Treatment for disease modification

Abstract

Systems and methods are disclosed for treating cancer in a subject comprising an extracorporeal circulation device. The systems and methods treat and return cleansed blood to the subject. The systems and methods include an extracorporeal blood circuit which treats cancer by removing cancer mediators, which mediators reduce or inhibit the subject's immune system.

Description

TREATMENT FOR DISEASE MODIFICATIONCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Nos. 63 / 606,316, filed December 5, 2023, 63 / 562,371, filed March 7, 2024, and 63 / 703,329, filed October 4, 2024, all of which are incorporated herein by reference for all purposes.BACKGROUND

[0002] Cancer is a difficult disease to treat. The primary cancer or primary solid tumor often responds to different treatments, with satisfactory results. However, in certain instances, the high mortality of cancer may come from metastatic disease, which originates from the primary cancer.

[0003] Once the disease spreads to distant sites such as the lung, liver, and bone, therapeutic options become limited and the treatment response unsatisfying, making this metastatic disease one of the leading causes of death in oncologic patients.

[0004] Circulating tumor cells (CTCs) are cells that can be found in the peripheral blood of patients suffering from cancer. Most patients with metastatic disease are treated with systemic agents, which prolong survival and improve symptoms but are typically not curative, and patients are unable to achieve long-term survival. In addition, it is well known that cytostatic drugs show a low safety profile and severe side effects, since they are not specific to tumor cells (Anttila JV, et al., PLoS Comput Biol. 2019 Nov; 15(11)).

[0005] In recent years, immunotherapy has become an important focus for cancer treatment, and it appears that immunotherapy combined with classical treatments, such as surgery, radiotherapy, and chemotherapy, can better improve patient survival rates (See, Zhong X, et al., Mol Cancer. 2020 Jan 24;19(I): 15).

[0006] Despite the advances to date of immunotherapy treatments, there remains a need for new and improved immunotherapy treatments. The present disclosure satisfies this need and offers other advantages as well.BRIEF SUMMARY

[0007] The present disclosure provides systems and methods for treating cancer in a subject, the system comprising: an extracorporeal circulation device that is for treating and returning a cleansed blood sample of the subject, and which system includes a blood circuit, a cartridge comprising adsorption media and a blood pump. The extracorporeal blood circuit treats cancer by removing cancer mediators, which reduce or overwhelm or inhibit the subject’s immune system, to thereby treat the cancer.

[0008] In one embodiment, the disclosure provides an extracorporeal method for treating a cancer in a subject in need thereof, the method comprising: treating the subject by contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent such as heparin, heparan sulfate or mixtures thereof, with a whole blood sample from the subject to form a cleansed blood sample, wherein the adsorption media has a binding affinity for circulating cancer cells and mediators that inhibit an immune response; and reducing the inhibition of the immune response by increasing the bioavailability of immune cells by removing the cancer mediators in the whole blood sample; and returning the cleansed blood sample to the subject to thereby treat the cancer.

[0009] In certain aspects, the cancer is a solid tumor cancer. The solid tumor cancers include, but are not limited to, lung, colorectal, prostate, testicular, pancreatic, adenocarcinoma, renal, breast, ovarian, endometrial cervical, urothelial, thyroid, stomach, or liver cancer.

[0010] In certain aspects, the method disclosed herein treat cancer metastasis.

[0011] In certain aspects, the method reduces or retards inhibition of the subject’s immune system.

[0012] In certain aspects, the method removes circulating tumor cells, circulating cancer stem cells, noncellular debris and other cancer byproducts and / or mediators from the blood sample.

[0013] In certain aspects, the method reduces or eliminates T-cell exhaustion and Regulatory T-cells.

[0014] In one embodiment, the present disclosure provides a method for treating a cancer in a subject in need thereof, the method comprising: treating the subject by contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent with a whole blood sample from the subject ex vivo to form a cleansed sample, wherein the adsorption media has a binding affinity for circulating cancer cells and mediators that inhibit an immune response; and reducing the inhibition of the immune response by increasing the bioavailability of immune cells to thereby treat the cancer.

[0015] In certain aspects, the method increases local inflammation.

[0016] In certain aspects, the cancer is a solid tumor cancer or a metastatic lesion.

[0017] In certain aspects, the solid tumor cancer or metastatic lesion is selected from lung, colorectal, prostate, testicular, pancreatic, adenocarcinoma, renal, breast, ovarian, endometrial cervical, urothelial, thyroid, stomach, liver cancer, and / or melanoma.

[0018] In another embodiment, the present disclosure provides a method for profiling disease characteristics and / or functional activity of a subject having a cancer, the method comprising: contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent with a whole blood sample from the subject ex vivo to make an adhering complex between the adsorption media and cancer cells; releasing the adhering complex of cancer cells; and interrogating the cancer cells to generate a disease profile.

[0019] In certain aspects, the disease profile comprises a gene expression profile indicative of diagnosis and prognosis of the cancer in the subject.

[0020] In certain aspects, the method comprises selecting a treatment regimen or monitoring disease status by evaluating the likelihood of efficacious treatment or the status of treatment of a subject having a cancer.

[0021] In another embodiment, the present disclosure provides a method for inhibiting neovascularization of a solid tumor in a subject in need thereof, the method comprising:treating the subject by contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent comprising heparin, heparan sulfate or a mixture thereof, with a whole blood sample from the subject conveyed through an extracorporeal circuit to form a cleansed blood sample, wherein the adsorption media has a binding affinity for vascular endothelial growth factor (VEGF) such as VEGF- A; and reducing the amount of VEGF (e.g., VEGF -A) in the whole blood sample to inhibit neovascularization; and returning the cleansed blood sample to the subject.

[0022] In still another embodiment, the present disclosure provides a method for treating sepsis in a subject in need thereof, the method comprising: treating the subject by contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent with a whole blood sample from the subject ex vivo to form a cleansed sample, wherein the adsorption media has a binding affinity for inflammatory mediators that inhibit an immune response; and reducing the inhibition of the immune response by increasing the bioavailability of immune cells to thereby treat sepsis.

[0023] These and other aspects, objects and embodiments will become more apparent with the detailed description and figures that follow.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 illustrates an embodiment of the present disclosure.

[0025] FIG. 2 illustrates an embodiment of the present disclosure.

[0026] FIG. 3 A-B illustrate an embodiment of the present disclosure.

[0027] FIG. 4A-B illustrate an embodiment of the present disclosure.

[0028] FIG. 5A-D illustrate an embodiment of the present disclosure.

[0029] FIG. 6 illustrates an embodiment of the present disclosure.

[0030] FIG. 7A-C illustrate an embodiment of the present disclosure.

[0031] FIG. 8A-B illustrate an embodiment of the present disclosure.

[0032] FIG. 9 illustrates an embodiment of the present disclosure.

[0033] FIG. 10 shows a liquid biopsy of blood samples collected at 0, 30 and / or 45 minutes after initiation of Seraph® treatment showed rapid reduction of live, malignant tumor cells to below the 10 CTC / mL detection limit (assumed to be zero concentration) in 6 of the 10 patients. Mean CTC reduction after Seraph® 100 treatment for all patients was 91 ± 11.9%. Patient 01 shown here with exponential fit.

[0034] FIG. 11 shows an optical micrograph of multiple circulating tumor cells (CTCs) bound to a single adsorbent bead removed from a Seraph® 100 Filter after a clinical treatment, with staining to enhance visibility.

[0035] FIG. 12 illustrates a system of the present disclosure.DETAILED DESCRIPTIONI. Definitions

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein the following terms have the following meanings.

[0037] As used herein, the terms “about” and “approximately equal” are used herein to modify a numerical value and indicate a defined range around that value. If “X” is the value, “about X” or “approximately equal to X” generally indicates a value from 0.90X to 1.10X. Any reference to “about X” indicates at least the values X, 0.90X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.10X. Thus, “about X” is intended to disclose, e.g., “0.98X.” When “about” is applied to the beginning of a numerical range, it applies to both ends of the range. Thus, “from about 6 to 8.5” is equivalent to “from about 6 to about 8.5.” When “about” is applied to the first value of a set of values, it applies to all values in that set. Thus, “about 7, 9, or 11%” is equivalent to “about 7%, about 9%, or about 11% .”

[0038] As used herein, the terms “comprising” or “comprises” are intended to mean that the compositions, devices, and methods include the recited elements, but do not exclude others. “Consisting essentially of’ refers to those elements required for a given embodiment. The phrase permits the presence of additional elements that do not materially affect the basic and novel or functional character! stic(s) of the given embodiment (e.g., compositions, devices, and methods). “Consisting of’ refers to compositions, devices, methods, and respective components thereof, as described herein, which are exclusive of any element notrecited in that description of the embodiment. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0039] As used herein, the terms “adsorbent” or “adsorption media” refer to an at least one polysaccharide such as a glycosaminoglycan, attached to (e.g., linked, coupled or bound) a substrate (e.g., solid substrate). For example, at least one polysaccharide or a glycosaminoglycan adsorbent is a polymer resin with a polysaccharide or glycosaminoglycan mixture bound thereto. The polysaccharide or glycosaminoglycan mixture includes heparin, heparan sulfate, and mixtures thereof, and, optionally, one or more additional glycosaminoglycans, such as, for example, chondroitin sulfate, dermatan sulfate, keratan sulfate, sialic acid / sialylated glycans, and / or hyaluronic acid.

[0040] As used herein, the terms “analyte” and “adsorbate” are used interchangeably to refer to any molecule which inhibits an immune cell’s natural ability to express an immune response. In the context of the present disclosure, a sample obtained from a subject suffering from cancer will contain adsorbates. When contacted with the adsorption media of the present disclosure, the adsorbates bind to the surface of the adsorption media and are thus removed from the sample (e.g., whole blood). This allows clinically detectable tumors to be recognized by the subject’s immune system.

[0041] As used herein, the term “circulating tumor cells” (CTCs) are tumor cells that shed from the primary tumor and intravasate into the peripheral blood circulation system responsible for metastasis.

[0042] As used herein, the term acute “inflammation” refers to a protective response of tissue to injury or destruction in order to eliminate or cordon off any injurious agent and the injured tissue and initiate tissue repair. Inflammation can cause pain, heat, redness, swelling, and loss of function. In the context of tumor tissue, acute inflammation is a sign the immune system recognizes the tumor tissue as being foreign and will eliminate the tumor.

[0043] As used herein, the terms “polysaccharide(s)” or “glycosaminoglycan(s),” “GAG(s),” and “glycosaminoglycan chain(s)” are used interchangeably to refer to a glycan. In certain aspects, the glycosaminoglycans are structurally diverse, long, unbranched carbohydrate polymers of from about 5 to about 200,000, or more, repeating disaccharide units (e.g., from about 1 kDa to about 100,000 kDa, or more) and are negatively charged under physiological conditions. Each repeating disaccharide unit (i.e., “A-B”) includes a hexose or a hexuronic acid (i.e., “A”) glycosidically linked to a hexosamine (i.e., “B”),wherein the geometry of the glycosidic linkage can either be in the a or P configuration and each of the A and B components of every A-B unit can be independently modified or unmodified, the details of which are provided below. Non-limiting examples of GAGs which can be used in the embodiments described herein include chondroitin, chondroitin sulfate (CS), dermatan, dermatan sulfate (DS), heparan sulfate (HS), heparin, keratin, keratan sulfate, sialic acid / sialylated glycans, and hyaluronic acid (HA), the details of which are described herein.

[0044] As used herein, the terms “heparan sulfate” and “HS” are used interchangeably to refer to the glycosaminoglycan polymer of repeating disaccharide A-B units which can include GlcA, IdoA, and IdoA2S residues as possible A components, and GlcN, GlcNAc, GlcNAc6S, GlcNS, GlcNS6S, and GlcNS3S6S residues as possible B components. In general, heparan sulfate contains about 40-70% of non-sulfated disaccharide sequences, about 30-65% of various mono-sulfated disaccharide sequences, and about 1-10% of di- and / or trisulfated disaccharide sequences. Heparan sulfate polymers may have about 65 A-sulfates per every 100 disaccharide, an A-sulfation to (9-sulfation ratio of about 2:3 to about 3:4, and an average of about 0.8 to about 1.8 sulfate groups per every disaccharide unit. The most prevalent disaccharide sequence in heparan sulfate is 4GlcAP(l— >4)GlcNAcal, which may constitute up to about 50% or more of a HS chain. While GlcA is the more common hexuronic acid, IdoA may constitute about 30% to about 50% of heparan sulfate and may be present as the non-sulfated unit 4IdoAa(l— >4)GlcNAcal and / or any mono-, di-, and / or trisulfated disaccharide units, such as, for example, 4IdoAa(l— >4)GlcNSal,4IdoAa( l ^4)GlcNAc6Sal , and 4IdoAa( l ^4)GlcNS6Sa l . Heparan sulfate polymers can have from about 20 to about 200, or more, repeating disaccharide units and can have a molecular weight of from about 10 kDa to about 100 kDa. See, e.g., Shriver, S. el al. Handb. Exp. Pharmacol. 2012, 207, 159-176; Zhang, F. et al. Chapter 3 - Glycosaminoglycans.2010. In: Richard D. Cummings, J. Michael Pierce, et l., editors. Handbook of Glycomi cs, Academic Press, 2010, 59-80; Gandhi, N. et al. Che . Biol. Drug. Des. 2008, 72, 455-482; Lindahl, U, et al. Proteoglycans and Sulfated Glycosaminoglycans. 2017, In: Varki A, Cummings RD, Esko ID, et al., editors. Essentials of Glycobiology [Internet], 3rd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory’ Press; 2015-2017. Chapter 17.

[0045] As used herein, the terms “heparin” and “Hep” are used interchangeably to refer to the glycosaminoglycan polymer of repeating disaccharide A-B units which can include GlcA, IdoA, and IdoA2S residues as possible A components, and GlcN, GlcNAc, GlcNAc6S,GlcNS, GlcNS6S, and GlcNS3S6S residues as possible B components. In general, heparin contains about 70-90% of tri-sulfated disaccharide sequences, about 10-30% of various non-, mono-, and di-sulfated disaccharide sequences, and an average of about 1.8 to about 2.8 sulfate groups per every disaccharide unit. The most prevalent disaccharide sequence in heparin is 4IdoA2Sa(l— >4)GlcNS6Sal, which may constitute up to about 70% or more of a Hep chain. Heparin polymers can have from about 15 to about 60, or more, repeating disaccharide units and can have a molecular weight of from about 10 kDa to about 35 kDa. See, e.g., Shriver, S. et al. Handb. Exp. Pharmacol. 2012, 207, 159-176; Zhang, F. et al. Chapter 3 - Glycosaminoglycans. 2010. In: Richard D, Cummings, J. Michael Pierce, et al., editors. Handbook of Glycomics, .Academic Press, 2010, 59-80; Gandhi, N. et al. Chem. Biol. Drug. Des. 2008, 72, 455-482; Lindahl, U, el al. Proteoglycans and Sulfated Glycosaminoglycans. 2017. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology [Internet], 3rd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press, 2015-2017. Chapter 17.

