p. 22 / 121 19 / 94 The ranges established in this section include the indicated endpoints and all 0.001 nM increments covered by them.

[0083] The CAIX ligand may be or may comprise 3-((3-(cyclo-octylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (orthoCAL) or derivatives or analogs thereof. The CAIX ligand may be or may comprise 3-((2-(cyclo-octylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (meta-CAL) or derivatives or analogs thereof. The CAIX ligand may be or may comprise acetazolamide (Aza) or derivatives or analogs 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 radical thereof may target a CAIX-expressing cancer in an individual.

[0084] The bispecific adapter can be designed and synthesized specifically to achieve a particular bonding affinity for CAIX.

[0085] Binders

[0086] The ligands of the bispecific adapters described herein are arranged between the targeting ligand (e.g., a radical thereof) and the targeting fraction of the CAR T cell (e.g., comprising fluorescein, FITC, or NHS-fluorescein). The ligand may be any suitable ligand.

[0087] The term “ligand” includes a chain of atoms that is biofunctionally adapted 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 may include carbon, nitrogen, oxygen, sulfur, silicon (Si), and phosphorus (P), such as C, N, O, S, and P, or C, N, O, and S.

[0088] The linker may comprise a wide variety of linkages, such as in the range of about 2 to about 100 atoms in the contiguous back chain. The linker may comprise a releasable form of PEG, a non-releasable form of PEG, polyproline, an amino acid Petition 870250087052, dated 09 / 25 / 2025, page 23 / 121 20 / 94 hydrophilic, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone or a triblock copolymer comprising a central hydrophobic block of polypropylene glycol flanked on each side by a hydrophilic block of PEG.

[0089] The ligand may comprise PEG or a PEG derivative. The ligand may be (PEG)3.

[0090] The ligand may be non-releasable, i.e., non-labile. However, in some embodiments, it may be desirable that a ligand in a bispecific adapter be releasable, i.e., labile, such as, for example, photocleavable, acid-labile, base-labile, or enzyme-cleavable. The term “releasable,” in the context of a ligand, means a ligand that includes at least one bond that can be readily broken (e.g., chemically or enzymatically hydrolyzed) under physiological conditions, such as, for example, a multivalent releasable bond based on a reducing acid, pH-labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile, enzyme-labile, or p-aminobenzyl-based.It is recognized that the physiological conditions resulting in the breaking of the bond do not necessarily include a biological or metabolic process, but may instead include a standard chemical reaction, such as a hydrolysis reaction, for example, at physiological pH, or as a result of compartmentalization in a cellular organelle, such as an endosome with a pH lower than cytosolic pH. A cleavable bond may connect two adjacent atoms within the releasable ligand and / or connect other parts of the ligand or the targeting fraction and / or the targeting fraction of the CAR T cell, as described herein, for example, at one or both ends of the releasable ligand. In some cases, the releasable ligand is broken into two or more fragments. In some cases, the releasable ligand is separated from the targeting fraction of the CAR T cell.

[0091] In some embodiments, the ligand is formed in such a way that the CAR T cell-directed ligand (i.e., fluorescein) is cleaved from the cancer-directed fraction (i.e., the CAIX ligand) only. Petition 870250087052, dated 09 / 25 / 2025, page 24 / 121 21 / 94 after sufficient time for the bispecific adapter to circulate in an individual's systemic circulation following administration (e.g., to allow time for it to be captured and internalized by the target cell and / or receptor). In some embodiments, the release period will vary (e.g., from individual to individual (e.g., based on a variety of factors)). In some embodiments, a releasable ligand may be designed such that it is not cleaved / released until at least 24 hours after administration or even over a period of one week. In some embodiments, the bispecific adapter may safely pass through the individual's system, and any amount not captured by target cells (e.g., those expressing CAIX) may be excreted.

[0092] In contrast, the term “non-releasable” in the context of a ligand means a ligand that includes at least one bond that is not easily or rapidly broken under physiological conditions. In some embodiments, a non-releasable ligand comprises a structure that is stable under physiological conditions (e.g., the structure is not susceptible to hydrolysis (e.g., aqueous hydrolysis or enzymatic hydrolysis)). In some embodiments, a bispecific adapter comprising a non-releasable ligand does not release any component of the bispecific adapter (e.g., a cancer-targeted ligand or a CAR T-cell-targeted ligand). In some embodiments, the non-releasable ligand does not possess a disulfide bond (e.g., SS) or an ester in the structure.In some embodiments, bispecific adapters comprise a cancer-targeted ligand or a CAR-T cell-targeted ligand connected by a substantially stable structure throughout the duration of the bispecific adapter's circulation (e.g., during endocytosis in the target cell endosome). The non-releasable ligand may comprise: an amide, an ester, an ether, an amine, and / or a thioether (e.g., thiomaleimide). Although specific examples are provided, it should be understood that any molecule may be used in the non-releasable ligand. Petition 870250087052, dated 09 / 25 / 2025, p. 25 / 121 22 / 94 provided that at least one bond is formed that is not easily or quickly broken under physiological conditions.

[0093] Perhaps more specifically, a non-releasable ligand may comprise a ligand that, at neutral pH, for example, 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%) will be hydrolyzed into an aqueous solution (e.g., buffered (e.g., phosphate buffer)) within a period of time (e.g., 24 hours). In some embodiments, where a non-releasable ligand is employed, less than about ten percent (10%), and preferably less than five percent (5%) or none, of the administered bispecific adapter releases a fraction to which it is bound (e.g., into the systemic circulation prior to uptake by target cells / tissues).

[0094] In some embodiments, a cancer-targeted ligand does not separate from the CAR-T cell-targeted ligand of the bispecific adapter in vivo. This can be advantageous as it allows the bispecific adapter to bind to and deliver a CAR-T cell to a target cancer cell.

[0095] The length of a ligand can be selected to optimize the separation of molecules imposed by the ligand on the target cell surface, which, in turn, can facilitate the uptake of a CAR-bound T cell into the target cell (e.g., when the bispecific adapter is delivered). The ligand can have a chain length of at least about 5 nm. In certain embodiments, each ligand is approximately 5 nm to 15 nm long. In some embodiments, the ligand is at least about 7 nm long. In certain embodiments, each ligand is approximately 7 nm long and is flexible. In certain embodiments, each ligand is approximately 7-10 nm long. In some embodiments, the ligand is at least about 14 nm long. In some embodiments, the ligand is about 15 nm long. In some Petition 870250087052, dated 09 / 25 / 2025, p. 26 / 121 23 / 94 modalities, the ligand is between about 7 nm and about 31 nm in length (for example, about 7 to 31, 7 to about 31 or 7 to 31), between about 7 nm and about 24 nm in length (for example, about 7 to 24, 7 to about 24 or 7 to 24) or between about 7 nm and about 20 nm in length (for example, about 7 to 20, 7 to about 20 or 7 to 20). In some embodiments, the ligand is between about 14 nm and about 31 nm in length (for example, about 14 to 31, 14 to about 31 or 14 to 31), between about 14 nm and about 24 nm in length (for example, about 14 to 24, 14 to about 24 or 14 to 24) or between about 14 nm and about 20 nm in length (for example, about 14 to 20, 14 to about 20 or 14 to 20).In some embodiments, the ligand has 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 from 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 ligand is selected to facilitate the micro-clustering of linked molecules on a cell surface, providing a separation of approximately 7 to 10 nm between them (such as about 7 nm to about 10 nm, 7 nm to about 10 nm, about 7 nm to 10 nm, or 7 nm to 10 nm). The ranges specified in this paragraph include the indicated endpoints and all 1 nm increments covered by the indicated ranges.

[0096] A 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 configurations, natural or non-natural amino acids, or a combination of any of the above.

[0097] In certain embodiments, a ligand is a group comprising one or more covalently connected structural units.

[0098] The ligand can be designed to optimize biodistribution, bioavailability and PK / PD (e.g., of the bispecific adapter) Petition 870250087052, dated 09 / 25 / 2025, page 27 / 121 24 / 94 and / or to increase uptake (e.g., of a CAR-T cell connected to it and / or of the bispecific adapter itself), as previously described in the target tissue, according to methodologies commonly known in the art or developed subsequently, such as through PEGlaytion and similar methods.

[0099] In some embodiments, the ligands may comprise one or more spacers (e.g., to facilitate a specific release time, facilitate increased uptake in a target tissue, and / or optimize the biodistribution, bioavailability, and / or PK / PD of a bispecific adapter provided herein). A spacer may comprise one or more alkyl chains, PEGs, peptides, sugars, peptidoglycans, clickable ligands (e.g., triazoles), rigid ligands such as polyprolines and polypiperidines, and the like.

[0100] In some embodiments, a bispecific adapter binder comprises PEG, a PEG derivative, or any other binder known in the art or subsequently developed that may achieve the purpose set forth herein. In some embodiments, the binder is repeated n times, where n is a positive integer. For example, and without limitation, n may be any integer selected from a range of 1 to 16, 1 to 32, 1 to 64, or 1 to 96. The number of repetitions in the binder (i.e., n) may be selected to achieve the desired functionality, size, and / or power of the conjugate and / or with a view to the desired application. In some embodiments, the binder comprises one or more spacers (e.g., which may also be used to specifically design bispecific adapter features).

[0101] In certain embodiments, the linker comprises, consists of, or consists essentially of PEG1 - PEG9. In certain embodiments, the linker comprises, consists of, or consists essentially of PEG2 PEG8. In certain embodiments, the linker comprises, consists of, or consists essentially of PEG3 - PEG9. In certain embodiments, the linker comprises, consists of, or consists essentially of PEG4 PEG8. In certain embodiments, the linker comprises, consists of, or Petition 870250087052, dated 09 / 25 / 2025, page 28 / 121 25 / 94 essentially consists of PEG5-PEG7. In certain embodiments, the binder comprises, consists of, or essentially consists of PEG3-PEG18. In certain embodiments, the binder comprises, consists of, or essentially consists of PEG4-PEG17. In certain embodiments, the binder comprises, consists of, or essentially consists of PEG5-PEG16. In certain embodiments, the binder comprises, consists of, or essentially consists of PEG6-PEG15. In certain embodiments, the binder comprises, consists of, or essentially consists of PEG7-PEG14. In certain embodiments, the binder comprises, consists of, or essentially consists of PEG8-PEG13. In certain embodiments, the binder comprises, consists of, or essentially consists of PEG9-PEG12. In certain embodiments, the binder comprises, consists of, or essentially consists of PEG10-PEG11. The binder may comprise (or consist essentially of) PEG3 to PEG9, such as PEG3, PEG4, PEG5, PEG6, PEG7, PEG8 or PEG9.The ligand may comprise (or consist essentially of) PEG6. The ligand may comprise an alkyl group. The ligand may be, comprise, or consist essentially of (CH2)4. All ranges indicated in this paragraph include the indicated endpoints.

[0102] The ligand may be or comprise (or consist essentially of) PEG1. The ligand may be or comprise (or consist essentially of) PEG6. In certain embodiments, the ligand comprises, consists of, or consists essentially of PEG3. In certain embodiments, the ligand comprises, consists of, or consists essentially of PEG4. In certain embodiments, the ligand comprises, consists of, or consists essentially of PEG12. In certain embodiments, the ligand comprises, consists of, or consists essentially of PEG16.

[0103] In some embodiments, the binder is a hydrolyzable binder. In some embodiments, the ligand is a non-hydrolyzable ligand. In some embodiments, the ligand is an optionally substituted heteroalkyl. In some embodiments, the ligand is a heteroalkyl. Petition 870250087052, dated 09 / 25 / 2025, p. 29 / 121 26 / 94 substituted comprising at least one substituent selected from the group consisting of alkyl, hydroxyl, oxo, PEG, carboxylate and halo. In some embodiments, the ligand comprises a spacer (for example, as described elsewhere in this document).

[0104] In some embodiments, the ligand is a substituted heteroalkyl with at least one disulfide bond in its main structure. In some embodiments, the ligand is a peptide with at least one disulfide bond in its main structure.

[0105] In some embodiments, the ligand comprises -CONHCH(COOH)-CH2-SS-CH2-CRaRb-O-CO-, -CONH-CH(COOH)CRaRb-OCO-, -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)CRaRb-O-CO- or C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)-CH2-SS-CH2-CRaRb-OCO-, wherein Ra and Rb are independently H, alkyl or heteroalkyl (e.g., PEG).

[0106] In some forms, the linker comprises a structure of: or where n or m (when applicable) is from 0 to 10.

[0107] In some forms, the linker comprises a Petition 870250087052, dated 09 / 25 / 2025, page 30 / 121 27 / 94 structure of: or where neither are they each independently rated from 0 to 10.

[0108] In some forms, the linker comprises a structure of: where n is from 1 to 32. In at least one exemplary embodiment, n is from 1 to 30 and w is from 0 to 5 (when applicable).

[0109] In some forms, the linker comprises the structure of: Petition 870250087052, dated 09 / 25 / 2025, p. 31 / 121 28 / 94 where n is from 1 to 16.

[0110] In certain forms, the ligand may comprise the structure of: where n is from 1 to 30 and w is from 0 to 5.

[0111] Bispecific Adapters

[0112] The bispecific adapter, or its pharmaceutically acceptable salt or hydrate, can be used with an antifluorescein CAR-T cell in the treatment of a CAIX-expressing cancer and comprises a fluorescein-ligand CAIX ligand. The bispecific adapter may have a structure with the formulas shown in Figure 1.

