KR20230016032A - System and method for removal of immune inhibitors from biological fluids - Google Patents

Abstract

The systems and methods are useful for removing immunosuppressive agents, such as soluble TNF receptors, from body fluids of cancer patients. In some embodiments, soluble TNF-receptors 1 and 2 are selectively removed from plasma with an efficiency of 80% or greater. In some embodiments, the system includes an immobilized capture ligand of single chain TNF alpha. The systems and methods are useful for the treatment of various cancer types, stages and severities.

Description

Systems and methods for removing immunosuppressants from biological fluids {SYSTEM AND METHOD FOR REMOVAL OF IMMUNE INHIBITORS FROM BIOLOGICAL FLUIDS}

1. field

The present disclosure relates to systems and methods for removing immunosuppressive agents from plasma.

2. Introduction

Harnessing the immune system to kill cancer has been a major concern of oncologists and cancer researchers for over a century. Observation that patient tumors go into remission after infection with immune stimulatory bacteria (see, e.g., Coley, W. B. (1991). Clin Orthop Relat Res, 3-11; Hughes, W. T., and Smith, D. R. (1973) Cancer 31, 1008-1014; Yates, J. W., and Holland, J. F. (1973) Cancer 32, 1490-1498; Muller, H. E. (1974) (author's translation), Pathol Microbiol (Basel) 40, 297-304) as well as Correlation between immune cell infiltration and survival (see, eg, Lipponen, P. K., et al. (1992) Eur J Cancer 29A, 69-75; Ma, D., and Gu, M. J. (1991) J Tongji Med Univ 11, 235-239; Pastrnak, A., and Jansa, P. (1989) Acta Univ Palacki Olomuc Fac Med 124, 7-71; Di Giorgio, et al. (1992) Int Surg 77, 256-260); Di Giorgio, A., et al. (1992) Int Surg 77, 256-260) suggested the possibility of immunological modulation of neoplasia. However, it is only in the last decade that practitioners in the field of cancer immunotherapy have been able to claim significant improvements in patients' prognosis. A major achievement in this field has been the development of antibodies that inhibit negative regulators or checkpoints of T-cell activation. These antibodies belong to a class of drugs called “immune checkpoint inhibitors”. The first antibody approved by the FDA, ipilimumab, an antagonistic antibody targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), improved overall survival in patients with metastatic melanoma in 2010. A related study was unresectable stage III or A total of 676 HLA-A*0201-positive patients with stage IV melanoma were evaluated. Median overall survival was 10.0 months for patients receiving ipilimumab plus gp100 and 6.4 months for patients receiving gp100 only (hazard ratio for death, 0.68; P<0.001). Median overall survival with ipilimumab alone was 10.1 months (hazard ratio for death compared with gp100 alone, 0.66; P=0.003). No difference in overall survival was detected between the ipilimumab groups (hazard ratio of ipilimumab+gp100, 1.04; P=0.76) (see, e.g., Hodi, F. S., et al. (2010) N Engl J Med 363, 711-723). Following the success of anti-CTLA-4 therapy, antibodies targeting programmed cell death protein 1 (PD-1) or its ligand PD-L1 have proven effective in improving overall survival in a variety of cancers (e.g. , Hamid, O., et al., (2013) N Engl J Med 369, 134-144; Herbst, R. S., et al. (2014) Nature 515, 563-567; Powles, T., et al. (2014) Nature 515, 558-562; Topalian, S. L., et al. (2014) J Clin Oncol 32, 1020-1030; Ribas, A., and Wolchok, J. D. (2018) Science 359, 1350-1355) . For example, in one study, 296 patients were treated with an anti-PD-1 ligand antibody. Of the 236 patients whose response could be evaluated, an objective response (complete or partial response) was observed in patients with non-small cell lung cancer, melanoma or renal cell carcinoma. The cumulative response rate (all doses) was 18% (14 of 76) in patients with non-small cell lung cancer, 28% (26 of 94) in patients with melanoma, and 27% in patients with renal cell carcinoma (9 out of 33). The response was persistent; 20 of 31 responses lasted more than 1 year in patients who received more than 1 year of follow-up (see, eg, Topalian, S. L., et al. (2012) N Engl J Med 366, 2443-2454) . The encouraging results of these studies have generated interest in the field of cancer research and inspired further research into the targeting of alternative immune checkpoint molecules.

Although checkpoint blockade represents a breakthrough in cancer therapy, most cancer patients do not respond to such treatment, and some tumor types appear to be intrinsically resistant. This treatment is designed to enhance a sustained immune response and is ineffective in cases where initial immune activation is lacking, including in tumors lacking infiltrating T-cells. Thus, development of therapeutic strategies to enhance immune cell recruitment may increase the proportion of patients who respond to immune checkpoint blockade. Limitations of checkpoint inhibitors include the inability to consistently elicit a response as well as systemic exposure of the patient to the antibody used.

TNFα (tumor necrosis factor-alpha or interchangeably referred to herein as TNF) promotes anticancer activity and, as its name implies, is a potent cytokine initially characterized as an antitumor agent (e.g. , Carswell, E. A., et al. (1975) Proc Natl Acad Sci USA 72, 3666-3670). Subsequently, TNF has been shown to have both pro-tumor and anti-tumor effects depending on its contextual activity within the tumor microenvironment (see, e.g., Wang, X., and Lin, Y. (2008) Acta Pharmacol Sin 29, 1275-1288]). In the tumor microenvironment, expression of low levels of TNF contributes to angiogenesis, vascular permeability and metastatic potential; At high levels and during therapeutic delivery to tumors, TNF has exhibited anti-tumor effects including disruption of vascular integrity through apoptosis, direct tumor killing and induction of anti-tumor immune responses (see, e.g., Berberoglu, U. (2004) Int J Biol Markers 19, 130-134 Michalaki, V., et al. (2004) Br J Cancer 90, 2312-2316 Talmadge, J. E., et al. 47, 2563-2570). Beneficial effects of elevated TNF in clinical settings have been reported. For example, a study of TNF expression in 61 non-small cell lung carcinoma patients demonstrated expression of TNF in 45.9% of cases directly correlated with a more favorable clinical outcome (see, e.g., Boldrini, L ., et al., G. (2000) Br J Cancer 83, 480-486). TNF administration has been approved for isolated limb administration and has shown clinical benefit in isolated liver procedures for liver cancer.

sTNF-R (a soluble receptor for TNF) suppresses anticancer immune responses and contributes to the regulation of TNF toxicity. The natural modulation or attenuation of TNF antitumor effects is due to the presence of inhibitory molecules, including released soluble TNF receptors, present in plasma and binding/neutralizing TNF (see, e.g., Xanthoulea, S., et al. (2004) J Exp Med 200, 367-376; Aderka, D., et al. (1998) J Clin Invest 101, 650-659; Aderka, D., et al. (1991) Cancer Res 51, 5602-5607 Selinsky, C. L., et al. (1998) Immunology 94, 88-93; Selinsky, C. L., and Howell, M. D. (2000) Cell Immunol 200, 81-87). The cancer-promoting activity of these soluble inhibitors was discovered after early observations of cancer regression in patients undergoing plasmapheresis (see, eg, Israel, L., et al. (1976) Lancet 2, 642-643; Israel, L., et al. (1977) Cancer 40, 3146-3154). Subsequent studies showed that these observations were due to sTNF-R clearance. Molecular cloning of cDNA and studies of recombinant proteins have confirmed their anti-TNF activity and pro-tumor function (see, e.g., Schall, T. J., et al. (1990) Cell 61, 361-370; Engelmann, H., et al. (1990) J Biol Chem 265, 1531-1536).

At low doses of TNF, normal concentrations of sTNF-R inhibitors are able to bind and inactivate the small amount of TNF administered. However, administration of increased amounts or stimulation of TNF production to higher than normal levels can induce sTNF-R shedding, which antagonizes TNF's ability to reach therapeutic anti-tumor concentrations without toxic effects. . Thus, the ability to overcome TNF inhibition to achieve anticancer effects requires administration of TNF in amounts that are too close to the maximum tolerated dose (MTD). For this reason, systemic TNF therapy, although probably effective, has been toxic in numerous human clinical trials. Due to these adverse risk/benefit considerations, systemic therapy with TNF has largely been abandoned. However, isolated limb procedures that block systemic exposure to TNF have been performed with chemotherapeutic agents (see, e.g., Deroose, J. P., et al. (2012) Ann Surg Oncol 19, 627-635; Verhoef, C., et al. (2007) Curr Treat Options Oncol 8, 417-427).

Attempts have been made to use medical devices that remove tumor-generating immune “blockers” outside the body. Unfortunately, to date these devices do not have a) non-specific binding of other biological substances; b) "leaching" of the immunoadsorbent material from the device into the patient's circulatory system; and c) ineffective clearance of target proteins from the circulation. The present invention overcomes these and other limitations of prior systems.

outline

The following is a non-exhaustive list of some aspects of the present technology. These and other aspects are described in the following disclosure.

Accordingly, one or more aspects of the present disclosure relate to a system for removing at least one target component of a bodily fluid. The system includes an inlet configured to receive bodily fluid from a patient and an isolation chamber coupled to the inlet and configured to receive bodily fluid from the inlet. The isolation chamber includes a capture support configured to engage at least one target component of the bodily fluid to capture the at least one target component in the isolation chamber in response to contact between the capture support and the bodily fluid. The capture support is configured to bind the at least one target component to reduce the amount of the at least one target component in bodily fluid. The isolation chamber includes first and second access ports configured to provide access to the isolation chamber separate from the inlet. The first and second access ports are configured to facilitate insertion and/or removal of the capture support into and/or from the isolation chamber. The system is configured to pass the bodily fluid having the reduced amount of the at least one target component from the isolation chamber to selectively reintroduce some or all of the bodily fluid having the reduced amount of the at least one target component back to the patient. exit; and one or more filters configured to separate the capture support of the isolation chamber from the inlet and outlet. One or more filters are configured to retain the capture support within the isolation chamber.

