Blood filtration device and method for treating cancer patients having circulating tumor cells and soluble targets

Abstract

In accordance with at least one aspect of this disclosure, a method for treating a cancer patient via a blood filtration procedure comprises obtaining blood from a patient having a cancer: wherein the blood has at least one type of circulating tumor cells (CTCs) or at least one type of soluble targets; contacting the blood with a modified membrane to remove the CTCs or the soluble targets; wherein the modified membrane comprises at least one type of ligand composition attached to at least one of its surfaces.

Description

[0001] Docket No. 1515138.118WO2

[0002] BLOOD FILTRATION DEVICE AND METHOD FOR TREATING CANCER PATIENTS HAVING CIRCULATING TUMOR CELLS AND SOLUBLE TARGETS IN

[0003] BLOOD

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 733,672 (Atty Doc. No. LL 1515138.118US0), filed on December 13, 2024. The disclosures of the applications are incorporated herein by reference.

[0006] SEQUENCE LISTING

[0007] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on December 6th2025, is entitled “1515138_118US2_SL.xml”, and is 6069 bytes in size.

[0008] FIELD OF THE INVENTION

[0009] The present invention relates to blood filtration device and method for treatment of a cancer patient having circulating tumor cells and cancer-related soluble targets in his or her blood.

[0010] BACKGROUND

[0011] Circulating tumor cells (CTCs) are cancer cells that shed from primary or metastatic tumors into the bloodstream, serving as a critical intermediary in the process of metastasis. Metastasis, the spread of cancer to distant organs, is a hallmark of malignancy and a leading cause of cancer-related mortality. CTCs navigate the complex microenvironment of the bloodstream, evading immune surveillance and resisting mechanical stress. Once they reach distant sites, they extravasate and establish secondary tumors, aided by interactions with the local tissue microenvironment. CTCs are valuable therapeutic targets as well as diagnostic and prognostic tools in oncology. Docket No. 1515138.118WO2

[0012] Moreover, CTCs from the same patient can express varying levels or combinations of cell surface receptors, leading to a diverse population of CTCs with different molecular characteristics, which can impact their behavior and response to therapy; essentially, not all CTCs from a single patient will have the same receptor profile, contributing to tumor heterogeneity. This tumor heterogeneity poses significant challenges to cancer treatment by allowing subsets of cancer cells to adapt and resist therapies that may effectively target other parts of the tumor. Heterogeneity also complicates the identification of universal biomarkers for diagnosis and therapy.

[0013] In addition to CTCs, many cancer patients have cancer-related soluble targets in their plasma due to proteolytic cleavage, over-expression, or aberrant expression. While cell- associated targets can be used for cancer diagnostic and treatment drugs, when soluble in plasma they also act as a sink for the drugs because they would sequester an injected drug before it reaches the site of disease e.g. tumor site, and change its pharmacokinetics and clearance. In many cases, the concentration of soluble targets in plasma is high enough that it creates an unfavorable gradient between the blood and the cancer target sites. In certain instances, the soluble targets in plasma can be too large to be filtered by the kidney. As such, these soluble targets in a cancer patient’s blood significantly impact the efficiency of cancer treatment.

[0014] Therefore, lowering and / or removing the CTCs and / or cancer-related soluble targets from the patient’s blood may effectively contribute to the treatment of cancers.

[0015] Accordingly, there remains a need in the art for device and method for diagnosis and treatment of metastatic tumors by capturing, removing, and / or lowering CTCs as well as soluble targets from bloodstream of a patient.

[0016] SUMMARY OF INVENTION

[0017] The present invention is directed, at least in part, to a method for treating a cancer patient via a blood filtration procedure. The method comprises obtaining blood from a patient having a cancer; wherein the blood has at least one type of circulating tumor cells (CTCs) or at least one type of soluble targets; contacting the blood with a modified non-porous membrane to remove Docket No. 1515138.118WO2 the CTCs or the soluble targets; wherein the modified membrane comprises at least one type of ligand composition attached to at least one of its surfaces.

[0018] In one embodiment, the at least one type of CTCs has at least one type of receptor on their surfaces.

[0019] In one embodiment, the ligand composition comprises at least one type of targeting ligand, wherein the at least one type of targeting ligand binds to the at least one type of receptor on the CTCs or at least one type of soluble targets.

[0020] In one embodiment, the at least one type of receptor comprises a first type of receptor, a second type of receptor, and a third type of receptor.

[0021] In one embodiment, the at least one type of targeting ligand comprises a first type of targeting ligand, a second type of targeting ligand, and a third type of targeting ligand, wherein the first type of targeting ligand binds to the first type of receptor; the second type of targeting ligand binds to the second type of receptor; and the third type of targeting ligand binds to the third type of receptor.

[0022] In one embodiment, at least one of the first type of targeting ligand, the second type of targeting ligand, and the third type of targeting ligand binds to the at least one type of soluble targets.

[0023] In one embodiment, the at least one ligand composition comprises a conjugate having more than one targeting ligand.

[0024] In one embodiment, the conjugate comprises a nexus, a first targeting ligand, a second targeting ligand, and a third targeting ligand conjugated to the nexus.

[0025] In one embodiment, the first targeting ligand binds to a first type of receptor on the CTCs; the second targeting ligand binds to a second type of receptor on the CTCs; and the third targeting ligand binds to a third type of receptor on the CTCs.

[0026] In one embodiment, the at least one of the first targeting ligand, second targeting ligands, and third targeting ligand binds to the at least one type of soluble targets.

[0027] In one embodiment, the first targeting ligand comprises JMV594, the second targeting ligand comprises KUE, and the third targeting ligand comprises FAPI.

[0028] In one embodiment, the first targeting ligand comprises cRGDyK, the second targeting ligand comprises DOTATATE, and the third targeting ligand comprises EpCAM. Docket No. 1515138.118WO2

[0029] In one embodiment, the conjugate is a zwitterionic multimeric targeted anti-cancer conjugate.

[0030] In one embodiment, the at least one ligand composition comprises a targeting ligand binds to FAPI, a targeting ligand binds to a5pi, a targeting ligand binds to GRPR, and a targeting ligand binds to MUC1.

[0031] In one embodiment, the conjugate comprises a nexus conjugates to a targeting ligand binds to FAPI, a targeting ligand binds to a5pi, a targeting ligand binds to GRPR, and a targeting ligand binds to MUC1.

[0032] In one embodiment, both of the at least one type of circulating tumor cells (CTCs) and the at least one type of soluble targets are removed from the blood.

[0033] In one embodiment, the blood is whole blood of the patient.

[0034] In one embodiment, the CTCs are captured by the modified membrane after the blood contacts the modified membrane, such that an amount of CTCs is lowered after the blood contacts the modified membrane.

[0035] In one embodiment, the method further comprises collecting the blood after it contacts the modified membrane for a contacting period; and returning the collected blood to the patient.

[0036] In another aspect of the invention, a blood filtration device for treating a cancer patient comprises a casing; and a membrane cartridge having one or more modified non-porous membranes; wherein the membrane cartridge is removably disposed inside the casing; wherein the modified membrane comprises at least one type of conjugate attached to at least one of its surfaces.

[0037] In one embodiment, the conjugate comprises a nexus and multiple targeting ligands linked to the nexus.

[0038] In one embodiment, the multiple targeting ligands comprise a first targeting ligand, a second targeting ligand, and a third targeting ligand.

[0039] In one embodiment, the first targeting ligand comprises JMV594, the second targeting ligand comprises KUE, and the third targeting ligand comprises FAPI.