[0113] The bispecific adapter may have the structure with the following formulas: Petition 870250087052, dated 09 / 25 / 2025, p. 32 / 121 29 / 94 ortho-CAL-(CH2)4-FITC Or it may be a pharmaceutically acceptable salt or hydrate of any of the preceding structures.

[0114] The bispecific adapter may have the structure of the following formula or be a pharmaceutically acceptable salt or hydrate thereof: OH Petition 870250087052, dated 09 / 25 / 2025, p. 33 / 121 30 / 94

[0115] In certain embodiments, the bispecific adapter is for use with an antifluorescein CAR-T cell in the treatment of a CAIX-expressing cancer, wherein the fluorescein comprises FITC, the CAIX ligand radical is ortho-CAL and the ligand comprises (or essentially consists of) PEG, or wherein the adapter is a pharmaceutically acceptable salt or hydrate of the foregoing.

[0116] In certain embodiments, the bispecific adapter is for use with an antifluorescein CAR-T cell in the treatment of a CAIX-expressing cancer, wherein the fluorescein comprises FITC, the CAIX ligand radical is ortho-CAL and the ligand comprises (or consists essentially of) PEG1, PEG6 or PEG9, or wherein the adapter is a pharmaceutically acceptable salt or hydrate of the foregoing.

[0117] In certain embodiments, the bispecific adapter is for use with an antifluorescein CAR-T cell in the treatment of a CAIX-expressing cancer, wherein the fluorescein comprises FITC, the CAIX ligand radical is ortho-CAL and the ligand comprises (or consists essentially of) (CH2)4, or wherein the adapter is a pharmaceutically acceptable salt or hydrate of the foregoing.

[0118] In certain embodiments, the bispecific adapter is for use with an antifluorescein CAR-T cell in the treatment of a CAIX-expressing cancer, wherein the fluorescein comprises FITC, the CAIX ligand radical is meta-CAL and the ligand comprises (or consists essentially of) PEG3, PEG6 or PEG9, or wherein the adapter is a pharmaceutically acceptable salt or hydrate of the foregoing.

[0119] A bispecific adapter for use with an antifluorescein CAR-T cell in the treatment of a CAIX-expressing cancer is also provided, wherein the fluorescein comprises fluorescein, FITC or NHS-fluorescein, the radical of the CAIX ligand is Aza and the ligand comprises (or consists essentially of) PEG, or wherein the adapter is a pharmaceutically acceptable salt or hydrate of the foregoing. In this case, the ligand may comprise (or consist essentially of) PEG1 to PEG9. The ligand may comprise (or consist essentially of) PEG6. The Petition 870250087052, dated 09 / 25 / 2025, p. 34 / 121 31 / 94 binder may comprise (or consist essentially of) PEG9.

[0120] The bispecific adapter may have a structure of the following formulas or be a pharmaceutically acceptable salt or hydrate thereof:

[0121] The bispecific adapter may have a structure of the following formulas, or comprise a pharmaceutically acceptable salt or hydrate thereof: Chemical Formula: C40H47N7O12S3 Chemical Formula: (34^59^01583 Molecular Weight: 1046.19 OR Petition 870250087052, dated 09 / 25 / 2025, p. 35 / 121 32 / 94 s Chemical Formula: Molecular weight: Aza-rEG9-rlTC SR-392 C52H71N7O18S3 1178.35 The 'S, (í

[0122] The bispecific adapter may contain one or more chiral centers or may otherwise be capable of existing as multiple stereoisomers. Consequently, various embodiments of the bispecific adapter may include pure stereoisomers as well as mixtures of stereoisomers, such as enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures. The bispecific adapter may be capable of existing as geometric isomers, as pure geometric isomers, or mixtures of geometric isomers.

[0123] The conjugates of the bispecific adapter can be synthesized according to methods known in the art. Several synthesis methods are exemplified in the Examples.

[0124] Salts

[0125] The bispecific adapters described herein may be presented as a pharmaceutically acceptable salt. A “pharmaceutically acceptable salt” of a bispecific adapter refers to salts whose counter-ions can be used in pharmaceutical products. Such salts include (i) acid addition salts, which may be obtained by reacting the free base of the original conjugate with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid and the like, or with organic acids such as acetic acid, oxalic acid, malic acid (D) or (L), 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 Petition 870250087052, dated 09 / 25 / 2025, page 36 / 121 The 33 / 94 present in the original conjugate is replaced by a metal ion, for example, an alkali metal ion, an alkaline earth ion, or an 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 skilled in the art, and any pharmaceutically acceptable salt of this type is contemplated herein.

[0126] In several embodiments, suitable basic salts are formed from bases that form non-toxic salts. Illustrative examples include salts of arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc. Hemisals of acids and bases can also be formed, for example, hemisulfate and hemicalcium salts.

[0127] Pharmaceutically acceptable salts can be synthesized from the original bispecific adaptor conjugate containing a basic or acidic moiety by conventional chemical methods. In some cases, such salts can be prepared by reacting the free acidic or basic forms of these conjugates with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, the description of which is incorporated herein by reference.

[0128] The bispecific adapter, or its pharmaceutically acceptable salt or hydrate, may exist in undissolved forms as well as in solvated forms, including hydrated forms. Solvated forms may be equivalent to non-solvated forms. In each embodiment described herein, it should be understood that the formulas include and represent not only all pharmaceutically acceptable salts of the bispecific adapters, but also include any and all hydrates and / or solvates of the conjugated formulas or their salts. The term Petition 870250087052, dated 09 / 25 / 2025, p. 37 / 121 34 / 94 “solvate” means a compound, or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of solvent bound together by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0129] Certain functional groups, such as hydroxyl, amino and the like, can form coordination complexes and / or conjugates with water and / or various solvents. Consequently, the formulas should be understood as including and representing these various hydrates and / or solvates. Non-hydrates and / or non-solvates of the bispecific adaptors are also included.

[0130] Pharmaceutical Compositions

[0131] In view of the foregoing, a composition (e.g., a pharmaceutical composition) for the treatment of cancer (e.g., a cancer expressing CAIX) is also provided comprising at least one bispecific adaptor and a pharmaceutically acceptable carrier or excipient. “Pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as, but not limited to, a buffering agent, a preservative agent, an anesthetic agent, a solubilizing agent, an isotonic agent, a humectant, and a stabilizer. The term also covers any of the agents approved by a regulatory agency, such as the U.S. Food and Drug Administration (FDA), or listed in the U.S. Pharmacopoeia for use in animals (e.g., mammals, such as humans). The carrier may be a phosphate-buffered saline solution, water, or an emulsion, such as an oil / water or water / oil emulsion.

[0132] A pharmaceutical composition is also provided for use in the treatment of a cancer expressing CAIX, comprising any of the bispecific adaptors described herein (for example, a fluorescein-CAIX ligand) and a pharmaceutically acceptable carrier or excipient.

[0133] Bispecific adaptors can be formulated as pharmaceutical compositions and administered to a mammalian host, Petition 870250087052, dated 09 / 25 / 2025, page 38 / 121 35 / 94 as a human patient, in a variety of forms adapted to the chosen route of administration. For example, the pharmaceutical composition may be formulated and administered orally or parenterally, intravenously, intra-arterially, intraperitoneally, intrathecally, epidurally, intracerebroventricularly, intraurethrally, intrasternally, intracranially, intratumorally, intramuscularly, topically, by inhalation and / or subcutaneously. In fact, in at least one embodiment, a bispecific adapter and / or composition, as described herein, may be administered directly into the bloodstream, muscle or internal organ.

[0134] For example, in at least one embodiment, the present bispecific adapters may 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 adapter can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, scotch tablets, 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(s) and a binder, excipients, a disintegrating agent, a lubricant, and / or a sweetening agent (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.

[0135] The bispecific adaptors and pharmaceutical compositions described herein may be formulated as parenteral formulations. Parenteral formulations are typically aqueous solutions, which may contain carriers or excipients such as salts, carbohydrates, and buffering agents (preferably at pH 3 to 9), but may be more appropriately formulated as a sterile, non-aqueous solution or as a dry solution to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water or sterile saline solution. Preparation under sterile conditions, by lyophilization to produce a powder. Petition 870250087052, dated 09 / 25 / 2025, p. 39 / 121 The preparation of sterile lyophilized 36 / 94 for a parenteral formulation can be carried out using well-known methods in the art. The solubility of the bispecific adapter, or of a pharmaceutically acceptable salt or hydrate thereof, for parenteral formulation can be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.

[0136] Bispecific adapters / compositions can also be administered by infusion or injection (e.g., using needle injectors (including microneedles) and / or needleless injectors). Solutions of the composition may be aqueous, optionally mixed with a non-toxic surfactant and / or may contain carriers or excipients such as salts, carbohydrates and buffering agents (preferably at pH 3 to 9), but for some applications they may be more appropriately formulated as a sterile non-aqueous solution or as a dry form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water or phosphate-buffered saline (PBS). For example, dispersions may be prepared in glycerol, liquid PEGs, triacetin and mixtures thereof, and in oils. Under normal storage and use conditions, these preparations may also contain a preservative to prevent the growth of microorganisms.

[0137] Suitable pharmaceutical forms for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredients adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final pharmaceutical form must be sterile, flowable and stable under manufacturing and storage conditions. The liquid vehicle or carrier may be a solvent or liquid dispersion medium containing, for example, and without limitation, water, ethanol, a polyol (e.g., glycerol, propylene glycol, liquid PEG(s) and the like), vegetable oils, non-toxic glyceryl esters and / or suitable mixtures thereof. In at least one Petition 870250087052, dated 09 / 25 / 2025, page 40 / 121 In the 37 / 94 modality, adequate fluidity can be maintained by the formation of liposomes, by maintaining the necessary particle size in the case of dispersions, or by the use of surfactants. The action of microorganisms can be prevented by the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In certain cases, it will be desirable to include one or more isotonic agents, such as sugars, buffers, or sodium chloride. Prolonged absorption of injectable compositions can be achieved by incorporating agents formulated to delay absorption, for example, aluminum monostearate and gelatin.

[0138] Sterile injectable solutions can be prepared by incorporating the bispecific adapter(s) and / or composition in the required amount of appropriate solvent with one or more of the other ingredients mentioned above, as needed, followed by sterilization by filtration. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and lyophilization techniques, which produce a powder of the active ingredient plus any desired additional ingredient present in the previously filtered sterile solutions.

[0139] For topical administration, it may be desirable to administer the bispecific skin adaptants as compositions or formulations in combination with a dermatologically acceptable carrier, which may be a solid or a liquid. For example, in certain embodiments, solid carriers may include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Similarly, useful liquid carriers may comprise water, alcohols, or glycols, or water-alcohol / glycol mixtures, in which the present conjugates may be dissolved or dispersed to effective levels, optionally with the aid of non-toxic surfactants. Additionally or alternatively, adjuvants such as fragrances and antimicrobial agents may be added to optimize the properties for a particular use. Liquid compositions Petition 870250087052, dated 09 / 25 / 2025, page 41 / 121 38 / 94 resulting products can be applied using absorbent pads, used to impregnate bandages and / or other dressings, sprayed onto the target area using pump or aerosol sprayers, or simply applied directly to the desired area of ​​the individual.

[0140] Thickeners such as synthetic polymers, fatty acids, salts and esters of fatty acids, fatty alcohols, modified celluloses or modified mineral materials may also be used with liquid vehicles to form pastes, gels, ointments, soaps and the like for direct application to the subject's skin.

[0141] The amount of bispecific adapter (or its pharmaceutically acceptable salt or hydrate) to be administered to an individual may vary significantly 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” mean (unless specifically indicated otherwise) an amount of a bispecific adapter that, when administered once or over a course of treatment, affects an individual’s health, well-being, or mortality (e.g., and without limitation, delays the onset and / or reduces the severity of one or more symptoms associated with a 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 from mice and other animals to human subjects are known in the art. In fact, the dosage of the bispecific adapter can vary significantly depending on the condition of the host individual, the cancer being treated, the degree of advancement of the pathology, the route of administration of the bispecific adapter and tissue distribution, and the possibility of co-use of other therapeutic treatments (such as radiotherapy or additional medications in combination therapies, such as, for example, CAR T-cell therapy). The amount of the composition. Petition 870250087052, dated 09 / 25 / 2025, page 42 / 121 39 / 94 required for use in treatment (e.g., the therapeutically or prophylactically effective amount or dose) will vary not only with the specific application but also with the salt selected (if applicable) and the individual's characteristics (such as age, condition, sex, body surface area and / or mass, drug tolerance) and will ultimately be at the discretion of the attending physician, clinician, or other professional.

[0142] The amount to be administered to a subject may vary, for example, from 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 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 from 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.A professional with adequate technical knowledge will readily understand that the dose may vary within the different ranges provided above, based on the factors mentioned above, and may be at the discretion of the attending physician.

[0143] Therapeutically effective or prophylactically effective amounts or doses may vary, for example, from about 0.05 mg / kg of patient body weight to about 30.0 mg / kg of patient body weight, or from about 0.01 mg / kg of patient body weight to about 5.0 mg / kg of 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, where all kg are body weight of Petition 870250087052, dated 09 / 25 / 2025, page 43 / 121 40 / 94 patient. The total therapeutically or prophylactically effective amount of the bispecific adapter may be administered in single or divided doses and may, at the physician's discretion, fall outside the typical range presented here.