In one embodiment, (a) the capture efficiency of the capture scaffold that binds to the at least one target component is at least 80% at a flow rate of plasma flow of 45 mL per minute or less, and optionally (b) the capture scaffold for the one or more target components has a binding affinity of at least about 10 ⁻⁷ K _D and/or (c) a leaching rate of the capture support through the outlet of less than about 100 ng/mL/min.

In one embodiment, (b) the binding affinity of the capture scaffold to the at least one target moiety is greater than 10 ⁻⁷ K _D , and optionally (a) the capture efficiency of the capture scaffold that binds to the at least one target moiety is 45 mL 80% or greater at a flow rate of less than /min, and/or (c) a leaching rate of the capture support through the outlet of less than about 100 ng/mL/min.

In one embodiment, (c) the leaching rate of the capture support through the outlet is less than about 100 ng/mL/min, and optionally (a) the capture efficiency of the capture support that binds to the at least one target component is 45 mL/min. and/or (b) the binding affinity of the capture support to one or more target moieties is greater than or equal to 10 ⁻⁷ K _D .

In one embodiment, the bodily fluid includes plasma.

In one embodiment, the at least one target component comprises a protein, complex, assembly or cell.

In one embodiment, the at least one target component comprises one or more plasma components that function to suppress an anti-cancer immune response in a patient.

In one embodiment, at least one target component comprises one or more immunosuppressive agents.

In one embodiment, the at least one targeting component comprises a soluble TNF-α receptor.

In one embodiment, the at least one targeting component comprises a sTNF-R1 receptor and/or a sTNF-R2 receptor.

In one embodiment, the capture support comprises an affinity chromatography support material. In other embodiments, the capture support includes hollow fiber membranes, sheet or rolled sheet membranes, membrane cassettes and/or beads.

In one embodiment, the capture support comprises an affinity chromatography support material, and the affinity chromatography support material comprises sepharose, agarose or acrylamide.

In one embodiment, the entrapment support comprises a porous or non-porous matrix material including but not limited to a ceramic material.

In one embodiment, the capture scaffold is configured to bind to more than one target component of a bodily fluid.

In one embodiment, the capture support comprises a solid support on which an antibody, antibody fragment, conjugated peptide, aptamer or avimer is immobilized.

In one embodiment, the antibody is selected from the group consisting of IgA, IgD, IgE, IgG, IgM and combinations thereof.

In one embodiment, the capture scaffold comprises TNFα, multimers of TNFα, single-chain TNFα, fragments of TNFα, multimers of fragments of TNFα, or combinations thereof.

In one embodiment, the multimer of TNFα comprises TNFα monomers in which one or more monomers are amino-terminus to carboxyl-terminal linkages.

In certain embodiments, multimers of TNFα may exclude or include spacers between monomers.

In certain embodiments, a spacer comprises one or more amino acid residues.

In one embodiment, the spacer comprises one or more glycine, serine and/or alanine amino acids.

In one embodiment, the capture scaffold comprises the entire sequence or partial sequence of a sc-TNFα ligand, optionally SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

In one embodiment, the capture scaffold comprises a trimeric form of a TNFα ligand.

In one embodiment, the trimeric form of the scTNFα ligand comprises the sequence of SEQ ID NO:2 or SEQ ID NO:3 with or without spacer amino acids.

In one embodiment, the capture scaffold comprises a ligand bound to a bead.

In one embodiment, the ligand has a given density and orientation in a given bead. The density and/or orientation is configured to enhance binding between the ligand and the at least one target component of the bodily fluid.

In one embodiment, the size, number, density and/or concentration of the beads is configured to promote laminar flow of bodily fluid through the beads to enhance binding between the ligand and at least one target component of the bodily fluid.

In one embodiment, beads are quenched in ethanolamine to enhance binding specificity.

In one embodiment, the bodily fluid is whole blood.

In one embodiment, the inlet, isolation chamber and outlet form an extracorporeal closed circuit column.

In one embodiment, the extracorporeal closed circuit column is configured to remain sterile during operation.

In one embodiment, the system includes a target component exit port configured to facilitate sampling or removal of all or a portion of the captured at least one target component without damaging the extracorporeal closed circuit column.

In one embodiment, the system includes an elution reagent port configured to facilitate introduction of an elution reagent into the isolation chamber without damaging the extracorporeal closed circuit column.

In one embodiment, the elution reagent port is further configured to receive a conditioning agent configured to prepare the system for reuse.

In one embodiment, the system further includes a pump configured to drive the regenerant through the inlet, the isolation chamber, and the outlet.

In one embodiment, the pump comprises a syringe pump, a peristaltic pump, a piston pump, a diaphragm pump, or a combination thereof.

In one embodiment, the one or more filters have an average pore diameter of between about 3 microns and about 100 microns.

In one embodiment, the system further comprises one or more additional isolation chambers comprising a capture support having the same function.

In one embodiment, one or more additional isolation chambers are combined with the isolation chamber to form a multi-stage isolation circuit configured to engage a plurality of different target components.

In one embodiment, the patient is a human or veterinary subject. Veterinary subjects include livestock such as dogs, cats, and the like; farm or ranch animals such as horses, pigs, cattle, etc.; and/or other animals.

According to yet another embodiment, a method for removing at least one target component of a bodily fluid with the system of any of the embodiments described above is provided. The method includes passing bodily fluid from a patient through an inlet into an isolation chamber; binding the at least one target component of the bodily fluid to capture the at least one target component in the isolation chamber to reduce the amount of the at least one target component in the bodily fluid; and optionally passing some or all of the bodily fluid having the reduced amount of the at least one target component through the outlet for reintroduction from the isolation chamber back to the patient.

In one embodiment, the method further comprises measuring the reduced amount of the at least one target component in the bodily fluid reintroduced to the patient.

In one embodiment, the measurement is liquid chromatography-mass spectrometry (LC-MS), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UHPLC), resistivity measurement, luminescence measurement, chemiluminescence, electroluminescence, electrochemical Luminescence, chromatographic monitoring, positron emission tomography (PET), x-ray computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, gamma camera, single photon emission computed tomography (SPECT), ELISA, surface plasmon resonance (SPR) and/or biolayer interferometry (BLI).

In one embodiment, the method further comprises measuring the leaching rate of the entrapment scaffold in bodily fluid reintroduced to the patient.

In some embodiments, the method is for human, veterinary, livestock/companion animals, ranch/farm animals, and/or other uses.

These and other objects, features and characteristics of the system or method disclosed herein, as well as the methods of operation and function of the relevant elements of the structure and the combination of manufacturing parts and economy, will be considered in the following description and appended claims with reference to the accompanying drawings. As will become clearer when, all of which form part of this specification, like reference numbers indicate corresponding parts in the various drawings. However, it should be clearly understood that the drawings are for illustration and description only and are not intended to define the limitations of the present invention. As used in the specification and claims, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise.

The above-mentioned and other aspects of the present technology will be better understood upon reading this application in view of the following figures in which like numbers represent like or identical elements.
1 illustrates an exemplary embodiment of the present system according to one or more embodiments.
2 provides a more detailed view of the system according to one or more embodiments.
3 illustrates a corresponding cross-sectional view of the present system according to one or more embodiments.
4 illustrates a capture support (e.g., in this embodiment, in an isolation chamber of a housing of a system that binds to a target component of a bodily fluid (e.g., blood plasma in this embodiment) to capture the target component in the isolation chamber, according to one or more embodiments. Ligand-coated beads in) are exemplified.
5 is an enlarged view of the capture support shown in FIG. 4 according to one or more embodiments. The enlarged view shows the capture scaffold comprising a bead and capture ligand.
6 illustrates an exemplary regeneration mechanism in accordance with one or more embodiments.
7 illustrates an exemplary embodiment according to one or more embodiments in which the system includes two or more internal isolation chambers that may be configured in series or parallel for inlet to outlet fluid flow.
8 illustrates an exemplary method of use according to one or more embodiments in which multiple systems may be utilized in a series and/or parallel configuration within a single plasmapheresis flow circuit.
9 illustrates multiple isolation chambers each having independently controllable flow valves in a single housing, according to one or more embodiments.
10 illustrates a method for removing target components of blood, plasma, and/or other bodily fluids with a system, according to one or more embodiments.
11 illustrates representative system performance characteristics including column capture efficiency and sTNF-R1 and sTNF-R2 reduction from a patient's blood pool as a function of procedure time, according to one or more embodiments.

While this invention is capable of many modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will be described in detail herein. The drawing may not be in proportion. However, the drawings and detailed description thereof are not intended to limit the invention to the specific forms disclosed, but on the contrary, all modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the appended claims. What you want to include should be understood.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

In order to alleviate the problems described herein, the inventors have had to invent solutions and, in some cases just as importantly, to recognize problems that have been overlooked (or not yet foreseen) by others in the field. Indeed, the inventors seek to highlight the difficulty of recognizing such problems, which are nascent problems and will become even more evident in the future if industry trends continue as the inventors anticipate. Moreover, because multiple problems are addressed, many of which are addressed simultaneously, some embodiments are problem specific, and all embodiments address all problems of existing systems described herein or provide all advantages described herein. You have to understand that you don't. That said, improvements addressing various variations of this problem are described below.