[0040] In one embodiment, the first targeting ligand comprises cRGDyK, the second targeting ligand comprises DOTATATE, and the third targeting ligand comprises EpCAM. Docket No. 1515138.118WO2

[0041] In one embodiment, the multiple targeting ligands comprise a first targeting ligand binds to FAP, a second targeting ligand binds to a501, and a third targeting ligand binds to GRPR, and a fourth targeting ligand binds to MUC1.

[0042] In one embodiment, the conjugate is a zwitterionic multimeric targeted anti-cancer conjugate.

[0043] In one embodiment, the membrane cartridge is recyclable.

[0044] DETAILED DESCRIPTION

[0045] The present invention discloses one or more blood filtration devices and blood filtration methods for capturing, lowering, and / or removing CTCs as well as cancer-related soluble targets directly from bloodstream of a patient. Once captured, the CTCs, together with any cancer- related soluble targets in plasma if also captured, can be removed from a patient’s circulation, potentially reducing the risk of metastasis and / or treating the metastasis, as well as enhancing the efficiency of cancer diagnostic and treatment drugs. Additionally, the present invention discloses an effective device and method for capturing, lowering, and / or removing CTCs having more than one biomarker of tumors on their cell membrane surfaces, and therefore improving the efficiency of treatment of tumors demonstrating heterogeneity. This is particularly important for certain tumor therapies, such as targeted alpha therapy (TAT), where reducing single metastatic cells in the blood can improve outcomes. Moreover, the present invention discloses a blood filtration soluble targets, can be removed from the blood filtration device, and being replaced with a new one or recycled for use in the same patient

[0046] Comparing to a traditional hemodialysis process, the membranes used in the present invention are non-porous membranes instead of semi-permeable membranes. Comparing to a traditional immunoadsorption process, the present invention avoids the separation step for separating plasma from the blood cells. By using the blood filtration device and the method of the present invention, the CTCs, together with the cancer-related soluble targets in plasma, are removed from the whole blood of a patient in a one-step process.

[0047] Definitions

[0048] The following definitions will be useful in understanding the instant invention. Docket No. 1515138.118WO2

[0049] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. “Consisting essentially of’, when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.

[0050] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0051] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23. 24. 25. 26. 27. 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39. 40. 41. 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0052] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive.

[0053] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0054] As used herein, the term “subject” or “patient” encompasses mammals and nonmammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes monkeys, cattle, horses, sheep, goats, swine; rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, parasites, microbes, and the like. Docket No. 1515138.118WO2

[0055] As used herein, the term “administration” or “administering” of the subject compound refers to providing a combination composition of the invention and / or prodrugs thereof to a subject in need of diagnosis or treatment.

[0056] As used herein, the term “ligand”, “targeting vector”, or “targeting ligand” refers to a moiety which is bound to or coordinated to the imaging agents or zwitterionic metal chelators of the combination compositions of the invention to provide enhanced binding to particular cell types or an increased concentration in the presence of particular cell types. In certain embodiments, the targeting vector can be bound to the imaging agents or zwitterionic metal chelators of the combination compositions in addition to the zwitterionic groups thereon. In still other embodiments, the targeting vector can be bound to the zwitterionic metal chelator in place of one or more zwitterionic groups provided that the zwitterionic metal chelator retains at least one zwitterionic group.

[0057] As used herein, the term “carrier” refers to chemical compounds or agents that facilitate the incorporation of a compound described herein into cells or tissues.

[0058] As used herein, the term “acceptable” with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated.

[0059] As used herein, the term “blood filtration” refers to the process of selectively removing specific components or substances from the blood using mechanical, chemical, or biological methods.

[0060] As used herein, the term “diluent” refers to chemical compounds that are used to dilute a compound described herein prior to delivery. Diluents can also be used to stabilize compounds described herein.

[0061] As used herein, the term “circulating tumor cells” (CTCs) refers to cancer cells that detach from a primary tumor or metastatic site and enter the bloodstream or lymphatic system of a patient. Docket No. 1515138.118WO2

[0062] As used herein, the term “combination” refers to a mixture of more than one type of compounds.

[0063] As used herein, the term “contacting” refers to the bringing together of substances in physical contact such that the substances can interact with each other. For example, when an agent is “contacted” with tissue or cells, the tissue or cells can interact with the agent, for example, allowing the possibility of binding interactions between the agent and molecular components of the tissue or cells. “Contacting” is meant to include the administration of a substance such as an agent of the invention to an organism. Administration can be, for example, oral or parenteral.

[0064] As used herein, the term “hemodialysis” or “dialysis” refers to a process involves diverting the patient’s blood to an external device, where it passes through a membrane in a dialysis filter (dialyzer). This membrane allows waste molecules, such as urea and creatinine, and excess ions to diffuse into a dialysis solution, while retaining essential components like blood cells and proteins. The cleaned blood is then returned to the body, helping maintain proper chemical balance and fluid levels.

[0065] As used herein, the term “ionic group” refers to a moiety comprising one or more charged substituents. The “charged substituent” is a functional group that is generally anionic or cationic when in substantially neutral aqueous conditions (e.g. a pH of about 6.5 to 8.0 or about physiological pH (7.4)). As recited above, examples of charged anionic substituents include anions of inorganic and organic acids such as sulfonate (-SO31), oxide, sulfinate, carboxylate, phosphinate, phosphonate, phosphate, and esters (such as alkyl esters) thereof. In some embodiments, the charged substituent is sulfonate or oxide. Examples of charged cationic substituents include quaternary ammonium ions (-NRs+) and phosphonium ions (-PRs+), where R is independently selected from C1-6 linear alkyl, C4-6 branched alkyl, C3-6 cycloalkyl, aryl, heteroaryl and arylalkyl or heteroarylalkyl. Other charged cationic substituents include protonated primary, secondary, and tertiary amines, and as well as guanidinium or amidinium or pyridinium or other protonated, alkylated or oxygenated nitrogen heterocycles. In some embodiments, the charged substituent is -N(CH3)3+. Docket No. 1515138.118WO2

[0066] As used herein, the phrase “membrane modification” refers to a process of alteration or enhancement of the membrane used in the blood filtration process to improve its functionality, biocompatibility, or therapeutic capabilities. In some embodiments, the membrane modification refers to a process of adding functional groups or molecules, such as antibodies, ligands or any conjugates having the antibodies / ligands, to target specific cells or substances in the blood, such as circulating tumor cells (CTCs), soluble targets, inflammatory mediators, or toxins.

[0067] As used herein, the phrase "non-porous membrane" and “non-semi-permeable membrane” refer to a membrane that lacks any visible pores. The phrase "semi-permeable membrane" refers to a membrane that selectively allows some substances to pass through. The semi-permeable membrane includes porous membranes with visible channels.

[0068] As used herein, the phrase “non-ionic oligomeric or polymeric solubilizing groups” refers to soluble polymers such as, for example, polyethylene glycol, polypropylene glycol, polyethylene oxide and propylene oxide copolymer, a carbohydrate, a dextran, polyacrylamide, a peptide and the like. The solubilizing group can be attached by any desired mode. The point of attachment can be, e.g., a carbon-carbon bond, a carbon-oxygen bond, or a nitrogen-carbon bond. The attachment group can be. e.g., an ester group, a carbonate group, an urea group, an alcohol group, an ether group, a sulfide group, an amino group, an alkylene group, an alkyne group, an azide group, a tetrazine, an amide group, a carbonyl group, or a phosphate group.

[0069] Some examples of solubilizing groups include polyethylene glycols, such as -(CH2CH2O)a-H, -OC(=O)O(CH2CH2O)aH, -OC(=O)O(CH2CH2O)aCH3, -O(CH2CH2O)aCH3, and -S(CH2CH2O)2CH3, “a” being an integer between about 2 and about 250. In some embodiments, “a” is 4 to 12 or 5 to 10. In further embodiments, “a” is 6, 7, or 8. Other examples of solubilizing groups include dextrans such as -OC(=O)O(dextran).