[0144] In another embodiment, the bispecific adapter can be administered in a therapeutically or prophylactically effective amount of about 0.5 g / m2 to about 500 mg / m2, about 0.5 g / m2 to about 300 mg / m2, or about 100 g / m2 to about 200 mg / m2. In other embodiments, the amounts may range from about 0.5 mg / m2 to about 500 mg / m2, from about 0.5 mg / m2 to about 300 mg / m2, from about 0.5 mg / m2 to about 200 mg / m2, from about 0.5 mg / m2 to about 100 mg / m2, from about 0.5 mg / m2 to about 50 mg / m2, from about 0.5 mg / m2 to about 600 mg / m2, from about 0.5 mg / m2 to about 6.0 mg / m2, from about 0.5 mg / m2 to about 4.0 mg / m2 or from about 0.5 mg / m2 to about 2.0 mg / m2. The total amount may be administered in single or divided doses and may, at the discretion of the physician, be outside the typical range presented here. These quantities are based on square meters of body surface area.All ranges specified in this paragraph include the indicated endpoints and include all 0.5 g / m2 increments covered within each specified range.

[0145] In other embodiments, the amount of bispecific adapter (or pharmaceutically acceptable salt or hydrate thereof) to be administered to a subject may vary, for example, from 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, 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, approximately 50 nmol / kg to approximately 300 nmol / kg, approximately 50 nmol / kg to approximately 200 nmol / kg, approximately 50 nmol / kg to approximately 100 Petition 870250087052, dated 09 / 25 / 2025, p. 44 / 121 41 / 94 nmol / kg, approximately 100 nmol / kg to approximately 300 nmol / kg, approximately 100 nmol / kg to approximately 500 nmol / kg, approximately 100 nmol / kg to approximately 1,000 nmol / kg, or approximately 100 nmol / kg to approximately 2,000 nmol / kg of the subject's body weight. In other modalities, the dose may be approximately 100 nmol / kg, approximately 150 nmol / kg, approximately 200 nmol / kg, approximately 250 nmol / kg, approximately 300 nmol / kg, approximately 450 nmol / kg, approximately 700 nmol / kg, approximately 350 nmol / kg, approximately 500 nmol / kg, approximately 800 nmol / kg, approximately 400 nmol / kg, approximately 600 nmol / kg, approximately 900 nmol / kg, approximately 1,000 nmol / kg, approximately 2,000 nmol / kg, or approximately 3,000 nmol / kg of the subject's body weight. In other modalities, doses between approximately 20 μg / kg and approximately 3 mg / kg of the subject's body weight may be administered. The amount may vary between approximately 0.2 mg / kg and approximately 0.4 mg / kg of the subject's body weight or approximately 50 μg / kg of the subject's body weight. All ranges specified in this paragraph include the indicated endpoints and all increments of 1 nmol / kg or 10 μg / kg, as applicable, encompassed within each specified range.

[0146] Uses and Methods

[0147] A method is further provided for treating cancer in a subject (e.g., a cancer expressing CAIX). The method comprises administering to the subject cancer-treatment effective amounts of (i) antifluorescein CAR-T cells (e.g., fluorescein, FITC or NHS-fluorescein) or a pharmaceutical composition containing them and a pharmaceutically acceptable carrier or excipient, and (ii) a bispecific adapter or a pharmaceutical composition containing them and a pharmaceutically acceptable carrier or excipient.

[0148] The terms “treat”, “treating”, “treated” and “treatment” refer to therapeutic treatment. Such treatment may have a prophylactic effect. Cancer is treated when the symptoms or signs of cancer are alleviated, such as reducing the size of a tumor, completely or partially eliminating a tumor, stabilizing the cancer, for example. Petition 870250087052, dated 09 / 25 / 2025, page 45 / 121 42 / 94 by inhibiting the progression of cancer (for example, increasing the size of a tumor or increasing the number of tumors, such as due to metastasis), or any other effect on cancer that a physician would consider to constitute therapeutic (or prophylactic) treatment.

[0149] The term “subject,” as used herein, means an animal, such as a mammal, and in particular a human being. In veterinary applications, the subject may be a laboratory animal, a farm animal, a domestic animal, or a wild animal. Examples of such animals include, but are not limited to, a rodent, a rabbit, a monkey, a chimpanzee, a dog, a cat, a cow, a horse, a pig, a sheep, a goat, a bear, a panda, a lion, a tiger, a leopard, an elephant, a zebra, a giraffe, a gorilla, a dolphin, or a whale.

[0150] Antifluorescein CAR-T cells (e.g., fluorescein, FITC, or NHS-fluorescein) are T cells (alternatively, NK cells may be used) engineered to express a CAR that recognizes and binds to fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) on the bispecific adapter.

[0151] CAR is a fusion protein comprising at least three domains, which include (i) a recognition region (e.g., a single-chain fragment variable (scFv) region of an antibody), which recognizes and binds to fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein) with specificity, (ii) a co-stimulation domain, which enhances T lymphocyte proliferation and survival, and (iii) an activation signaling domain, which generates an activation signal for cytotoxic T lymphocytes.

[0152] scFv regions of antibodies that bind to fluorescein (e.g., in FITC) can be used and prepared from (i) an antibody known in the art that binds to fluorescein (e.g., fluorescein, FITC, or NHS-fluorescein), (ii) a newly prepared anti-fluorescein antibody, or (iii) sequence variants derived from the scFv regions of such antibodies, e.g., scFv regions Petition 870250087052, dated 09 / 25 / 2025, p. 46 / 121 43 / 94 with 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 with the amino acid sequence of the scFv region from which they are derived. The CAR-binding portion may be, for example, an scFv of an antibody, a Fab, Fv, Fc, or (Fab')2 fragment.

[0153] “Percentage sequence identity (%)” with reference to a polypeptide or nucleotide sequence is defined as the percentage of amino acid or nucleic acid residues, respectively, in a candidate sequence that are identical to the residues in the reference sequence, after sequence alignment and the introduction of gaps, if necessary, to achieve maximum percentage sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage sequence identity can be achieved in various ways that are within technical capability, for example, using publicly available computer software.For example, determining the percentage identity or similarity between sequences can be done using, for instance, the GAP program (Genetics Computer Group software; now available online via Accelrys), and alignments can be performed using, for example, the ClustalW algorithm (VNTI software, InforMax Inc.). Additionally, a sequence database can be searched using the nucleic acid or amino acid sequence of interest. Algorithms for searching databases are typically based on the BLAST software (Altschul et al., 1990), but experts in the field can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment along the entire length of the compared sequences. In some modalities, the percentage identity can be determined along the entire length of the nucleic acid or amino acid sequence. Petition 870250087052, dated 09 / 25 / 2025, p. 47 / 121 44 / 94

[0154] In several embodiments, the CAR has a recognition region and the recognition region is an scFv region of an antifluorescein antibody, which can bind to fluorescein, FITC or NHS-fluorescein (see, for example, the antifluorescein antibody E2 described in Vaughan et al., Nature Biotechnol 14(3): 309-314 (1996), and the exemplary CAR construct, which expresses a CAR comprising the antifluorescein antibody E2, shown in Fig. 1 and described on page 66, line 16, to page 69, line 12, of International Patent Application Publication No. WO 2019 / 144091, both of which are incorporated herein by reference for their teachings thereon).The CAR has a co-stimulatory domain, and the co-stimulatory domain 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, CD27 (cluster of differentiation 27), CD30 (cluster of differentiation 30), CD150 (cluster of differentiation 150), DAP10, NKG2D, CD278 (cluster of differentiation 278; ICOS), a co-stimulatory molecule of the CD28 superfamily expressed on activated T cells, a family of signaling lymphocyte activating molecule-related receptors (SLAMs) (such as 2B4), or any combination thereof. Variants in the sequence of co-stimulation domains, which have activity equal to or similar to that of the domain in which they are modeled, can also be used without negatively impacting the method.The CAR has an activation signaling domain, and the activation signaling domain can be a CD3α chain of T cells, a CD3 delta receptor protein, an MBL receptor protein, a B29 receptor protein, or an Fcγ receptor. Sequence variants of the activation signaling domains, which have activity equal to or similar to that of the domain in which they are modeled, can also be used without negatively impacting the method. Such co-stimulation domains and variants of such co-stimulation domains and activation signaling domains. Petition 870250087052, dated 09 / 25 / 2025, page 48 / 121 45 / 94 may 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 with the amino acid sequence of the domain from which they are derived.

[0155] In one embodiment of a CAR comprising an antifluorescein E2 antibody fragment, the CAR comprises an IgG4 hinge domain and a CD28 transmembrane domain. The co-stimulation domain is CD137 (4-1BB) and the activation signaling domain is CD3Ç.

[0156] In certain embodiments, a CAR comprises an scFv of an antifluorescein antibody as a recognition region, a CD137 (4-1BB) co-stimulation domain, and CD3Z as an activation signaling domain.

[0157] Constructs encoding CARs are prepared using genetic engineering techniques. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference. By way of example, a plasmid or viral expression vector (e.g., a lentiviral vector, a retroviral vector, a Sleeping Beauty vector, and a piggyback vector (transposon / transposase systems that include a virus-nonmediated CAR gene delivery system)) can be prepared to encode a fusion protein comprising a recognition region, one or more co-stimulation domains, and an activation signaling domain in structure and linked in the 5' to 3' direction. Other arrangements may be acceptable and may include a recognition region, an activation signaling domain, and one or more co-stimulation domains.The positioning of the recognition region on the fusion protein will generally be such that the region is displayed on the exterior of the cell. The CAR may also include additional elements, such as a signal peptide, to ensure proper export of the fusion protein. Petition 870250087052, dated 09 / 25 / 2025, page 49 / 121 46 / 94 for the cell surface, a transmembrane domain to ensure that the fusion protein is maintained as an integral membrane protein, and a hinge domain that confers flexibility to the recognition region and allows strong binding to the target fraction of CAR.

[0158] T lymphocytes (e.g., cytotoxic T lymphocytes) can be genetically modified to express CAR constructs 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.

[0159] T lymphocytes can be autologous, although heterologous cells can be used, such as when the patient undergoing treatment has received high-dose chemotherapy or radiotherapy to destroy the patient's immune system. In several modalities, allogeneic cells can be used.

[0160] T lymphocytes can be obtained from a patient by well-known means in the art. For example, T cells can be obtained by collecting peripheral blood from the patient, subjecting the blood to Ficoll density gradient centrifugation, and then using a negative T cell isolation kit (such as the EasySep™ T Cell Isolation Kit) to isolate a population of cytotoxic T cells from the peripheral blood. In several embodiments, the cytotoxic T lymphocyte population need not be pure and may contain other cells, such as other T cells, monocytes, macrophages, natural killer cells, and B cells. The collected cell population may comprise at least about 90% of the selected cell type, such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the selected cell type.

[0161] After obtaining T lymphocytes, the cells can be cultured under conditions that promote their activation. The culture conditions can be such that the cells can be administered to a Petition 870250087052, dated 09 / 25 / 2025, page 50 / 121 47 / 94 patient without concern regarding reactivity against culture medium components. For example, culture conditions may not include bovine serum products such as bovine serum albumin (BSA). Activation can be achieved by introducing known activators into the culture medium, such as anti-CD3 antibodies in the case of cytotoxic T cells. Other suitable activators include anti-CD28 antibodies. The lymphocyte population can be cultured under conditions that promote activation for approximately 1 to 4 days. The appropriate level of activation can be determined by cell size, proliferation rate, or activation markers determined by flow cytometry.

[0162] After the cytotoxic T lymphocyte population 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 to the patient immediately or cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more days, or between about 5 and about 12 days, between about 6 and about 13 days, between about 7 and about 14 days, or between about 8 and about 15 days, for example, to allow the cells to recover from transfection. Suitable culture conditions may be similar to the conditions under which the cells were cultured for activation, with or without the agent that was used to promote activation.

[0163] After transfection and activation of the cells, a composition containing the CAR-T cells can be prepared and administered to the individual. Culture media free of animal products, such as BSA, can be used. Tissue culture conditions typically used in the technique can be employed to avoid contamination by bacteria, fungi, and mycoplasma. The cells can be pelleted, washed, and resuspended in a pharmaceutically acceptable carrier, diluent, or excipient.

[0164] Exemplary compositions containing CAR-T cells include compositions containing the cells in sterile 290 mOsm saline solution, Petition 870250087052, dated 09 / 25 / 2025, page 51 / 121 48 / 94 infusible cryomedia (containing Plasma-Lyte A, dextrose, sodium chloride, human serum albumin (HSA), and dimethyl sulfoxide (DMSO)) in 0.9% NaCl with 2% HSA, or in any other sterile 290 mOsm infusible material. Depending on the identity of the culture medium, CAR-T cells may be administered in the culture medium as a composition or concentrated and resuspended in the culture medium before administration.

[0165] The CAR-T cell composition may be administered to the subject by any suitable means, such as parenteral administration, for example, intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, or intrathecally.