[0046] As used herein, the term “contacting” refers to the process of bringing into contact at least two distinct species such that they are touching or of immediate or local proximity. In the context of the instant disclosure, a sample from a subject suffering from cancer is contacted with adsorption media. As such, the analytes / adsorbates within this sample contact the adsorption media. Once in contact with the adsorption media, the analytes / adsorbates remain attached to the adsorption media, thereby removing the analytes / adsorbates from the sample.

[0047] As used herein, the term “sample” refers to any biological sample that could contain an analyte / adsorbate obtained from a subject suffering from cancer. Typically, the sample is in liquid form or can be changed into a liquid form. Non-limiting examples of samples include whole blood, serum, and plasma. In the context of the instant disclosure, a sample that has not been contacted with adsorption media is regarded as an “untreated sample.” A “treated sample” is a sample that has been contacted with adsorption media. The treated sample will contain a reduced amount of the analytes / adsorbates and, thus, can be considered “cleansed sample” or “clean sample.”

[0048] As used herein, the term “rigid polymer bead” refers to a bead, granule, pellet, sphere, particle, microcapsule, sphere, microsphere, nanosphere, microbead, nanobead,microparticle, nanoparticle, and the like that is made from a polymer resin or other biocompatible substrate material.

[0049] As used herein, “patient” or “subject” refers to a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research.

[0050] As used herein, “Reference” level, standard or profile refers to the source of the reference nucleic acid or protein (e.g., RNA, DNA, mRNA, miRNA or protein). In one aspect, the reference standards are obtained from biological samples selected from a reference healthy human subject or a population having a cancer.

[0051] As used herein, the term “diagnosing” or “diagnosis” refers to a clinical or other assessment of the condition of a subject based on observation, testing, or circumstances for identifying a subject having a disease, disorder, or condition based on the presence of at least one sign or symptom of the disease, disorder, or condition. Typically, diagnosing using the methods of the disclosure include the observation of the subject for other signs or symptoms of a cancer, a disease, disorder, or condition.

[0052] As used herein, the terms “prognosticating” or “prognosis” refers to the determination of probability, risk or possibility of developing a disease, disorder, or condition, such as cancer, or the risk of severity of the disease in a subject.

[0053] As used herein, the terms “treat” or “treating” as used herein means to administer a therapy according to the present disclosure to a subject or patient having a cancer such as contacting a whole blood sample with adsorption media, to achieve at least one positive therapeutic effect, such as, for example, complete response, complete remission, partial response, partial remission, improved overall survival, improved overall response, or stable disease. The term “treating” also includes adjuvant treatment or combination therapy of a subject. The treatment regimen for a combination of the disclosure that is effective to treat a subject may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the therapy to elicit an anti-cancer response in the subject.

[0054] The terms “treatment regimen”, “dosing protocol” and “dosing regimen” are used interchangeably to refer to the dose and timing of administration of a therapeutic agent or treatment process. The term “treating” may also encompass the term “ameliorating” which, as used herein, means a lessening or improvement of one or more symptoms as compared tonot administering a a method or regimen of the disclosure. “Ameliorating” also includes shortening or reduction in duration of a symptom.

[0055] A “control population” or “control group”, used interchangeably herein, refer to a population of individuals who are matched to the subject but who differ in some aspect, such as disease state. For example, a control group may be matched to the subject by diagnosis and treatment regimen but may differ in disease state. As an alternative example, a control group may be matched to the subject by diagnosis and treatment regimen, but may have a different disease profile, for example a different mutation status.

[0056] The extracorporeal treatment system 100 containing a filter device is shown in FIG. 1. The filter device or cartridge 102 contains for example, endpoint attached heparin. The system 100 contains a vascular access 105 to the patient 101 for removing blood to be treated. A first conduit 107 is connected to the vascular access 105 and conveys blood to the filter 102. A blood pump 115 draws the blood from the patient through the first conduit 107 via 122 to the filter 102. The filter or cartridge 102 can be SERAPH® 100 Microbind® Affinity Blood Filter from ExThera Medical Inc. An arterial pressure sensor 110 is optionally connected to the first conduit to measure the pressure of the blood leaving the patient. A pressure sensor 118 is in-line and is disposed between the blood pump 115 and the filter 102. This sensor can ensure the pressure is within a defined range and optionally alarm if out of range.

[0057] Prior to attaching the system 100 to a patient, the system can be primed. The vascular access 105 is attached to for example, a blood bag comprising saline (not shown), a hemofiltration fluid, a buffer, or a flushing fluid. Priming the system removes air from the filter and blood pump lines. Once the system is primed, the arterial line 107 is connected to the arterial end of a large bore vascular access 105 catheter, and the venous line is connected to the venous end 151 of the same vascular access catheter.

[0058] The extracorporeal treatment system 100 removes circulating tumor cells, cancer stem cells, bloodborne acellular debris, and / or other cancer mediators. The treatment regimen can be initiated by attaching the inflow port of the first conduit 107 to the vascular access 105 of the arterial limb. The blood pump 115 is capable of delivering a blood flow rate between about 50-450 mL / min. The outflow port of filter 102 contains treated or cleansed blood wherein cancer mediators have been removed and is conveyed via second conduit 125, which is connected to the venous limb access 151 via conduit 145. The secondconduit 125, which carries treated or cleansed blood and comprises a venous pressure sensor 131, an optional venous drip chamber 135 and an air trap containing an air detector 141.Although a blood pump is useful, in other embodiments, gravity can also be used.

[0059] Treatment is initiated by starting the extracorporeal pump 115 and slowly increasing the blood pump speed to achieve a blood flow rate of between about 50 to about 450 mL / min. The increase in flow is performed slowly and in conjunction with intensive monitoring of the patient’s arterial blood pressure via the pressure sensor 110 to prevent hypotension, shock, and cardiovascular collapse when initially starting an extracorporeal procedure. Furthermore, the arterial sensor 110 and venous sensor 131 pressures on the blood pump 115 are monitored to detect and correct any flow restrictions and inappropriate pressure readings in the circuit. Once a steady state blood flow has been reached, the treatment time may be extended for up to 24 hours in order to optimize sufficient exposure of the patient’s 101 blood to the filter 102 (e.g., Seraph® 100 adsorption media). The entire (e.g., 5000 mL) blood volume of the patient can be treated during one or more passes through the filter.

[0060] After the treatment period is complete, the blood that is in the extracorporeal circuit is returned back to the patient 101 in order to prevent hypotension and unnecessary wastage of the patient’s blood. A blood rinse back procedure is performed, whereby the pump 115 is paused. The arterial limb of the circuit is disconnected from the patient’s access 105 catheter and connected to a bag of normal saline (not shown). This prevents any additional blood from entering the extracorporeal circuit. The blood pump 115 is then resumed at a low flow rate of ~50mL / min and the remaining blood in the extracorporeal circuit is then rinsed back to the patient 101 through the venous limb access 151 of the circuit. When the venous limb is clear of the patient’s blood, the pump 115 is stopped, and the venous limb access 151 of the circuit is disconnected from the patient 101, thus terminating the extracorporeal procedure.

[0061] The administration of the treatment procedure can be documented in the medical record by recording the hourly blood flow rate through the blood filter. This allows the clinician to calculate the total volume of blood filtered / refiltered by the device, which is in certain aspects, how the treatment procedure is dosed.

[0062] Table 1 is merely an example of a treatment regimen, after the extracorporeal system is attached to the patient.Table 1

[0063] By way of example only, the foregoing regimen allows optimization and sufficient exposure of the patient’s blood to the adsorption media, comprising the duration and blood flow rate.

[0064] Turning now to FIG. 2, an embodiment of the present disclosure is schematically shown. As shown therein, a solid tumor 220 (e.g. primary tumor) can be for example, lung, colorectal, prostate, testicular, pancreatic, adenocarcinoma, renal, breast, ovarian, endometrial cervical, urothelial, thyroid, stomach, liver, or a melanoma lesion. The blood of a subject having a cancer will have circulating tumor cells (CTCs) 225 infiltrating the bloodstream and the CTCs being conveyed 250. The blood vessels 218 surrounding tumor will have erythrocytes 230, leukocytes 233 circulating tumor cells and cancer stem cells (CSCs) 250 and cells which form metastatic lesions 245. A filter 202 comprising a polysaccharide adsorbent media such as heparin (e.g., SERAPH® 100 Microbind® Affinity Blood Filter), will remove cancer cells and cancer mediators from whole blood to form a cleansed blood sample. The filter comprising the adsorption media 202 has a binding affinity for circulating cancer cells and mediators that inhibit an immune response from the subject. The removal of the cancer mediators reduces or eliminates the inhibition of the immune response by increasing the bioavailability of immune cells to treat the cancer by for example, reducing the tumor size. The methods of the present disclosure re-enables a robust immune response from the patient.

[0065] By removing circulating acellular debris, cancer cells and cancer stem cells 245, CTCs 250 and mediators such as Regulatory T (Treg) cells, the subject’s immune system secretes Th-1 cytokines (e.g., IFN-y, IL-2, and IL-12), recruits NK cell, and cytotoxic T lymphocytes (CTLs) to inhibit tumorigenesis. The secretion of cytokines, NK cell recruitment, and the presence of CTLs induces acute inflammation locally at the site of a cancerous tumor 220 and successfully treats the cancer. The methods reduce or reverse tumor growth, suppress tumorigenesis, and eliminate tumor cells.

[0066] In certain instances, the methods herein remove Regulatory T (Treg) cells, which are a subset of CD4+ T cells that are required for immunosuppression, and which inhibit inflammation caused by the immune response to pathogens. Treg cells can promote the development and progression of numerous types of malignancies and tumors and aid the evasiveness of the immune system. By removing Regulatory T (Treg) cells, the methods employ the subject’s immune system to reduce tumor growth, suppress tumorigenesis, and eliminate tumor cells.

[0067] In certain aspects, the methods herein target the removal of epithelial cell adhesion molecule (EpCAM). EpCAM is well-recognized as a biomarker of cancer stem cells (CSCs) and circulating tumor cells (CTCs). By targeting the removal of EpCAM, the sensitivity of tumor cells to immune surveillance is increased. By removing EpCAM, macrophage infiltration in the tumor microenvironment is increased which efficiently eliminates tumor cells using the subject’s innate immune system.

[0068] The systems and methods remove moieties that cause immunosuppression such as blocking leukocyte activation (e.g., cytotoxic T-cells, NK natural killer cells) which would otherwise interfere with the recognition and killing of cancer cells by leukocytes.

[0069] In certain aspects, the methods reduce or remove a cancer mediator such as circulating growth factors that contribute to angiogenesis of tumors, circulating cytokines that contribute to angiogenesis, circulating heparanases that degrade heparan sulfate segments on endothelial cell walls that can lead to tumor cell invasion, and circulating fibrin or thrombin that leads to venous thromboembolism. In certain instances, the cancer mediator is vascular endothelial growth factor (VEGF), such as VEGF-A, basic fibroblast growth factor (bFGF), IL-8, heparanases, pro-uPA (pro-urinary plasminogen activator protein), plasminogen, or thrombin.

[0070] In certain aspects, the circulating tumor cells (CTC), cancer stem cells and cancer mediators are adsorbed onto the adsorption media or beads during extracorporeal treatment. The methods remove and eliminate these cancer cells and mediators from circulation via capture by the filter within the extracorporeal circuit. In certain instances, CTCs are released in the blood, killer T-cells may be misdirected towards the CTCs in circulation instead of the solid tumor. This may lead to T-cell exhaustion. The extracorporeal treatment removes mediators including pathogens, circulating tumor cells (CTCs), DAMPs, and PAMPs. By clearing the bloodstream, increasing the bioavailability of killer T-cells and potentially reversing T-cell exhaustion, the treatment recruits or employs the subject’s immune system towards the local sites of infection and / or solid tumor cancers. In certain instances, the methods remove acellular debris and / or neutrophil extracellular traps (NETs), which are made of a network of extracellular strings of DNA that bind pathogenic microbes. By removing these disease agents, the process generates or facilitates an immune inflammatory response surrounding the local site of the primary tumor’s microenvironment.

[0071] The disclosure provides methods to stop tumor growth and reduce existing tumor size. FIG. 3 A shows a CT image of a lung tumor from a female patient 01 with Stage 4 lung cancer. The image illustrated in FIG. 3 A is an adenocarcinoma before blood treatment with the adsorption media described and disclosed. After 3-4 weeks of the disclosed treatment, FIG. 3B clearly shows the solid tumor reduction. In certain aspects, the methods herein will reduce the tumor size (e.g., as measured by volume in cm3) by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and / or about 100%. In certain instances, the tumor is eliminated.

[0072] FIG. 4A (before treatment) and FIG. 4B (after treatment) are closeups of FIG. 3 A and FIG. 3B, respectively. Clearly the adenoma has been reduced is size (e.g., volume).

[0073] The methods herein promote NK cell recruitment, and the presence of cytotoxic T- lymphocytes. This in turn induces acute inflammation locally at the site of a cancerous tumor and successfully treats the cancer. As shown in FIG. 5A-D, there is elevation of a patient’s immune response which includes NK cell recruitment (FIG. 5A), Helper T-Cells (FIG. 5B), cytotoxic T-lymphocytes (FIG. 5C), and CD3+cells (FIG. 5D). Helper T cells are important cells in adaptive immunity, as they activate B cells to secrete antibodies and macrophages to destroy the tumor and activate cytotoxic T cells to kill the target tumor cells.

[0074] In addition, the present disclosure provides methods that reduce or eliminate the formation of metastases by removing circulating epithelial tumor cells (CETC), which cells are considered to be responsible for metastatic activity. Using a diagnostic technique such as maintrac®, which allows a direct assessment of tumor activity, the opportunity to check the effectiveness of the adsorption media therapy can be performed, maintrac® cell counts provide information on whether a cancer therapy is effective. If the number of CETC cells decreases during therapy, this is an indication that the therapy is having a positive therapeutic effect.

[0075] In this regard, FIG. 6 shows the results of Patient 7, which indicates the number of live and dead cells over three treatment regimens. The number of live CETC cells stays relatively constant, but the number of dead CETC cells increases.

[0076] In certain aspects, adsorption media is provided packed within a container or housing, such as a column or as a filter, that is designed to hold adsorption media. In certain aspects, the adsorption media is designed so that it will not be carried away in the flowing blood (‘preventing media migration’) and permits the flow of blood past essentially all of the media’s surface.