The systems and methods are useful for immune modulation in cancer patients and may provide relatively useful immune modulation for other diseases including, but not limited to, autoimmune and inflammatory disorders. In some embodiments, in vitro removal of immunosuppressive factors from a patient's blood using an immunosorbent means is provided. In some embodiments, the systems and methods provide efficient removal of soluble tumor necrosis factor receptor (sTNF-R) from cancer patients. TNF is an endogenous cytokine that regulates tumor growth and suppression as part of the body's natural immune response to cancer. However, in many cancers, the antitumor effects of TNF are blocked by the presence of circulating inhibitory molecules known as soluble TNF receptors (sTNF-R1 and sTNF-R2; see, e.g., Gatanaga, T., et al. ( 1990) Lymphokine Res 9, 225-229; Gatanaga, T., et al. (1990) Proc Natl Acad Sci USA 87, 8781-8784; Schall, T. J., et al. (1990) Cell 61, 361-370; Berberoglu , U. et al. (2004) Int J Biol Markers 19, 130-134). These receptors that block the therapeutic antitumor effects of endogenous TNF have been shown to be increased in cancer and correlated with disease stage (see, e.g., Aderka, D., et al. (1991) Cancer Res 51, 5602 -5607]). sTNF-R is also a prognostic marker for breast cancer, malignant melanoma, colorectal cancer and osteosarcoma, and is negatively correlated with patient survival (see, e.g., Langkopf, F., and Atzpodien, J. (1994)). Lancet 344, 57-58; Viac, J., et al. (1996) Eur J Cancer 32A, 447-449). Selective removal of sTNF-R from a patient's blood by plasma apheresis, a process known as Immunopheresis™, enhances the patient's natural anti-tumor immune response by revealing the anti-tumor effects of endogenous TNF, which results in a reduction in tumor burden. and can improve patient survival.

1 illustrates an exemplary embodiment of the present system (item 10). In FIG. 1 , system 10 is shown coupled to apheresis machine 12 . The system 10 may obtain sTNF-R (e.g., soluble tumor necrosis factor receptor 1 (sTNF- R1); The system 10 includes a highly selective binding matrix within a housing 16 having various ports to facilitate filling and plasma flow during use. Blood drawn from the patient 14 may be processed to obtain plasma, which may be processed by placing the system 10 in the plasma flow line 18 of the apheresis machine 12 for a plasmapheresis procedure. can

Examples of commercially available apheresis machines 12 include, for example, the Terumo BCT Spectra Optia system. Other manufacturers of apheresis machines include, but are not limited to, Fresenius, Haemonetics, Baxter, Nigale and Asahi. Apheresis can then be performed according to the manufacturer's instructions.

As shown in FIG. 1 , apheresis machine 12 may facilitate intravenous removal of blood 20 from patient 14 followed by separation 22 of the blood into plasma and cell fractions ( eg using centrifugal forces, membrane filters and/or other components). The plasma fraction is then pumped to system 10, where the plasma is transferred to a capture support (comprising a binding matrix as described herein) that captures sTNF-R using, for example, a TNF ligand (as described herein). ) pass through. The plasma is then pumped back out of system 10, where some or all of the processed plasma can be recombined with the patient's 14 isolated cells and then reintroduced back into the patient's 14 circulatory system. (24).

In some embodiments, the treated plasma may be discarded and replaced with fresh plasma. For example, plasma exchange can occur concurrently with the exchanged plasma being further processed to remove the inhibitor. Immunopheresis (eg, as described herein) can be performed, eg, after plasma exchange.

Although blood, plasma, sTNF-R and TNF ligands are specifically referred to throughout this application, the components and/or principles described herein may be applied to other body fluids, other target components of body fluids, and/or other capture or binding elements. It should be noted that there are

2 provides a more detailed view of system 10. 3 illustrates a corresponding cross-sectional view. Referring to FIGS. 2 and 3 , system 10 includes housing 16 , inlet 200 , capture support 204 , isolation chamber 202 including access ports 206 and 208 , outlet 210 . , one or more filters (eg, 212 and 214 shown in FIG. 3), end caps 250 and 252, and/or other components. As shown in FIGS. 2 and 3 , system 10 may form an extracorporeal closed circuit column, for example.

A closed circuit column may be configured to remain sterile during operation. In some embodiments, components of system 10 are washed prior to assembly, for example with 70% isopropyl alcohol, to remove particulates. Filters 212 and 214 may be fitted to end caps 250 and 252 , which may be pressed into the ends of a barrel or tube (eg) forming housing 16 . Caps 254 and 256 may be threaded and/or otherwise coupled to inlet 200 and outlet 210 . This subassembly can be packaged and sterilized using, for example, ethylene oxide (EtO). EtO residues can be dissipated before continuing with the production phase. The EtO sterilized subassemblies can be aseptically filled into the capture support 204 via access ports 206 and 208, which are then sealed with polycarbonate (eg) lures (eg eg) tightly capped with a cap (eg, as described herein). The assembled devices can then be individually packaged and final sterilized using E-beam irradiation with 17.5-30 kGy (for example). In some embodiments, other means of sterilization that may be used include gamma irradiation, ethylene oxide, hydrogen peroxide, bleach, heat sterilization, steam sterilization, ozone, and/or other sterilization operations, depending on the stability of the capture support 204 and/or other factors. includes

Housing 16, inlet 200, outlet 210, and/or other components of system 10 may be used in an apheresis machine (e.g., machine 12 shown in FIG. 1) (e.g., eg plasma) flow line. Housing 16 , inlet 200 , outlet 210 , and/or other components of system 10 may separate the patient's blood into, e.g., cellular and plasma fractions (e.g., as described herein). ) and then coupled with the flow line of the apheresis machine at a point in the flow line. The housing 16 may form a fluid channel or flow path for conveying a patient's bodily fluid between the inlet 200 and the outlet 210 .

Housing 16 may provide structural support for capture support 204 and/or other components of system 10 . The housing 16 may form an elongated tubular body having a circular cross-sectional shape and/or other cross-sectional shapes. The housing 16 may house an isolation chamber 202 that includes a capture support 204, one or more filters 212 and/or 214, and/or other components. In some embodiments, housing 16 and/or other components of system 10 may be manufactured by injection molding and/or other operations.

Housing 16 can accommodate filters 212 and 214 such that filters 212 and 214 are substantially perpendicular to the direction of fluid flow between inlet 200 and outlet 210 . Filters 212 and 214 may be configured to separate capture support 204 within isolation chamber 202 from inlet 200 and outlet 210 . Filters 212 and 214 may be configured to retain capture support 204 within isolation chamber 202 and/or perform other functions.

In some embodiments, filters 212 and/or 214 may form a porous barrier mounted substantially perpendicular to the direction of fluid flow through housing 16 . A filter 212 may be positioned proximate the inlet 200 and a filter 214 may be positioned proximate the outlet 210, thereby forming an isolation chamber 202 inside the housing 16. can Filters 212 and/or 214 may, for example, allow some (including most and all) of capture supports 204 (eg, one or more beads as described herein) to escape system 10. to prevent passage into the patient's circulatory system. In some embodiments, filters 212 and/or 214 may include, for example, porous frit. In some embodiments, filters 212 and 214 can have average pore diameters and/or other average pore diameters, for example, between about 3 microns and about 100 microns. In some embodiments, filters 212 and 214 have a diameter greater than the inside diameter of housing 16 such that filters 212 and 214 fit snugly within housing 16 and do not move as bodily fluid flows through system 10. can have In some embodiments, filters 212 and 214 are end caps that press filters 212 and 214 against a rim of housing 16 (eg, at either end of a tube formed by housing 16 ). It is held in place by pressure from 250 and 252. In some embodiments, filters 212 and 214 are secured in place within housing 16 by other mechanisms such as adhesives, washers, gaskets, stitching, overmolding, ultrasonic welding, and/or other components and/or processes. do. In some embodiments, filters 212 and 214 may be formed of polyethylene and/or other materials.

In some embodiments, housing 16 includes end caps 250 and 252 at opposite ends of housing 16 that form and/or include inlet 200 and outlet 210 . End caps 250 and 252 can be screwed to housing 16 and/or otherwise coupled to housing 16 (eg, via clips, clamps, adhesives, ultrasonic welding, pressure fit, etc.) It can be. In some embodiments, end caps 250 and/or 252 may be attached on housing 16 to make system 10 substantially airtight. In other words, the system 10 is configured to withstand internal and external pressures (both air and fluid) to ensure sterility during storage, transportation, and use. In some embodiments, end caps 250 and 252 are at the ends of inlet 200 and outlet 210, respectively. In some embodiments, inlet 200 and/or outlet 210 may include caps 254 and 256 . In some embodiments, end caps 250 and/or 252 may be formed from polypropylene and/or other materials. In some embodiments, caps 254 and/or 256 may be formed of, for example, high density polyethylene and/or other materials.