[0166] The total number of CAR-T cells and the concentration of cells in the composition administered to the subject will vary depending on a number of factors, including the type of CAR-T cells used, the binding specificity of the CAR, the identity of the target fraction of the CAR (in the examples described herein, FITC) and the identity of the small molecule ligand / target ligand of the bispecific adapter (e.g., a CAIX ligand), the identity of the cancer, the location of the cancer in the subject, the means used to administer the compositions to the subject, and the health, age, and weight of the subject to be treated. Suitable compositions comprising transduced CAR-T cells include those with a volume between about 5 ml and about 200 ml, containing from about 1 x 10⁵ to about 1 x 10¹⁵ transduced CAR-T cells. Typical compositions comprise a volume between approximately 10 ml and approximately 125 ml and contain from approximately 1 x 107 to approximately 1 x 1010 CAR-T cells.An exemplary composition comprises approximately 1 x 10⁹ CAR-T cells in a volume of approximately 100 ml. A single dose or multiple doses of CAR-T cells may be administered to the individual. Compositions may comprise approximately 1 million (M), 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M, 10 M, 11 M, 12 M, 12.5 M, 13 M, 14 M, or 15 M CAR-T cells per kg of the patient's body weight. When the CAR-T cell composition is administered by injection into the subject's bloodstream, the CAR-T cells in the subject's bloodstream are at least 5%, 7%, 10%, 11%, 12%, 13%, 14%. Petition 870250087052, dated 09 / 25 / 2025, page 52 / 121 49 / 94 or 15% of the subject's total T cells in the subject's bloodstream approximately four weeks after injection, at least 20%, 25%, 30%, 35%, 40%, or 50% of the subject's total T cells in the subject's bloodstream approximately two weeks after injection, or at least 85%, 90%, or 95% of the subject's total T cells approximately one week after injection.

[0167] The bispecific adapter (or its pharmaceutically acceptable salt or hydrate) or the pharmaceutical composition comprising it, or a combination thereof, and the antifluorescein CAR-T cells or the pharmaceutical composition comprising the antifluorescein CAR-T cells may be administered to the patient by any suitable method known in the field. The terms “administer,” “administering,” “administered,” and “administration” refer to methods of introducing the bispecific adapter (or its pharmaceutically acceptable salt or hydrate) or a pharmaceutical composition comprising the bispecific adapter (or its pharmaceutically acceptable salt or hydrate) and methods of introducing the antifluorescein CAR-T cells or a pharmaceutical composition comprising the antifluorescein CAR-T cells. Examples of suitable routes of administration include, but are not limited to, oral, intravenous, intramuscular, subcutaneous, and transdermal.The components can be administered directly into the bloodstream, muscle, or internal organ. Suitable routes for parenteral administration include, but are not limited to, intravenous, intra-arterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous. Needle injectors, including microneedles, needleless injectors, and infusions may be used. The components may be administered in unit-dose forms and / or formulations containing conventional and non-toxic pharmaceutically acceptable carriers or excipients (or vehicles or adjuvants).

[0168] In the method, CAR-T cells antifluorescein (or Petition 870250087052, dated 09 / 25 / 2025, page 53 / 121 The 50 / 94 pharmaceutical composition comprising antifluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient) and the bispecific adapter may be administered simultaneously or sequentially, in any order, by the same or different routes. When administered simultaneously by the same route, the formulations may be the same or different. In several embodiments, the bispecific adapter may be administered to the individual after the CAR-T cells.The interval between CAR-T cell administration and bispecific adapter administration can vary widely depending on factors including the type of CAR-T cells used, the binding specificity of the CAR, the identity of the CAR targeting fraction (in the examples described here, a fluorescein) and the small molecular ligand / targeting fraction of the bispecific adapter (i.e., CAIX ligands), the identity of the cancer, the location of the cancer in the individual, the means used to administer the CAR-T cells and the bispecific adapter to the individual, as well as the patient's health, age, and weight.

[0169] The bispecific adapter(s) may be administered before or after CAR-T cells, such as at approximately 3, 6, 9, 12, 15, 18, 21 or 24 hours, or at approximately 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 or more days. The rate of tumor lysis may be regulated by adjusting the rate of administration of the bispecific adapter, for example (e.g., depending on the dosing schedule, such as continuous, once daily, twice daily, three times daily, once weekly, twice weekly or three times weekly). Continuous means at least one hour, at least four hours, at least six hours, at least eight hours, at least 10 hours, at least 12 hours or at least 24 hours, or a daily or weekly administration schedule, such as once a day, twice a day, three times a day, on alternate days, once a week, twice a week, three times a week or any other suitable schedule.In this method, CAR-T cells are treated with antifluorescein (or a composition). Petition 870250087052, dated 09 / 25 / 2025, page 54 / 121 51 / 94 pharmaceutical comprising antifluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient) and the bispecific adapter can be administered intravenously. The cancer may 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 may be endometrial cancer or glioma (e.g., stage 3-4 glioma).

[0170] Cytokine release syndrome (CRS) can be controlled by varying the dose of the bispecific adapter. See, for example, International Patent Application Publication No. WO 2017 / 177149.

[0171] Such combination therapy methods can be performed using any modified cell that is suitable for cancer treatment and may include the use of more than one of these types of agents. In certain modalities, the modified cell used in this combination therapy is CAR T cells and may also (or alternatively) comprise modified stem cells and other cells.

[0172] The modified cells used in combination with bispecific adaptors or compositions may be any CAR T cells, stem cells or other modified cells, or a combination thereof. Several adoptive cell therapies (also called cellular immunotherapy) are known in the art for use in cancer treatment, and T-cell immunotherapy, in particular, has received much attention. Some non-limiting examples of such therapies include modified T-cell receptor (TCR) therapy, CAR T-cell therapy and natural killer (NK) cell therapy.

[0173] In certain approaches, the administration of both bispecific adaptor conjugates and modified cell therapy results in a greater than additive inhibition of cancer growth.

[0174] When multiple therapeutic agents and / or therapies are Petition 870250087052, dated 09 / 25 / 2025, page 55 / 121 52 / 94 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 targeted bispecific adapter is administered at least once during a course of treatment involving the administration of cell therapy to an individual.

[0175] The cancer treatment methods described herein may include administering any of the bispecific adapters to the patient and administering any of the modified cell compositions or modified cell therapy to the patient.

[0176] In certain embodiments, a method of treating cancer is provided to an individual. The method comprises administering to the individual cancer-treatment effective amounts of: (i) antifluorescein CAR-T cells or a pharmaceutical composition comprising antifluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient; and (ii) any bispecific adapter or any pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier or excipient. Steps (i) and (ii) may be administered simultaneously or sequentially, in any order, by the same route or by different routes.

[0177] Antifluorescein CAR-T cells may comprise any CAR T cells suitable for use as described. In certain embodiments, antifluorescein CAR-T cells comprise a recognition region comprising an scFv region of an antifluorescein antibody; a co-stimulation domain, wherein the co-stimulation domain is CD28, CD137 (4-1BB), CD134 (OX40) or CD278 (ICOS); and / or an activation signaling domain that is a CD3Z chain of T cells or an Fc γ receptor.

[0178] In certain modalities, both steps (i) and (ii) of the method Petition 870250087052, dated 09 / 25 / 2025, page 56 / 121 53 / 94 are administered intravenously.

[0179] The bispecific adapter fluorescein can bind to the antifluorescein-binding CAR-T cell with affinity after exposure to it, and the bispecific adapter targeting ligand can bind the antifluorescein-binding CAR-T cell to a target cancer cell by binding the bispecific adapter targeting ligand to a receptor on that target cancer cell with affinity. In this way, the conjugates and compositions described herein facilitate increased efficacy of CAR-T cell therapy.

[0180] In certain embodiments, the receptor on the target cancer cell is an overexpressed CAIX. The cancer may be a CAIX-expressing cancer and at least one bispecific adapter of (ii) may comprise a radical of a CAIX ligand.

[0181] Methods are also provided for treating CAIX-expressing cancer in an individual. In certain embodiments, the method comprises administering to the individual cancer-treatment-effective amounts of (i) antifluorescein CAR-T cells or a pharmaceutical composition comprising antifluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient; and (ii) any bispecific adapter or a pharmaceutical composition comprising a bispecific adapter and a pharmaceutically acceptable carrier or excipient. As noted above, the CAR may have a recognition region, and the recognition region is an scFv region of an antifluorescein antibody. In certain embodiments, the CAR comprises: a co-stimulation domain, and the co-stimulation domain is CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS); and / or an activation signaling domain, and the activation signaling domain is a CD3α chain of T cells or an Fcγ receptor.

[0182] The methods described herein may also include obtaining images of the cancer in the individual. Cancer imaging may include optical imaging, positron emission tomography (PET), or single-photon emission computed tomography. Petition 870250087052, dated 09 / 25 / 2025, page 57 / 121 54 / 94 (SPECT), for example.

[0183] In the methods described herein, cancer may additionally be visualized before administration to the individual of the bispecific adapter, or of its pharmaceutically acceptable salts or hydrates, or of the modified cell composition (e.g., a composition of cytotoxic lymphocytes expressing CAR or a composition of CAR-NK cells). Cancer may additionally or alternatively be visualized during or after administration to assess metastasis, for example, and treatment efficacy. For example, imaging may be obtained by means of PET, magnetic resonance imaging (MRI) or SPECT / computed tomography (CT). The imaging method may be any suitable imaging method known in the art.

[0184] Cancer can be any cancer. Cancer has its simple and common meaning when read in light of the specification and may include, but is not limited to, a group of diseases involving abnormal cell growth with 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 stroma, endometrium, breast, cervix, ovary, colon, or prostate, leukemias, lymphomas, other blood-related cancers, and head and neck cancer. In certain modalities, the cancer being treated is a tumor. In certain modalities, the cancer is malignant. In certain modalities, the cancer is ovarian cancer, endometrial cancer, breast cancer, glioma (such as, optionally, stage 3-4 glioma), or clear cell renal cell carcinoma (such as, optionally, stage 3-4 clear cell renal cell carcinoma).

[0185] In some aspects of these modalities, cancer is a cancer that expresses CAIX.

[0186] In some modalities, the cancer is visualized before administration of (i) and (ii) to the individual. Imaging can be done by PET, Petition 870250087052, dated 09 / 25 / 2025, page 58 / 121 55 / 94 MRI or SPECT / CT, for example.

[0187] In certain embodiments, the use of a bispecific adapter, a pharmaceutically acceptable salt, hydrate or solvate of the bispecific adapter, or a composition thereof, is provided in the manufacture of a medicament for the treatment of cancer in an individual. The bispecific adapter may be any conjugate. The medicament may be for use in combination with the administration of a cell therapy designed for the individual, such as, for example, CAR T-cell therapy, in which the CAR T cells express antifluorescein.

[0188] In addition, a method is provided for increasing CAR T cell activation. The method may comprise providing a bispecific adapter, a pharmaceutical composition comprising the same, or a combination thereof (e.g., a therapeutically effective amount of any of the foregoing); and exposing antifluorescein CAR T cells or a pharmaceutical composition comprising antifluorescein CAR T cells and a pharmaceutically acceptable carrier or excipient to the bispecific adapter(s), pharmaceutical composition or combination; wherein the CAR-T cell exhibits increased activation after exposure, compared with a CAR-T cell not exposed to the bispecific adapter. Antifluorescein CAR-T cells may be in systemic circulation in an individual when exposed to the bispecific adapter, pharmaceutical composition or combination, for example.Alternatively, antifluorescein CAR-T cells can be exposed in vitro to the bispecific adapter, pharmaceutical composition, or combination (e.g., prior to administration to an individual).

[0189] Kits

[0190] A kit is also provided. The kit may comprise (i) a bispecific adapter or a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier or excipient, and (ii) antifluorescein CAR-T cells (e.g., anti-FITC CAR-T cells) or a pharmaceutical composition comprising the same and a Petition 870250087052, dated 09 / 25 / 2025, page 59 / 121 56 / 94 carrier or pharmaceutically acceptable excipient. In certain embodiments, the bispecific adapter (or pharmaceutical composition comprising the same) and the CAR-T cells (or pharmaceutical composition comprising the same) are stored in separate containers.

[0191] General

[0192] Experts in the field will recognize that numerous modifications can be made to the specific implementations described above. Implementations should not be limited to the particular modalities described. Other implementations may be possible.

[0193] Although the bispecific adapters and pharmaceutical compositions are illustrated and described in detail in the preceding description, they should be considered as illustrative and not restrictive in character, it being understood that only certain embodiments have been shown and described and that all alterations and modifications that fall within the spirit of the invention should be protected.

[0194] The scope of the present bispecific adapters, compositions and methods is intended to be defined by the following Claims. However, this disclosure may be practiced in a manner different from that specifically explained and illustrated without departing from its spirit or scope. Those skilled in the art will understand that various alternatives to the embodiments described herein may be employed in the practice of the Claims without departing from the spirit and scope as defined in the following Claims.

[0195] Any use of section headings is intended to aid in reading the document and should not be interpreted as limiting. Furthermore, information relevant to a section heading may occur within or outside that specific section.

[0196] All publications, patents, patent application publications, journal articles, textbooks and other publications mentioned in this document are indicative of the skill level of those in the art to which the disclosure relates. All of these Petition 870250087052, dated 09 / 25 / 2025, pp. 60 / 121 57 / 94 publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated for incorporation by reference. In case of inconsistent uses between this document and the documents thus incorporated by reference, the use in the incorporated reference shall be considered supplementary to that in this document; for irreconcilable inconsistencies, the use in this document shall prevail.