[0077] In certain aspects, the adsorption media substrate is in the form of solid beads or particles. The ‘beads’ can be made of materials that are sufficiently rigid to resist deformation / compaction under the encountered flow rates and pressures (such as polymer beads). Resistance to deformation is an advantage to maintain the interstitial dimensions and overall free volume and subsequent low pressure drop of the packed bed contactor. The dimensional stability of the packed bed is also important in maintaining sufficient inter-bead separation to avoid filtering out blood cells. The substantial lack of accessible pores in the bulk of the substrate eliminates the need for adsorbates to diffuse into the pores prior to adsorption / binding. The adsorption sites of the present disclosure are primarily on the surface of the media and are thus positioned to be accessible to adsorbates in the blood delivered to that surface largely by convective transport. Suitable substrates need not be perfectly smooth on their surface since roughness produces a desirable increase in surface area for attachment of binding sites, e.g., by ionic or covalent bonding of heparin. Accessible internal pores with molecular dimension, on the other hand, are largely avoided to eliminate the need for molecular adsorbates to diffuse into the pores before attaching to binding sites.

[0078] In certain aspects, the adsorption media of the present disclosure comprises heparin covalently linked to the surface of a solid substrate. Various methods can be used to attach heparin to the desired substrate, such as described in a review article by Wendel and Ziemer. (See, H.P Wendel and G. Ziemer, European Journal of Cardio-thoracic Surgery 16 (1999) 342- 350). In one aspect, the heparin is linked to the solid substrate by covalent end-point attachment. This method increases the safety of the device by reducing or eliminating the release of heparin from the substrate surface that could enter the blood stream. Covalent attachment of heparin to a solid substrate provides better control of parameters such as surface density and orientation of the immobilized molecules as compared to non-covalent attachment. The surface concentration of heparin on the solid substrate can be in the range of about 1-10 pg / cm2. Covalent end-point attachment means that heparin is covalently attached to the solid substrate via the terminal residue of the heparin molecule. Heparin can also be bound at multiple points i.e., multi-point attachment.

[0079] Various kinds of beads can be used in the present disclosure. Suitable beads have sufficient size and rigidity to avoid deformation / compaction during use in the method and have sufficient surface area to be capable of being coated with heparin for use in the method.

[0080] In certain aspects, the substrate comprises a plurality of rigid polymer beads.

[0081] In certain aspects, the rigid polymer beads are rigid polyethylene beads.

[0082] The beads or other high-surface-area substrates may be made from a number of different biocompatible materials, such as natural or synthetic polymers or non-polymeric material including glasses, ceramics, and metals, that are essentially free of leachable impurities. Some exemplary polymers including polyurethane, polymethylmethacrylate, polyethylene or co-polymers of ethylene and other monomers, polyethylene imine, polypropylene, and polyisobutylene. Examples of useful substrates include nonporous Ultra High Molecular Weight PolyEthylene (UHMWPE). Other suitable beads are optionally cross-linked polystyrene, high density and low density polyethylene, silica, polyurea, and chitosan.

[0083] Methods for making such beads are known in the art. Polyethylene beads and other polyolefin beads are produced directly during the polymer synthesis process and can often be used without further size reduction. Other polymers may need to be ground or spray dried and classified, or otherwise processed to create beads of the desired size distribution and shape.

[0084] In certain aspects, the size of the channels or interstitial space between individual beads for extracorporeal blood filtration reduce or eliminate a high-pressure drop between the inlet and outlet of the cartridge, to permit safe passage of the blood cells between the individual beads in a high flow environment, and to provide appropriate interstitial surface area for binding of the polysaccharide adsorbent to the cancer mediators in the blood.

[0085] In a close packed bed of 300-micron, roughly spherical beads, an appropriate interstitial pore size is approximately 68 microns in diameter. Useful beads have a size ranging from about 100 to above 500 microns in diameter such as about 100 microns, 125 microns, 150 microns, 175 microns, 200 microns, 225 microns, 250 microns, 275 microns, 300 microns, 325 microns, 350 microns, 375 microns, 400 microns, 425 microns, 450 microns, 475 microns, and / or 500 microns. The average size of the beads can be from 150 to 450 microns. For example, polyethylene beads having an average diameter of 0.3 mm are suitable. The interstitial pore is a function of bead size. For use, the suitable beads are housed in a container, such as a column or filter housing.

[0086] In certain aspects, the beads have a diameter ranging from about 100 to about 450 microns.

[0087] In one embodiment, the present disclosure provides a method for treating a cancer in a subject in need thereof, the method comprising: treating the subject by contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent with a whole blood sample from the subject ex vivo to form a cleansed sample, wherein the adsorption media has a binding affinity for circulating cancer cells and mediators that inhibit an immune response; and reducing the inhibition of the immune response by increasing the bioavailability of immune cells to thereby treat the cancer.

[0088] In certain aspects, a substrate for the adsorption media is a solid substrate. Suitable substrates include, but are not limited to, non-porous rigid beads including polymer beads, particles, or packing, reticulated foams, a rigid monolithic bed (e.g. formed from sintered beads or particles), a column packed with woven or non-woven fabric, a column packed with a yarn or solid or hollow dense (not microporous) monofilament fibers, a spiral wound cartridge formed from flat film or dense membrane, or a combination of media such as a mixed bead / fabric cartridge. A suitable substrate for use in the present disclosure is one thatis initially microporous but becomes essentially nonporous when the surface is treated before, during or after the creation of adsorption sites, e.g., coated with a polysaccharide such as end- point-attached heparin, multipoint attachment, or ionic bound heparin.

[0089] In certain aspects, the adsorption media comprises at least one polysaccharide selected from the group of sulfated polysaccharides, glycosaminoglycans, heparin, heparan sulfate, hyaluronic acid, sialic acid, carbohydrates with mannose sequences, chitosan and combinations thereof. In certain aspects, the adsorption media comprises heparin, heparan sulfate or mixtures thereof.

[0090] In certain aspects, the surface area of the adsorption media relevant to advection kinetics is between about 5 m2and about 50 m2or even higher, such as between about 10 m2and about 20 m2.

[0091] In certain aspects, adsorption media is provided packed within a container or housing, such as a column or as a filter, that is designed to hold adsorption media. In certain aspects, the adsorption media is designed so that it will not be carried away in the flowing blood (‘preventing media migration’) and permits the flow of blood past essentially all of the media’s surface.

[0092] In certain aspects, the adsorption media substrate is in the form of solid beads or particles. The ‘beads’ can be made of materials that are sufficiently rigid to resist deformation / compaction under the encountered flow rates and pressures (such as polymer beads). Resistance to deformation is an advantage to maintain the interstitial dimensions and overall free volume and subsequent low pressure drop of the packed bed contactor. The dimensional stability of the packed bed is also important in maintaining sufficient inter-bead separation to avoid filtering out blood cells. The substantial lack of accessible pores in the bulk of the substrate eliminates the need for adsorbates to diffuse into the pores prior to adsorption / binding. The adsorption sites of the present disclosure are primarily on the surface of the media and are thus positioned to be accessible to adsorbates in the blood delivered to that surface largely by convective transport. Suitable substrates need not be perfectly smooth on their surface since roughness produces a desirable increase in surface area for attachment of binding sites, e.g., by ionic or covalent bonding of heparin. Accessible internal pores with molecular dimension, on the other hand, are largely avoided to eliminate the need for adsorbates to diffuse into the pores before attaching to binding sites.

[0093] Various kinds of beads can be used in the present disclosure. Suitable beads have sufficient size and rigidity to avoid deformation / compaction during use in the method and have sufficient surface area to be capable of being coated with heparin for use in the method.

[0094] In certain aspects, the adsorption media are beads.

[0095] In certain aspects, the substrate comprises a plurality of rigid polymer beads.

[0096] In certain aspects, the rigid polymer beads are rigid polyethylene beads.

[0097] The beads or other high-surface-area substrates may be made from a number of different biocompatible materials, such as natural or synthetic polymers or non-polymeric material including glasses, ceramics, and metals, that are essentially free of leachable impurities. Some exemplary polymers including polyurethane, polymethylmethacrylate, polyethylene or co-polymers of ethylene and other monomers, polyethylene imine, polypropylene, and polyisobutylene. Examples of useful substrates include nonporous Ultra High Molecular Weight PolyEthylene (UHMWPE). Other suitable beads are optionally cross-linked polystyrene, high density and low density polyethylene, silica, polyurea, and chitosan.

[0098] Methods for making such beads are known in the art. Polyethylene beads and other polyolefin beads are produced directly during the polymer synthesis process and can often be used without further size reduction. Other polymers may need to be ground or spray dried and classified, or otherwise processed to create beads of the desired size distribution and shape.

[0099] In certain aspects, the size of the channels or interstitial space between individual beads for extracorporeal blood filtration reduce or eliminate a high-pressure drop between the inlet and outlet of the cartridge, to permit safe passage of the blood cells between the individual beads in a high flow environment, and to provide appropriate interstitial surface area for binding of the polysaccharide adsorbent to the toxins, cytokines or pathogens in the blood. In a close packed bed of 300-micron, roughly spherical beads, an appropriate interstitial pore size is approximately 68 microns in diameter. Useful beads have a size ranging from about 100 to above 500 microns in diameter such as about 100 microns, 125 microns, 150 microns, 175 microns, 200 microns, 225 microns, 250 microns, 275 microns, 300 microns, 325 microns, 350 microns, 375 microns, 400 microns, 425 microns, 450 microns, 475 microns, and / or 500 microns. The average size of the beads can be from 150 to450 microns. For example, polyethylene beads having an average diameter of 0.3 mm are suitable. The interstitial pore is a function of bead size. For use, the suitable beads are housed in a container, such as a column or filter.

[0100] In certain aspects, the beads have a diameter ranging from about 100 to about 450 microns.

[0101] In certain aspects, the adsorption media has a macroporous structure that presents a high surface area to the blood or serum, while preventing a large pressure drop and high shear rates. In addition to the potential for damaging the blood by hemolysis, high pressure drops should be avoided because they can shut down extracorporeal circuits equipped with automatic shut offs that respond to pressure drop. The substrate may also take the form of a dense barrier membrane, in a spiral wound configuration, for example. In certain aspects, the surface of a non-porous film is modified by a coating such as by binding heparin, heparan sulphate or another adsorbent polysaccharide together with optional adsorbing groups not derived from heparin, heparan sulphate, or the adsorbent polysaccharide to the membrane’s surface.

[0102] In certain aspects, adsorption media is a surface coated solid substrate. Suitable surface coatings include heparin, polyethyleneimine (PEI), sialic acid, hyaluronic acid, polyvinylpyrrolidone (PVP), and combination thereof.

[0103] In certain aspects, other suitable forms of substrates include reticulated foams. Reticulated foams have open cells and can be made from, for example, polyurethanes and polyethylenes. Control of pore size can be achieved by controlling the manufacturing method. In general, reticulated foams can have between 3 and 100 pores / inch and can exhibit a surface area of >66 cm2.

[0104] In certain aspects, beads can be sintered into a monolithic porous structure through either chemical or physical means. Polyethylene beads can be sintered by heating the beads above their melting temperature in a cartridge and applying pressure. The resulting interstitial pore size is slightly reduced from the interstitial pore size of a packed bed of nonsintered beads of equal size. This reduction can be determined empirically and used to produce the desired final interstitial pore size.

[0105] In certain aspects, the adsorbent polysaccharide of the adsorbent media can be bound to the surface of the solid substrate (e.g., bead) by various methods, including covalentattachment or ionic attachment. The adsorption media can comprise heparin covalently linked to the surface of the solid substrate. In one embodiment, the heparin is linked to the solid substrate by covalent end-point attachment. This method increases the safety of the device by reducing or eliminating the release of heparin from the substrate surface that could enter the blood stream. Leaching of heparin by and into the blood is to be avoided because it can increase the risk of bleeding and heparin-induced thrombocytopenia. Covalent attachment of the polysaccharide, such as heparin, to a solid substrate provides better control of parameters such as surface density and orientation of the immobilized molecules as compared to non-covalent attachment. These parameters have been shown to be advantageous in order to provide optimal Antithrombin III, cytokine or pathogen binding to the immobilized carbohydrate molecules. The surface concentration of heparin on the solid substrate is often in the range of 1-10 pg / cm2. Covalent end-point attachment means that the polysaccharide, such as heparin is covalently attached to the solid substrate via the terminal residue of the heparin molecule. Heparin can also be bound to the surface at multiple points or ionic attachment. End-point attachment is beneficial due to the extended surface area for adsorbates.

[0106] In certain aspects, the heparin is full length heparin having a mean molecular weight in the range of 15-25 kDa, such as about a mean molecular weight of 21 kDa or more.

[0107] In certain aspects, the heparin has a surface concentration of 1-20 pg / cm2, such as about 5-15 pg / cm2.

[0108] In certain aspects, the heparin is full length heparin covalently attached to a solid substrate via stable secondary amino groups.

[0109] In certain aspects, a total surface area of the solid substrate is in the range of 0.5-3 m2. In other aspects, the total surface is about 5 m2to about 50 m2, such as between about 10 m2and about 20 m2.

[0110] In certain aspects, the beads may be hydrophilized prior to attachment of the polysaccharide, such as heparin, or other compounds. Possible methods of preparing the beads include acid etching, plasma treating, and exposure to strong oxidizers such as potassium permanganate.[OHl] In certain aspects, the adsorption media is sized to be larger than the inner diameter of the hollow fiber membrane.

[0112] In certain other instances, the adsorption media contains materials similar to the filter or filtration device of the Seraph® Microbind® Affinity Blood Filter (ExThera Medical Corporation, Martinez, California) , which is a filter that allows body fluids to pass over microbeads coated with molecular receptor sites that mimic the receptors on human cells which pathogens use to colonize when they invade the body. The adsorption media is a flexible platform that uses covalently-bonded, immobilized heparin or heparan sulfate for its unique binding capacity. See, for example, US Patent Nos. 8,663,148, 8,758,286 or 9,173,989, disclosing at least one polysaccharide adsorbent, or immobilized heparin, each of which is incorporated by reference.

[0113] In certain other instances, the adsorption media can be for example, similar material as the extracorporeal hemoadsorption filter device to remove cytokines from circulating blood such as a biocompatible, sorbent bead technology e.g., CytoSorb™, CytoSorbents™, Inc. CytoSorb hemoadsorption beads are polystyrene-divinylbenzene porous particles (450 pm avg. particle diameter, 0.8-5 nm pore diameter, 850 m2 / g surface area) with a biocompatible polyvinyl-pyrrolidone coating. See for example, US Patent No. 8,647,666 which claims a method of using a composition comprising polystyrene divinyl benzene copolymer and a polyvinyl pyrrolidone polymer.

[0114] In one embodiment, the disclosure provides a device, the device comprising a filtration media, which filtration media includes a combination of (i) a hollow fiber membrane and (ii) adsorption media.

[0115] In certain instances, the beads are in a fluidized bed as disclosed in PCT / US2024 / 057258, filed November 25, 2024. A “fluidized bed” refers to a solid adsorption media dispersed in a fluid and acting as a fluid. A fluidized bed refers to a mixture state of two different phases, solid and fluid. The fluidized bed behaves as a fluid material. A fluidized bed is a physical phenomenon that occurs when solid such as an adsorption media such as heparinized media, disposed within a container or cartridge under the right conditions behaves like a fluid including a gaseous fluid. For example, the adsorption media being loosely packed without intentional compression within the container, or an inert gas being pumped into the container, or the velocity of the fluid being pumped into the container is high enough to create a fluidized bed, or when pressurized fluid enters the adsorption media are all illustrative ways to achieve fluidization. In one aspect, the fluidized bed comprises solid particles (such as ridged beads) kept in motion by a downward or anupward flow of a fluid, such as whole blood. In certain aspects, fluidized beds operate by balancing the downward gravity forces of the weight of the adsorption particles with the upward forces created by high gas and / or fluid flow. Fluidization or suspension can be carried out on a large range of material densities and particle sizes.