In some embodiments, housing 16 may be formed of plastic and/or other materials. For example, housing 16 may be made of ECTFE (ethylene-chlorotrifluoroethylene copolymer, halar ECTFE, ETFE (ethylene-tetrafluoroethylene), tefzel ethylene tetrafluoroethylene (ETFE) , FEP (Fluorinated Ethylene Polypropylene), HDPE (High Density Polyethylene), LDPE (Low Density Polyethylene), PC (Polycarbonate), Makrolon Polycarbonate, PEI (Polytetherimide), PET (Polyethylene terephthalate), PETG (polyethylene terephthalate copolymer), PFA (polyfluoroalkoxy), Teflon PFA, PMMA (polymethyl methacrylate), PMP (polymethypentene), polypropylene, PPCO (polypropylene) copolymer), polystyrene, PSF (polysulfone), PTFE (polytetrafluoroethylene), SAN (styrene acrylonitrile), TFE (tetrafluoroethylene), Teflon TFE, TMX (termanox), PMX (permanox) ), and/or other materials In some embodiments, housing 16 may be formed from a metallic material (eg, iron, iron alloy, steel, stainless steel, aluminum, aluminum alloy), glass and/or other materials.

Inlet 200 may be configured to receive blood, plasma and/or other bodily fluids from a patient (eg, patient 14 shown in FIG. 1 ). An outlet 210 may be used for reintroducing blood, plasma, and/or other components having reduced amounts of one or more target components from the isolation chamber 202 for reintroduction back to the patient (eg, patient 14 shown in FIG. 1 ). It can be configured to pass bodily fluids. In some embodiments, inlet 200 and/or outlet 210 may be luer fittings and/or other inlet or outlet fluid connector types or configurations. In some embodiments, inlet 200 and/or outlet 210 is an apheresis machine tubing set commonly used in closed loop patient fluid line assemblies, intravenous tubing extension sets, fluid tubing adapters, filters, stopcocks, and/or fluidly coupled to another element. In some embodiments, inlet 200 and/or outlet 210 allows blood, plasma, and/or other bodily fluids from the patient to enter the system ( 10) can be configured to flow through. In some embodiments, the flow rate may be between about 10 mL/min and about 100 mL/min. In some embodiments, the flow rate may be between about 25 mL/min and about 75 mL/min. In some embodiments, the flow rate may be between about 35 mL/min and about 70 mL/min. In some embodiments, the flow rate may be between about 40 mL/min and about 60 mL/min. These exemplary flow rates are in a range that can be accommodated by the systems described herein. For some procedures, flow rates below 5 mL/min may require excessive time to complete. Conversely, flow rates greater than 300 mL/min may limit the capture efficiency of system 10 when used with an apheresis machine. However, flow rates of 300 mL/min are expected to be possible. The inlet 200 and/or outlet 210 may have a particular sized diameter and/or other feature that facilitates such a flow rate.

In some embodiments, inlet 200 and/or outlet 210 may be configured to flow human plasma containing up to about 200 micrograms (eg) of sTNF-R protein through system 10. . This example is based on the patient's expected total plasma amount of sTNF-R. The range of concentrations of sTNF-R (sTNF-R1 and sTNF-R2 in combination) in human plasma is about 3-10 ng/mL, and the plasma volume of an exemplary patient can range from 50 cc/kg body weight (W). . The total amount of sTNF-R is about (W(Kg)x50mL/Kgx(3-10ng/mL)/1000ng/μg). Thus, for a 70 Kg individual (eg), the amount of sTNF-R would be in the range of 10.5 to 35 micrograms. In some embodiments, the ability of system 10 to capture excess is sufficient based on laboratory bench tests for excess sTNF-R with plasma flow through system 10 at a rate of up to about 45 mL/min. Efficiency boundaries can be created. In some embodiments, inlet 200 and/or outlet 210 may be formed of polypropylene, polycarbonate, Macrolon™, and/or other materials.

Isolation chamber 202 may be coupled to inlet 200 . Isolation chamber 202 may be configured to receive blood (eg, whole blood), plasma, and/or other bodily fluids from inlet 200 . Isolation chamber 202 may include capture support 204 , access ports 206 and 208 , and/or other components.

The capture support 204 may be configured to bind at least one target component of blood, plasma and/or other bodily fluid to capture the at least one target component in the isolation chamber 202 . The capture support 204 can be and/or can include, for example, a binding matrix (including bead ligands and/or other components as described herein). Entrapment can occur in response to contact between the capture support 204 and blood, plasma, and/or other bodily fluids. The capture scaffold 204 can be configured to bind at least one target component to reduce the amount of the at least one target component in blood, plasma and/or other bodily fluids.

In some embodiments, at least one target component may comprise a complex, assembly or cell. In some embodiments, the at least one target component may include one or more blood products such as plasma or serum components that function to suppress an anticancer immune response in a patient. In some embodiments, at least one target component may include one or more immunosuppressive agents. For example, the at least one target component may include soluble TNFα receptor, sTNF-R1 receptor, sTNF-R2 receptor, sTNF-R1 and sTNF-R2 receptors, and/or other receptors and receptor combinations.

In some embodiments, the capture moiety can be selected to bind to and capture other specific molecules in plasma. Examples of these other molecules or targets include, but are not limited to, acetyl-choline receptors, adenosine receptors, adrenergic receptors, GABA receptors, angiotensin receptors, cannabinoid receptors, cholecystokinin receptors, dopamine receptors, glucagon receptors, glucocorticoid receptors, glutamate receptors, histamine receptors, mineralocorticoid receptors, olfactory receptors, opioid receptors, purinergic receptors, secretin receptors, serotonin receptors, somatostatin receptors, steroid hormone receptors, calcium sensing receptors, hormone receptors, erythropoietin receptors, and natriuretic peptide receptors or their ligands am. Other examples include type I cytokine receptors such as type I interleukin receptor, erythropoietin receptor, GM-CSF receptor, G-CSF receptor growth hormone receptor, oncostatin M receptor, myostatin receptor, leukemia inhibitory factor receptor; type II cytokine receptors, eg, type II interleukin receptors, interferon-α/β receptors, interferon-γ receptors or their ligands; members of the immunoglobulin superfamily, such as interleukin-1 receptor, CSF1, ckit receptor, interleukin-18 receptor or their ligands; CD27, CD40 and lymphotoxin receptors or their ligands; chemokine receptors, including serpentine CCR and CXCR receptors, such as CCR1 and CXCR4, and interleukin 8 receptors or their ligands; TGF β receptors including TGF β receptor 1 and TGF β receptor 2 or their ligands; galectin; and/or other structures (see Ozaki and Leonard, J. Biol. Chem 277:29355-29353, 2002).

In some embodiments, capture support 204 may include a solid support and/or other components. The solid support can be an affinity chromatography support material, a hollow fiber membrane, a sheet membrane, a membrane cassette, a rolled sheet membrane, and/or other materials. In embodiments where the capture support 204 comprises an affinity chromatography support material, the affinity chromatography support material is a sugar, carbohydrate or polysaccharide such as sepharose, agarose, or a polymer such as acrylamide; and / or other materials.

In some embodiments, capture support 204 may include a porous or non-porous matrix material. In some embodiments, the capture scaffold 204 can be configured to bind to more than one target component of blood, plasma, and/or other bodily fluids.

In some embodiments, capture support 204 is an affinity chromatography matrix comprising different capture moieties, including but not limited to, affinity reagents (eg, ligands as described herein) bound to the support. can Affinity chromatography matrices include, but are not limited to, cyanogen bromide, tresyl, triazine, vinyl sulfone, aldehydes, epoxides, or Linking groups such as activated carboxylic acids may alternatively be included. Chromatographic matrices can be obtained by chemically activating the solid support as necessary and by contacting the solid support with a methyl-lysine affinity reagent such that the affinity reagent is covalently attached to the solid support, thereby obtaining a methyl-lysine affinity to the solid support having a linking group. It can be prepared by coupling reagents. Additionally, the affinity reagent can be coupled to the solid support via a linker to make the affinity reagent more accessible for binding to methylated proteins and peptides.

In some embodiments, capture support 204 may comprise a solid support having antibodies, antibody fragments, conjugated peptides, aptamers, avimers, and/or other components immobilized thereon. In some embodiments, the antibody is one or more of IgA, IgD, IgE, IgG, or IgM, immunoglobulin subclasses and mixtures thereof, combinations thereof, and/or other antibodies.

In some embodiments, the capture scaffold 204 is a ligand, such as TNFα (as described above, TNF and TNFα are used interchangeably herein), a multimer of TNF, single chain (sc) TNF, TNF It may include an affinity reagent comprising a fragment of a fragment, a multimer of a fragment of TNF, or a combination thereof. In some embodiments, the capture support 204 can be and/or can include a TNF ligand bound to one or more solid supports. In some embodiments, a linkage can be, for example, a covalent linkage and/or other linkage.

Types of TNF include mammalian TNF, such as primate TNF and human TNF. Exemplary human TNF sequences include:

One.

2. Trimeric form:

3. Trimeric form:

Exemplary TNF is a monomer of the sequence of SEQ ID NO:1, a dimer of the sequence of SEQ ID NO:1, a trimer or trimeric form of the sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO: 3 or a partial sequence thereof. Monomers comprising SEQ ID NO:2 or SEQ ID NO:3 may optionally be covalently linked by a spacer sequence of glycine or serine such as GGGS, or a spacer multimer such as (GGGS) ₄ . Amino acids, spacer sequences and spacer multimers may or may not be incorporated in dimeric or trimeric form.

Variants of naturally occurring and non-naturally occurring TNF are included. Such variants include gain and loss of function variants.