[0197] Various techniques and mechanisms sometimes describe a connection or link between two components. Words such as attached, linked, coupled, connected, and similar terms with their inflectional morphemes are used interchangeably unless the difference is noted or otherwise clarified by the context. These words and expressions do not necessarily mean direct connections, but include connections through mediate components. It should be noted that a connection between two components does not necessarily mean a direct and unimpeded connection, since a variety of other components may reside between the two notable components. Consequently, a connection does not necessarily mean a direct and unimpeded connection unless otherwise indicated.

[0198] Certain Definitions

[0199] 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 commonly understood by someone with common knowledge in the field.

[0200] The term “about” or “approximately” means within an acceptable range for the specific value, as determined by someone with common knowledge in the field, which will depend in part on how the value is measured or determined, for example, the limitations of the measurement system. For example, “about” might mean a range up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. By way of further example, “about” or “approximately” might mean Petition 870250087052, dated 09 / 25 / 2025, page 61 / 121 58 / 94 within 90%, within 95%, 99%, 99.5%, 99.9%, 99.99% or at least approximately 99.999% or more of a stated value or a stated limit of a range. Alternatively, particularly with regard to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times, and more preferably within 2 times, of a value. Unless otherwise indicated, the term “approximately” means within an acceptable error range for the specific value, such as ± 1-20%, preferably ± 1-10% and more preferably ± 1-5%.

[0201] When a range of values ​​is provided, it is understood that each intermediate value, between the upper and lower limits of that range and any other declared or intermediate value within that declared range, is covered. The upper and lower limits of those smaller ranges may be included independently within the smaller ranges and are also covered, subject to any limit specifically excluded in the declared range. When the declared range includes one or both limits, ranges that exclude one or both limits are also included.

[0202] A phrase that refers to “at least one of” a list of items refers to any combination of those items, including individual members. As an example, “at least one of a, b, or c” is intended to cover: a, b, c, ab, ac, bc, and abc.

[0203] The terms “a,” “an,” or “the” are used to include one or more of one, unless the context clearly indicates otherwise. The term “or” is used to refer to a non-exclusive “or,” unless otherwise indicated. Furthermore, it should be understood that the phraseology or terminology employed herein, and not otherwise defined, is for descriptive purposes only and not limiting purposes.

[0204] The term “or” is used to refer to a non-exclusive “or,” unless otherwise indicated. Furthermore, it should be understood that the phraseology or terminology employed herein, and not otherwise defined, is for descriptive purposes only and not limiting purposes.

[0205] The terms and expressions employed are used as terms Petition 870250087052, dated 09 / 25 / 2025, page 62 / 121 59 / 94 Descriptive and not limiting. When certain terms are defined and described or otherwise discussed elsewhere in the “Detailed Description,” all such definitions, descriptions, and discussions shall be attributed to those terms. Nor is there any intention, in the use of such terms and expressions, to exclude any equivalents of the features shown and described, or parts thereof. Furthermore, although subheadings may be used in the “Detailed Description,” such use is only for ease of reference and is not intended to limit any disclosure made in a section to that section alone; rather, any disclosure made under a subheading is intended to constitute a disclosure under all other subheadings.

[0206] It is recognized that various modifications are possible within the scope of the claimed invention. Thus, although the present invention has been specifically disclosed in the context of preferred embodiments and optional features, those skilled in the art may resort to modifications and variations of the concepts disclosed herein. Such modifications and variations are considered within the scope of the invention as claimed herein. EXAMPLES

[0207] The following examples serve to illustrate the present disclosure. The examples are not intended to limit the scope of the claimed invention in any way. Example 1 General procedure for the synthesis of ortho-CAIX-PEGl-FITC and ortho-(CH2)4-FITC conjugates.

[0208] The ortho-CAIX-PEG1-FITC and ortho-(CH2)4-FITC conjugates can be synthesized according to Scheme 1. Petition 870250087052, dated 09 / 25 / 2025, p. 63 / 121 60 / 94 Scheme 1 ortho-CAIX-PEGi-FITC h2n PyBOP, DIPEA, DMF, 37% TFA:CH2CI2(1:3) . FITC, DIPEA, DMF 65% Chemical Formula: C42H44F3N5OÍJS3 Molecular Weight: 916.02 ortho-(CH2)4-FITC h2n N HBOC PyBOP, DIPEA, DMF, 32% Petition 870250087052, dated 09 / 25 / 2025, page 64 / 121 61 / 94 1. TFA:CH2CI2(1:3) ---------------------------*- 2. FITC, DIPEA. DMF 64% Chemical Formula: C42H44F3N5OgS3 Molecular Weight: 900.02

[0209] PyBOP (1.2 eq) + DIPEA (2.0 eq) were added to a stirred solution of acidic compound (1.0 eq) in dimethylformamide (DMF). After 10 minutes of stirring, BocNH-PEGl-NH2 or BocNH(CH2)4NH2 (1.2 eq) were added to the above reaction mixture, and stirring was continued for a further 2 hours. After complete conversion of the starting materials (measured by liquid chromatography-mass spectrometry (LC-MS)), the reaction mixture was diluted with water and then extracted over dichloromethane (DCM) (2 x 20 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure. Next, the crude residue was obtained and purified by combiflash using (A = 10 mM ammonium acetate, pH = 7.5, B = acetonitrile) for 40 minutes, using method 595. The desired fractions obtained were kept in the lyophilizer for 48 hours to produce the coupled product as white solids.

[0210] Trifluoroacetic acid (TFA) (1 mL) was added to the lyophilized solids (0.0133 mmol) in DCM (3 mL) and stirring was maintained for Petition 870250087052, dated 09 / 25 / 2025, p. 65 / 121 62 / 94 minutes. Then, the reaction mixture was evaporated under reduced pressure to give the free amine as a brown gummy solid. The amine obtained was used in the next step without purification. To a stirred solution of amine (1 eq) in DMF (500 pL), N,N-diisopropylethylamine (DIPEA) (10 eq) followed by fluorescein isothiocyanate isomer 1 (1 eq) were added and stirring was continued at room temperature for a further 2 hours. The reaction mixture was diluted with water and purified by ultra-high performance liquid chromatography (U-HPLC) using (A = 10 mM ammonium acetate, pH = 7.5, B = acetonitrile) for 60 minutes using method 5-35 and the desired fractions obtained were quickly held in a lyophilizer for 48 hours to provide the desired ortho-CAIX-FITC compounds as yellow solids. Example 2 Binding affinities of bispecific adapters directed to carbonic anhydrase IX

[0211] MDA-carbonic anhydrase IX (CAIX) cells (0.2 million) were incubated with serially diluted bispecific adaptors twice, starting with 250 nM in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) for one hour at room temperature. The cells were washed once to remove unbound adaptors. The fluorescence intensity of the cell-bound adaptors was analyzed by flow cytometry. The results are shown in Figures 2A-2B.

[0212] For ortho-CAL, the binding affinity to CAIX+ cells decreased with increasing ligand length. For meta-CAL, the binding affinity to CAIX+ cells decreased slightly with increasing ligand length, but all bispecific meta-CAL adapters showed increased binding. Petition 870250087052, dated 09 / 25 / 2025, page 66 / 121 63 / 94 Example 3 Full connection and surface exposure of CAIX bispecific adapters

[0213] HT29 cells or MDA-CAIX cells were incubated with 1 μM bispecific adapters with different CAIX ligands and different polyethylene glycol (PEG) ligands in complete RPMI medium (RPMI + 10% FBS) for one hour at room temperature. Free compounds were removed by washing and the fluorescence intensity of fluorescein isothiocyanate (FITC) was analyzed by flow cytometry.

[0214] To analyze the surface exposure of the FITC fraction, stained cells were incubated with allophycocyanin anti-FITC antibody (APC) on ice for 30 minutes. Then, the cells were washed and the fluorescence intensity of the 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 exhibited better total binding to HT29 cells than the other adapters.As shown in Figure 3B, all three meta-CALFITC adapters exhibited better surface binding and FITC exposure in HT29 cells. As shown in Figure 3C, meta-CAL-FITC with different ligands and Aza-FITC exhibited better overall binding to MDA-CAIX cells. As shown in Figure 3D, all meta-CAL-FITC adapters exhibited better surface binding and FITC exposure than the other adapters tested in MDA-CAIX cells. Example 4 Bispecific adapters targeting CAIX mediated the cytotoxicity of anti-FITC CAR-T cells and the release of IFNγ in MDA-CAIX cells.

[0215] Anti-FITC CAR-T cells were incubated with an equal number of MDA-CAIX cells in the presence of bispecific adaptors at different concentrations. The number of MDA-CAIX cells was Petition 870250087052, dated 09 / 25 / 2025, page 67 / 121 64 / 94 determined at the end of co-culture, and the percentage of cell lysis was calculated using the following formula: [(number of untreated cells / number of treated cells) / number of untreated cells] * 100%.

[0216] The secretion of interferon gamma (IFNy) from antiFITC CAR-T cells was analyzed by enzyme-linked immunosorbent assay (ELISA) using the supernatants of the co-cultured medium at the end of co-culture. The results are shown in Figures 4A-4B.

[0217] As shown in Figure 4A, the efficacy of meta-CAL-PEG6-FITC is greater than that of ortho-CAL-PEG6-FITC and Aza-FITC between 0.001 nM and 10 nM. As shown in Figure 4A. 4B, meta-CAL-PEG6-FITC mediated a higher level 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. Example 5 Effect of ligand length on the cytotoxicity of anti-FITC CAR-T cells mediated by the bispecific meta-CAL-FITC adaptor and on IFNY release when co-cultured with MDA-CAIX cells.

[0218] Anti-FITC CAR-T cells were incubated with an equal number of MDA-CAIX cells in the presence of bispecific adaptors at different concentrations. The number of MDA-CAIX cells was determined at the end of co-culture, and the percentage of cell lysis was calculated using the following formula: [(number of untreated cells - number of treated cells) / number of untreated cells] * 100%.

[0219] IFNγ secretion from anti-FITC CAR-T cells was analyzed by ELISA using the supernatants of the co-cultured medium at the end of the co-culture. The results are shown in Figures 5A-5B. As shown in Figure 5A, meta-CAL-PEG6-FITC and meta-CALPEG9-FITC showed greater efficacy 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 Petition 870250087052, dated 09 / 25 / 2025, page 68 / 121 65 / 94 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). Example 6 Effect of ligand length on the cytotoxicity of anti-FITC CAR-T cells mediated by the bispecific adapter ortho-CAL-FITC and on IFNY release when co-cultured with MDA-CAIX cells.

[0220] Anti-FITC CAR T cells were incubated with an equal number of MDA-CAIX cells in the presence of bispecific adaptors at different concentrations. The number of MDA-CAIX cells was determined at the end of the co-culture and the percentage of cell lysis was calculated using the following formula: [(number of untreated cells - number of treated cells) / number of untreated cells] *100%.

[0221] IFNγ secretion from anti-FITC CAR T cells was analyzed by ELISA using the supernatants from the co-cultured medium at the end of the co-culture study. The results are shown in Figures 6A-6B.

[0222] As shown in Figure 6A, the bispecific ortho-CAL-FITC adapters showed similar efficacy, although longer PEG spacers were slightly better than the (CH2)4 spacer. As shown in Figure 6B, the bispecific ortho-CAL-FITC adapters showed similar efficacy, although longer PEG spacers were slightly superior to the (CH2)4 spacer. Example 7 In vivo efficacy of CAIX-targeted bispecific adapters in an HT29 tumor model.

[0223] HT29 cells (1.5 million) were implanted into each NOD scid gamma (NSG) mouse by subcutaneous injection. When tumor volumes reached approximately 100 mm3, the treatment groups Petition 870250087052, dated 09 / 25 / 2025, page 69 / 121 66 / 94 were injected with 10 million anti-FITC CAR-T cells and bispecific adapters, as shown in FIG. 7A. Tumor volume and body weight were monitored regularly. Tumor volume was calculated using the formula: (length * width²) / 2. The results are shown in FIGS. 7B-7C.

[0224] As shown in FIG. In Figure 7B, Aza-FITC and metaCAL-PEG3-FITC slightly inhibited HT29 tumor growth, while meta-CAL-PEG9-FITC significantly inhibited HT29 tumor growth. As shown in Figure 7C, only mice treated with meta-CAL-PEG9-FITC lost body weight. The body weight loss may be due to the release of cytokines from expanded CAR-T cells, which is an indication of improved metaCAL-PEG-9-FITC functionality. Toxicity can be minimized by optimizing the adapter dosage. Example 8 Retention and surface exposure of CAIX bispecific adapters in HT29 tumor cells

[0225] HT29 tumor-bearing mice were injected with CAIX bispecific adapters at 500 nmol / kg. After injection (24 hours later), the tumors were dissected and digested into individual cells. Total retention of the bispecific adapters was determined by the fluorescence intensity of FITC, analyzed by flow cytometry. To analyze the exposure of the bispecific adapters to FITC on the surface of tumor cells, the digested tumor cells were stained with APC-anti-FITC antibody on ice for 30 minutes. After washing away the unbound antibody, the fluorescence intensity of the APC was analyzed by flow cytometry. The results are shown in Figures 8B-8C.