[0116] The sample from the subject can be any bodily fluid taken from the subject. In some aspects, the sample comprises whole blood. In some aspects, the sample comprises serum. In some embodiments, the sample comprises plasma. In some embodiments, the sample comprises cerebrospinal fluid. The sample can be taken from the subject in the form of a discrete sample to be treated with the method. The sample can be taken from the subject in the form a continuous or semi-continuous stream. The method includes treating the subject by contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent with a sample from the subject ex vivo or extracorporeal. The method forms a treated or cleansed sample, wherein the adsorption media has a binding affinity for circulating cancer cells and cancer mediators. These mediators inhibit the cell derived immune response.

[0117] The amount of sample that can be used in the claimed methods is not intended to be limited. It can range from less than 1 mL to above 1 L, up to and including the entire blood volume of the subject when the sample comprises blood and when continuous recirculation back to the subject is employed. One or more ‘passes’ through the adsorption media or bed may be used if needed. The term “adsorption bed” refers to a container, chamber, column, etc. which holds the adsorption media. The adsorption bed may be part of a dialysis or extracorporeal circuit. In other aspects, it can be part of a blood bag. The adsorption bed or media may comprise a cartridge.

[0118] In some embodiments, the sample is taken from the subject at a rate of about 5 mL / min, about 10 mL / min, about 15 mL / min, about 20 mL / min, about 25 mL / min, about 30 mL / min, about 35 mL / min, about 40 mL / min, about 45 mL / min, about 50 mL / min, about 60 mL / min, about 70 mL / min, about 80 mL / min, about 90 mL / min, about 100 mL / min, about 150 mL / min, about 200 mL / min, about 250 mL / min, about 300 mL / min, about 350 mL / min, about 400 mL / min, about 450 mL / min, about 500 mL / min, about 550 mL / min, about 600 mL / min, about 700 mL / min, about 800 mL / min, about 900 mL / min, about 1000 mL / min, or even about 2000 to 6000 mL / min.

[0119] The sample from the subject suffering from cancer can be contacted with the adsorption media for any length of time sufficient to remove cancer cells and mediators. In some aspects, the sample contacts the adsorption media for a duration of from 1 minute to 12 hours, or longer. In some embodiments, the sample is in contact with the adsorption media for a duration of 1 minute, 5 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours. In some embodiments, the sample contacts the adsorption media as an unbroken or contiguous stream, wherein the sample continuously flows over, on, or through the adsorption media. The treatment period may be 1 day up to 1, 2, 3, 4, 5 or more weeks. In certain instances, the frequency is 1-3 times for the first week of treatment followed by once weekly for the following 3 weeks, for a total of up to 6 treatments over the first 28 days; and biweekly monthly thereafter or as needed.

[0120] The continuous-flow of a sample includes a constant or variable fluid flow having a set velocity or rate at which the sample is in contact with the adsorption media. In some embodiments, the sample is in contact with the adsorption media at a rate of about 5 mL / min, about 10 mL / min, about 15 mL / min, about 20 mL / min, about 25 mL / min, about 30 mL / min, about 35 mL / min, about 40 mL / min, about 45 mL / min, about 50 mL / min, about 60 mL / min, about 70 mL / min, about 80 mL / min, about 90 mL / min, about 100 mL / min, about 150 mL / min, about 200 mL / min, about 250 mL / min, about 300 mL / min, about 350 mL / min, about 400 mL / min, about 450 mL / min, about 500 mL / min, about 550 mL / min, about 600 mL / min, about 700 mL / min, about 800 mL / min, about 900 mL / min, or about 1000 mL / min.A. Cancer Treatment

[0121] As described herein, treatment of a cancer patient with the adsorption media removes circulating cancer cells, circulating cancer stem cells and cancer mediators that inhibit an immune response and de facto acts as a checkpoint inhibitor. Cancer cells are often making protein called PD-L1 which binds to PD-1 receptor(s) on T cells effectively sending a signal to the immune system to power-down, slow or stop T cell production, so the cancer can continue spreading without being attacked. The adsorption media effectively disables this switch, by eliminating CTCs from the blood, which than restores the T-cell ability to attack the cancer cell(s). This in turn, increases the bioavailability of immune cells, antibodies and / or chemoagents, which target the cancer tumor to increase local inflammation,and treat the cancer. Advantageously, the adsorption media has a binding affinity for circulating cancer cells and mediators that inhibit an immune response.

[0122] Inflammation has a dual role in cancer. Inducing acute inflammation locally at the site of a cancerous tumor can successfully treat the cancer tumor or lesion. Hence, with the infiltration of leukocytes and subsequent inflammation, the impact from inflammatory mediators can eliminate tumor cells and prevent tumor development. On the other hand, chronic inflammation is a necessary consequence of cancer progression. Most tumors progress to a state of chronic inflammation that promotes different aspects of tumor progression, including genomic instability, immune evasion, and metastasis.

[0123] Without being bound by any particular theory, it is believed that the methods of treatment described herein promote a T-cell response against tumor-derived antigens and thereby controls tumor progression. This is accomplished by removing circulating cancer cells and mediators such as Regulatory T (Treg) cells. This removal aids cancer immunosuppression and instead promotes the secretion of Th-1 cytokines (e.g., IFN-y, IL-2, and IL- 12), NK cell recruitment, and the presence of cytotoxic T lymphocytes (CTLs) to inhibit tumorigenesis. The secretion of cytokines, NK cell recruitment, and the presence of CTLs induces acute inflammation locally at the site of a cancerous tumor and successfully treats the cancer. The methods reduce tumor burden by reducing or eliminating tumor cells.

[0124] In certain instance, the methods herein remove Regulatory T (Treg) cells, which are a subset of CD4+T cells that are required for immunosuppression, and which inhibit inflammation caused by the immune response to pathogens. Treg cells can promote the development and progression of numerous types of malignancies and tumors and aid the evasiveness of the immune system.

[0125] The method disclosed herein are useful for all types of cancers including a solid tumor cancer or a metastatic lesion. Solid tumor cancer or metastatic lesion is selected from the group of lung, colorectal, prostate, testicular, pancreatic, adenocarcinoma, renal, breast, ovarian, endometrial cervical, urothelial, thyroid, stomach, liver, and melanoma.

[0126] In certain aspects, the methods include immune system activation and migration of immune cells into tumors, which results in acute inflammation, tumor cell death and tumor reduction.

[0127] In certain aspects, the method removes circulating tumor cells and circulating cancer stem cells from the blood sample.

[0128] In certain aspects, the methods comprise measuring circulating tumor cells and circulating cancer stem cells before, during and after treatment. CTCs are a population of cancer cells that detach from a primary tumor and enter the circulation. CTCs are rare cells.

[0129] In certain aspects, the methods remove cancer stem cells. Cancer stem cells (CSC) may be the founder cells of metastasis.

[0130] In certain aspects, the method reduces or completely eliminates T-cell exhaustion. In patients with cancer, due to long-term exposure to persistent antigens and an inflammatory microenvironment, T-cells are continuously stimulated and gradually differentiate into an exhausted state.

[0131] In certain aspects, the disclosed methods promote immune cells and cytotoxic T- cells infiltration into the solid tumor and promote acute inflammation. The methods reduce or prevent metastasis.

[0132] In certain aspects, treatment using the adsorption media impacts both cellular and humoral immunity in ways that facilitates infiltration or influx of T cell subtypes e.g. Killer T cells (also referred to as “cytotoxic” or “cytolytic” or NK cells) into cancerous tumors. The T-cells can then directly eliminate, attack and / or destroy cancerous cells displaying targets they recognize. Once NK and T cells destroy cancerous cells, cellular immunity via macrophage activations triggers macrophage clearance of the interstitial environment of extraneous cancerous cellular material.

[0133] The VEGF (vascular endothelial growth factor) family members are involved in various biological processes, such as angiogenesis, lymphopoiesis, inflammation, oxidative stress, lipid metabolism. VEGF family includes the following members: VEGF-A, VEGFB, VEGFC, VEGFD, placental growth factor (P1GF), and EG- VEGF.

[0134] VEGF-A binds vascular endothelial growth factor receptor VEGFR1 or VEGFR2 and then activates downstream signals to promote angiogenesis. In response to ligand binding, VEGFR1 and VEGFR2 dimerize to form homodimers or heterodimers. VEGFR2 is the main binding receptor of VEGF-A and the key regulator for angiogenesis. With the removal of VEGF-A using the methods of the present disclosure, it is believed that there isless dimerization and thus, less downstream signals to promote angiogenesis. This reduces tumor angiogenesis as well as tumor size.

[0135] In the methods of the present disclosure, VEGF-A binds to the adsorbent and is removed. The removal of VEGF-A prevents VEGF-A from interacting with VEGFR-2 and thus prevents or reduces tumor angiogenesis.B. Combination Therapy

[0136] In certain aspects, the methods further comprise administering an immunostimulatory agent such as small molecule anti-cancer agents, INF-y, IL-7 and checkpoint inhibitors. In certain aspects, the checkpoint inhibitor is a member selected from the group of pembrolizumab (Keytruda), ipilimumab (Yervoy), nivolumab (Opdivo) and atezolizumab (Tecentriq).

[0137] In certain aspects, the checkpoint inhibitor is a member selected from the group consisting of anti-PDl, anti-PDLl, anti-CTLA4, anti-TIM3, and anti-LAG3 antibodies.

[0138] The therapeutic agent can be for instance an immunomodulatory such as an immune checkpoint inhibitor, a T-cell-based cancer immunotherapy including adoptive cell transfer (ACT), genetically modified T-cells or engineered T-cells such as chimeric antigen receptor cells (CAR-T cells), a conventional chemotherapeutic, radiotherapeutic or anti-angiogenic agent, or targeted immunotoxin.

[0139] The disclosure provides combined therapies in which contacting the adsorption media of the disclosure is administered to patients with, before, simultaneously, or after an immunomodulator such as an immune checkpoint inhibitor (CI). In one aspect, the immunomodulator is an inhibitor of the PD-1 / PD-L1 pathway.

[0140] In certain aspects, the immunomodulator is an inhibitor of an immune checkpoint molecule. In one aspect, the immunomodulator is an inhibitor of PD-1, PD- LI, PD-L2, CTLA-4, TIM-3, LAG-3, NKG2D, NKG2L, KIR, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and / or TGFRbeta. In one aspect, the inhibitor of an immune checkpoint molecule inhibits PD-1, PD-L1, LAG-3, TIM-3, TIGIT or CTLA-4, or any combination thereof. The term “inhibition” or “inhibitor” includes a reduction in a certain parameter, e.g., an activity, of a given molecule, e.g., an immune checkpoint inhibitor. For example, inhibition of an activity, e.g., a PD-1 or PD-L1 activity, of at least 5%, 10%, 20%, 30%, 40%, 50% or more is included by this term. Thus, inhibition need not be 100%.

[0141] Some non-limiting examples of chemotherapeutic drugs which can be combined with adsorption media method therapies of the present disclosure are carboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), cyclophosphamide (Cytoxan, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (VePesid), fluorouracil (5-FU), gemcitabine (Gemzar), imatinib mesylate (Gleevec), irinotecan (Camptosar), methotrexate (Fol ex, Mexate, Am ethopterin), paclitaxel (Taxol, Abraxane), sorafinib (Nexavar), sunitinib (Sutent), topotecan (Hycamtin), vincristine (Oncovin, Vincasar PFS), and vinblastine (Velban).

[0142] In some embodiments, the anti-cancer therapy comprises a kinase inhibitor. In some embodiments, the methods provided herein comprise administering to the individual a kinase inhibitor, e.g., in combination with adsorption media therapy. Examples of kinase inhibitors include those that target one or more receptor tyrosine kinases, e.g., BCR-ABL, B-Raf, EGFR, HER-2 / ErbB2, IGF-IR, PDGFR-a, PDGFR-P, cKit, Flt-4, Flt3, FGFR1, FGFR3, FGFR4, CSF1R, c-Met, RON, c-Ret, or ALK; one or more cytoplasmic tyrosine kinases, e.g., c-SRC, c-YES, Abl, or JAK-2; one or more serine / threonine kinases, e.g., ATM, Aurora A & B, CDKs, mTOR, PKCi, PLKs, b-Raf, S6K, or STK11 / LKB1; or one or more lipid kinases, e.g., PI3K or SKI. Small molecule kinase inhibitors include PHA-739358, nilotinib, dasatinib, PD166326, NSC 743411, lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sutent (SU1 1248), sorafenib (BAY 43-9006), or leflunomide (SU101). Additional non -limiting examples of tyrosine kinase inhibitors include imatinib (Gleevec / Glivec) and gefitinib (Iressa).

[0143] In certain aspects, the adsorption media methods comprise contacting the whole blood sample or a cancer subject ex vivo between 1-5 times (1, 2, 3, 4, or 5 times) every 7 days (1 week) for up to 28 days or 30 days.

[0144] In certain aspects, the adsorption media methods comprise contacting the whole blood sample ex vivo up to 3 times for the first week of treatment followed by once weekly for the following 3 weeks, for a total of up to 6 treatments over the first 28 days; and biweekly monthly thereafter or as needed.

[0145] In certain aspects, adsorption media methods are treatment modifying methods, wherein the method is performed as an adjunct therapy before, during or after a primary therapy such as immunotherapy as disclosed herein.

[0146] In certain aspects, the adsorption media methods further comprises administering an angiogenesis inhibitor.

[0147] In certain aspects, the angiogenesis inhibitor is a member selected form the group consisting of bevacizumab, ramucirumab, ziv-afhbercept, sunitinib, sorafenib, axitinib, pazopanib and vandetanib.C. Cancer Diagnostics

[0148] In another embodiment, the present disclosure provides a method for profiling cancer disease characteristics and / or functional activity of a subject, the method comprising: contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent with a whole blood sample from the subject ex vivo to make an adhering complex between the adsorption media and cancer cells; releasing the adhering complex of cancer cells; and interrogating the cancer cells to generate a disease profile.

[0149] In certain aspects, the circulating tumor cells (CTC) are used as a liquid biopsy to prepare a disease profile. A liquid biopsy from the sample already collected is an alternative to a surgical biopsy of the tumor. The interrogation of the CTCs allows for a range of information about the tumor, including diagnosis, prognosis, relapse potential and metastatic potential. The CTCs can be counted and enumerated to ascertain the extent of tumor burden. Further, therapeutic targets on the cell can be identified and the treatments most likely to work for that patient can be identified.