[0070] The solubilizing moiety can have an absolute molecular weight of from about 500 amu to about 100,000 amu, e.g., from about 1,000 amu to about 50,000 amu or from about 1,500 to about 25,000 amu. Docket No. 1515138.118WO2

[0071] Further examples of solubilizing groups include: -(CH2)c-(OCH2CH2)d-ORa, wherein “c” is 0 to 6, “d” is 1 to 200, and Rais H or Ci-6 alkyl. In some embodiments, “c” is 1 to 4, “d” is 1 to 10, and Rais H. In some embodiments, “d” is 6 or 7.

[0072] See WO 2008 / 017074, U.S. Ser. No. 12 / 376,243 (filed February 3, 2009), and U.S. Ser. No. 12 / 376,225 (filed February 3, 2009), each of which is incorporated herein by reference in its entirety, for a further description of suitable non-ionic oligomeric or polymeric solubilizing groups, and method for incorporating them into dyes.

[0073] It is further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

[0074] Compounds of the invention can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0075] The chemical substances represented herein by name, chemical formula, or structure are meant to include all stereoisomers, geometric isomers, tautomers, resonance structures, and isotopes of the same, unless otherwise specified.

[0076] The chemical substances described herein may be charged or include substituents with formal charges. When such chemical substances are represented as charged, it is understood that, unless otherwise specified, the charges are generally countered with an appropriate counterion. For example, chemical substances or functional groups having a charge of -I are understood to be countered with an ion have a +1 charge. Suitable counterions with +1 charge include Na+, K+, tetraalkylammonium ions, and the like. Conversely chemical substances or functional groups having a charge of +1 are understood to be countered with an ion having a -1 charge. Suitable counterions with -1 charge include F-, C1-, Br-, I-, perchlorate, acetate, trifluoroacetate, and the like. Docket No. 1515138.118WO2

[0077] As used herein, the term “whole blood” refers to blood that is drawn directly from the body of a subject and contains all its components in their natural, unaltered state, including blood cells, platelets, plasma, and etc. For patients having cancers, the whole blood may also include circulating tumors cells, cancer-related soluble targets, and other cancer-related matters.

[0078] As used herein, the term “zwitterionic group,” “zwitterionic ligand,” or “zwitterion” refer to one or more charged moieties with balanced (electrically neutral) polyionicity. The zwitterionic feature could be part of the linker and / or targeting ligand. The zwitterionic multimeric drugs of the claimed invention are decorated by one or more zwitterionic groups.. In the case of a payload that is a metal chelator, these zwitterionic groups are distinct from metal chelator cores, which typically has negative charges to chelate positively-charged metals. For example, a zwitterionic metal chelator with a chelator core of -4 that binds a +4 metal would have a total net charge of zero. Although a total net charge of zero is considered ideal, a zwitterionic metal chelator with a chelator core of -4 that binds a +2 metal, resulting in an overall charge of -2, would still be expected to exhibit improved properties in vivo because of shielding of the chelator core / metal complex by one or more zwitterionic groups. In the absence of zwitterionic groups, the molecule would have no such shielding or expanded water of hydration and would be more likely to bind non- specifically.

[0079] A particular active agent molecule may have several attached "zwitterionic groups” or charge pairs. In general, the anion portion and the cation portion of the zwitterionic group (charge pair) will be part of the same moiety, though it is possible for two ionic groups to be used as separate moieties to form a zwitterionic group. In particular embodiments, the zwitterionic group is covalently bound to the base structure via a carbon-carbon bond, a carbonoxygen bond, or a nitrogen-carbon bond. Examples of zwitterionic groups (charge pairs) that can be included in the compounds and complexes of the claimed invention include, but are not limited to, ammoniophosphates, ammoniophosphonates, ammoniophosphinates, ammoniosulfonates, ammoniosulfates, ammoniocarboxylates, ammoniosulfonamides, ammonio- sulfon-imides, guanidiniocarboxylates, pyridiniocarboxylates, pyridiniosulfonates, ammonio(alkoxy)dicyanoethenolates, ammonioboronates, sulfoniocarboxylates, phophoniosulfonates, and phosphoniocarboxylates. The charged groups in these zwitterions can be separated by suitable spacer groups like linear or branched alkyl chains, aryl or heteroaryl Docket No. 1515138.118WO2 moieties. In certain embodiments, the zwitterionic groups can be derivatives of amino acids, such as amino carboxylic acids, amino phosphonic acids, amino phosphinic acids or amino sulfonic acids, furthermore, aminoalkyl substituted sulfates or phosphates. Zwitterions can also be derivatives of betaines, such as carboxybetaines, sulfobetaines, sulfabetaines, phosphobetaines or phosphabetaines or N-oxides or derivatives of sulfamic acid. Particular examples of zwitterionic groups include ammonium sulfobetaines or N-oxides. A simple example of a zwitterionic group at physiological pH is the charge pair of a carboxylic acid (deprotonated at physiological pH) and an amine (protonated at physiological pH).

[0080] In some embodiments, the targeting ligands of the zwitterionic multimer drugs of the invention can also comprise a targeting vector for an agricultural process, chemical process, disease, or tissue- specific epitope, such as the cyclic peptide cRGDyK (aka cRGD). cRGD is a cyclic derivative of the tripeptide Arg-Gly-Asp which can be conjugated to one or more aims of the metal chelators of the invention. In still other embodiments, the targeting vector is octreotide or bombesin. In other embodiments, the targeting vector is KUE or dPSMA-617, a small molecule capable of targeting Fibroblast Activation Protein (FAP) also called FAP-inhibitor or FAPI, an amino acid or combination of amino acids, or derivatives thereof. In such embodiments, the targeting vector-conjugates can be formed in place of one or more zwitterionic groups. In certain embodiments, the targeting ligand includes one or more of LyP-1 peptide having a sequence of CGQKRTRGC (SEQ ID NO: 1) and binding to P32 for diagnosing / treating melanoma; K237 peptide having a sequence of HTMYYHHYQHHL(SEQ ID NO: 2) and binding to VEGFR-2 for diagnosing / treating breast tumor; IL4RPep-l peptide having a sequence of CRKRLDRNC(SEQ ID NO: 3) and binding to IL4R for diagnosing / treating lung tumor, breast tumor, colon tumor; mUNO peptide having a sequence of CSPGAK (SEQ ID NO: 4) and binding to CD206 for diagnosing / treating breast tumor; folate receptors for diagnosing / treating ovarian and lung cancer; GE11, a dodecapeptide, binding to epidermal growth factor receptor (EGFR or ErbBl) for diagnosing / treating tumors of epithelial origin.

[0081] An ideal active agent conjugated to a targeting vector would adopt the total net charge of the targeting vector, which is purposeful because in most cases the charges on the targeting vector are crucial for the ability to bind its target. Targeted zwitterionic multimer drugs thus retain the major advantage of minimizing non-specific binding while maximizing specific Docket No. 1515138.118WO2 binding. It should be apparent to those skilled in the art that additional charges can be added to the zwitterionic multimer drugs, if needed, to balance overall surface charge to zero.