[0226] As shown in Figure 8B, all compounds showed very low retention in the HT29 tumor; this may be due to Petition 870250087052, dated 09 / 25 / 2025, pp. 70 / 121 67 / 94 to low CAIX expression in HT29 tumor cells. As shown in Figure 8C, meta-CAL-FITC with different ligands showed better surface retention and FITC exposure than the other compounds. meta-CAL-PEG6-FITC and meta-CAL-PEG-9-FITC showed better FITC exposure than meta-CAL-PEG3-FITC in vivo. Example 9 T-cell counts in blood and HT29 tumors in mice at the end of treatments.

[0227] At the end of the in vivo study with the HT29 solid tumor model described in Figures 7A-7C, blood was collected from the mice and the red blood cells present 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 the T cells infiltrating the tumor, the tumors were dissected 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 in the total number of live cells was analyzed by flow cytometry. The results are shown in Figures 9A-9B.

[0228] As shown in Figure 9A, the blood of mice treated with meta-CAL-PEG-9-FITC showed more T cells than the blood of other treatment groups. As shown in Figure 9B, there were more T cells infiltrating the tumor in the tumors of mice treated with meta-CAL-PEG9-FITC. Example 10 In vivo efficacy of CAIX targeting bispecific adapters in a model Petition 870250087052, dated 09 / 25 / 2025, pp. 71 / 121 68 / 94 tumoral MDA-CAIX

[0229] MDA-CAIX cells (5 million) were implanted into each NSG mouse by subcutaneous injection. When tumor volumes reached approximately 100 mm3, treatment groups were injected with 10 million anti-FITC CAR T cells and bispecific adapters were indicated, as shown in Figure 10A. Tumor volume and body weight were monitored regularly. Tumor volume was calculated using the formula: (length * width2) / 2. The results are shown in Figures 10B-10C.

[0230] As shown in FIG. 10B, meta-CAL-PEG3-FITC slightly inhibited the growth of MDA-CAIX tumors, while ortho-CAL-PEG6-FITC significantly inhibited the growth of MDA-CAIX tumors. As shown in FIG. 10C, neither of the bispecific adapters showed toxicity to mice during treatment. Example 11 Retention and surface exposure of CAIX bispecific adapters in MDA-CAIX tumor cells

[0231] MDA-CAIX tumors from mice in different treatment groups were dissected and digested into individual cells 24 hours after the last injections with bispecific CAIX adapters at 500 nmol / kg. Total retention of bispecific adapters 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. Exposure of bispecific adapters to FITC on the tumor cell surface was analyzed by flow cytometry after staining with APC-antiFITC antibody on ice for 30 minutes. Surface exposure of FITC also Petition 870250087052, dated 09 / 25 / 2025, page 72 / 121 69 / 94 was analyzed in cells stained with 100 nM meta-CAL-PEG6-FITC. The results are shown in FIGS. 11B-11E.

[0232] As shown in FIG. In Figure 11B, tumors treated with Aza-FITC and meta-CAL-FITC with PEG3 or PEG6 ligands showed similar total FITC retention in the cells. As shown in Figure 11C, tumor cells treated with Aza-FITC showed slightly higher CAIX levels than tumors treated with meta-CAL-FITC with PEG3 or PEG6. As shown in Figure 11D, meta-CAL-PEG6-FITC showed 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 showed virtually no anti-FITC antibody staining, indicating that tumor cells expressing CAIX were killed by CAR-T cells. Example 12 General procedure for the synthesis of acetazolamide (Aza)PEG-FITC conjugates Scheme 2 H Acetazolamide Chemical Formula: C4H5N4O3S2 1. 1 M HCl, reflux 4h 2. Et^N, MeOH, 1h Quantitative yield Chemical Formula: C2H4N4O2S2 Molecular Weight: 80.20 Molecular Weight: 222.24 Petition 870250087052, dated 09 / 25 / 2025, page 73 / 121 70 / 94 Molecular Weight: 543.66 1. 20% piperidine in DM F 2. FITC, DIPEA, DMF HO Petition 870250087052, dated 09 / 25 / 2025, page 74 / 121 71 / 94 Example 13 Effects of ligand length on the binding affinity of bispecific Aza-FITC adapters.

[0233] MDA-CAIX cells (0.2 million) were incubated with bispecific adapters diluted in quadruple series, starting with 1000 nM in PBS containing 2% FBS, for 1 hour at room temperature. 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 12, which is a graph of concentration (nM) versus normalized MFI, showing that PEG spacers do not have significant effects on the binding affinity of acetazolamide (Aza)-FITC bispecific adapters. Example 14 Effects of binder length on total bond and surface exposure of Aza-FITC bispecific adapters.

[0234] MDA-CAIX cells were incubated with 500 nM of bispecific Aza-FITC adapters with different PEG spacers in complete RPMI medium (RPMI + 10% FBS) for 1 hour at room temperature. Free compounds were removed by washing, and the fluorescence intensity of FITC was analyzed by flow cytometry. To analyze the surface exposure of the FITC fraction, stained cells were incubated with APC-anti-FITC antibody on ice for 30 minutes. Then, the cells were 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 bispecific adapter vs. FL MFI, showing the total binding of Aza-FITC bispecific adapters to MDA-CAIX cells. Petition 870250087052, dated 09 / 25 / 2025, page 75 / 121 72 / 94 The length of the PEG spacer did not significantly affect the total binding. Figure 13B is a plot of bispecific adapter vs. MFI of APC-antiFITC, showing the surface exposure of FITC fractions in Aza-FITC bispecific adapters with different PEG binders after binding to MDA-CAIX cells. Increasing the length of the PEG spacer increased the surface exposure of FITC after binding to MDA-CAIX cells. Example 15 Effects of ligand length on IFNγ release from anti-FITC CAR-T cells mediated by the bispecific adapter Aza-FITC when co-cultured with HT29 cells

[0235] Anti-FITC CAR-T cells were incubated with HT29 cells at a 1:5 ratio in the presence of bispecific adaptors at different concentrations for 40 hours. IFNγ secretion from anti-FITC CAR-T cells was analyzed by ELISA using the supernatants of the co-cultured medium at the end of the co-culture study. The results are shown in Figure 1.14, which is a graph of concentration (nM) vs. IFNγ (pg / ml), showing the effect of ligand length on IFNγ release mediated by the bispecific adaptor Aza-FITC in anti-FITC CAR-T cells when co-cultured with HT29 cells.

[0236] Aza-PEG3-FITC and Aza-PEG9-FITC mediated higher levels of IFNγ released from anti-FITC CAR-T cells was higher than that released from Aza-PEG0-FITC. Aza-PEG6-FITC mediated the highest level of IFNγ released from anti-FITC CAR-T cells. Example 16 Bispecific adapters targeting CAIX cells with optimal ligands mediated IFNγ release in anti-FITC CAR-T cells.

[0237] Anti-FITC CAR-T cells were incubated with HT29 cells Petition 870250087052, dated 09 / 25 / 2025, page 76 / 121 73 / 94 in a 1:5 ratio in the presence of bispecific adaptors at different concentrations for 40 hours. IFNγ secretion from anti-FITC CAR T cells was analyzed by ELISA using co-culture medium supernatants at the end of the co-culture study. The results are shown in Figure 15, which is a graph of CA9-PEG(n)-FITC ligand concentration (nM) vs. IFNγ (pg / ml), showing bispecific adaptors targeting CAIX with optimal ligands mediating IFNγ release from anti-FITC CAR T cells. MetaCAL-PEG9-FITC mediated the highest level of IFNγ released from anti-FITC CAR T cells at low concentrations (< 0.1 nM). Aza-PEG6-FITC mediated similar levels of IFNγ release from anti-FITC CAR T cells as metaCAL-PEG9-FITC at concentrations above 0.1 nM. Lower levels of IFN mediated by OrthoCAL-PEG6-FITC released from anti-FITC CAR-T cells. Example 17 In vivo efficacy of Aza-PEG6-FITC and orthoCAL-PEG6-FITC

[0238] KB cells (1 million) were implanted into each NSG mouse by subcutaneous injection. When tumor volumes reached approximately 50 mm3, treatment groups were injected with 10 million anti-FITC CAR-T cells and indicated bispecific adaptors, as shown in Figure 16A. Tumor volume and body weight were monitored regularly. Tumor volume was calculated using the formula: (length * width2) / 2. The results are shown in Figures 16B-16C.

[0239] Figure 16B is a graph of days after cell injection. CAR-T versus tumor volume (mm3), showing the tumor growth curves of different treatment groups. Aza-PEG6-FITC and orthoCAL-PEG6-FITC slightly inhibited the growth of KB tumors. The efficacy of Aza-PEG6-FITC is slightly superior to that of orthoCAL-PEG6-FITC. Figure 16C is a graph of days after cell injection. Petition 870250087052, dated 09 / 25 / 2025, page 77 / 121 74 / 94 CAR-T versus change in body weight (%), which shows the changes in body weight of mice in different treatment groups. Neither Aza-PEG6-FITC nor orthoCAL-PEG6-FITC induced significant body weight loss. Example 18 Aza-FITC Synthesis

[0240] Synthesis of 5-amino-2-sulfamoyl-1,3,4-thiadiazole monohydrochloride B: Hydrochloric acid (1 M aq.) (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 for 3 hours under reflux (Scheme 3). Then, the water was evaporated by rotary evaporator. The crude material (dissolved in a small amount of methanol with the aid of TEA for solid loading) was purified by column chromatography (CHCl3 / MeOH: 100 / 0 to 70 / 30) to yield product B (2.768 g, 96%). Scheme 3 o ii q H H2N-S^ / bx^N n11 \\ / / ° NN / AO c 0 H2N-S^ / S\^r II \\ / / 0 NN O Hydrolysis H2N-S^S'v^NH2.HC ------------------------------------------------* \\ / / 1MHCI, reflux· 3h O NN B ^NHFmoc Coupling Ethyl chloroformate N-methyl morpholine DMF, rt, 8-12h ίη2 D Petition 870250087052, dated 09 / 25 / 2025, p. 78 / 121 75 / 94 Lack of protection 20% piperidine in DMF rt, 2 h Coupling FITC, DIPEA DMSO RT, 3 h

[0241] Synthesis of (9H-fluoren-9-yl)methyl (8-oxo-8-((5-sulfamoyl,3,4-thiadiazol-2-yl)amino)octyl)carbamate D: N-methyl morpholine (0.063 mL, 1.1 equiv) was added at room temperature and under an argon atmosphere 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 dried DMF (1.5 mL), and then stirred for 30 minutes (Scheme 3). 5-amino-2-Petition 870250087052, dated 25 / 09 / 2025, p. 79 / 121 76 / 94 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 minutes at 13 mL / minute). The pure fractions were pooled and lyophilized, yielding (9H-fluoren-9-yl)methyl (8-oxo-8-((5-sulfamoyl-1,3,4-thiadiazole-2-yl)amino)octyl)carbamate D.

[0242] 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 / minute). The pure fractions were pooled and lyophilized, yielding 8-amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)octanamide E.

[0243] Synthesis of Aza-FITC F: DIPEA (3.0 equiv.) at room temperature was added 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., added in parts of 0.80 equiv. + 0.20 equiv. over 1 hour) in dimethyl sulfoxide (DMSO) (1 mL) and stirred continuously for 3 hours under argon. 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). The pure fractions were combined and lyophilized, yielding the Aza-FITC F. LCMS (ESI) (m / z): (M + H)+ calculated for C31H30N6O8S3 + H = 711.13 found (m / z): (M + H)+ 710.80. Example 19 Petition 870250087052, dated 09 / 25 / 2025, pp. 80 / 121 77 / 94 Synthesis of CAL-PEGs-FITC Conjugates Scheme 4 M NH3in MeOH -10 °C - rt, 4 h THE TEA, MeOH reflux, 24 h AcOH:H2O: (2:1) H2O2, h2o 70 °C - rt, 4 h

[0244] Synthesis of the meta-CAL-P3-FITC conjugate

[0245] meta-CAL-PEG3 NHBoc (8). The meta-CA 6 ligand was synthesized by the known procedure described in the literature. More specifically, to a stirred solution of meta-CA 6 ligand (0.039 g, 1.0 equiv), t-Boc-N-amido-PEG3 amine 7 (0.027 g, 1.1 equiv), and azabenzotriazole tetramethyluranium hexafluorophosphate (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 to 4 hours, as mentioned 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 Petition 870250087052, dated 09 / 25 / 2025, page 81 / 121 78 / 94 minutes). The pure fractions were pooled and lyophilized, yielding meta-CAL-PEG3 NHBoc 8.

[0246] meta-CAL-PEG3 NH2 (9). The meta-CAL-PEG3 NHBoc 8 (0.050 g) was dissolved in 30% TFA in moist DCM (1.5 mL) at room temperature and stirred for 1 to 2 hours (Scheme 5). The reaction was monitored by LCMS and volatiles were removed by rotary evaporator. The residue was dissolved in DCM and purified by column chromatography on silica gel (DCM: Methanol: TFA = 89:10:1). In addition, residual impurities were purified by preparative thin-layer chromatography (TLC) (DCM: Methanol: TFA = 89:10:1) to provide meta-CAL- PEG3NH2 9.