[0150] In certain aspects, the disease profile comprises the type and the stage of cancer. In this manner, the primary type of cancer is identified along with its stage, metastatic potential, and prognosis. In order to interrogate CTCs, one can utilize DNA, RNA, or protein analytical techniques to assess genotype, gene expression and / or protein expression. Various staining and imaging techniques allow for phenotype determination. Nucleic acid analysis and amplification allows for mutations to be identified.

[0151] In certain aspects, a disease profile comprises therapeutic options. For example, nucleic acid and protein techniques, interrogation of CTCs allows for identification of therapeutic targets. For example, CTCs from breast cancer will allow determination of HER2, PR and ER status. It is also possible to determine whether KRAS, or BRCA1 and / or BRCA2 are mutated. Using the methods disclosed herein, the disease profile optionally includes identified therapeutic targets and treatments.

[0152] In certain aspects, circulating tumor cells are analyzed in a multiwell plate each well with an anticancer drug. For example, in one aspect, CTCs are loaded in a 96-well plate and various therapeutic options are used to determine the most efficacious option for the individual.

[0153] In certain aspects, the CTCs are analyzed for therapeutic efficacy of the anticancer drug. Combination treatments and dosing regimens can be established and tested before administration into the subject and optionally included in the disease profile.

[0154] In certain aspects, the cancer cells are tumor cells, circulating tumor cells or cancer stem cells. All of these cells are useful in the present methods.

[0155] In certain aspects, the anticancer drug is a series of anticancer drugs or combinations of anticancer drugs. In certain aspects, drug cocktails are tested for efficacy and a treatment regimen is established.

[0156] In certain aspects, CTCs are defined as tumor cells that have been sloughed from the primary tumor and are swept away by the circulatory or lymphatic systems. The enhanced liquid biopsy disclosed herein allow for significantly increased sensitivity and specificity which drive positive predictive value and increases potential of CTC for general population screening, disease diagnosis, monitoring of disease progression and surveillance for recurrence or relapse of the disease. Once screening identifies at risk patient, the disease can be monitored for progression or be utilized as surveillance for recurrence or relapse.

[0157] In certain aspects, disease profile comprises a gene expression profile indicative of diagnosis and prognosis of cancer in the subject. Using gene expression, the cancer is evaluated, and the most efficacious therapy is selected to be administered.

[0158] In certain aspects, the method comprises selecting a treatment regimen or monitoring disease status by evaluating the likelihood of efficacious treatment or the status of treatment of a subject having cancer at different time points.

[0159] In certain aspects, the disease profile is a patient specific result (a personalized approach) based upon the diagnosis and therapeutic options.

[0160] In certain aspects, the disease profile is generated using a neural network model trained on the genotype and phenotype of cancer cells from a population have a certain cancer.

[0161] In certain aspects, the disease profile is assessed before, during and after treatment. The disease profile can be compared to a healthy control or positive control or reference. The disease profiles can be used to diagnose and classify cancer and to monitor therapeutic response.D. Systems and Devices

[0162] In another embodiment, the present disclosure provides systems and methods for specimen extraction and cellular analysis. Turning to FIG. 12, in one aspect of the disclosure, system 1200 is useful for extracting a sample from the adsorption media device or filter 1205 and interrogating the cells.

[0163] In certain aspects, the disclosure provides an extractor 1215, which is a vacuum extractor or an external energy augmented extraction. In one aspect, the specimen extraction device 1215 comprises a spring loaded 1231 connector 1221 configured for insertion into the adsorption media device 1205 comprising a substrate having at least one polysaccharide adsorbent. The extractor 1215 for removing the substrate, which has at least one polysaccharide adsorbent; and a barrel 1218 for storing the substrate.

[0164] In certain aspects, the barrel 1218 is optionally two barrels 1228 or optionally three barrels 1229 for testing and analyzing the substrate.

[0165] In certain aspects, the specimen extraction device 1215 is shown in the figure, which comprises a barrel 1218 or a chamber that has a first end 1212 and a second end 1207 with connectors such as a luer connector on each end. On one end, the specimen extraction device connects via a connector to the adsorption media column (e.g., the blood filter) and on the other side to either single or multiplex specimen collection 1241 and or processing chambers.

[0166] In certain aspects, once the specimen extraction device 1215 is connected to blood filter 1205 via luer connector 1207, a spring loaded 06-20 G non-coring needle 1221 is depressed piercing the retention plate or screen of the blood filter 1205. Thereafter, the noncoring needle 1221 is inserted into the beads, vacuum activation via an external energy source (capsule with pressurized gas and / or battery) will aspirate blood filter contents into a chamber 1218 (up to 3 1218, 1228, 1229 chambers) that can serve the purpose of i) storage, ii) processed for qualitative or semiqualitative analysis or iii) extracted into a multiplex chamber 1241 for sensitivity analysis.

[0167] The cells from extraction device and / or patient, can be used to determine the status of the patient’s disease and whether it is early or late stage. In addition, it is possible to remove pathogens or CTCs from the patient and assess and interrogate the cell type and / or primary tumor source.

[0168] In one aspect, cancer cells released from a patient undergoing blood filtration are placed in a multiwall plate (96-well plate) 1252 with a variety of immune or anti-cancer drugs. The wells having the best anti-cancer result can then be used for the patient. This personalized treatment ensures the most efficacious treatment is employed to treat the patient. In one approach, the beads can be released from the device and contacted with a variety of anti-cancer treatments and using this protocol, a tailored personalized drug treatment regimen can be analyzed, determined, and employed.

[0169] In certain aspects, after the cancer is assessed and categorized, the treatment protocol can be determined. With regard to cancer treatment, the current process can take the place of a tumor “assessment board” that regularly meets at many hospitals to determine treatment protocols for new patients. As shown a computer database (Al enabled) 1263 with the treatment protocols based on the results of the device.

[0170] The systems and devices include machine learning, wherein computers learn complex data patterns to make predictions. These algorithms assist medical practitioners through analyses of liquid biopsies and blood tests to improve risk stratification and early diagnosis. For example, an artificial neural network (ANNs) can be used to predict cancer risk, outcome and therapeutic efficacy by using clinical parameters. ANNs comprise (1) an input layer, (2) a ‘hidden layer’, consisting of multiple nodes which multiply the input by weights and add a bias value, and (3) the output layer, passing the weighted sum of hidden layer nodes to an activation function to make predictions.

[0171] A multivariate index assay (MIA) is based on Al. It combines the values of multiple variables using an artificial intelligence interpretation function to yield a single, patient-specific result (e.g., a “classification,” “score,” “index,” and the like.), that is intended for use in the diagnosis of the type and severity of cancer, the cure, mitigation, treatment or prevention of relapse. MIA results for the blood filter can be binary (dichotomous) (such as yes or no), categorical (such as disease type), ordinal (such as low, medium, high) or a continuous scale.

[0172] In addition to cancer, the systems and methods disclosed herein can be used to treat sepsis. Sepsis is a life-threatening syndrome caused by an abnormal immune response induced by for example, a blood stream infection. Once a pathogen enters the blood stream, the nonspecific immune system (aka the innate immune system), recognizes a foreign body and activates a nonspecific inflammatory response, primarily composed of several white blood cells and plasma proteins. The innate immune system resists the invading pathogenic bacteria using immunologically active substances such as lysozyme and antibacterial peptides.

[0173] The innate immune system is the body’s first line of defense against germs entering the body. It responds in the same way to all germs and foreign substances, which is why it is sometimes referred to as the “nonspecific” immune system. If the infection persists, the infected cells can recruit innate immune cells such as neutrophils and monocytes in the blood by releasing chemokines such as C-XC motif chemokine ligand 1 (CXCL1) and C-C motif ligand 8 (CCL8). These cells migrate from the blood vessels to the local tissue to exert an inflammatory effect. Toll-like receptors (TLRs) are important receptors associated with innate immunity. They specifically recognize and bind to pathogen-associated molecular patterns (PAMPs), which are derived from microorganisms (Pradeu and Cooper, 2012 The Danger Theory: 20 Years Later. Front. Immunol. 3, 287; Netea et al., 2017 A Guiding Map for Inflammation. Nat. Immunol. 18, 826-831.), and which in turn, prompt multitude of signaling pathways called damage-associated molecular patterns (DAMPs) that lead to inflammatory factor release and ultimately activates the adaptive immune system (Fitzgerald et al., LPS-TLR4 Signaling to IRF-3 / 7 and NF-kappaB Involves the Toll Adapters TRAM and TRIF. J. Exp. Med. 198, 1043-1055, 2003; Kawai and Akria, 2010 The Role of Pattern- Recognition Receptors in Innate Immunity: Update on Toll-like Receptors. Nat. Immunol. 11, 373-384 ).

[0174] T-cell exhaustion is present in both sepsis and cancer which prevents an adequate immune response to the disease which contributes to immunosuppression. During sepsis, many disease mediators circulate in the bloodstream and overwhelm the potential immune response. As such, the signaling molecules typically released at the site of infection are obscured by the presence of similar signaling molecules in the bloodstream. Therefore, the immune system cannot precisely target the local infection and may lead to tissue damage and multiorgan failure.

[0175] In another embodiment, the disclosure provides a method for treating sepsis in a subject in need thereof, the method comprising: treating the subject by contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent with a whole blood sample from the subject ex vivo to form a cleansed sample, wherein the adsorption media has a binding affinity for inflammatory mediators that inhibit an immune response; and reducing the inhibition of the immune response by increasing the bioavailability of immune cells to thereby treat sepsis.

[0176] In certain aspects, the inflammatory mediator is a member selected from the group consisting of a cytokine, a pathogen-associated molecular patterns (PAMPs) and a damage- associated molecular patterns (DAMPs).

[0177] In addition, when CTCs are released in the blood, killer T-cells are misdirected towards the CTCs instead of the solid tumor. Both sepsis and cancer can lead to T-cell exhaustion. The adsorption media treatment removes pathogens, circulating tumor cells (CTCs), DAMPs, and PAMPs. Therefore, the adsorption media can be a disease modifier by clearing the bloodstream, increasing the bioavailability of killer T-cells and potentially reversing T-cell exhaustion. The treatment therefore enables recruiting or employing the subject’s immune system towards the local sites of infection and / or solid tumor cancers. By removing these disease agents, the process generates or facilitates an immune inflammatory response surrounding the local site of infection and / or tumor’s microenvironment.Examples

[0178] Cancer patients were treated with a blood filter (Seraph® 100, ExThera Medical) comprised of beads surfaced modified with heparin. Cancer cell concentrations were measured before, during and after the treatment.Example 1

[0179] A cancer patient with lung adenocarcinoma (patient 6) is treated with a blood filter (Seraph®100, ExThera Medical) comprised of beads surfaced modified with heparin. CTC concentrations were measured before, during and after the treatment.

[0180] (i) Before treatment, tumor cells were detected; (ii) 45 minutes after treatment initiation, the number of detectable tumor cells was less than 10; (iii) 3 hours after treatmentinitiation the number of detectable tumor cells remained less than 10; (iv) at the end of treatment, there are substantially no cells detectable.

[0181] At the end of treatment, there were numerous EpCAM specific cell fragments detected. Specific cell fragments may occur as a part of an immune response and indicate damaged / non viable cells.QHi)(iii)

[0182] The results indicate that the treatment is efficacious.Example 2

[0183] A cancer patient with lung adenocarcinoma (patient 1) is treated with a blood filter (Seraph®100, ExThera Medical) comprised of beads surfaced modified with heparin. After finding elevated number of live, malignant tumor cells circulating in the blood at start of second therapy, 30 min. of treatment, there is no indication of live, potentially malignant tumor cells above detection limit circulating in blood.

[0184] However, there were some EpCAM specific cell fragments detected. Specific cell fragments may occur as a part of an immune response and indicate damaged / non viable cells.(i) Initially, at start of second treatment, tumor cell numbers are at elevated level, (ii) after 30 minutes of treatment, there are no longer cells seen.Q

[0185] The results indicate that the treatment is efficacious.Example 3

[0186] A cancer patient is treated with a blood filter (Seraph®100, ExThera Medical) comprised of beads surfaced modified with heparin. As shown in FIG. 7A, cancer stem cells (Spheres) are markers of tumor aggressiveness. FIG. 7B is an enlargement of 7A. As shown in FIG. 7C, the number of spheres decreases. The therapy is effective when the number of cells decreases.Example 4

[0187] A study and analysis was performed under the direction of ExThera Medical to ascertain whether circulating tumor cells (CTCs) can bind to heparinized beads. The heparinized beads used in this study were made of polyethylene with covalently end-point- attached heparin. The resin was tested for CTC-clearance by single cartridge-passages as well as by cartridge-integration into a small extracorporeal circuit.

[0188] Three types of cell lines were used. Cells from metastasizing prostate cancer (DU145), neuroblastoma cells (UKF-NB-3) as well as leukemia cells (Molm-13) were cultured according to standard procedures, counted and checked for vitality. 5000 cells / ml were spiked into complete medium (IMDM with 10% FCS, Pen / Strep and glutamine, Sigma Aldrich, Taufkirchen, Germany) as well as heparinized plasma.

[0189] The extracorporeal circuit was made of MicroPerpex tubing (i.d. 1.3 mm, o.d. 3.4 mm, silicone, GE Healthcare, Freiburg, Germany) and a LKB 2232 Microperpex peristaltic pump (LKB, Bromma, Sweden). The pump was set at a flow rate of 1 ml / min. Prior to contact to plasma, the cartridges were equilibrated with 5 ml of sterile phosphate-buffered saline (PBS). Samples were taken every 30 minutes for 120 min; each was analyzed by flow cytometry (FACS Canto II, Becton-Dickinson, Heidelberg, Germany). The amount of cells was detected in each sample (in duplicates), a sample only with cells served as control, thus allowing accurate quantification of cell amount reduction (FIG. 8A-B). The experiments in 8 A and 8B above show a time dependent CTC clearance by the heparinized beads. DU145 showed similar results.

[0190] Next, the heparinized beads were removed from the mini-cartridges under sterile conditions and cultured in complete medium. CTCs from Molm-13, UKF-NB and DU145 could be grown from Seraph resin.Example 5

[0191] In this example, a patient with small cell lung cancer, and tremors due to a fungal infection from Candida tropicalis was treated for 5 hours with a Seraph® 100 MicroBind® Affinity Blood Filter (ExThera Medical, Martinez, CA). ‘Seraph 100’ consists of a transparent column filled with small plastic beads which have been surface modified with heparin. Blood was continuously filtered outside the patient’s body and returned in a procedure resembling hemodialysis. The patient’s Circulating Tumor Cell (CTC) concentration was measured before, during and after the treatment. CTCs cause metastasis when they travel to other regions of the body distant from the original tumor or lesion, and itis well known that a patient’s bloodstream concentration of CTCs is a determinant of overall survival.