[0082] In certain embodiments, the zwitterionic multimeric drugs comprise a reactive conjugation group. Such reactive conjugation groups are typically an activated derivative of a carboxylic acid, such as an n-hydroxysuccinimide (NHS) ester, a sulfo-NHS ester, a pentafluorophenyl (PFP) ester, a hydroxybenzotriazole (HOBt) ester, a hydroxyazabenzotriazole (HO At) ester, a tetrafluorophenyl (TFP) ester, an acid anhydride, an acid azide or an acid halide. Such reactive conjugation groups can be bound or substituted onto the chelator at any suitable structural location as would be understood by one of ordinary skill in the synthesis of such compounds. Reactive conjugation groups also include, but are not limited to, alkynes, azides, maleimides, thiols, amines, alkohols, phenols, carbonyls, phosphanes, alkenes and tetrazines.

[0083] As used herein, the phrase “core”, “central core”, “branching point” or “nexus” refers to a chemical structure which is located at the center of the drug with four substituents for binding zwitterionic linkers and / or targeting ligands.

[0084] As used herein, the phrase “linker” refers to a chemical structure connects two or more functional groups.

[0085] Other definitions appear in context throughout the disclosure.

[0086] Blood Filtration Device for Capturing CTCs and Soluble Targets

[0087] The present invention discloses a blood filtration device efficiently capturing, lowering and / or removing the CTCs and cancer-related soluble targets from a cancer patient’s blood.

[0088] The blood filtration device facilitates the extracorporeal removal of the CTCs and cancer- related soluble targets from the blood during a blood filtration process. Its design incorporates Docket No. 1515138.118WO2 key structural components to ensure efficient blood filtration while maintaining biocompatibility and minimizing complications.

[0089] In certain embodiments, the blood filtration device includes a casing, a membrane cartridge having one or more non-porous membranes disposed in the casing, ports for blood flow into and out of the casing, and any internal support structures to hold the components securely.

[0090] In certain embodiments, the blood filtration device may further include one or more pumps for pumping the blood through the device.

[0091] In certain embodiments, the blood filtration device may further include one or more sensors for monitoring the blood filtration process.

[0092] Casing

[0093] In certain embodiments, the casing of the blood filtration device is of a shape proper for disposing the membrane cartridge. In certain embodiments, the casing of the blood filtration device is cylindrical. The cylindrical shape of the casing provides a compact and ergonomic design, optimizing the flow dynamics and pressure distribution within the device.

[0094] In certain embodiments, the casing of the blood filtration device is made from medicalgrade materials, such as polycarbonate, to ensure durability and chemical resistance. In certain embodiments, the casing of the blood filtration device is made from transparent medical-grade plastic to ensure visual monitoring of blood flow and other solutions movement. Such materials also permit sterilization using gamma-rays and other techniques.

[0095] In certain embodiments, the casing is fitted with inlet and outlet ports for blood and other solutions involved in the blood filtration process, as well as clamps or connectors for integration into the extracorporeal circulation system.

[0096] In certain embodiments, the casing includes an opening for disposing and removing the membrane cartridge.

[0097] Membranes and Cartridge Docket No. 1515138.118WO2

[0098] The membrane(s) is the functional core of the blood filtration device of the present invention, as it is responsible for the selective removal of the CTCs and cancer-related soluble targets from the blood. The membrane(s) of the present invention is modified by one or more of targeting ligands or conjugates of the targeting ligands described below, such that the CTCs and cancer-related soluble targets are selectively bound to the membranes modified, and therefore being lowered and / or removed from the blood after the blood passes through the membrane cartridge.

[0099] In certain embodiments, the membranes used in the blood filtration device are non- porous membranes having no visible pores. It should be noted that one major difference between the blood filtration device of the present invention and a traditional hemodialysis machine is the membranes in the casing. While the traditional hemodialysis machine uses semi-permeable membranes made of hollow fibers or other materials having visible pores so as to allow molecules to pass through the membranes, the membranes used in the present invention are non- porous membranes without visible pores.

[0100] The present device is also different from traditional immunoadsorption procedures which requires separation of plasma to remove the soluble targets therein. In the present invention, the whole blood from a cancer patient is contacted with the non-porous membranes without any preceding separation step, and both the CTCs and soluble targets in plasma can be efficiently removed by contacting the membranes as components in the whole blood.

[0101] In certain embodiments, the membranes used in the blood filtration device may include one or more semi-permeable membranes in addition to the non-porous membranes. In certain embodiments, the semi-permeable membrane selectively allows some substances to pass through. In certain embodiments, the structure of the membrane has a dense non-porous inner layer that controls selective permeability and a porous outer layer that supports the inner structure and promotes flow.

[0102] In certain embodiments, a membrane cartridge includes multiple non-porous membranes which are arranged in a certain manner and attached together by a cartridge structure. In certain embodiments, membranes, when attached together, are spaced from each other for a certain Docket No. 1515138.118WO2 distance so as to permits the passage of the blood without clogging the membrane cartridge. In certain embodiments, the arranging manner permits the efficient removing of the CTCs and soluble targets from the whole blood. In certain embodiments, the arranging manner facilitates the flow of the blood through the membrane cartridge.

[0103] In certain embodiments, the membrane cartridge includes 1-10 membranes. In certain embodiments, the membrane cartridge includes 11-20 membranes. In certain embodiments, the membrane cartridge includes 21-50 membranes. In certain embodiments, the membrane cartridge includes 51-100 membranes.

[0104] In certain embodiments, the membrane cartridge is disposable. That is, a new and unused membrane cartridge is inserted into the casing of the blood filtration device before each use, and is removed and abandoned after the use.

[0105] In certain embodiments, the membrane cartridge is recyclable and / or reuseable. That is, after each round of the use, the membrane cartridge is treated with reagents and protocols for removing the bound CTCs and soluble targets from the membrane before being reused for another round.

[0106] In certain embodiments, this recycle process is accomplished without removing the membrane cartridge from the casing. That is, the reagents for removing the bound CTCs and soluble targets from the membrane enter into the blood filtration device, flow through the membrane cartridge to remove the bound CTCs and soluble targets from the membrane, and flow out of the blood filtration device. In certain embodiments, this recycle process is accomplished by removing the membrane cartridge from the casing and treated with the removal reagents and protocols outside of the casing.

[0107] Support Structures

[0108] In certain embodiments, the internal structure of the blood filtration device includes headers at both ends of the membrane cartridge to direct blood flow into and out of the membrane cartridge. These headers are sealed to prevent blood from leaking. Docket No. 1515138.118WO2

[0109] In certain embodiments, the blood filtration device includes one or more pumps locates outside the casing for pumping the blood flow through the blood filtration device. In certain embodiments, the blood filtration device includes one or more pumps locates inside of the casing for pumping the blood flow through the blood filtration device.

[0110] Blood Filtration Process for Capturing CTCs and Soluble Targets

[0111] CTCs are rare cells shed from primary or metastatic tumors into circulation, where they play a key role in the spread of cancer, while the cancer related soluble targets in the blood impair cancer drugs’ efficiency. In the present invention, the principles of blood filtration are adapted to capture CTCs and cancer-related soluble targets from the bloodstream of the patient, offering a novel approach to cancer therapy. Traditional hemodialysis semi-permeable membranes are designed for the filtration of small molecules. However, to capture CTCs and / or soluble targets, the membranes of the present invention are structurally different from the one being used for traditional hemodialysis, and have to be engineered and / or modified with specific functionalities.

[0112] The present invention discloses modifying the surfaces of non-porous membranes with targeting ligands and / or conjugates that can selectively bind to receptors expressed on the surface of CTCs, as well as cancer-related soluble targets in plasma. These functionalized membranes enable the isolation of CTCs and cancer-related soluble targets bound to the ligands or conjugates, as blood flows through the blood filtration device, effectively "filtering out" the CTCs and cancer-related soluble targets without harming normal blood components. Once the blood passes through the membranes in the blood filtration device, the CTCs and cancer-related soluble targets in the blood are lowered or eliminated, before the filtered blood is then returned to the patient’s circulation system.