[0247] meta-CAL-P3-FITC (11). The meta-CAL-PEG3NH2 8 (0.041 g, 1.0 equiv.) was dissolved in DMF (1.0 mL) under an argon atmosphere. Then, FITC 10 (0.024 g, 1.0 equiv., added in parts of 0.80 equiv. + 0.20 equiv. over 2 hours) and DIPEA (1.0 equiv.) was added at room temperature in the dark (Scheme 5). The reaction mixture was stirred for another hour. 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 minutes). The pure fraction was collected, frozen at -80 °C for three hours, and lyophilized to produce the meta-CAL-P3-FITC 11 conjugate. The purity of the meta-CAL-P3-FITC 11 conjugate compound was analyzed by LCMS. Scheme 5 Petition 870250087052, dated 09 / 25 / 2025, page 82 / 121 79 / 94 Η ACT. DI PEA DMF, rt, 3 to 4 h Coupling NHBoc H2N NHBoc Lack of protection 30% TFA in DCM, rt, 1-2 h Coupling FITC. 10 DIPEA, DMF rt, 2 to 3 h Petition 870250087052, dated 09 / 25 / 2025, page 83 / 121 80 / 94

[0248] Synthesis of the meta-CAL-P6-FITC conjugate

[0249] meta-CAL-PEG6 NHBoc (13). DIPEA (0.0177 mL, 1.2 equiv) was added at room temperature under an argon atmosphere to a stirred solution of meta-CA 6 ligand (0.040 g, 1.0 equiv), t-Boc-N-amidoPEG6 amine 12 (0.039 g 1.1 equiv) and HATU (0.037 g, 1.15 equiv) in DMF (1.5 mL), and the solution was stirred for 3 to 4 hours, as mentioned in Scheme 6. 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 minutes). The pure fractions were pooled and lyophilized, yielding meta-CAL-PEG6 NHBoc 13.

[0250] meta-CAL-PEG6 NH2 (14). The meta-CAL-PEG6 NHBoc 13 (0.056 g, 1.0 equiv.) was dissolved in 30% TFA in moist DCM (1.5 mL) at room temperature and stirred for 1 to 2 hours (Scheme 6). The reaction was monitored by LCMS and volatiles were removed by rotary evaporator. The residue was dissolved in DCM and purified by silica gel column chromatography (DCM: Methanol: TFA = 94:5:1). In addition, residual impurities were purified by preparative TLC (DCM: Methanol: TFA = 94:5:1) to give the meta-CALPEG6 NH2 14.

[0251] meta-CAL-P6-FITC (15). The meta-CAL-PEG6NH2 14 (0.057 g, 1.0 equiv.) was dissolved in DMF (1.0 mL) under an argon atmosphere. Then, FITC 10 (0.028 g, 1.0 equiv., added in parts of 0.80 equiv. + 0.20 equiv. over 2 hours) and DIPEA (1.0 equiv.) were added at room temperature in the dark (Scheme 6). The reaction mixture was stirred for another hour. 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 minutes). The pure fraction was collected, frozen at -80 °C for three hours, and lyophilized to produce the meta-CAL-P6-FITC 15 conjugate. The purity of the meta-CAL-P6-FITC 15 conjugate compound was analyzed by LCMS. Scheme 6 Petition 870250087052, dated 09 / 25 / 2025, page 84 / 121 81 / 94 NHBoc NHBoc Lack of protection 10%TFA in DCM, rt, 1 to 2 h Petition 870250087052, dated 09 / 25 / 2025, page 85 / 121 82 / 94

[0252] Synthesis of the meta-CAL-P9-FITC conjugate

[0253] meta-CAL-PEG9 NHBoc (17). To a stirred solution of meta-CA 6 ligand (0.031 g, 1.0 equiv), t-Boc-N-amido-PEG9 amine 16 (0.040 g 1.1 equiv) and HATU (0.028 g, 1.15 equiv) in DMF (1.0 mL) was added DIPEA (0.014 mL, 1.2 equiv) at room temperature under argon, and the solution was stirred for 3 to 4 hours, as mentioned in Scheme 7. 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 minutes). The pure fractions were pooled and lyophilized, yielding meta-CAL-PEG9 NHBoc 17.

[0254] meta-CAL-PEG9 NH2 (18). The meta-CAL-PEG9 NHBoc 17 (0.067 g, 1.0 equiv.) was dissolved in 30% TFA in moist DCM (1.5 mL) at room temperature and stirred for 1 to 2 hours (Scheme 7). The reaction was monitored by LCMS and volatiles were removed by rotary evaporator. The residue was dissolved in DCM and purified by column chromatography on silica gel (DCM: Methanol: TFA = 94:5:1). In addition, residual impurities were purified by preparative TLC (DCM: Methanol: TFA = 94:5:1) to provide the meta-CALPEG9 NH2 18.

[0255] [meta-CAL-P9-FITC (19). The meta-CAL-PEG9NH2 18 (0.041 g, 1.0 equiv.) was dissolved in DMF (1.0 mL) under an argon atmosphere. Then, F1TC 10 (0.017 g, 1.0 equiv., added in parts of 0.80 equiv. + 0.20 equiv. over 2 hours) and DIPEA (1.0 equiv.) were added at room temperature in the dark (Scheme 7). The reaction mixture was stirred for another hour. 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 minutes). The pure fraction was collected, frozen at -80 °C for three hours, and lyophilized to produce the meta-CAL-P9-FITC 19 conjugate. The purity of the meta-CAL-P9-FITC 19 compound was analyzed by LCMS. Scheme 7 Petition 870250087052, dated 09 / 25 / 2025, page 86 / 121 83 / 94 NH2 NHBoc NHBoc Lack of protection 30% TFA™ DCM, rt, 1-2 h NH2 Coupling FITC, 10 DIPEA, DMF rt, 2 to 3 h

[0256] Synthesis of the ortho-CAL-P6-FITC conjugate

[0257] ortho-CAL-PEG6 NHBoc (21). To a stirred solution of Petition 870250087052, dated 09 / 25 / 2025, p. 87 / 121 84 / 94 ortho-CA 20 ligand (0.033 g, 1.0 equiv), t-Boc-N-amido-PEG6 amine 12 (0.032 g, 1.1 equiv) and HATU (0.033 g, 1.15 equiv) in DMF (1.5 mL) was added to DIPEA (0.015 mL, 1.2 equiv) at room temperature under argon, and the solution was stirred for 3 to 4 hours, as mentioned in Scheme 8. 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 minutes). The pure fractions were pooled and lyophilized, yielding ortho-CAL-PEG6 NHBoc 21.

[0258] ortho-CAL-PEG6 NH2 (22). Ortho-CAL-PEG6 NHBoc 21 (0.110 g, 1.0 equiv.) was dissolved in 25% TFA in moist DCM (1.5 mL) at room temperature and stirred for 2 hours (Scheme 8). The reaction was monitored by LCMS, and volatiles were removed by rotary evaporator. The residue was dissolved in DMF purified by reversed-phase C18 column (mobile phase: A = 20 mM ammonium acetate, pH = 7, B = ACN; method: 5% B to 95% B in 60 minutes). The pure fractions were pooled and lyophilized, yielding ortho-CAL-PEG6 NH2 22.

[0259] ortho-CAL-P6-FITC (23). ortho-CAL-PEG6NH2 22 (0.060 g, 1.0 equiv.) was dissolved in DMF (1.0 mL) under an argon atmosphere. Then, FITC 10 (0.028 g, 0.9 equiv., added in parts of 0.70 equiv. + 0.20 equiv. over one hour) and DIPEA (1.25 equiv.) were added at room temperature in the dark (Scheme 8). The reaction mixture was stirred for another hour. 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 minutes). The pure fraction was collected, frozen at -80 °C for three hours, and lyophilized to produce the ortho-CAL-P6-FITC 23 conjugate. The purity of the ortho-CAL-P6-FITC 23 compound was analyzed by LCMS. Scheme 8 Petition 870250087052, dated 09 / 25 / 2025, pp. 88 / 121 85 / 94 Cycloctylamine DMSO, 60 °C, 16 h Coupling HATU, DI PEA DMF, rt, 3 to 4 h H2N NHBoc DCM, rt, 2 h Petition 870250087052, dated 09 / 25 / 2025, p. 89 / 121 86 / 94

[0260] Synthesis of the ortho-CAL-P9-FITC conjugate

[0261] ortho-CAL-PEG9 NHBoc (24). The ortho-CA 20 ligand was synthesized by the known procedure described in the literature. Specifically, to a stirred solution of ortho-CA 20 ligand (0.022 g, 1.0 equiv), t-Boc-N-amido-PEG9 amine 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 to 4 hours, as mentioned 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 minutes). The pure fractions were pooled and lyophilized, yielding ortho-CAL-PEG9 NHBoc 24.

[0262] ortho-CAL-PEG9 NH2 (25). Ortho-CAL-PEG9 NHBoc 24 (0.098 g, 1.0 equiv.) was dissolved in 25% TFA in moist DCM (1.5 mL) at room temperature and stirred for 2 hours (Scheme 9). The reaction was monitored by LCMS and volatiles were removed by rotary evaporator. The residue was dissolved in DMF and purified by reverse phase on a C column. Petition 870250087052, dated 09 / 25 / 2025, pp. 90 / 121 87 / 94 (mobile phase: A = 20 mM ammonium acetate, pH = 7, B = ACN; method: 5% of B to 95% of B in 60 minutes). The pure fractions were pooled and lyophilized, yielding ortho-CAL-PEG9 NH2 25.

[0263] ortho-CAL-P9-FITC (26). ortho-CAL-PEG9N hour (0.071 g, 1.0 equiv.) was dissolved in DMF (1.0 mL) under an argon atmosphere. Then, FITO 10 (0.028 g, 0.9 equiv., added in parts of 0.70 equiv. + 0.20 equiv. over one hour) and DIPEA (1.25 equiv.) were added at room temperature in the dark (Scheme 9). The reaction mixture was stirred for another hour.

[0264] 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 minutes). The pure fraction was collected, frozen at -80 °C for 3 hours and lyophilized to produce the ortho-CAL-P9-FITC 26 conjugate. The purity of the ortho-CAL-P9-FITC 26 compound was analyzed by LCMS. Scheme 9 HATU, DIPEA DMF, rt, 3 to 4 h NHBoc Coupling Petition 870250087052, dated 09 / 25 / 2025, pp. 91 / 121 88 / 94 LISTED EMBODIMENTS OF THE CLAIMED INVENTION

[0265] Clause 1. A bispecific adapter or a pharmaceutically acceptable salt or hydrate thereof, comprising a fluorescein, fluorescein isothiocyanate (FITC) or N-hydroxysuccinimide (NHS)-fluorescein conjugated to a radical of a Petition 870250087052, dated 09 / 25 / 2025, pp. 92 / 121 89 / 94 carbonic anhydrase IX (CAIX) ligand by means of a ligand, wherein the ligand comprises, consists essentially of, or consists of polyethylene glycol (PEG).

[0266] Clause 2. The bispecific adapter of clause 1, wherein the CAIX ligand is or comprises 3-((3-(cyclo-octylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL), 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (metaCAL), acetazolamide (Aza) or a derivative or analogue of any of the above.

[0267] Clause 3. The bispecific adapter of clause 1, in which the binder comprises, consists essentially of, or consists of, PEG1 to PEG9.

[0268] Clause 4. The bispecific adapter of clause 1, wherein the CAIX binder is or comprises ortho-CAL or a derivative or analogue thereof and the binder comprises, consists essentially of, or consists of, PEG1 to PEG9.

[0269] Clause 5. The bispecific adapter of clauses 1 to 3, in which the binder comprises, consists essentially of, or consists of, PEG3 to PEG9.

[0270] Clause 6. The bispecific adapter of clauses 1 to 3, wherein the CAIX binder is or comprises meta-CAL or a derivative or analogue thereof and the binder comprises, consists essentially of, or consists of PEG3 to PEG9.

[0271] Clause 7. The bispecific adapter of clause 3, wherein the CAIX binder is or comprises Aza or a derivative or analogue thereof.

[0272] Clause 8. The bispecific adapter of any of clauses 4, 6 and 7, in which the binder comprises, consists essentially of or consists of PEG6.

[0273] Clause 9. The bispecific adapter of clause 6 or clause 7, in which the binder comprises, consists essentially of or consists of PEG9.

[0274] Clause 10. The bispecific adapter of clause 7, where Petition 870250087052, dated 09 / 25 / 2025, pp. 93 / 121 90 / 94 the binder comprises, consists essentially of, or consists of PEG6.

[0275] Clause 11. The bispecific adapter of any of clauses 1 to 10, in which the binder comprises, consists essentially of, or consists of an alkyl.

[0276] Clause 12. The bispecific adapter of any of clauses 1 to 10, in which the binder comprises, consists essentially of or consists of (CH2)4.

[0277] Clause 13. The bispecific adapter of clause 1, comprising a structure of one of the following formulas: or a pharmaceutically acceptable salt or carbohydrate of any of the above.

[0278] Clause 14. The bispecific adapter of clause 1 comprising a structure of the following formulas: Petition 870250087052, dated 09 / 25 / 2025, pp. 94 / 121 91 / 94 OH or a pharmaceutically acceptable salt or carbohydrate thereof.