[0192] During the first hour of treatment this patient’s tremors stopped, and CTC counts dropped from 700 / mL to an undetectable level (<10 / mL). CTCs were found adherent to the filter media when it was disassembled after use, as was Candida tropicalis fungus. Followup CT scans showed shrinkage of the primary tumor. A post-treatment biopsy was negative and the patient’s tremors did not return.Discussion

[0193] Seraph 100 Extracorporeal Blood Filtration (‘EBF’) does not have the side effects of radiation or chemotherapy. This novel therapy can quickly reduce the concentration of cancer cells and the amount of miscellaneous ‘debris’ generated from dead cells found in the bloodstream of cancer and pathogenemia patients. Once these impurities are reduced below a specific threshold level, the immune system quickly becomes (re)activated, and is thereby capable of mounting an effective defense against cancer. Blood purification with simultaneous pathogen removal is designed to treat cancer at the same time it treats potentially fatal cancer-related ‘superinfections’, which are opportunistic bloodstream infections which afflict most cancer patients due to their compromised immune response. Unlike chemotherapy, Seraph 100 EBF therapy makes patients feel better immediately, often during the first treatment.

[0194] There are many meaningful signs, symptoms and biomarkers that support the premise that rapid removal of CTCs, Cancer Stem Cells (CSCs) and pathogens from the bloodstream with an appropriate adsorption media leads to ‘immune system (re-)activation’ . In the ca. 15 clinical cases performed to date, the following have been observed: i. Very rapid decrease in CTCs and CSCs by the procedure with many cancer cells bound to the adsorption media after use ii. Tumor shrinkage iii. Positive-to-negative biopsies within days following treatment. iv. New inflammation surrounding cancerous tissue after treatment. v. Increase in killer T-cells after treatment.vi. Pathogens, ‘Neutrophil Extracellular Traps’, exosomes and unidentified noncellular debris trapped in and on the adsorption media, especially during first treatment. vii. Dramatic improvement in patient signs and symptoms during and after treatment. a. Reduced demand for pain medication b. Reduced peripheral cyanosis c. General improvement in energy level and well-being d. Outpatient or in home treatment viii. Increasing antibody levels with decreasing viral load (in COVID-19)

[0195] A ‘painless’ therapy that can lower CTCs, and / or shrinks tumors, and / or causes positive biopsies to become negative constitutes an improvement in cancer treatment. Given this, a blood compatible EBF filter that can safely reduce CTC and CSC levels to undetectable levels in less than an hour can become an exciting new tool in cancer treatment. As mentioned, these positive responses to EBF therapy have been accompanied by often dramatic improvement in patient’s signs and symptoms including tremors, peripheral cyanosis, pain / need for painkillers, energy level and general feeling of wellbeing.

[0196] The delivery of anti-cancer therapy by Seraph 100 EBF can become similar to common hemodialysis, a life-extending therapy that patients with chronic kidney failure receive three times every week. The likely difference is that after a few short well-tolerated Seraph EBF treatments, therapy may be temporarily discontinued while the body attacks the original tumor and the metastatic sites it has produced. Later, a regular but infrequent, perhaps monthly, schedule of ‘tune-up’ treatments may be used to prevent recurrence.

[0197] After sufficient manufacturing scale-up, the inherently low cost of the EBF Therapy will reduce the cost of cancer care, making it available to a much larger patient population, including underserved or disadvantaged groups for whom cancer survival today is unnecessarily low.

[0198] Because Seraph 100 EBF filter media is designed to mimic sites within the body that are targeted by CTCs, CSCs, bacteria, viruses and fungi, the therapy is efficacious against many di seasing-causing adsorbates. Biomimicry is achieved by permanent modification of the large blood-contacting surface area within the filter with heparin, which performs at least three important functions:1. By mimicking the internal (glycocalyx) lining of blood vessels the filter can bind disease-causing substances and remove them from the bloodstream2. It augments the heparin-containing ‘mast cells’, which are immune cells present in the skin, the nasal passages, the lungs, and the gut, which provide the first line of defense by the immune system against invasion.3. It provides a surface that actually prevents blood from clotting on contact, which contributes to the safety of the procedure.

[0199] The Seraph 100 EBF filter is a ‘depth-type filter’ with a ‘tortuous path’ through which the blood flows around and between small heparin-modified beads. Their very high surface area provides plenty of contact for interactions between the blood and the surface of the beads. A less blood-compatible material could cause clotting and clog the filter with thrombus. With heparin-coated beads of controlled size, blood cells pass through the filter unharmed, while larger aggregates or micro-clots are unable to pass. That is, in addition to cancer cells and other pathogens binding to the filter media by specific chemical and physical interactions, aggregates and clots can also be trapped by ‘size exclusion’ through the filter media because of their size or inability to deform.

[0200] In the near future, the treatment of cancer should look very different. Today multiple chemotherapy sessions initially make patients feel worse, causing fatigue, loss of appetite, nausea, bowel issues, hair loss, mucositis, and skin problems. These side effects can be so bad that patients sometimes opt out of therapy and succumb to their cancer and associated superinfections. Superinfections which afflict most cancer patients can ultimately be fatal, as can ‘thrombotic complications’. Based on the disassembly of used EBF filters and visualization of the retained reside, blockage of capillaries, e.g., in various organs, may actually be caused by debris from dead cells including Neutrophil Extracellular Traps (NETs) that are present in large numbers in the blood of patients with cancer and pathogenemia.

[0201] With Seraph 100 EBF therapy, typically patients are hooked up to a simple blood pump for 2 to 5 hours, during which they can read or watch TV without experiencing any negative side effects. Single needle peripheral vein access works well at moderate flowrates, so the therapy can be safely administered at home without indwelling catheters or central lines. Convenient and safe nocturnal treatment will be an option. Symptoms of opportunistic infections may subside or disappear, and the patient’s energy level is likely to improve. Even reduced use of pain killers has been reported.

[0202] Based on clinical experience, a typical protocol will be as follows: During the first week of treatment the patient may receive two or three Seraph 100 EBF procedures before a one-week break, during which the patient’s immune system will be fighting the cancer without the burden of large numbers of CTCs, CTSs or cell debris in the bloodstream. One or two procedures will be given during the following couple of weeks, all of which can be followed by imaging, liquid or surgical biopsies and / or histology.

[0203] What is removed from the blood during a therapeutic procedure is identified to determine the patient’s progress and / or the need for additional procedures. Alternatively, the still viable cancer cells captured on the filter can be grown in culture to produce more cells for studying their susceptibility to drugs: a form of personalized medicine. This aspect of the procedure can be thought of as Intelligent Therapy™, because the therapeutic treatment itself is ‘interrogating’ the entire blood volume of the patient in the first hour, and the captured cancer cells can be made to multiply in culture for use in optimizing the patient’s therapy, if required.

[0204] The ability of the Seraph 100 EBF Procedure to remove CTCs is close to 100% of the incoming amount for every pass of blood through the filter. At even moderate blood flowrates this translates to rapid reduction to undetectable levels, often in an hour or two. We have observed that the speed of reduction greatly exceeds the rate predicted by in vitro lab tests (Figure 9), which cannot be enhanced by the patient’s immune system. The most likely explanation for this is that the patient’s immune system becomes more effective against the cancer during and after the treatment. That is, the Seraph 100 EBF filter itself removes CTCs, etc., but the immune system is also activated. Thus, by debulking the blood of cancer- related substances, the patient’s body is again able to resume the job of fighting cancer and curing associated bloodstream infections.

[0205] FIG. 9 shows reducing the concentration of circulating tumor cells, cancer stem cells (CTCs and CSCs) and miscellaneous ‘debris’ in the bloodstream to a level that no longer overwhelms the patient’s immune system. Once this happens, the immune system appears to become ‘reactivated’ and thereby capable of mounting an effective defense against the cancer in combination with the EBF Therapy. The upper blue line in the graph is the expected CTC reduction rate for the filter alone, based on analysis of spiked lab samples and extracorporeal clinical samples. The lower red line is the measured rate of CTC reductionduring a typical clinical EBF treatment. It strongly suggests that the filter is working together with the patient’s immune system to rapidly reduce CTC level.Example 6

[0206] Primary cancers often respond to treatment, while high mortality is associated with treatment-resistant metastatic lesions. Detached cells from solid malignant tumors that enter circulation are a primary cause of metastases. Most Circulating Tumor Cell (CTC) research has focused on diagnostics. A compelling question is whether there is a therapeutic benefit from high-yield CTC removal in metastatic cancer.

[0207] We conducted a first-in-human, trial of the Seraph® 100 Microbind® Affinity Blood Filter [1] (ExThera Medical, Martinez, CA) evaluating safety, and potential for combined pathogen and CTC removal in ten patients with cancers positive for epithelial cell adhesion molecule (EpCAM). Seraph 100 is a single-use filter containing ultra-high molecular weight polyethylene beads surface modified with heparin that mimics the glycocalyx surface of naturally occurring mammalian cells [1, 2].

[0208] Study protocol was approved by the Institutional Review Board Ethical Committee, and patients gave written informed consent before participation. Liquid biopsy measured CTCs in the blood of ten consecutive cancer patients (Lung Adeno, Small Cell Neuroendocrine, Colorectal, Pancreatic, Prostate, Endometrial, Ovarian and ductal carcinoma in situ, estrogen and progesterone negative receptors, at stages from TNM 0 to Late Stage IV) all of whom also had positive pathogen cultures. Automated microfluorimetric image analysis of EpCAM-positive cells with visual control (MAINTRAC) was used for CTC enumeration and characterization as previously described [3, 4],

[0209] We measured elevated concentrations of live, malignant tumor cells circulating in the blood of all enrolled patients before Seraph 100 treatment. Depending on cancer type and stage, baseline CTCs ranged from 50 to 2600 live cells / mL. Seraph 100 filtration media removed from used filters was evaluated by cell culture isolation and identification in a molecular pathology laboratory. FIG. 10 shows a Liquid biopsy of blood samples collected at 0, 30 and / or 45 minutes after initiation of Seraph treatment showed rapid reduction of live, malignant tumor cells to below the 10 CTC / mL detection limit (assumed to be zero concentration) in 6 of the 10 patients. Mean CTC reduction after Seraph 100 treatment for all patients was 91 ± 11.9%. Patient 01 shown here with exponential fit.

[0210] The effectiveness and extent of Seraph CTC removal was further confirmed by cell culture isolation of malignant cancer cells from Seraph filters. Blood cultures became negative for all confirmed pathogens (Candida tropicalis, Aspergillus fumigatus,Staphylococcus aureus., Staphylococcus epidermidis, Escherichia coli, Klebsiella aerogenes) within 4 hours of initiating Seraph 100 EBF.

[0211] Seraph 100 EBF has potential in at least three areas a. Slowing metastatic disease progression by rapid adsorptive CTC removal. b. Possible immunomodulatory effects from debulking the blood of both CTCs and pathogens, enabling immune system reactivation. c. Diagnostic and personalized treatment of thq cancer using quells captured by the filter

[0212] CTC level can be a biomarker in metastatic cancer treatment.1. Schmidt JJ, Borchina DN., van't Klooster M et al. Interim analysis of the COSA (COVID-19 patients treated with the Seraph® 100 Microbind® Affinity filter) registry. Nephrol Dial Transplant. 2022 Mar 25;37(4):673-680.2. Single-Center Experience With the Seraph-100® Microbind® Affinity Blood Filter in Patients With SARS-CoV-2 Infection and Septic Shock at a Military Treatment Facility; Military Medicine, Volume 188, Issue 7-8, July / August 2023, Pages e2670-e2674.3. Pachmann, K.; Clement, JH.; Schneider, CP.et al. Standardized quantification of circulating peripheral tumor cells from lung and breast cancer. Clin. Chem. Lab. Med. 2005, 43, 617-627.4. Pachmann, K. Current and potential use of MAINTRAC method for cancer diagnosis and prediction of metastasis. Expert Rev Mol Diagn. 2015 May;15(5):597-605.Example 7

[0213] This example illustrates simultaneous removal of circulating tumor cells and pathogens by extracorporeal blood filtration (EBF).

[0214] A patient with Stage 4 small-cell lung cancer, and tremors due to an undiagnosedCandida tropicalis fungal infection from was treated for 5 hours with a Seraph® 100 MicroBind® Affinity Blood Filter (ExThera Medical, Martinez, CA). ‘Seraph 100’comprises a transparent column filled with small beads which have been surface modified with heparin (FIG. 1).

[0215] Blood was filtered outside the body and returned to the patient in a continuous procedure resembling hemodialysis. The patient’s Circulating Tumor Cell (CTC) concentration was measured before, during and after the treatment. CTCs cause metastasis when they travel to other regions of the body distant from the original tumor or lesion, and a patient’s bloodstream concentration of CTCs is a determinant of overall survival.

[0216] FIG. 1 shows a schematic of The Seraph® 100 MicroBind® Affinity Blood Filter in use in the Extracorporeal Blood Filtration Procedure. During the first hour of treatment the patient’s tremors stopped, and CTC counts dropped from 700 / mL to an undetectable level of <10 / mL. CTCs were found adherent to the filter media (FIG. 11.) when it was disassembled after use, as were Candida tropicalis fungal spores. Follow-up CT scans showed shrinkage of the primary tumor. A post-treatment biopsy was negative, and the patient’s tremors did not return.

[0217] FIG. 11 shows an optical micrograph of multiple Circulating Tumor Cells bound to a single adsorbent bead removed from a Seraph 100 Filter after a clinical treatment, with staining to enhance visibility.

[0218] ‘Seraph 100 EBF’ does not have the side effects of radiation or chemotherapy. This novel therapy can simultaneously reduce the concentration of cancer cells, the amount of miscellaneous ‘debris’ in the bloodstream of cancer and pathogenemia patients e.g. that is generated from dead cells. Once these impurities are reduced below a specific threshold level, the immune system appears able to quickly become capable of mounting an effective defense. Seraph 100 EBF with simultaneous pathogen removal is designed to treat cancer at the same time it treats potentially fatal cancer-related ‘superinfections’, which are opportunistic bloodstream infections which afflict most cancer patients due to their compromised immune response. Unlike chemotherapy, Seraph 100 EBF therapy makes patients feel better immediately, often during the first treatment.

[0219] There are many meaningful signs, symptoms and biomarkers that support our belief that rapid removal of CTCs, Cancer Stem Cells (CSCs) and pathogens from the bloodstream with our unique filter leads to immune system (re-)activation. In the 10 clinical cases we have performed in a recent OUS study of patients with several different cancers (Lung Adeno, Small Cell Neuroendocrine, Colorectal, Pancreatic, Prostate, Endometrial, Ovarianand ductal carcinoma in situ, estrogen and progesterone negative receptors, at stages from TNM 0 to Late Stage IV, all of whom also had positive pathogen cultures) we observed: i. Very rapid decrease in CTCs and CSCs by the procedure, with many cancer cells bound to the filter after use ii. Tumor shrinkage iii. Positive-to-negative biopsies within days following treatment iv. New inflammation surrounding cancerous (pancreatic) tissue after treatment. v. Increase in killer T-cells after treatment. vi. Pathogens, apparent Neutrophil Extracellular Traps, exosomes and unidentified noncellular debris trapped in and on the filter, especially during the first treatment vii. Dramatic improvement in patient signs and symptoms during and after treatment. a. Reduced demand for pain medication b. Reduced peripheral cyanosis c. General improvement in energy level and well-being d. Outpatient or in home treatment viii. Increasing antibody levels with decreasing pathogen load (in COVID-19)

[0220] A ‘painless’ minimally-invasive therapy that can lower CTCs, shrink tumors, and cause positive biopsies to go negative is definitely new and noteworthy. Given this, an adsorption filter that safely reduces CTC and CSC levels to undetectable levels in less than an hour should become an exciting new tool in cancer treatment, once sufficient clinical evidence is available.