[0113] In certain embodiments, the blood filtration process of the present invention includes steps of obtaining a patient’s whole blood, contacting the whole blood with the membranes by allowing the whole blood flow through the device, and retuning the filtered blood back to the patient’s body. The CTCs and soluble targets are captured by the membranes and removed fully or partially from the whole blood during the contact of the whole blood and the membranes. Docket No. 1515138.118WO2

[0114] In certain embodiments, the whole blood of a patient is obtained by an arteriovenous (AV) fistula, a graft, or a central venous catheter.

[0115] In certain embodiments, the whole blood is drawn from the patient via a tube connected to the blood filtration device. A pump regulates the blood flow, ensuring a steady rate (usually 200-500 mL / min) for efficient filtration.

[0116] In certain embodiments, the whole blood enters the blood filtration device, which contains multiple non-porous membranes modified by the targeting ligands or conjugates having the targeting ligands. The whole blood contacts the modified membranes when flow through the membrane cartridge, while the CTCs and soluble targets bind to the targeting ligands or the conjugates attached to the modified membranes, and thus are removed from the whole blood.

[0117] In certain embodiments, after passing through the blood filtration device, the filtered blood is returned to the patient through a second tube. Anticoagulants, such as heparin, are often administered during the process to prevent clotting in the extracorporeal circuit.

[0118] In certain embodiments, the membranes are engineered and / or modified for capturing CTCs and cancer-related soluble targets directly from a patient’s whole blood.

[0119] In certain embodiments, the membranes are modified with one or more ligands as functional molecules that selectively bind to receptors expressed on the surface of CTCs, as well as cancer-related soluble targets. In certain embodiments, the membranes are modified with one or more conjugates as functional molecules that selectively bind to receptors expressed on the surface of CTCs, as well as cancer-related soluble targets.

[0120] In certain embodiments, as described below, each of the conjugates has more than one ligand conjugated together. In certain embodiments, more than one ligand of each of the conjugates are the same. In certain embodiments, more than one ligand of each of the conjugates are different. Docket No. 1515138.118WO2

[0121] In certain embodiments, the conjugates are or include small-zwitterionic molecule having a core chemical structure located at a center and two or more targeting ligands conjugated to the core via linkers.

[0122] In certain embodiments, these functionalized cartridges provide providing both therapeutic and diagnostic benefits.

[0123] In certain embodiments, the cartridges are integrated with biosensors monitoring filtration efficiency and detect abnormalities in real time.

[0124] In certain embodiments, the membranes are modified to reduce protein fouling and prevent clotting, with coatings like heparin or hydrophilic substances that improve biocompatibility.

[0125] In certain embodiments, the membranes are equipped with endotoxin-retentive layers to prevent contamination of the blood from the dialysate, ensuring patient safety.

[0126] In certain embodiments, the whole blood diverted from the patient undergoes blood filtration process of the present invention for removing the CTCs and / or soluble targets captured by the ligands or conjugates having the ligands directly from the whole blood.

[0127] This approach of the present invention offers multiple benefits. First, capturing CTCs in real-time could prevent their colonization at distant sites, thereby reducing the risk of metastasis, while removing the soluble targets in blood enhances the efficiency of cancer diagnostic and treatment drugs. Additionally, the isolated CTCs and / or soluble targets can be analyzed for their molecular and genetic characteristics, providing valuable insights for personalized cancer therapy. This dual-purpose application — therapeutic removal and diagnostic analysis — represents a promising advancement in oncology. Moreover, by modifying the membrane with conjugates having more than one ligand, the device / method of the present invention can efficiently remove CTCs having more than one type of targets on their surface and demonstrating heterogeneity.

[0128] Membrane Modification For Blood Filtration Device

[0129] Ligands and Conjugates Docket No. 1515138.118WO2

[0130] The present invention discloses membranes modified by targeting ligands and / or conjugates having one or more targeting ligands used in the blood filtration process to capture CTCs and / or soluble targets in the blood of a patients with tumors.

[0131] In certain embodiments, the one or more targeting ligands capable of binding to one or more surface receptors of CTCs, as well as one or more soluble targets, are attached to the membranes.

[0132] In certain embodiments, the one or more conjugates capable of binding to one or more surface receptors of CTCs, as well as one or more soluble targets, are attached to the membranes.

[0133] Ligands

[0134] Certain targeting ligands are effective in capturing and treating CTCs in a cancer patient’s blood. Specific embodiments of said targeting ligands and their specific structures are described in further detail in U.S. Patent Application Serial No. 18 / 640,169 filed on April 19, 2024, U.S. Patent Application Serial No. 18 / 641 ,097 filed on April 19, 2024, U.S. Patent Application Serial No. 18 / 641,149 filed on April 19, 2024, U.S. Patent Application Serial No. 18 / 732,982 filed June 4, 2024, U.S. Patent Application No. 18 / 640,620, filed April 19, 2024, U.S. Patent Application No. 19 / 209,293, filed on May 15, 2025, which are incorporated herein by reference in their entirety.

[0135] In certain embodiments, the membrane is modified with one of the following targeting ligands: cRGD, cRGDyK, PSMA (e.g., KUE or dPSMA-617), FAP, octreotide (Tyr-Phe substituted octreotide), (SEQ ID No: 5), DOTATATE, bombesin (e.g., JMV594) (SEQ ID No: 6), EpCAM peptide Pep 10.

[0136] In certain embodiments, the targeting ligands which can be used for modifying the membrane further include LyP-1 peptide having a sequence of CGQKRTRGC (SEQ ID NO: 1) and binding to P32 for diagnosing / treating melanoma; K237 peptide having a sequence of HTMYYHHYQHHL(SEQ ID NO: 2) and binding to VEGFR-2 for diagnosing / treating breast tumor; IL4RPep-l peptide having a sequence of CRKRLDRNC(SEQ ID NO: 3) and binding to IL4R for diagnosing / treating lung tumor, breast tumor, colon tumor; mUNO peptide having a Docket No. 1515138.118WO2 sequence of CSPGAK (SEQ ID NO: 4) and binding to CD206 for diagnosing / treating breast tumor; folate receptors for diagnosing / treating ovarian and lung cancer; GE11, a dodecapeptide, binding to epidermal growth factor receptor (EGFR or ErbBl) for diagnosing / treating tumors of epithelial origin.

[0137] In certain embodiments, the membrane is modified with a mixture of more than one of the targeting ligands described above.

[0138] In certain embodiments, the membrane is modified with a particular combination of the targeting ligands. In one embodiment, the particular combination of the targeting ligands includes JMV594 (bombesin), KUE (PSMA), FAPI. In one embodiment, the particular combination of the targeting ligands includes cRGDyK, DOTATATE, and anti-EpCAM peptide Pep 10.

[0139] Zwitterionic Multimeric Anti-Cancer Conjugates

[0140] Certain small zwitterionic multimeric targeted anti-cancer conjugates are effective in capturing and treating CTCs demonstrating tumor heterogeneity. Specific embodiments of said conjugates and their specific structures are described in further detail in U.S. Provisional Patent Application No. 63 / 648,305, filed May 16, 2024, U.S. Patent Application No. 19 / 209,293, filed on May 15, 2025, which are incorporated herein by reference in its entirety.

[0141] In certain embodiments, a zwitterionic multimeric anti-cancer conjugate or a mixture of multiple different zwitterionic multimeric anti-cancer conjugates is used for modifying the membrane of the blood filtration device of the present invention, such that the CTCs (together with soluble targets) in the blood are captured by the conjugates, and thus the CTCs as well as soluble targets are lowered and / or eliminated from the blood before the filtered blood is returned to the patient’s body.