[0279] Clause 15. The bispecific adapter of clause 1, comprising a structure of the following formulas: Chemical Formula: C46H5gN7O15S3 Molecular Weight: 1046.19 or THE. 's, d'nh2 SR-392 Chemical Formula: Molecular Weight: 1178.35 Petition 870250087052, dated 09 / 25 / 2025, pp. 95 / 121 92 / 94 or a pharmaceutically acceptable salt or hydrate of any of the above.

[0280] Clause 16. The bispecific adapter of any of clauses 1 to 15 for use with an anti-chimeric fluorescein antigen receptor (CAR) T cell in the treatment of cancer.

[0281] Clause 17. The bispecific adapter of any of clauses 1 to 15 for use with a fluorescein anti-CAR T cell in the treatment of a CAIX-expressing cancer.

[0282] Clause 18. A pharmaceutical composition for the treatment of a cancer that expresses CAIX, comprising the bispecific adapter of any of clauses 1 to 16 and a pharmaceutically acceptable carrier or excipient.

[0283] Clause 19. A kit comprising: (i) at least one dosage unit of a bispecific adapter of any of clauses 1 to 17 or a pharmaceutical composition of clause 18; and (ii) at least one dosage unit of fluorescein anti-chimeric antigen receptor (CAR) T cells or a pharmaceutical composition comprising fluorescein anti-CAR T cells and a pharmaceutically acceptable carrier or excipient; where (i) and (ii) are optionally in separate containers.

[0284] Clause 20. The kit of clause 19, wherein the CAIX binder of the bispecific adapter is or comprises ortho-CAL or a derivative or analogue thereof.

[0285] Clause 21. The kit of clause 19, wherein the CAIX binder of the bispecific adapter is or comprises meta-CAL or a derivative or analogue thereof. Petition 870250087052, dated 09 / 25 / 2025, pp. 96 / 121 93 / 94

[0286] Clause 22. The kit of clause 19, wherein the BOX of the bispecific adapter is or comprises Aza or a derivative or analogue thereof.

[0287] Clause 23. A method for treating cancer in an individual, comprising administering to the individual amounts effective for the treatment of cancer of: (i) antifluorescein CAR-T cells or a pharmaceutical composition comprising antifluorescein CAR-T cells and a pharmaceutically acceptable carrier or excipient; and (ii) a bispecific adapter of any of clauses 1 to 17 or a pharmaceutical composition of Claim 18; in which the individual is treated for cancer.

[0288] Clause 24. The method of clause 23, wherein the CAR comprises: a recognition region comprising a single-chain fragment (scFv) variable region of an antifluorescein antibody; (a co-stimulation domain, which is CD28, CD137 (41BB), CD134 (OX40) or CD278 (ICOS); and / or an activation signaling domain, which is a T cell CD3α chain or an Fcγ receptor.

[0289] Clause 25. The method of clause 23, wherein the fluorescein of the bispecific adapter binds to the antifluorescein-binding CAR-T cell with affinity upon exposure to it, and the CAIX ligand of the bispecific adapter binds the antifluorescein-binding CAR-T cell to a CAIX-expressing cancer cell after the bispecific adapter binds to a receptor on that CAIX-expressing cancer cell with affinity.

[0290] Clause 26. The method of clause 23, wherein (i) and (ii) are administered simultaneously or sequentially, in any order, by the same route or by different routes.

[0291] Clause 27. The method of any of clauses 23 to 26, where (i) and (ii) are administered intravenously. Petition 870250087052, dated 09 / 25 / 2025, pp. 97 / 121 94 / 94

[0292] Clause 28. The method of any of clauses 23 to 27, further including obtaining images of the cancer in the individual.

[0293] Clause 29. The method of clause 28, wherein the cancer image comprises optical images, positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0294] Clause 30. The method of any of clauses 23 to 29, where cancer is a cancer that expresses BOX.

[0295] Clause 31. The method of any of clauses 23 to 30, where the cancer is ovarian cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer, or clear cell renal cell carcinoma, as optionally, stage 3-4 clear cell renal cell carcinoma.

[0296] Clause 32. A method for increasing chimeric antigen receptor (CAR) T cell activation, comprising: (i) provide a bispecific adapter of any of clauses 1 to 17 or a pharmaceutical composition of clause 18; and (ii) expose antifluorescein CAR T cells or a pharmaceutical composition comprising antifluorescein CAR T cells and a pharmaceutically acceptable carrier or excipient to the bispecific adapter or pharmaceutical composition; in which the CAR T cell exhibits increased activation against cancerous cells after exposure, compared to a CAR T cell not exposed to the bispecific adapter.

[0297] Clause 33. The method of clause 32, in which the T cells Antifluorescein CAR antibodies are in systemic circulation in an individual when exposed to the bispecific adapter.

[0298] Clause 34. The method of clause 32, wherein the cancer cells are cancer cells that express CAIX. Petition 870250087052, dated 09 / 25 / 2025, pp. 98 / 121

Claims

1 / 8 CLAIMS 1. Bispecific Adapter, or pharmaceutically acceptable salt or hydrate thereof, characterized in that it comprises a fluorescein, fluorescein isothiocyanate (FITC) or N-hydroxysuccinimide (NHS)-fluorescein conjugated to a carbonic anhydrase IX (CAIX) ligand radical by means of a ligand, wherein the ligand comprises, essentially consists of or consists of polyethylene glycol (PEG).

2. Bispecific Adapter, according to Claim 1, characterized in that the CAIX ligand is or comprises 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL), 3-((2-(cyclo-octylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (meta-CAL), acetazolamide (Aza) or a derivative or analogue of any of the foregoing.

3. Bispecific Adapter, according to Claim 1, characterized in that the binder comprises, consists essentially of, or consists of PEG1 to PEG9.

4. Bispecific Adapter, according to Claim 1, characterized in that the CAIX binder is or comprises ortho-CAL or a derivative or analogue thereof and the binder comprises, consists essentially of, or consists of PEG1 to PEG9.

5. Bispecific Adapter, according to Claim 1, characterized in that the binder comprises, consists essentially of, or consists of PEG3 to PEG9.

6. Bispecific Adapter, according to Claim 1, characterized in that the CAIX binder is or comprises meta-CAL or a derivative or analogue thereof and the binder comprises, consists essentially of, or consists of PEG3 to PEG9. Petition 870250079905, dated 05 / 09 / 2025, pp. 176 / 186 2 / 8 7. Bispecific Adapter, according to Claim 3, characterized in that the CAIX binder is or comprises Aza or a derivative or analogue thereof.

8. Bispecific Adapter, according to any one of Claims 4, 6 and 7, characterized in that the binder comprises, consists essentially of, or consists of PEG6.

9. Bispecific Adapter, according to Claim 6 or Claim 7, characterized in that the binder comprises, consists essentially of, or consists of PEG9.

10. Bispecific Adapter, according to Claim 7, characterized in that the binder comprises, consists essentially of, or consists of PEG6.

11. Bispecific Adapter, according to Claim 1, characterized in that the binder comprises, consists essentially of, or consists of an alkyl group.

12. Bispecific Adapter, according to Claim 1, characterized in that the binder comprises, consists essentially of, or consists of (CH2)4.

13. Bispecific Adapter, according to Claim 1, characterized in that it comprises a structure of one of the following formulas: Petition 870250079905, dated 05 / 09 / 2025, p. 177 / 186 3 / 8 or a pharmaceutically acceptable salt or hydrate of any of the above items.

14. Bispecific Adapter, according to Claim 1, characterized in that it comprises a structure of the following formulas: OH a pharmaceutically acceptable salt or hydrate thereof. or 15. Bispecific Adapter, as per Claim 1, Petition 870250079905, dated 05 / 09 / 2025, p. 178 / 186 4 / 8 characterized in that it comprises a structure of the following formulas: Aza-PEG3-FITC SR-402 Chemical Formula: C^FU / NyO^Sg Molecular Weight: 914.03 O' nh2 h2n nn Aza-PEG6-FITC compound code: SR-380 Chemical Formula: C46H59N7O15S3 Molecular Weight: 1046.19 OU O >N SR-392 Chemical Formula: C52H71N7O18S3 O Molecular Weight: 1178 35 , or a pharmaceutically acceptable salt or hydrate of any of the above items.

16. Bispecific Adapter, according to any one of Claims 1 to 7 and 10 to 15, characterized in that it is for use with a chimeric antigen receptor (CAR) antifluorescein T cell in cancer treatment.

17. Bispecific Adapter, according to any one of Claims 1 to 7 and 10 to 15, characterized in that it is for use with an antifluorescein T-CAR cell in the treatment of a CAIX-expressing cancer. Petition 870250079905, dated 05 / 09 / 2025, pp. 179 / 186 5 / 8 18. Pharmaceutical Composition, for the treatment of a cancer that expresses CAIX, characterized in that it comprises the bispecific adapter, as defined in any one of Claims 1 to 17, and a pharmaceutically acceptable carrier or excipient.

19. Kit, characterized in that it comprises: (i) at least one dosage unit of a bispecific adapter, as defined in any of Claims 1 to 17, or a pharmaceutical composition, as defined in Claim 18; and (ii) at least one dosage unit of antifluorescein chimeric antigen receptor (CAR) T cells or a pharmaceutical composition comprising antifluorescein CAR-T cells and a pharmaceutically acceptable excipient or carrier, wherein (i) and (ii) are optionally in separate containers.

20. Kit, according to Claim 19, characterized in that the CAIX binder of the bispecific adapter is or comprises 3-((3(cyclo-octylamino)-2,5,6-trifluoro-4-sulfamoylphenyl)thio)propanoic acid (ortho-CAL) or a derivative or analogue thereof.

21. Kit, according to Claim 19, characterized in that the CAIX ligand of the bispecific adapter is or comprises 3-((2(cyclo-octylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (meta-CAL) or a derivative or analogue thereof.

22. Kit, according to Claim 19, characterized in that the BOX of the bispecific adapter is or comprises acetazolamide (Aza) or a derivative or analogue thereof. Petition 870250079905, dated 05 / 09 / 2025, pp. 180 / 186 6 / 8 23. Method for Treating Cancer in an Individual, characterized in that it comprises administering to the individual cancer-treatment-effective amounts of: (i) antifluorescein chimeric antigen receptor (CAR) T cells or a pharmaceutical composition comprising antifluorescein CAR-T cells and a pharmaceutically acceptable excipient or carrier; and (ii) a bispecific adapter, as defined in any one of Claims 1 to 17, or a pharmaceutical composition, as defined in Claim 18; wherein the individual is treated for cancer.

24. Method for Treating Cancer in an Individual, according to Claim 23, characterized in that the CAR comprises: a recognition region comprising a single-chain fragment (scFv) variable region of an antifluorescein antibody; a co-stimulation domain, which is CD28, CD137 (4-1BB), CD134 (OX40) or CD278 (ICOS); and / or an activation signaling domain, which is a CD3Z T cell chain or an Fc γ receptor.

25. Method for Treating Cancer in an Individual, according to Claim 23, characterized in that the fluorescein of the bispecific adapter binds to the antifluorescein-bound T-CAR cell with affinity after exposing it, and the CAIX ligand of the bispecific adapter binds the antifluorescein-bound T-CAR cell to a cancer cell expressing CAIX after the bispecific adapter binds to a receptor on that cancer cell expressing CAIX with affinity. Petition 870250079905, dated 05 / 09 / 2025, pp. 181 / 186 7 / 8 26. Method for Treating Cancer in an Individual, according to Claim 23, characterized in that (i) and (ii) are administered simultaneously or sequentially, in any order, by the same route or by different routes.

27. Method for Treating Cancer in an Individual, according to any one of Claims 23 to 26, characterized in that (i) and (ii) are administered intravenously.

28. Method for Treating Cancer in an Individual, according to any one of Claims 23 to 27, characterized in that it further comprises obtaining images of the cancer in the individual.

29. Method for Treating Cancer in an Individual, according to Claim 28, characterized in that obtaining cancer images comprises optical imaging, positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

30. Method for Treating Cancer in an Individual, according to any one of Claims 23 to 29, characterized in that the cancer is a cancer that expresses CAIX.

31. Method for Treating Cancer in an Individual, according to any of Claims 23 to 30, characterized in that the cancer is ovarian cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer, or clear cell renal cell carcinoma, as optionally, stage 3-4 clear cell renal cell carcinoma.

32. Method for Enhancing Chimeric Antigen Receptor (CAR) T-Cell Activation, characterized in that it comprises: Petition 870250079905, dated 05 / 09 / 2025, pp. 182 / 186 8 / 8 providing a bispecific adapter, as defined in any of Claims 1 to 17, or a pharmaceutical composition, as defined in Claim 18; and exposing antifluorescein CAR-T cells or a pharmaceutical composition comprising antifluorescein CAR-T cells and a pharmaceutically acceptable excipient or carrier to the bispecific adapter or pharmaceutical composition; wherein the CAR-T cell exhibits increased activation against cancer cells after exposure, compared to a CAR-T cell not exposed to the bispecific adapter.

33. Method for Increasing Chimeric Antigen Receptor T Cell Activation, according to Claim 32, characterized in that antifluorescein CAR-T cells are in the systemic circulation of an individual when exposed to the bispecific adapter.

34. Method for Enhancing T-Cell Activation of Chimeric Antigen Receptor, according to Claim 32, characterized in that the cancer cells are cancer cells expressing CAIX. Petition 870250079905, dated 05 / 09 / 2025, pp. 183 / 186

Images