[0221] We reason that the delivery of anti-cancer therapy by EBF will become similar to common hemodialysis, a life-extending therapy that patients with chronic kidney failure receive three times every week. The likely difference is that after a few easily-tolerated cancer treatments, Seraph 100 EBF may be temporarily discontinued while the body attacks the original tumor and the metastatic sites it has created. Later, a regular but infrequent schedule of ‘tune-up’ treatments may be needed to prevent or respond to recurrence, although so far we have not seen the need in our limited cases.

[0222] The cost of the Seraph 100 EBF will reduce the cost of cancer care, making it available to a larger patient population, including underserved or disadvantaged groups for whom cancer survival today is unnecessarily low.

[0223] How can Seraph 100 EBF, a well -tolerated and simple procedure, have a positive effect on cancer, and at the same time treat associated bloodstream infections? Because it is designed to mimic sites within the body that are targeted by CTCs, CSCs, bacteria, viruses and fungi. This is done by permanent modification with heparin of the filter’s large bloodcontacting surface, which performs at least three important functions:1. By mimicking the internal (glycocalyx) lining of blood vessels the filter can bind disease-causing substances and remove them from the bloodstream.2. Seraph 100 augments the heparin-containing ‘mast cells’, which are immune cells present in the skin, the nasal passages, the lungs and the gut, and which provide the first line of defense by the immune system against invasion.3. Seraph 100 has a blood-compatible surface that does not cause clotting on contact, which contributes to the safety of the procedure.

[0224] The Seraph 100 filter is an adsorption filter, but it is also a blood-compatible ‘depth-type filter’ with a ‘tortuous path’ through which the blood flows around and between small heparin-functional beads. Their very high surface area provides plenty of contact for interactions between the blood and the surface of the beads. A more thrombogenic material could cause blood clotting and clog the filter with actual thrombus. With heparin-coated beads of controlled size, blood cells pass through the filter unharmed, while larger aggregates or micro-clots are unable to pass. That is, in addition to cancer cells and other pathogens binding to the filter media by specific chemical and physical interactions, aggregates and clots can also be trapped by ‘size exclusion’ through the filter media because of their size or inability to deform.

[0225] In the near future, the treatment of cancer may look very different. Today multiple chemotherapy sessions initially make patients feel worse, causing fatigue, loss of appetite, nausea, bowel issues, hair loss, mucositis, and skin problems. These side effects can be so bad that patients sometimes opt out of therapy and succumb to their cancer and associated superinfections. Superinfections which afflict most cancer patients can ultimately be fatal, as can ‘thrombotic complications’. Based on disassembly of used Seraph 100 filters and visualization of the retained residue, blockage of capillaries, e.g., in various organs, may actually be caused by debris from dead cells including Neutrophil Extracellular Traps (NETs) that are present in large numbers in the blood of patients with cancer and bloodstream infections. In fact, Sedimentation Rate, a simple ‘old school’ blood test increases in cancer patients, but Seraph 100 EBF can restore ‘Sed Rate’ to normal values.

[0226] With EBF therapy, patients can be hooked up to a simple blood pump for 2 to 5 hours, during which they can read or watch TV without experiencing any apparently negative side effects. Single needle peripheral venous access works well at flowrates <150 mL / min, so the therapy can eventually be administered at home without indwelling catheters or central lines. Convenient and safe nocturnal treatment will be an option. Symptoms of opportunistic infections are expected to subside or disappear, and the patient’s energy level is likely to improve. Even reduced use of pain killers has been reported.

[0227] Based on our limited clinical experience to date with several different cancers, we expect that a typical protocol could be as follows: During the first week of treatment the patient may receive two or three Seraph 100 EBF treatments before a one-week break, during which the patient’s immune system will be fighting the cancer without the burden of large numbers of CTCs, CTSs or bloodborne cell debris. One or two procedures will be given during the following couple of weeks, all of which are followed by imaging, liquid or surgical biopsies and / or histology.

[0228] What is removed from the blood during a therapeutic procedure can be identified to determine the patient’s progress and / or the need for additional procedures. Alternatively, the still viable cancer cells captured on the filter can be grown in culture to produce more cells for studying their susceptibility to e.g., chemotherapy drugs: a form of personalized medicine. We refer to this aspect of the procedure as Intelligent Therapy™, because the therapeutic treatment itself is ‘interrogating’ the entire blood volume of the patient in the first hour, and the captured cancer cells can be made to multiply in culture for use in optimizing the patient’s therapy, if required.

[0229] The ability of Seraph 100 to remove CTCs is close to 100% of the incoming amount for every pass of blood through the filter. At even moderate blood flowrates this translates to rapid reduction to undetectable levels, often in an hour or two. We have observed that the speed of reduction greatly exceeds the rate predicted by in vitro lab tests, which are not enhanced by the patient’s immune system. The most likely explanation for this is that the patient’s immune system becomes more effective against the cancer during and after the treatment. That is, the Seraph 100 filter itself removes CTCs, CSCs, etc., but the immune system is also reactivated. Thus, by debulking the blood of cancer-related substances, the patient’s body is again able to resume the job of fighting cancer and associated bloodstream infections.FIG. 9 Seraph 100 EBF Therapy is designed to reduce the concentration of pathogens, circulating tumor cells, cancer stem cells (CTCs and CSCs) and miscellaneous ‘debris’ in the bloodstream to a level that no longer overwhelms the patient’s immune system. Once this happens, the immune system appears to be ‘reactivated’ and thereby capable of mounting an effective defense against the cancer in combination with the Seraph 100 filter. The upper blue line in the graph is the expected CTC reduction rate for the filter alone, based on analysis of spiked lab samples and extracorporeal clinical samples. The lower red line is the measured rate of CTC reduction during a typical clinical treatment when blood is filtered and continuously returned to the patient. These results strongly suggest that the filter is working together with the patient’s immune system to rapidly reduce CTC level.Example 8

[0230] This example illustrates treatment results of a cancer patient with small cell lung cancer. A cancer patient with small cell lung cancer is treated for 5 hours with a blood filter (Seraph®100, ExThera Medical) comprised of beads coated with heparin. CTC concentrations were measured before, during and after the treatment.

[0231] Results showed that CTC counts became undetectable during the treatment. Follow-up examination found that the inflammatory response around the solid tumor increased as was evidenced by CT scans and a tumor biopsy. With CTCs and other cancer mediators removed from the subject, the innate immune system was refocused towards the tumor’s microenvironment. The blood filtration process thus enabled neutrophil infiltration of the tumor with resulting inflammation. The blood filtration process resulted in removal of CTCs and an immune system anti-tumor reaction. The immune system recognized the tumor as foreign matter and began to eradicate naturally, which resulted in tumor shrinkage.Example 9

[0232] This example illustrates treatment protocol and advantages of the methods of the disclosure. The treatment methods disclosed herein do not have the side effects of radiation or chemotherapy. The therapeutic procedures are designed to quickly reduce the concentration of cancer cells (CTCs and CSCs) and cancer-related ‘debris’ in the bloodstream to a level that no longer overwhelms the patient’s immune response. Once this happens, the immune system becomes activated and thereby capable of mounting an effective defense against cancer. The adsorption media therapy is designed to treat cancer at the same time it treats potentially fatal cancer-related ‘pathogenemias’, the opportunistic bloodstreaminfections which afflict most cancer patients. Unlike chemotherapy, the present therapy make patients feel better immediately.

[0233] There are many meaningful indicators and biomarkers that support the idea that rapid removal of CTCs and pathogens from the bloodstream with the adsorption media treatment leads to ‘immune system activation’. In the clinical cases we have performed to date we have observed the following: ix. Very rapid decrease in CTCs and Cancer Stem Cells (CSC) by the adsorption media procedure. x. Tumor shrinkage xi. Positive-to-negative biopsies following treatment. xii. Inflammation surrounding cancerous tissue after treatment. xiii. Increase in killer T-cells after cancer treatment. xiv. Increasing antibody levels with decreasing viral load (in COVID-19) xv. Pathogens, ‘NETs’, exosomes and noncellular debris trapped on the adsorption media filter. xvi. Overall improvement in clinical signs and symptoms during and after treatment.

[0234] Relatively small improvements in overall survival are often cited in the promotion of cancer therapies. In addition, it may be inaccurate to claim that any therapy alone is a ‘cure’ for cancer, or for a serious bloodstream infection, for that matter. However, a therapy that reduces a patient’s CTCs, and / or shrinks tumors, and / or causes biopsies to become negative is definitely noteworthy. Given this, a blood-compatible adsorption filter that can safely reduces CTC levels and cancer stem cells to an undetectable level in less than an hour is an exciting new tool in cancer treatment. These positive responses to adsorption media therapy disclosed herein have been accompanied by general improvement in the patient’s signs and symptoms, including energy level, tremors and discoloration of the extremities.

[0235] The delivery of anti-cancer therapy by blood purification is similar to common hemodialysis, a life-saving therapy that patients with chronic kidney failure receive three times every week. The likely difference is that after a few short cancer treatments, the adsorption media therapy may be temporarily discontinued while the body attacks the original tumor and the metastatic sites it has produced. Later, a regular but infrequent schedule of ‘tune-up’ treatments may be used to prevent recurrence. Initially the adsorption media procedure itself will be more costly than single hemodialysis sessions, which havebecome commoditized. However, a course of cancer treatment by the adsorption media procedure is expected to eventually cost only a fraction of the cost of many new or existing cancer therapies.

[0236] The inherently low cost of the therapeutic procedure will eventually reduce the cost of cancer care while making effective cancer treatment available to a much larger patient population, including underserved or disadvantaged groups for whom cancer survival today is unnecessarily low.

[0237] How can the adsorption media treatment, an apparently simple procedure that relies on blood filtration have a positive effect on cancer, and at the same time treat associated bloodstream infections? Because it is designed to mimic sites within the body that are targeted by CTCs, CSCs, bacteria, viruses and fungi. This is done by permanently modifying the blood-contacting surfaces of the filter with heparin, which performs at least three important functions:4. It augments the heparin-containing ‘mast cells’, which are immune cells present in the skin, the nasal passages, the lungs and the gut, and which provide the first line of defense by the immune system against invasion.5. By mimicking the internal (glycocalyx) lining of blood vessels the filter can bind disease-causing substances and remove them from the bloodstream6. It provides a surface that actually prevents the treated blood from clotting on contact, which contributes to the safety of the procedure.

[0238] The device disclosed herein is a depth-type filter with a ‘tortuous path’ through which the blood flows around and between small heparin-modified beads. Their very high surface area provides plenty of contact for interactions between the blood and the surface of the beads. A less blood-compatible filter would cause clotting and clog the filter. With heparin-coated beads of controlled size, blood cells pass through the filter unharmed, while aggregates and micro-clots are unable to pass. That is, in addition to becoming bound to the filter media by specific chemical and physical interactions, they can also be stopped by exclusion through the filter media because of their large size or inability to deform.

[0239] In the near future, the treatment of cancer will look very different. Today multiple chemotherapy sessions initially make patients feel worse, causing fatigue, loss of appetite, nausea, bowel issues, hair loss, mucositis, and skin problems. These side effects can be sobad that patients sometimes opt out of therapy and succumb to their cancer (and associated superinfections) before their time.

[0240] With the adsorption media procedure, patients are hooked up to a simple blood pump for 2-4 hours, during which they can read or watch TV without experiencing negative side effects. Peripheral vein access works well at low to moderate flowrates, so the therapy can be safely administered at home without indwelling catheters or central lines. Nocturnal treatment will be an option. Symptoms of opportunistic infections may subside or disappear, and the patient’s energy level is likely to improve.

[0241] During the first week of treatment the patient may receive two or three procedures with about a one-week break, during which the patient’s immune system will be fighting the cancer without the burden of large numbers of CTCs or CTSs in the bloodstream. This is followed by a few procedures during the following couple of weeks, all of which are followed by imaging, liquid or surgical biopsies and / or histology.

[0242] What is removed from the blood during a therapeutic procedure can be identified to determine the patient’s progress and / or the need for additional procedures. Alternatively, the cancer cells captured on the filter can be grown in culture to produce more cells for studying their susceptibility to drugs: a form of personalized medicine. We refer to this aspect of the procedure as Intelligent Therapy. This is because the therapeutic treatment itself is ‘interrogating’ the entire blood volume of the patient in the first hour and the captured CTCs can be made to multiply in culture for use in optimizing the patient’s therapy, if required.

[0243] The ability of the filter to bind and remove CTCs is close to 100% of the incoming amount for every pass of blood through the filter. At even moderate blood flowrates this translates to rapid reduction to undetectable levels, often in an hour or two. We have unexpectedly observed that the speed of reduction exceeds the rate predicted by lab tests which are not enhanced by the patient’s immune system. The most likely explanation for this is that the patient’s immune system becomes effective against the cancer during the treatment procedure. That is, the therapeutic procedure itself removes CTCs, etc., but the immune system is also activated. Thus, by debulking the blood of cancer-related substances, the patient’s body is again able to resume the job of fighting and eventually curing cancer and associated bloodstream infections.Example 10

[0244] This example relates to treating a central catheter related infection during continuous renal replacement therapy (CRRT) or an EECMO canula related blood stream infection. The methods and systems herein find utility in treating a central catheter related infection during continuous renal replacement therapy (CRRT) or an EECMO canula related blood stream infection.

[0245] A patient with confirmed central catheter / canula gram positive infection and clinical signs and symptoms of sepsis is treated for 4 hours with an adsorption media comprising beads coated with heparin. Results will indicate that gram positive bacteria become undetectable at the end of treatment with patient clinical symptomatology improving. The extracorporeal blood purification with the filter comprised of beads coated with heparin remove bacteria from the blood which enable innate and adaptive immunity to regain preblood stream infection homeostasis. This is evident by significant decrease in CRP as compared to a pre-treatment baseline.

[0246] Example 11

[0247] Further, increased VEGF-A expression leads to immunosuppression by inhibiting dendritic cell (DC) maturation, reducing T cell tumor infiltration, and promoting suppressive cell types in the tumor microenvironment. By removing VEGF-A, the immune system can take on a role in cancer treatment.