[0142] In certain embodiments, the structure of the zwitterionic multimeric targeted anti-cancer conjugate includes a nexus, more than one targeting ligands, one or more payload, linkers for conjugating the targeting ligands and the payloads to the nexus. In certain embodiments, the multimeric targeted anti-cancer conjugate is attached to a membrane of a blood filtration device. Docket No. 1515138.118WO2

[0143] In certain embodiments, the zwitterionic multimeric targeted anti-cancer conjugate includes a nexus, three different targeting ligands conjugated to the nexus, one or more payloads, and zwitterionic flexible linkers conjugating each of the three different targeting ligands and the payloads and to the nexus.

[0144] In certain embodiments, the zwitterionic multimeric targeted anti-cancer conjugate is attached to the membrane by a direct or indirect attachment to the center core.

[0145] In certain embodiments, the membrane and the nexus of the multimeric targeted anticancer conjugate are attached via covalent or non-covalent bonding. In certain embodiments, the zwitterionic multimeric targeted anti-cancer conjugate is attached to the membrane by a linker between the membrane and the nexus. In certain embodiments, the linker between the membrane and the nexus is a zwitterionic flexible linker.

[0146] Specific embodiments of said zwitterionic flexible linker and their specific structures are described in further detail in U.S. Provisional Patent Application No. 63 / 903,096, filed October 21, 2025, which is incorporated herein by reference in its entirety.

[0147] In certain embodiments, the zwitterionic multimeric targeted anti-cancer conjugate is attached to the membrane by a direct or indirect attachment to the one or more of the targeting ligands.

[0148] In certain embodiments, the membrane and the one or more of the targeting ligands are attached via covalent or non-covalent bonding. In certain embodiments, the zwitterionic multimeric targeted anti-cancer conjugate is attached to the membrane by a linker between the membrane and one or more of the targeting ligands. In certain embodiments, the linker between the membrane and one or more of the targeting ligands is a zwitterionic flexible linker.

[0149] In certain embodiments, the nexus, zwitterionic flexible linkers of the small zwitterionic multimeric targeted anti-cancer conjugate form a “force field” of charge-balanced polyionicity and water of hydration such that only the targeting ligands are exposed out of the force field. Docket No. 1515138.118WO2

[0150] In certain embodiments, the multiple targeting ligands conjugated to the nexus are different from each other, such that each of the three targeting ligands binds to a particular receptor on the tumor cells. Therefore, by having multiple different targeting ligands, the small zwitterionic multimeric targeted anti-cancer conjugate is capable of binding to multiple different receptors on the tumor cells, thus significantly improves the small zwitterionic multimeric targeted anti-cancer conjugate’s capability of capturing CTCs demonstrating heterogeneity. That is, at least one of the three possible receptors should be present on every malignant cell.

[0151] In certain embodiments, the conjugate includes 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting ligands conjugated to a nexus via linkers. In certain embodiments, each of the targeting ligands in a conjugate is different from the other targeting ligands of the same conjugate. In certain embodiments, at least two of the targeting ligands in a conjugate are the same but are different from the other targeting ligands of the same conjugate. In certain embodiments, all of the targeting ligands in a conjugate are the same.

[0152] In one embodiments, the conjugate includes a nexus, JMV594 (bombesin), KUE (PSMA), FAPI as three targeting ligands coupled to the core directly or via one or more linkers.

[0153] In one embodiment, the conjugate includes a core, cRGDyK, DOTATATE, and anti- EpCAM peptide Pep 10 as three targeting ligands coupled to the core directly or via one or more linkers.

[0154] In certain embodiments, a zwitterionic multimeric targeted anti-cancer drug includes a targeting ligand binding to FAP, a targeting ligand binding to a501 , a targeting ligand binding to GDPR, and a targeting ligand binding to MUC1.

[0155] Table 1 discloses examples of zwitterionic multimeric targeted anti-cancer drug, their respective targeting ligands and payload, clinical application, and diseases to be diagnosed or treated.

[0156] Table 1. Examples of zwitterionic multimeric targeted anti-cancer drug Docket No. 1515138.118WO2

[0157] As shown in the example above, a zwitterionic multimeric targeted anti-cancer drug includes one or more targeting ligands binding to cancer-independent targets, one or more Docket No. 1515138.118WO2 targeting ligands binding to career-dependent targets, a payload which include a base payload and a specific payload.

[0158] Membrane Modification Process

[0159] Membrane surface characteristics have a significant impact on membrane hemocompatibility and antifouling properties because the blood is directly in contact with the membrane. Surface modification is the process of changing the physical, chemical, or biological features of a membrane’s surface from those of the polymeric membrane.

[0160] Physical and chemical procedures are the two primary groups of these strategies. The classification of these significant groups is determined by whether or not a chemical bonding happens through the process. Generally, physical changes do not affect the chemical structure of the membrane. The use of grinding, polishing, and thermal treatment to change the surface characteristics of a membrane, such as roughness, grain boundaries, and size, is common in these procedures. Blending and coating, on the other hand, are the most prevalent physical-process- based approaches that affect the membrane chemical composition. In contrast to physical methods, chemical techniques such as grafting, ozone & plasma treatment, photochemical & radiation reaction, radical polymerization, click chemistry, and biomimetic treatment, definitely alter the final composition of the membrane surface.

[0161] In certain embodiments, one or more modification process is conducted to attach the targeting ligands or the conjugates to the membranes used for the blood filtration process of the present invention.

[0162] Chemical Modification Process

[0163] The chemical modification involves changing the surface of membranes by using chemical reactions to modify molecules with various functional groups that are then cross-linked or immobilized with biocompatible compounds. The surface chemistry of the membranes is altered as a result of chemical modifications. It provides long-term stability and can be applied to both polymers and inorganic membrane materials. Docket No. 1515138.118WO2

[0164] In certain embodiments, an activation process is conducted on the membrane surface to activate the membrane for introducing functional groups that can form stable bonds with the targeting ligands and conjugates.

[0165] In certain embodiments, the activation process includes one or more of plasma treatment in which the membrane is exposed to ionized gas, creating reactive groups (e.g., carboxyl, hydroxyl, or amine groups) on its surface, and chemical activation including oxidation treatments and cross-linker reaction.

[0166] In certain embodiments, the present invention uses a chemical modification process to attach the targeting ligands or the conjugates to the membranes.

[0167] In certain embodiments, chemical modification process includes one or more chemical techniques: chemical grafting, UV-induced grafting, controlled radical polymerization, click chemistry, mussel-inspired chemistry, initiated chemical vapor deposition (iCVD), plasma techniques, and enzymatic treatment.

[0168] Cross-Linking Chemistry

[0169] In certain embodiments, the present invention uses a cross-linking chemistry modification process to attach the targeting ligands or the conjugates to the membranes.

[0170] In certain embodiment, the present invention uses a carbodiimide chemistry (EDC / NHS) process to attach the targeting ligands or the conjugates to the membranes. In certain embodiments, the EDC / NHS process involves using EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide) to activate carboxylic acid groups on the membrane surface; using NHS (N- hydroxy succinimide) to stabilize the reaction intermediate, allowing the targeting ligands’ amine groups to covalently bind, and leading to bonding of the targeting ligands or the conjugates to the membrane surface. The EDC / NHS process is suitable for maintaining targeting ligand’s orientation and activity.

[0171] In certain embodiment, the present invention uses a click chemistry process to attach the targeting ligands or the conjugates to the membranes. In certain embodiments, the click Docket No. 1515138.118WO2 chemistry process involves using azide-alkyne cycloaddition for a highly specific and efficient reaction, and leading to bonding of the targeting ligands or the conjugates to the membrane surface.