Non-limiting examples of TNF variants include one or more amino acid substitutions of a reference TNF sequence (e.g., 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30 -40, 40-50, 50-100, or more residues), additions (e.g., insertions or 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100, or more residues) and deletions (eg, subsequences or fragments). In some embodiments, the variant TNF sequence retains at least some of the functions or activities of the unmodified sequence, such as the ability to bind to sTNF-R (eg, the sTNF-R1 receptor and/or the sTNF-R2 receptor).

A variant may have one or more non-conservative or conservative amino acid sequence differences or modifications, or both. A “conservative substitution” is the replacement of one amino acid with a biologically, chemically or structurally similar residue. Biologically similar means that the substitution does not destroy the biological activity. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine and serine or similar sizes. Chemical similarity means that the moieties have the same charge or both are hydrophilic or hydrophobic. Specific examples include substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, serine for threonine, etc. It involves substituting a polar moiety for another. Specific examples of conservative substitutions include the substitution of a hydrophobic residue such as isoleucine, valine, leucine, or methionine for another, a polar residue for another, such as lysine for arginine, aspartic acid for glutamic acid, or asparagine for glutamine. including substituting For example, conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. "Conservative substitutions" also include the use of a substituted amino acid in place of the parent unsubstituted amino acid.

Such variants include, for example, proteins or polypeptides that have been or can be modified using recombinant DNA techniques such that the protein or polypeptide has altered or additional properties. A variant may differ from a reference sequence such as a naturally occurring protein or peptide.

At the amino acid sequence level, a naturally occurring or non-naturally occurring variant protein will typically be at least about 70% identical, more typically about 80% identical, and even more typically about 90% or more identical to a reference protein, but with substantial non-identity. Regions are permitted (e.g. less than 70% identical, eg less than 60%, 50% or even less than 40%) that are not conserved. In other embodiments, the sequence has at least 60%, 70%, 75% or more identity to the reference sequence (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%). greater than % identity). Procedures for introducing amino acid changes into proteins or polypeptides are known to those skilled in the art (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual (2007)).

The term "identity" and its grammatical variations mean that two or more referenced entities are identical when they are in "aligned" sequences. Thus, for example, when two polypeptide sequences are identical, they have identical amino acid sequences, at least within the referenced region or portion. Identity may span defined regions (regions or domains) of a sequence. A "region" or "area" of identity refers to parts of two or more referenced entities that are the same. Thus, when two protein sequences are identical over one or more sequence regions or regions, they share identity within that region. An “aligned” sequence refers to a multiple protein (amino acid) sequence that often includes corrections for missing amino acids (gaps) compared to a reference sequence.

Identity may extend over the entire length of a sequence or over a portion of a sequence. For example, the length of sequences that share percent identity is 2, 3, 4, 5 or more contiguous amino acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, etc. contiguous amino acids. In another non-limiting example, the length of sequences that share identity is 20 or more contiguous amino acids, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, etc. contiguous amino acids. In a further non-limiting example, the length of sequences that share identity is at least 35 contiguous amino acids, e.g., 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45, 47, 48, 49, 50, etc. contiguous amino acids. In another specific non-limiting example, the sequences that share identity are 50 or more amino acids in length, e.g., 50-55, 55-60, 60-65, 65-70, 70-75, 75-80; 80-85, 85-90, 90-95, 95-100, 100-110, etc. contiguous amino acids.

The degree of identity between two sequences can be ascertained using computer programs and mathematical algorithms. Such algorithms that calculate percent sequence identity generally account for sequence gaps and mismatches for regions or regions of comparison. For example, the BLAST (eg, BLAST 2.0) search algorithm (see, eg, Altschul et al., J. Mol. Biol . 215:403 (1990), publicly available via NCBI) is It has exemplary search parameters: no match -2; gap open 5; Gap extension 2. For protein or polypeptide sequence comparisons, the BLASTP algorithm is typically used with a scoring matrix such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantify the degree of identity (Pearson et al., Proc. Natl. Acad. Sci . USA 85:2444 (1988); Pearson, Methods Mol Biol.132 :185 (2000) and Smith et al., J. Mol. Biol.147 :195 (1981)). A program to quantify protein structural similarity using Delaunay-based topology mapping has also been developed (Bostick et al., Biochem Biophys Res Commun . 304:320 (2003)).

Ligands and proteins such as TNF include additions and insertions, eg, heterologous domains. Additions (eg, heterologous domains) can be covalent or non-covalent attachment of molecules of any type. Typically, additions and insertions (eg, heterologous domains) confer complementary or distinct functions or activities.

Non-limiting examples of additions or insertions are amino acid spacers, spacers comprising two or more amino acids and multimers of such spacers comprising two or more amino acids. Non-limiting examples of amino acids that function as multimers of amino acids and spacers include glycine, serine and alanine.

Additions and insertions include chimeric and fusion sequences, which are protein sequences that have one or more molecules not normally present in a reference native (wild-type) sequence covalently attached to the sequence. The term "fusion" or "chimera" and its grammatical variants is meant to include other entities that are distinct from the molecule (heterologous) because a part or parts of the molecule do not normally coexist in nature. That is, for example, a portion of a fusion or chimera includes or consists of structurally distinct portions that do not coexist in nature.

In some embodiments, methods for covalently linking TNF ligands to the solid support(s) include amine reduction chemistry, cyanogen bromide (CNBr), N-hydroxy succinimide esters, carbonyl diimidazole, reductive amination , 2-fluoro-1-methylpyridinium (FMP) activation, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-mediated amide bond formation organic sulfonyl chloride tosyl chloride and tresyl chloride , divinylsulfone, azlactone, cyanuric acid chloride (trichloro-s-triazine), sulfhydryl reactive chemistry, iodoacetyl and bromoacetyl activation, maleimide, pyridyl disulfide, divinylsulfone, epoxy or Bisoxirane, TNF-thiol, carbonyl reactive chemistry, hydrazide, reductive amination, hydroxyl reactive chemistry, cyanuric acid chloride, active hydrogen reactive chemistry, diazonium, Mannich condensation, photoreactive crosslinking, CNBr activation fixed serum albumin, periodate activation, and/or other methods.

In some embodiments, the bonds can be, for example, ionic bonds, electrostatic bonds, van der Waals bonds, hydrophobic bonds, and/or other bonds. In some embodiments, electrostatic binding is performed by binding a TNF ligand to one or more solids using linking molecules such as immobilized avidin streptavidin and monomeric avidin linked to biotin, antibody-antigen complexes, ligand receptor complexes, and/or other linking molecules. It can be formed between supports.

In some embodiments, the solid support is agarose, sepharose, cellulose, pore glass, silica, acrylamide derivative polyacrylamide beads, trisacryl, sephacryl, Ultrogel® AcA chromatography adsorbent (Pall Corporation), azlactone beads , Methacrylate Derivatives, TSKgel® Chromatography Gel (Tosoh Corporation), TOYOPEARL® HW Polymer Gel (Tosoh Corporation), HEMA (2 Hydroxyethyl Methacrylate, Poly(2 Hydroxyethyl Methacrylate), Eupergit, Polystyrene and its derivatives, Poros, polyether sulfones, polysaccharides, polytetrafluoroethylene, polysulfones, polyesters, polyvinylidene fluoride, polypropylene, poly(tetrafluoroethylene-co-perfluoro(alkyl vinyl ethers) ), polycarbonate, polyethylene, glass, polyacrylate, polyacrylamide, poly(azolactone), polystyrene, polylactide, ceramic, nylon, metal, and/or other other materials. In some embodiments, the solid support may be formed by plates, membranes, beads, ceramics, and/or other components.

In some embodiments, the solid support can be or include, for example, a bead. In some embodiments, the capture scaffold 204 includes a ligand bound to a bead. As described above, in some embodiments, the ligand can be and/or include sc(single chain)-TNFα ligand. In some embodiments, a ligand may be and/or may include a dimeric or trimeric form of an sc-TNF ligand. In some embodiments, the ligand is a TNF ligand, e.g., a single chain polypeptide (sc-TNF) ligand (monomer, dimer or trimer) that binds and effectively captures sTNF-R from the patient's plasma. ) and / or may include them.

Ligands with conformational changes due to altered amino acid sequence or purity of the protein can cause substantial changes, such as enhancing or reducing binding affinity. Such mutations in the sequence may each improve or reduce the binding efficiency of the polypeptide to sTNF-R. Impurities in the TNF preparation will lower the amount of TNF used for coupling by lowering the amount of TNF coupled relative to the total amount of protein used. These ligands may have high target affinity or binding affinity for this target portion of the patient's biological material. Such binding affinity is denoted by K _D . (It should be noted that the lower the K _D value, the greater the binding affinity.) A representative target affinity of a ligand is, for example, greater than about 10 ⁻⁶ K _D , or greater than about 10 ⁻⁷ K _D , or about greater than 10 ^-8 K _D , or greater than about 10 ^-9 K _D , or greater than about 10 ^-10 K _D , or greater than about 10 ^-11 K _D , or greater than about 10 ^-12 K _D , or greater than about 10 ^-13 K _D may be excessive. The affinity of TNF for sTNF-R1 can be, for example, approximately 10 ^-11 K _D . The affinity of TNF for sTNF-R2 can be, for example, approximately 10 ^-10 K _D .