[0248] This example relates to vascular endothelial-derived growth factor A(VEGF-A) Vascular endothelial-derived growth factor A (VEGF-A) has been proven to be integral in the molecular pathogenesis of metastasis and tumor growth. It is known that VEGF is responsible for encoding a heparin-binding protein, which exists as a disulfide-linked homodimer which serves to promote the propagation and movement of vascular endothelial cells. This is essential for both pathological and physiological angiogenesis. Circulating epithelial tumor cells (CETC) and cancer stem cells are potent secretors of VEGF-A which is an essential cytokine that facilitates new vascular vessel formation, thus further enabling metastatic tumor growth.

[0249] Methods: We developed a new extracorporeal microbind affinity blood filter platform technology with active heparan sulfate receptors (onco seraph i.e., SERAPH® 100 Microbind® Affinity Blood Filter ). Heparin is cleaved into short chains which are permanently covalently bound to ultrahigh molecular weight polyethylene (UHMWPE) surfaces to create a new composition of matter. Liquid biopsy established high capacity forthe rapid removal of circulating epithelial tumor cells (CETC) and Pancreatic ductal adenocarcinoma (PDAC) cancer stem cells.

[0250] Onco seraph was investigated in a dose escalation phase I / II clinical trial in adult patients with advanced and relap sed / refractory solid tumor and / or PDAC after standard of care and other lines of therapy failed in preventing disease progression.Below is data for pancreatic neuroendocrine cancer (Sample B1-B3) and Lung adenocarcinoma patient (Sample C1-C3). VEGFA was measured using Olink from ThermoFisher Scientific.

[0251] Results: To date 12 patients have been treated with Onco seraph. No patient experienced treatment related adverse effects. Best responses include complete responses and stable disease at a low Onco seraph dose. Greatest efficacy was observed for patients whose liquid biopsy revealed significant and rapid reduction of CETC and cancer stem cells, followed by greater than 50% reduction in circulating VEGF-A.

[0252] Conclusions: This data supports a conclusion that the onco seraph platform technology is safe and has a high therapeutic value as an extracorporeal treatment for metastatic solid tumors refractory to standard of care.

[0253] The patents and references mentioned herein are incorporated by reference. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.Additional EmbodimentsEmbodiment 1. A method for treating a cancer in a subject in need thereof, the method comprising: treating the subject by contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent with a whole blood sample from the subject ex vivo to form a cleansed sample, wherein the adsorption media has a binding affinity for circulating cancer cells and mediators that inhibit an immune response; and reducing the inhibition of the immune response by increasing the bioavailability of immune cells to thereby treat the cancer.Embodiment 2. The method of embodiment 1, wherein the method increases local inflammation.Embodiment 3. The method of embodiment 1, wherein the cancer is a solid tumor cancer or a metastatic lesion.Embodiment 4. The method of embodiment 3, wherein the solid tumor cancer or metastatic lesion is a member selected from the group consisting of lung, colorectal, prostate, testicular, pancreatic, adenocarcinoma, renal, breast, ovarian, endometrial cervical, urothelial, thyroid, stomach, liver, and melanoma.Embodiment 5. The method of embodiment 1, wherein the at least one polysaccharide is a member selected from the group consisting of sulfated polysaccharides, glycosaminoglycans, heparin, heparan sulfate, hyaluronic acid, sialic acid, carbohydrates with mannose sequences, and chitosan.Embodiment 6. The method of embodiment 1, wherein the substrate comprises a plurality of rigid polymer beads.Embodiment 7. The method of embodiment 6, wherein the rigid polymer beads are rigid polyethylene beads.Embodiment 8. The method of embodiment 6, wherein the beads have a diameter ranging from about 100 to about 450 microns.Embodiment 9. The method of embodiment 5, wherein the at least one polysaccharide is heparin or heparan sulfate.Embodiment 10. The method of embodiment 9, wherein heparin has a mean molecular weight of about 2 kDa to about 15 kDa.Embodiment 11. The method of embodiment 9, wherein heparin is attached to the substrate by covalent end-point attachment, multipoint attachment or ionic bound heparin.Embodiment 12. The method of embodiment 1, wherein the substrate having at least one polysaccharide adsorbent is a cartridge.Embodiment 13. The method of embodiment 1, wherein the method reduces tumor burden by reducing or eliminating tumor cells.Embodiment 14. The method of embodiment 1, wherein the method includes immune system activation and migration of immune cells into tumors, which results tumor cells death and tumor reduction.Embodiment 15. The method of embodiment 1, wherein the method removes circulating tumor cells and circulating cancer stem cells from the blood sample.Embodiment 16. The method of embodiment 15, wherein the method comprises measuring circulating tumor cells and circulating cancer stem cells before, during and after treatment.Embodiment 17. The method of embodiment 1, wherein the method removes cancer stem cellsEmbodiment 18. The method of embodiment 1, wherein the method reduces or completely eliminates T-cell exhaustion.Embodiment 19. The method of embodiment 3, wherein the method includes immune cell, cytotoxic T-cells infiltration into the solid tumor and / or metastasis.Embodiment 20. The method of embodiment 1, wherein the method reduces Regulatory T- cells.Embodiment 21. The method of embodiment 1, further comprising administering an immunostimulatory agent.Embodiment 22. The method of embodiment 21, wherein the immunostimulatory agent is a member selected from the group consisting of anti-cancer agent, INF-y, IL-7 and checkpoint inhibitors.Embodiment 23. The method of embodiment 22, wherein the checkpoint inhibitor is a member selected from the group consisting of pembrolizumab (Keytruda), ipilimumab (Yervoy), nivolumab (Opdivo) and atezolizumab (Tecentriq).Embodiment 24. The method of embodiment 21, wherein the checkpoint inhibitor is a member selected from the group consisting of anti-PDl, anti-PDLl, anti-CTLA4, anti-TIM3, and anti-LAG3 antibodies.Embodiment 25. The method of embodiment 1, wherein the method comprises contacting the whole blood sample ex vivo between 1-5 times every 7 days for up to 28 days.Embodiment 26. The method of embodiment 1, wherein the method comprises contacting the whole blood sample ex vivo up to 3 times for the first week of treatment followed by once weekly for the following 3 weeks, for a total of up to 6 treatments over the first 28 days; and biweekly monthly thereafter or as needed.Embodiment 27. The method of embodiment 1, wherein the method is a treatment modifying method, wherein the method is performed as an adjunct therapy before, during or after a primary therapy.Embodiment 28. The method of embodiment 1, wherein the method is a disease modifying method, wherein the method is performed as an adjunct therapy before, during or after a primary therapy.

[0254] The patents and references mentioned herein are incorporated by reference. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0255] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview ofthis application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. An extracorporeal method for treating a cancer in a subject in need thereof, the method comprising: treating the subject by contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent comprising heparin, heparan sulfate or a mixture thereof, with a whole blood sample from the subject conveyed through an extracorporeal circuit to form a cleansed blood sample, wherein the adsorption media has a binding affinity for circulating cancer cells and mediators that inhibit an immune response; and reducing the inhibition of the immune response by increasing the bioavailability of immune cells by removing cancer mediators in the whole blood sample; and returning the cleansed blood sample to the subject to thereby treat the cancer.

2. The method of claim 1, wherein the cancer is a solid tumor cancer.

3. The method of any one of claims 1-2, wherein the solid tumor cancer is selected from lung, colorectal, prostate, testicular, pancreatic, adenocarcinoma, renal, breast, ovarian, endometrial cervical, urothelial, thyroid, stomach, or liver cancer.

4. The method any one of claims 1-3, wherein the method treats metastasis.

5. The method any one of claims 1-4, wherein the method reduces inhibition of the subject’s immune system.

6. The method any one of claims 1-5, wherein the method removes circulating tumor cells and circulating cancer stem cells from the blood sample.

7. The method any one of claims 1-6, wherein the method reduces or eliminates T-cell exhaustion and Regulatory T-cells.

8. The method any one of claims 1-7, wherein the method includes infiltration of immune cells and cytotoxic T-cells into the solid tumor.

9. The method any one of claims 1-8, wherein the method reduces or removes a cancer mediator selected from the group consisting of circulating growth factors that contribute to angiogenesis of tumors, circulating cytokines that contribute to angiogenesis, circulating heparanases that degrade heparan sulfate segments on endothelial cell walls that can lead to tumor cell invasion, and circulating fibrin or thrombin that leads to venous thromboembolism.

10. The method any one of claims 1-8, wherein the method reduces or removes bloodborne acellular debris or neutrophil extracellular traps.

11. The method of claim 9, wherein the cancer mediator is a member selected from the group consisting of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), IL-8, heparanases, pro-uP A (pro-urinary plasminogen activator protein), plasminogen, and thrombin.

12. The method any one of claims 1-11, wherein the method comprises contacting the whole blood sample of the subject between 1-5 times every 7 days for up to 28 days in a treatment regimen.

13. The method any one of claims 1-12, wherein the method is performed on the subject as an adjunct therapy before, during or after a primary therapy.

14. The method of claim 13, wherein the primary therapy is chemotherapy.

15. The method any one of claims 1-14, further comprising administering an immunostimulatory agent.

16. The method of claim 15, wherein the immunostimulatory agent is a member selected from the group consisting of anti-cancer agent, INF-y, IL-7 and checkpoint inhibitors.

17. The method of claim 16, wherein the checkpoint inhibitor is a member selected from the group consisting of pembrolizumab (Keytruda), ipilimumab (Yervoy), nivolumab (Opdivo) and atezolizumab (Tecentriq).

18. The method of claim 16, wherein the checkpoint inhibitor is a member selected from the group consisting of anti-PDl, anti-PDLl, anti-CTLA4, anti-TIM3, and anti- LAG3 antibodies.

19. The method any one of claims 1-18, wherein the blood sample flow rate through the extracorporeal circuit is between 50-450 mL / min.

20. A system for treating cancer in a subject, the system comprising: an extracorporeal circulation device that is for treating and returning a cleansed blood sample of the subject, wherein the system includes a blood circuit, a cartridge comprising adsorption media comprising a substrate having at least one polysaccharide adsorbent and a blood pump or gravity -based delivery system, wherein the extracorporeal blood circuit treats cancer by removing cancer mediators which reduce or inhibit the subject’s immune system, to thereby treat the cancer.

21. A method for profiling cancer disease characteristics and / or functional activity of a subject, the method comprising: contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent with a whole blood sample from the subject ex vivo to make an adhering complex between the adsorption media and cancer cells; releasing the adhering complex of cancer cells; and interrogating the cancer cells to generate a disease profile.

22. The method any one of claims 20-21, wherein the at least one polysaccharide is a member selected from the group consisting of heparin, heparan sulfate, hyaluronic acid, sialic acid, carbohydrates with mannose sequences, and chitosan.

23. The method of claim 21, wherein the substrate comprises a plurality of rigid polymer beads.

24. The method of claim 23, wherein the rigid polymer beads are rigid polyethylene beads.

25. The method of claim 21, wherein the disease profile comprises the type and stage of cancer.

26. The method of claim 21, wherein the disease profile comprises therapeutic options.

27. The method of claim 21, wherein the cancer cells are analyzed in a multiwell plate each well with an anticancer drug.

28. The method of claim 27, wherein the cancer cells are analyzed for therapeutic efficacy of the anticancer drug.

29. The method of claim 21, wherein the cancer cells are tumor cells, circulating tumor cells or cancer stem cells.

30. The method of claim 28, wherein the anticancer drug is a series of anticancer drugs or combinations of anticancer drugs.

31. The method of claim 21, wherein the disease profile comprises a gene expression profile indicative of diagnosis and prognosis of cancer in the subject.

32. The method of claim 21, wherein the method comprises selecting a treatment regimen or monitoring disease status by evaluating the likelihood of efficacious treatment or the status of treatment of a subject having cancer.

33. The method of claim 21, wherein the disease profile is a patient specific result based upon the diagnosis and therapeutic options.

34. The method of claim 21, wherein the disease profile is generated using a neural network model trained on the genotype and phenotype of the cancer cell.

35. The method of claim 21, wherein the disease profile is assessed before, during and after treatment.

36. A specimen extraction device, the device comprising: a spring-loaded connector configured for insertion into a cartridge comprising a substrate having at least one polysaccharide adsorbent; an extractor for removing the substrate having at least one polysaccharide adsorbent; and a barrel for storing the substrate.

37. The device of claim 36, wherein the extractor is a vacuum extractor or an external energy augmented extraction.

38. The device of claim 36, wherein the barrel is optionally 2 or optionally 3 barrels for testing and analyzing the substrate.

39. A method for inhibiting neovascularization of a solid tumor in a subject in need thereof, the method comprising: treating the subject by contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent comprising heparin, heparan sulfate or a mixture thereof, with a whole blood sample from the subject conveyed through an extracorporeal circuit to form a cleansed blood sample, wherein the adsorption media has a binding affinity for vascular endothelial growth factor (VEGF); reducing the amount of VEGF in the whole blood sample to inhibit neovascularization; and returning the cleansed blood sample to the subject.

40. The method of claim 39, wherein VEGF includes VEGF-A.

41. The method of claim 39, wherein the solid tumor cancer is selected from the group consisting of lung, colorectal, prostate, testicular, pancreatic, adenocarcinoma, renal, breast, ovarian, endometrial cervical, urothelial, thyroid, stomach, gastrointestinal stromal tumor (GIST), glioblastoma or liver cancer.

42. The method of claim 39, wherein the cancer is selected from the group consisting of pancreatic cancer, renal cell carcinoma (RCC), colorectal cancer, non-small cell lung cancer (NSCLC), gastrointestinal stromal tumor (GIST), and glioblastoma.

43. The method of claim 39, wherein the method treats metastasis.

44. The method of claim 39, wherein the method reduces the amount of VEGF to a normal level.

45. The method of claim 39, wherein the at least one polysaccharide further comprises a member selected from the group consisting of hyaluronic acid, sialic acid, carbohydrates with mannose sequences, and chitosan.

46. The method of claim 39, wherein the substrate comprises a plurality of rigid polymer beads.

47. The method of claim 46, wherein the rigid polymer beads are rigid polyethylene beads.

48. The method of claim 39, wherein treating the subject further comprises administering an angiogenesis inhibitor.

49. The method of claim 48, wherein the angiogenesis inhibitor is a member selected form the group consisting of bevacizumab, ramucirumab, ziv-aflibercept, sunitinib, sorafenib, axitinib, pazopanib and vandetanib.

50. The method of claim 39, wherein the blood sample flow rate through the extracorporeal circuit is between 50-450 mL / min.

51. The method of claim 39, wherein the solid tumor is reduced in size.

52. A method for treating sepsis in a subject in need thereof, the method comprising: treating the subject by contacting an adsorption media comprising a substrate having at least one polysaccharide adsorbent with a whole blood sample from the subject ex vivo to form a cleansed sample, wherein the adsorption media has a binding affinity for inflammatory mediators that inhibit an immune response; and reducing the inhibition of the immune response by increasing the bioavailability of immune cells to thereby treat sepsis.

53. The method for treating sepsis of claim 52, wherein the inflammatory mediator is a member selected from the group consisting of a cytokine, a pathogen-associated molecular patterns (PAMPs) and a damage-associated molecular patterns (DAMPs).

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