[0172] In certain embodiment, the present invention uses a maleimide chemistry process to attach the targeting ligands or the conjugates to the membranes. In certain embodiments, the maleimide chemistry process involves modifying maleimide-functionalized membranes by its reaction with thiol groups on the targeting ligands.

[0173] Chemical Grafting

[0174] In certain embodiments, the present invention uses a grafting modification process to attach the targeting ligands or the conjugates to the membranes.

[0175] In certain embodiment, the present invention used a UV-induced grafting process to attach the targeting ligands or the conjugates to the membranes. In certain embodiments, the UV- induced grafting process involves exposing the membrane to ultraviolet light in the presence of a monomer or bioactive molecule, leading to covalent bonding of the targeting ligands or the conjugates to the membrane surface.

[0176] In certain embodiment, the present invention used a plasma- induced grafting process to attach the targeting ligands or the conjugates to the membranes. In certain embodiments, the plasma -induced grafting process involves activating the membrane surface via plasma treatment, generating functional groups (e.g., hydroxyl, carboxyl) that can form covalent bonds with peptides, leading to covalent bonding of the targeting ligands or the conjugates to the membrane surface.

[0177] In certain embodiment, the present invention used a high-energy radiation grafting process to attach the targeting ligands or the conjugates to the membranes. In certain embodiments, the high-energy radiation grafting process involves using high-energy radiation, such as gamma rays or electron beams, to create reactive sites on the membrane surface, facilitating attachment of the targeting ligands or the conjugates to the membrane surface. Docket No. 1515138.118WO2

[0178] Physical Adsorption

[0179] In certain embodiment, the present invention used a physical adsorption process to attach the targeting ligands or the conjugates to the membranes. In certain embodiments, the physical adsorption process relies on electrostatic or hydrophobic interactions between the targeting ligands / conjugates and the membrane surface to attach the targeting ligands or the conjugates to the membrane surface.

[0180] Affinity-Based Immobilization

[0181] In certain embodiment, the present invention used an affinity-based immobilization process to attach the targeting ligands or the conjugates to the membranes. In certain embodiments, the affinity-based immobilization process involves biotin- streptavidin interaction which is a highly specific and strong non-covalent method. Biotinylated antibodies are captured on streptavidin-coated membranes.

[0182] EXAMPLES

[0183] Example 1: Mixture of Targeting Ligands

[0184] In one embodiment, a combination of the targeting ligands includes JMV594 (bombesin), KUE (PSMA), and FAPI, and the combination of targeting ligands is immobilized on the surfaces of membranes in the blood filtration device.

[0185] Whole blood from a patient containing CTCs having surface receptors binding to one or more of JMV594 (bombesin), KUE (PSMA), and FAPI and / or soluble targets binding to the same are contacted with one or more non-porous membranes having JMV594 (bombesin), KUE (PSMA), and FAPI immobilized on the surface(s). The CTCs and the soluble targets are captured by the one or more membranes , and thus are either lowered in the patient’s blood, or completely eliminated from the patient’s blood.

[0186] In one embodiment, a combination of the targeting ligands includes cRGDyK, DOTATATE, and anti-EpCAM peptide Pep 10, the combination of targeting ligands is immobilized on the surfaces of membranes in the hemodialyzer. Docket No. 1515138.118WO2

[0187] Whole blood from a patient containing CTCs having surface receptors binding to one or more of cRGDyK, DOTATATE, and anti-EpCAM peptide Pep 10 and / or soluble targets binding to the same are contacted with one or more non-porous membranes having cRGDyK, DOTATATE, and anti-EpCAM peptide Pep 10 immobilized on the surface(s). The CTCs and the soluble targets are captured by the one or more membranes in the hemodialyzer, and thus are either lowered in the patient’s blood, or completely eliminated from the patient’s blood.

[0188] In one embodiment, a combination of the targeting ligands includes a targeting ligand binding to FAP, a targeting ligand binding to a5pi, a targeting ligand binding to GDPR, and a targeting ligand binding to MUC1 and the combination of targeting ligands is immobilized on the surfaces of membranes in the blood filtration device.

[0189] Whole blood from a patient containing CTCs having one or more of the surface receptors FAP, a5pi, GDPR, MUC1 and / or one or more soluble targets of the same are contacted with one or more non-porous membranes having targeting ligands immobilized on the surface(s), which binding to FAP, a501, GDPR, MUC1. The CTCs and the soluble targets are captured by the one or more membranes in the hemodialyzer, and thus are either lowered in the patient’ s blood, or completely eliminated from the patient’s blood.

[0190] Example 2: Conjugates Having Multiple Targeting Ligands

[0191] In one embodiment, a JMV594-KUE-FAPI conjugate includes a nexus, JMV594 (bombesin), KUE (PSMA), FAPI as three targeting ligands coupled to the nexus directly or via one or more linkers (e.g. zwitterionic flexible linkers).

[0192] In one embodiment, a cRGDyK-DOTATATE-EpCAM conjugate includes a nexus, cRGDyK, DOTATATE, and anti-EpCAM peptide Pep 10 as three targeting ligands coupled to the nexus directly or via one or more linkers (e.g. zwitterionic flexible linkers).

[0193] In one embodiment, a FAPI, anti- a5pi, anti- GDPR, and anti-MUCl conjugate includes a nexus, a targeting ligand binding to FAP, a targeting ligand binding to a5pi. a targeting ligand Docket No. 1515138.118WO2 binding to GDPR, and a targeting ligand binding to MUC1 as four targeting ligands coupled to the nexus directly or via one or more linkers (e.g. zwitterionic flexible linkers).

[0194] The JMV594-KUE-FAPI conjugate, the cRGDyK-DOTATATE-EpCAM conjugate, the FAPE anti- a5 1, anti- GDPR, and anti-MUCl conjugate, or a combination thereof is immobilized on the surfaces of non-porous membranes in the blood filtration device.

[0195] Whole blood from a patient containing CTCs having surface receptors binding to one or more of cRGDyK, DOTATATE, anti-EpCAM peptide PeplO, JMV594 (bombesin), KUE (PSMA), FAPI , anti- a501, anti- GDPR, anti-MUCl, and / or soluble targets binding to the same are contacted with one or more non-porous membranes having the JMV594-KUE-FAPI conjugate, the cRGDyK-DOTATATE-EpCAM conjugate, and / or the FAPI, anti- ( 5|31 , anti- GDPR, and anti-MUCl conjugate immobilized on the surface(s). The CTCs and / or soluble targets are captured by the one or more membranes in the blood filtration device, and thus are either lowered in the patient’s blood, or completely eliminated from the patient’s blood.

[0196] Example 3: Protocols for attaching the targeting ligands or conjugates to the membrane of a blood filtration process

[0197] In certain embodiment, one or more conjugates and / or the combinations of targeting ligands are attached to the membranes in the blood filtration device using the carbodiimide chemistry process.

[0198] Carbodiimide chemistry is widely used for covalent attachment of ligands (e.g., peptides, antibodies, or other biomolecules) to surfaces such as hemodialysis membranes by coupling carboxyl (-COOH) groups on the surface to primary amine (-NH2) groups in the targeting ligand.

[0199] Materials Required

[0200] 1. Membrane Material: non-porous membrane with available carboxyl groups (or pretreated to introduce them). Docket No. 1515138.118WO2

[0201] 2. Carbodiimide Reagent: o EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride)

[0202] 3. Stabilizing Agent: o NHS (N-hydroxy succinimide) or Sulfo-NHS (for aqueous solubility)

[0203] 4. Ligands / Conjugates: The combinations of targeting ligands in Example 1, or the JMV594-KUE-FAPI conjugate, the cRGDyK-DOTATATE-EpCAM conjugate, and / or the FAPI, anti- a501, anti- GDPR, and anti-MUCl conjugate in the Example 2.