In some embodiments, the sc-TNF ligand comprises a sc-TNFα molecule. In some embodiments, a sc-TNF ligand comprises a TNFα monomer, a complex of one or more TNFα proteins, and/or other components. In some embodiments, complexes include dimers, trimers, multimers, muteins, and/or fragments thereof. In some embodiments, the capture scaffold 204 can include an sc-TNF protein ligand conjugated to a plurality of agarose beads (e.g., system 10 as shown in FIGS. 1-3). selectively binds to sTNF-R present in plasma circulating through

In some embodiments, the production of sc-TNF (and/or other ligands) can be accomplished through a variety of genetic engineering and protein expression means (see, e.g., Muller, R., et al. (1986) FEBS Lett 197, 99-104; Mori, T., et al. (1994) Gene 144, 289-293; Horwitz, A. H., et al. (1996) Protein Expr Purif 8, 28-40; Li, H., et al. (2019) World J Microbiol Biotechnol 35, 27 Ashman, K., et al. (1989) Protein Eng 2, 387-391 Su, X., et al. (1992) Biotechniques 13, 756-762; Li, C. B., et al. (1992) Sci China B 35, 319-328; Guo, D., et al. (1995) Biochem Biophys Res Commun 207, 927-932; Xiang, J., et al. (1997) ) J Biotechnol 53, 3-12 Tang, P., et al. (1996) Biochemistry 35, 8216-8225).

A ligand can have a given density and orientation for a given support (eg, such as a bead). The ligand can be covalently linked to the bead (and/or other support) via, for example, an amine or thiol moiety. Density and orientation can be configured to enhance binding between the ligand and the target component of the bodily fluid. In some embodiments, ligands can be configured to extend from a supporting matrix (eg, a given bead) at either the amino (N) or carboxyl (C) terminus for binding accessibility. In some embodiments, a linker may be placed between the bead (eg) surface and the ligand to extend the ligand into the bodily fluid being passed through as a means of reducing steric hindrance that would hinder binding.

Density can be expressed as milligrams of ligand per milligram of support. In some embodiments, a ligand can be a 54 KD protein. For example and without limitation, the support may be, for example, at least about 0.1 mg (1.8 x 10 ^-6 mmoles) ligand/mg of support (eg, beads), at least about 1 mg (18 x 10 ^-6 mmoles) ) mg of ligand/support (eg beads), at least about 5 mg mg of ligand/support (eg beads), at least about 7 mg (1.3 x 10 ^-4 mmoles) mg of ligand/support (eg eg, beads), at least about 10 mg ligand/mg of support (eg, beads), at least about 15 mg ligand/mg of support (eg, beads), or at least about 20 mg ligand/mg of support (eg, beads), or higher ligand densities.

The size, number, density and/or concentration of the support, such as a bead or beads, (e.g., in conjunction with the shape and size of housing 16) promotes laminar flow of blood, plasma and/or other bodily fluids through the beads to It can be configured to enhance binding between a ligand and a target component of a bodily fluid. Increasing the bead size can accommodate proportionately higher flow rates. Increasing the density of coupled ligands can increase the capture capacity while avoiding concentrations that can contribute to steric hindrances that would prevent effective binding of the target molecule. The size, number, density, orientation, and/or concentration of scaffolds (eg, beads) can be adjusted to, for example, blood plasma via system 10, which effectively balances capture rate with clinically practical procedure time. flow can be promoted. For example, a plasmapheresis procedure that includes one embodiment of system 10 is two patient plasma volumes through isolation chamber 202 to achieve a specific target concentration reduction of sTNF-R1/R2 from the patient's plasma. Whereas may require circulation of; An alternative embodiment with twice the capture rate efficiency of the first embodiment requires circulation of only one patient plasma volume through the isolation chamber to achieve a similar sTNF-R1/R2 concentration reduction, reducing the clinical procedure time to about 2 can be reduced by a factor of two. In some embodiments, the beads may include one or more different bead materials, such as commercially available agarose or polyacrylamide compositions.

In some embodiments, beads and/or other solid supports may be sized to prevent them from passing through filters 212 and 214 (see filter pore size discussion herein). In some embodiments, beads may be quenched with ethanolamine or ethylene diamine to enhance binding specificity (saturate occupied binding sites left after TNF coupling). Ethanolamine, for example, can be used as a quenching agent due to its biocompatible profile. In some embodiments, the beads may be pretreated with an agent such as immulon, polystyrene or polyethylene, and/or the beads may be pretreated with a commercially available crosslinker to maximize recovery and better control (or for other reasons). It can be. A cross-linker can be any chemical or substance used to facilitate attachment of molecules that capture one or more circulating immune complexes to a solid phase. Non-limiting examples of commercially available crosslinkers are, for example, poly-L-lysine, glutaraldehyde and cyanogen bromide.

In some embodiments, beads may form a binding matrix that may include covalently or non-covalently bound affinity molecules (eg, as described herein). In some embodiments, beads can range from about 20-1,000 μm in diameter, for example. In some embodiments, beads may range from about 25-500 μm in diameter. In some embodiments, beads may range from about 25-200 μm in diameter. In some embodiments, beads may range from about 40-180 μm in diameter. In some embodiments, beads may range from about 50-170 μm in diameter. In some embodiments, beads may range from about 65-160 μm in diameter. In some embodiments, beads may range from about 75-150 μm in diameter.

The beads are biocompatible and can be formed from materials to which various ligands are covalently linked or electrostatically bound (eg, as described above). Ligand binding is amine reduction chemistry, cyanogen bromide (CNBr), N-hydroxy succinimide ester, carbonyl diimidazole, reductive amination, FMP activation, EDC-mediated amide bond formation, organic sulfonyl chloride tosyl chloride and tresyl chloride, divinylsulfone, azlactone, cyanuric acid chloride (trichloro-s-triazine), sulfhydryl reactive chemistry, iodoacetyl and bromoacetyl activation methods, maleimide, pyridyl disulfide, di Vinylsulfone, epoxy or bisoxirane, TNF-thiol, carbonyl reactive chemistry, hydrazide, reductive amination, hydroxyl reactive chemistry, cyanuric acid chloride, active hydrogen reactive chemistry, diazonium, Mannich condensation, photoreactive cross-linking, and/or other operations. Coupling can also be performed using immobilized serum albumin with CNBr activation or periodate activation. In some embodiments, the binding is a non-covalent interaction, eg, a) immobilized avidin streptavidin and monomeric avidin bound to biotin; b) antibody-antigen complexes; and/or c) ligand-receptor complexes.

In some embodiments, the capture scaffold (204 ), the capture efficiency expressed as a percentage of sTNF-R1 and/or sTNF-R2 of at least 10% or more. In some embodiments, the capture efficiency can be at least 50% or higher. In some embodiments, the capture efficiency can be at least 80% or higher. In some embodiments, the capture efficiency can be at least 90%, 95%, 96%, 97%, 98%, 99% or higher.

As more of the target agent is cumulatively entrapped within the column (System 10), the available binding sites within the system's capture matrix decrease, so the capture efficiency value is dependent on a time-based factor (e.g., the first 5 min, 10 min of treatment, capture efficiency for 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes or more) can be considered. As a few non-limiting examples, in some embodiments, at least 80% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flow biological sample are removed from the isolation chamber 202, eg, within about 30 minutes. can bind to TNF ligands in In some embodiments, at least 90% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flow biological sample may bind to TNF ligands in the isolation chamber 202 within about 30 minutes. In some embodiments, at least 95% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flow biological sample may be bound to TNF ligands in the isolation chamber 202 within about 30 minutes. In some embodiments, at least 96% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flow biological sample may be bound to TNF ligands in the isolation chamber 202 within about 30 minutes. In some embodiments, at least 97% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flow biological sample may be bound to TNF ligands in the isolation chamber 202 within about 30 minutes. In some embodiments, at least 98% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flow biological sample may be bound to TNF ligands in the isolation chamber 202 within about 30 minutes. In some embodiments, at least 99% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flow biological sample may bind to TNF ligands in the isolation chamber 202 within about 30 minutes.

Without wishing to be bound by any theory, the capture efficiency values described herein may be related to the use of the sc-TNF ligand described above (e.g., having exceptionally high target affinity), the use of a trimeric form of the sc-TNF ligand, Purity of ligand, density of ligand on beads, orientation of ligand binding on beads, size of beads, number, density and concentration of beads in system 10, flow rate through system 10 balancing capture efficiency versus clinical procedure time, beads the physical size and construction of the housing 16 to create laminar flow through the , the process (e.g., chemical and/or electrostatic) used to couple the ligands to the beads, the sterilization technique and dose of radiation, and/or other can be attributed to the factor. In other words (again, not wishing to be bound by any theory), for example, the reason for the high efficiency of the column (System 10) may be due to the large amount of capture ligand on the bead matrix along with the high binding efficiency of the ligand.

In some embodiments, the high binding specificity and/or affinity of the capture support 204 for the target component may be due to the fact that the only known interaction of the capture ligand is exclusively with sTNF-R1 and/or sTNF-R2 in plasma. This binding specificity can be attributed to the use of the sc-TNF ligand described above, the trimeric form of the sc-TNF ligand, the materials used to form the beads and/or the specific bead matrix (e.g., their chemical composition), the methods used to quench the beads. use of ethanolamine (eg, used to reduce non-specific binding), optimization of target protein binding versus non-specific binding, pore size used in filters 212 and 214, and/or other factors. .