[0204] 5. Buffer Solutions: o MES buffer (2-(N-morpholino)ethanesulfonic acid), pH 4.5-6.0 o PBS (phosphate-buffered saline), pH 7.2-7.4

[0205] 6. Deionized Water.

[0206] 7. Additional Reagents (Optional): Blocking agents like BSA or ethanolamine to prevent non-specific interactions.

[0207] Equipment Required

[0208] 1. Stirring plate and magnetic stirrer.

[0209] 2. pH meter.

[0210] 3. Glassware (beakers, flasks, etc.).

[0211] 4. Pipettes and tips.

[0212] 5. Filtration system setup for washing.

[0213] Protocol Steps

[0214] 1. Preparation of the Membrane

[0215] 1. Rinse the membrane with deionized water to remove any surface impurities.

[0216] 2. If carboxyl groups are not present on the non-porous membrane, pre-treat with methods such as: o Plasma oxidation to introduce carboxylic acid groups. o Functionalization with carboxyl-containing molecules. Docket No. 1515138.118WO2 ation of Carboxyl Groups Dissolve EDC in MES buffer (pH 4.5-6.0) to a final concentration of 0.1-0.2 M. Add NHS or Sulfo-NHS to the solution to a final concentration of 0.05-0.1 M. Stir the solution gently until fully dissolved. Immerse the membrane in the activation solution and incubate for 15-30 minutes at room temperature with gentle agitation. This step forms a reactive NHS ester on the membrane. nds / Conjugates Coupling Prepare the ligands / conjugates solution: o Dissolve the ligands / conjugates in PBS (pH 7.2-7.4) or a suitable buffer to a concentration of 0.1-1 mg / mL. Remove the membrane from the activation solution and rinse briefly with PBS to remove excess EDC / NHS. Immediately immerse the activated membrane in the ligands / conjugates solution. Incubate for 1-2 hours at room temperature or at 4°C overnight for optimal coupling. ing Unreacted Sites (Optional) After coupling, rinse the membrane thoroughly with PBS to remove unbound ligand. Block remaining active sites on the membrane by immersing it in 0.1 M ethanolamine (pH 7.5) or 1% BSA solution in PBS for 30 minutes at room temperature. Wash the membrane thoroughly with PBS to remove blocking agents. ing and Storage Wash the membrane with: o PBS to remove any unreacted reagents. o Deionized water for final cleaning. Store the membrane in a sterile buffer (e.g., PBS with 0.01% sodium azide) at 4°C until use. Docket No. 1515138.118WO2

[0217] OTHER EMBODIMENTS

[0218] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0219] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.

[0220] Such equivalents are intended to be encompassed by the following claims.

Claims

Docket No. 1515138.118WO2What is claimed is:

1. A method for treating a cancer patient via a blood filtration procedure, the method comprising: obtaining blood from a patient having a cancer; wherein the blood has at least one type of circulating tumor cells (CTCs) or at least one type of soluble targets; contacting the blood with a modified non-porous membrane to remove the CTCs or the soluble targets; wherein the modified membrane comprises at least one type of ligand composition attached to at least one of its surfaces.

2. The method of claim 1, wherein the at least one type of CTCs has at least one type of receptor on their surfaces.

3. The method of claim 2, wherein the ligand composition comprises at least one type of targeting ligand, wherein the at least one type of targeting ligand binds to the at least one type of receptor on the CTCs or at least one type of soluble targets.

4. The method of claim 3, wherein the at least one type of receptor comprises a first type of receptor, a second type of receptor, and a third type of receptor.

5. The method of claim 4, wherein the at least one type of targeting ligand comprises a first type of targeting ligand, a second type of targeting ligand, and a third type of targeting ligand, wherein the first type of targeting ligand binds to the first type of receptor; the second type of targeting ligand binds to the second type of receptor; and the third type of targeting ligand binds to the third type of receptor.

6. The method of claim 4, wherein at least one of the first type of targeting ligand, the second type of targeting ligand, and the third type of targeting ligand binds to the at least one type of soluble targets.Docket No. 1515138.118WO27. The method of claim 2, wherein the at least one ligand composition comprises a conjugate having more than one targeting ligand.

8. The method of claim 7, wherein the conjugate comprises a nexus, a first targeting ligand, a second targeting ligand, and a third targeting ligand conjugated to the nexus.

9. The method of claim 8, wherein the first targeting ligand binds to a first type of receptor on the CTCs; the second targeting ligand binds to a second type of receptor on the CTCs; and the third targeting ligand binds to a third type of receptor on the CTCs.

10. The method of claim 9, wherein at least one of the first targeting ligand, second targeting ligands, and third targeting ligand binds to the at least one type of soluble targets.

11. The method of claim 9, wherein the first targeting ligand comprises JMV594, the second targeting ligand comprises KUE, and the third targeting ligand comprises FAPI.

12. The method of claim 9, wherein the first targeting ligand comprises cRGDyK, the second targeting ligand comprises DOTATATE, and the third targeting ligand comprises EpCAM.

13. The method of claim 6, wherein the conjugate is a zwitterionic multimeric targeted anticancer conjugate.

14. The method of claim 1, wherein the at least one ligand composition comprises a targeting ligand binds to FAPI, a targeting ligand binds to a5pi, a targeting ligand binds to GRPR, and a targeting ligand binds to MUC1.

15. The method of claim 2, wherein the conjugate comprises a nexus conjugates to a targeting ligand binds to FAPI, a targeting ligand binds to a501, a targeting ligand binds to GRPR, and a targeting ligand binds to MUC1.Docket No. 1515138.118WO216. The method of claim 1 , wherein both of the at least one type of circulating tumor cells (CTCs) and the at least one type of soluble targets are removed from the blood.

17. The method of claim 1, wherein the blood is whole blood of the patient.

18. The method of claim 2, wherein the CTCs are captured by the modified membrane after the blood contacts the modified membrane, such that an amount of CTCs is lowered after the blood contacts the modified membrane.

19. The method of claim 1 further comprises: collecting the blood after it contacts the modified membrane for a contacting period; and returning the collected blood to the patient.

20. A blood filtration device for treating a cancer patient, the device comprising: a casing; and a membrane cartridge having one or more modified non-porous membranes; wherein the membrane cartridge is removably disposed inside the casing; wherein the modified membrane comprises at least one type of conjugate attached to at least one of its surfaces.

21. The blood filtration device of claim 20, wherein the conjugate comprises a nexus and multiple targeting ligands linked to the nexus.

22. The blood filtration device of claim 21, wherein the multiple targeting ligands comprise a first targeting ligand, a second targeting ligand, and a third targeting ligand.

23. The blood filtration device of claim 22, wherein the first targeting ligand comprises JMV594, the second targeting ligand comprises KUE, and the third targeting ligand comprises FAPI.Docket No. 1515138.118WO224. The blood filtration device of claim 24, wherein the first targeting ligand comprises cRGDyK, the second targeting ligand comprises DOTATATE, and the third targeting ligand comprises EpCAM.

25. The blood filtration device of claim 21, wherein the multiple targeting ligands comprise a first targeting ligand binds to FAP, a second targeting ligand binds to a5pi, and a third targeting ligand binds to GRPR, and a fourth targeting ligand binds to MUC1.

26. The blood filtration device of claim 20, wherein the conjugate is a zwitterionic multimeric targeted anti-cancer conjugate.

27. The blood filtration device of claim 20, wherein the membrane cartridge is recyclable.

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