In some embodiments, the binding affinity of the capture support for at least one target moiety is at least about 10 ^-5 K _D or higher. In some embodiments, the binding affinity of the capture support for at least one target moiety is at least about 10 ⁻⁶ K _D . In some embodiments, the binding affinity of the capture support for at least one target moiety is at least about 10 ⁻⁷ K _D . In some embodiments, the binding affinity of the capture support for at least one target moiety is at least about 10 ⁻⁸ K _D . In some embodiments, the binding affinity of the capture support for at least one target moiety is at least about 10 ⁻⁹ K _D . In some embodiments, the binding affinity of the capture support for at least one target moiety is at least about 10 ^-10 K _D . In some embodiments, the binding affinity of the capture support for at least one target moiety is at least about 10 ^-11 K _D . In some embodiments, the binding affinity of the capture support for at least one target moiety is at least about 10 ^-12 K _D . In some embodiments, the binding affinity of the capture support for at least one target moiety is at least about 10 ^-13 K _D . In some embodiments, the binding affinity of the capture support for at least one target moiety is at least about 10 ^-14 K _D .

In some embodiments, system 10 may be configured to have a leaching rate of TNF that is less than 1/1000 the maximum allowable daily dose (MTD) limit (see, e.g., Goossens, V., et al. 1995) Proc. Natl Acad. Sci. USA, 92, 8115-8119). In some embodiments, system 10 may be configured to have a leaching rate less than 1/10000 of the MTD capacity limit. In some embodiments, system 10 may be configured to have a leaching rate of less than 1/500 of the MTD daily dose limit. In some embodiments, system 10 may be configured to have a leaching rate of less than 1/100 the MTD daily dose limit. This is one or more unbiased by side effects and/or clinical effects resulting from escape (eg, leaching) of a portion of the capture scaffold 204 (eg, a ligand described herein) into the patient's circulatory system. The clinical effectiveness of the system 10 including successful, efficient and specific capture of the target component can be ensured.

The leaching rate is the percentage of the capture support 204 that has escaped the system 10 relative to the total volume of the capture support 204 contained in the containment chamber 202 of the system 10 after production (e.g., ng/mL /min unit). For example, in some embodiments, system 10 may have a leaching rate of less than about 150 ng/mL/min. In some embodiments, system 10 may have a leaching rate of less than about 100 ng/mL/min. In some embodiments, system 10 may have a leaching rate of less than about 80 ng/mL/min. In some embodiments, system 10 may have a leaching rate of less than about 50 ng/mL/min. In some embodiments, system 10 may have a leaching rate of less than about 40 ng/mL/min. In some embodiments, system 10 may have a leaching rate of less than about 30 ng/mL/min. In some embodiments, system 10 may have a leaching rate of less than about 20 ng/mL/min. In some embodiments, system 10 may have a leaching rate of less than about 10 ng/mL/min.

The leaching rate depends on the strength and integrity of the (e.g., chemical and/or electrostatic) bond between the sc-TNF ligand and the bead, the use of reductive amination chemistry with the particular ligand, and the use of the trimeric form of the sc-TNF ligand. , the process (e.g., chemical and/or electrostatic) used to couple the ligand to the beads, the clinical pretreatment approach to prepare the system 10 for patient treatment (e.g., the volume of flushing prior to use) and flow rates), clinically realistic flow rate through the system 10 that balances capture efficiency versus clinical procedure time, cleaning of the housing 16 during manufacturing, sterilization techniques and radiation doses used in production, and/or other factors. can be attributed to In some embodiments, the clinical pretreatment preparation (eg, volume and flow rate of flushing prior to use) of system 10 includes flushing 1 liter of physiological saline at a flow rate of about 100 mL/min.

As a non-limiting example, FIGS. 4 and 5 show bodily fluids (in this example plasma 404 (sTNF-R1 402) and sTNF-R2) to capture capture target components 402 and 403 within the isolation chamber 202. A housing (including 403) that binds to the targeting component 402 (sTNF-R1) and/or the second targeting component 403 sTNF-R2 (these are only examples - more targeting components are possible) 16) illustrates the capture support 204 (eg, the bead 400 in this example) within the isolation chamber 202. Figure 5 illustrates the bead 400 (eg, the capture support) shown in FIG. 5 is an enlarged view of a bodily fluid (e.g., plasma 404) flowing through system 10 (FIGS. 1-3) from inlet 200 to outlet 210 (FIGS. 1-3). ) shows the direction 500. In some embodiments, the system 10 can be symmetrical and bi-directional so that the clinical user can select either end of the device to use as an upstream entry or a downstream exit.

As shown in Figures 4 and 5, capture occurs when there is contact between the capture scaffold 204 and a bodily fluid (plasma 404). Specifically, capture is a ligand bound (coupled) to sTNF-R1 and sTNF-R2 molecules (402, 403) and beads 400 in plasma 404 (eg, the sc-TNF ligand described above) ( 410) in response to proximity and/or direct contact between them. A bond 412 between ligand 410 and bead 400 is also shown. Capture support 204 (eg, a combination of beads 400 and ligand 410) is used to reduce the amount of one or more target components 402, 403 in a bodily fluid (eg, plasma 404). It can be configured to bind to one or more target components 402, 403 (sTNF-R1 and R2 molecules).

In some embodiments, once the processed sTNF-R deficient plasma passes through outlet 210 ( FIGS. 2 and 3 ), some or all of the treated sTNF-R deficient sample may be reinfused into the patient (e.g., eg, via the apheresis machine shown in Figure 1). As sTNF-R is eliminated, the stores of uncomplexed sTNF-R in the patient's blood are reduced, resulting in a concentration equilibrium shift towards increased availability of TNF to promote anticancer effects at the tumor site(s).

As described herein, system 10 (FIGS. 1-3) can be configured to selectively remove immunosuppressive factors associated with an immunosuppressive state. In some embodiments, the immunosuppressive factor is soluble TNF receptor 1 and soluble TNF receptor 2 (sTNF-R). Removal of sTNF-R from a patient's blood, plasma and/or other bodily fluids may increase the availability of endogenous TNF, which promotes anticancer effects at the tumor site(s).

Returning to FIGS. 2 and 3 , access ports 206 and 208 may be configured to provide access to isolation chamber 202 . This access may be separate from access via inlet 200, outlet 210 and/or other access points. Access ports 206 and 208 may be configured to facilitate insertion and/or removal of capture support 204 into or from isolation chamber 202 . This may be performed, for example, during manufacture of system 10, and/or at other times.

In some embodiments, capture scaffold 204 may be suspended in a preservative buffer solution for storage prior to use in system 10 . In some embodiments, the preservative buffer solution includes bacteriostatic saline, bacteriostatic phosphate, and/or other solutions. In some embodiments, the preservative buffer solution can be bacteriostatic phosphate buffered saline (PBS), which can contain 0.9% benzyl alcohol. The capture support 204 may be refrigerated in solution at 2-8° C. until ready for subsequent aseptic filling of the housing 16 . In some embodiments, system 10 may be stored at 2-8° C. between manufacturing, shipping, and/or clinical use.

In some embodiments, the preservative buffer solution is flushed from system 10 (eg, via inlet 200 and outlet 210) immediately prior to clinical use of system 10. In some embodiments, access ports 206 and 208 include luer fittings with caps and/or other components. In some embodiments, access ports 206 and/or 208 have corresponding caps and/or other components. Corresponding caps may be formed of polycarbonate and/or other materials.

6 depicts an exemplary regeneration mechanism 600 configured to couple with system 10 . In some embodiments, the regeneration mechanism may be considered an additional part and/or extension of system 10 . As shown in FIG. 6 , mechanism 600 may include a source of cleaning solution 602 , a source of regeneration solution 604 , a pump 606 , a waste line 608 , and/or other components. In some embodiments, cleaning solution 602 and regeneration solution 604 may be alternately pumped through system 10 by pump 606 to waste line 608 .

In some embodiments, system 10 is configured to facilitate sampling or removal of all or a portion of the entrapped target component (eg, without damaging an extracorporeal closed circuit column formed by system 10). An exit port may be included.

In some embodiments, system 10 may be configured to facilitate reuse by alternating capture and dissociation steps. Dissociation of the target prior to further capture can regenerate the column to approximately its original specifications and functions. To achieve this dissociation, the captured complexes of the bead-bound proteins and their ligands are disrupted, resulting in dissociated complexes. As such dissociation steps may be application dependent, specific dissociation conditions may depend on the particular subtype of circulating protein or complexed moiety that is captured. Agents that cause high salt concentrations, such as chaotropic agents or low pH, can be effective dissociation agents. To dissociate the capture complex, high salts such as sodium chloride (300 mM-1,5 M) or chaotropic agents such as guanidine hydrochloride 3-8 M concentrations can be used. In some embodiments, the salt solution may have a pH of approximately 7.2 and may include 500 mM NaOH, 2 mM EDTA and 50 mM Tris buffer, or 500 mM NaOH, 2 mM EDTA and 50 mM sodium phosphate. Alternatively, captured immune complexes can be dissociated with a low pH solution. An effective pH for the dissociation step may be, for example, approximately 2.8. An exemplary pH range may be 1.5 to 2.5. This can be achieved with pH adjusted citrate or glycine solutions. In some embodiments, the pH range may be 2.0 to 2.5. In some embodiments, the pH range may be 2.5 to 3.5. However, it should be recognized that the lower the pH, the shorter the dissociation time required. For example, acids such as acetic acid, citric acid and hydrochloric acid can be used to lower the pH. Besides raising the salt concentration or lowering the pH, other methods of dissociating immune complexes are possible. In addition to raising the salt concentration or lowering the pH, dissociation conditions can be configured to occur for a short period of time and can include bovine serum albumin (BSA) and/or other ligand or receptor competing components.

In some embodiments, system 10 will include an elution reagent port configured to facilitate introduction of an elution reagent into the isolation chamber (eg, without damaging an extracorporeal closed circuit column formed by system 10). can The elution reagent port may be further configured to receive a conditioning agent configured to prepare system 10 for reuse. In some embodiments, the elution reagent port(s) may be the same as one or both of the access ports 206 and/or 208.

In some embodiments, pump 606 may be configured to drive eluent/regenerant through inlet 200 (FIG. 2), isolation chamber 202 (FIG. 2), and outlet 210 (FIG. 2). there is. Pump 606 may include a syringe pump, a peristaltic pump, a piston pump, a diaphragm pump, combinations thereof, and/or other pumps.

In some embodiments, system 10 may include one or more additional isolation chambers 202 as shown in FIG. 7 . 7 illustrates two exemplary embodiments 700 and 750 of system 10 having an additional isolation chamber 202 . In the exemplary embodiment 700, the isolation chambers 202A and 202B are disposed in series within the housing 16. In exemplary embodiment 750, chambers 202C, 202D, and 202E are positioned in parallel within housing 16. The different chambers (eg, 202A and B, and 202C-E) may be separated by filters 212, 214 (and/or other filters), separation membranes 710, and/or other components. In some embodiments, separation membrane 710 can be, for example, filters 212 and/or 214 and vice versa.

These additional isolation chambers 202A-E may include capture supports similar and/or identical to capture supports 204 (FIG. 2) described above, or different capture supports. Additional isolation chambers 202 (A-E) and capture supports may be configured to function similarly and/or identically to isolation chambers 202 and capture supports 204. In some embodiments, one or more additional isolation chambers 202A-E can be combined (eg, 202A and B, and 202C, D and E) to form a multi-stage isolation circuit configured to engage a plurality of different target components. there is. In some embodiments, multiple isolation chambers are arranged in series in which the total volume of bodily fluid (eg, plasma) flowing through system 10 passes through each of the plurality of isolation chambers sequentially (eg, 202A then 202B). configuration (eg, embodiment 700). In some embodiments, the plurality of isolation chambers is such that the volume of bodily fluid (e.g., plasma) flowing through system 10 is divided into equal or unequal fractions of these discrete sub-volumes of fluid in the plurality of isolation chambers. (eg, 202C, 202D, and 202E) may be configured in a parallel configuration (eg, implementation 750) to pass in parallel through each.

FIG. 8 illustrates additional embodiments 800 and 850 in which multiple systems (eg, multiple systems 10) can be simultaneously configured into a single plasmapheresis flow circuit 802 or 852 during a treatment procedure. do. In one embodiment (eg, 800), multiple systems 10 may be cascaded in series, for example, with blood plasma flowing through one system 10 and then flowing into the next. In other embodiments (eg, 850), two or more systems 10 may be configured in parallel, wherein the volume of bodily fluid (eg, plasma) flowing through the plasmapheresis flow circuit is equal or can be divided into unequal fractions such that these fractionated sub-volumes of fluid can be passed in parallel through each of a plurality of systems (e.g., two systems 10 are shown in embodiment 850). but is not intended to be limiting). In one embodiment involving such a parallel system configuration, the total volume of fluid in the plasmapheresis flow circuit can be initially directed through one or more parallel system isolation chambers (eg, 202 as described above). and then, at subsequent times during the same treatment procedure, the fluid in the circuit may be redirected to pass through other systems or subdivided to pass through different combinations of parallel systems coupled within the flow circuit. In any of the configurations described above, each individual system may contain the same or different capture matrices that target the same or different target agents in bodily fluids.

9 shows a fluid inlet 200 for fluid to enter the system 10, a plurality of isolation chambers 902, 904, 906 and 908 positioned in parallel, each at the inlet 200 of the system 10 at the outlet ( 210) with independent flow paths 903-909 (eg, which may be formed by male Luer ports), each "on" or "off" an individual isolation chamber. An embodiment (900) of a system (10) comprising multiple isolation chambers having their own flow control valves (910, 912, 914, 916) (e.g., stopcocks and/or other components) that can be )" ) exemplifies. In this embodiment, fluid may be directed simultaneously through any one chamber or any combination of multiple chambers. An exemplary flow path 950 is shown in the bottom portion of FIG. 9 . Fluid present in one or more chambers being used at a time is recombined at the system fluid outlet 210 for return to the apheresis machine. In this embodiment, each of the isolation chambers 902-908 can include one or more different capture molecules configured to target one or more different target moieties in bodily fluid (e.g., included in the capture scaffold 204 described above). there is. As shown in FIG. 10 , a plurality of isolation chambers 902-908 and their corresponding flow control valves 910-916 may be contained within a single integral external housing (eg, 16 as described above). there is.

10 illustrates a method 1000 for removing target components of blood, plasma and/or other bodily fluids with system 10 (FIGS. 1-3). The operation of the method 1000 presented below is for illustrative purposes only. In some embodiments, method 1000 can be accomplished with one or more additional acts not described, and/or without one or more of the discussed acts. Additionally, the order in which the operation of method 1000 is illustrated in FIG. 10 and described below is not intended to be limiting.

In operation 1002, blood, plasma and/or other bodily fluids are drawn from a patient (e.g., patient 14 shown in FIG. 1) through an inlet 200 (FIGS. 2-3).

In operation 1004, the target component of blood, plasma and/or other bodily fluid is removed from the isolation chamber 202 (FIGS. 2-3) to reduce the amount of the target component in the blood, plasma and/or other bodily fluid. Can be bound to capture.

In operation 1006, bodily fluid having a reduced amount of the target component is passed from the isolation chamber 202 (FIGS. 2-3) to be reintroduced back to the patient through an outlet 210 (FIGS. 2-3).

In operation 1008, a reduced amount of the target component in the bodily fluid is reintroduced back to the patient, where it may or may not be measured quantitatively (eg, operation 1008 may be optional). In some embodiments, when quantitative measurements are used, the measurements include liquid chromatography-mass spectrometry (LC-MS), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UHPLC), resistivity measurements, luminescence measurements, chemical Luminescence, electroluminescence, electrochemiluminescence, chromatographic monitoring, positron emission tomography (PET), x-ray computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, gamma camera, single photon emission computed tomography ( SPECT), enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) measurements, and/or performing other tasks.

In operation 1010, the rate of leaching of the capture support from bodily fluid reintroduced back to the patient may or may not be measured (eg, operation 1010 may be optional). The concentration of the capture agent (eg, TNF) can be determined from a fluid sample extracted from inlet 200 and outlet 210 and/or from a fluid connector attached thereto. The difference between the two measured concentration values can be used to determine the rate and amount of trapping agent that escapes the containment chamber. In some embodiments, the flow rate in mL/min can be used to determine the total amount of capture agent leached from the system and into the patient's circulatory system over a period of time (eg, the duration of a single treatment procedure).

Although the system(s) or method(s) of this disclosure have been described in detail for purposes of illustration based on what is presently believed to be the most practical and preferred implementation, such detail is for that purpose only and the disclosure is limited to the disclosed implementation. It should be understood that, rather than not, it is intended to cover modifications and equivalent arrangements within the spirit and scope of the appended claims. For example, it should be understood that this disclosure contemplates that one or more features of any implementation may be combined with one or more features of any other implementation, to the extent possible.

The following appended examples provide various illustrations of the successful use of various embodiments of the systems and methods described herein.

Example 1 - Materials and Assemblies of the present system

Material—The immobilized solid support matrix (capture support 204) used in this embodiment of the system was an agarose-based bead (ie, a strongly cross-linked support resin).

Chemistry—The solid support matrix (capture support 204) was activated using sodium metaperiodate and then coupled to single-chain TNF ligands by reductive amination. An amount of 1 mg of TNF was coupled per mL of solid support matrix.

Assembly—The binding matrix (capture support 204) contains single-chain TNF (SC-TNF) proteins covalently linked to agarose beads. The device housing includes a polycarbonate tube with two side ports for filling, capped with a non-vented luer cap. The end cap has a male luer port and is rigidly coupled to the tube to create a fluid tight enclosure terminated at each end by an end cap luer port. Inside the housing at the junction between each end cap and the tube is a filter frit with an average pore size of 15-50 μm to retain the TNF ligand-coupled beads, the minimum diameter of which exceeds the pore size of the filter ( eg as described above).

Example 2 - Parameters for Utilizing the Present System and Method

1. Attach 3-way stopcock valves to the inlet and outlet of the column end cap, in configurations where pre- and post-column plasma concentration measurements are performed to determine capture efficiency and/or leaching (not required for routine clinical applications) This enables periodic plasma sampling. 2. The system is connected to the plasma circuit of the apheresis machine with a stopcock attached. 3. The apheresis unit is configured to have procedure-independent and/or procedure-specific operating parameters in accordance with the apheresis machine's Instructions for Use for use in completing steps 4, 6, and 10 below. 4. Flush the system with 1 L of normal saline before connecting the patient to the apheresis machine. 5. Connect the patient to the apheresis machine. 6. Treat the patient with the system. 7. If applicable, blood and plasma samples are collected according to the study protocol and/or clinical treatment procedure plan. 8. Continuously monitor the patient's vital signs according to typical apheresis treatment practice. 9. Patients are continuously monitored for side effects before, during, and after treatment. 10. Flush/rinse the device with physiological saline after treatment, recap the column with retained end caps, and ensure proper storage and/or disposal of used devices.

Columns of the present invention are intended for use with apheresis machines such as the Terumo BCT or Spectra Optia systems designed to accommodate, for example, plasma processing columns such as system 10. Such systems automate calculations based on operator configured patient data and device parameters. If your apheresis machine does not automate such calculations, the formulas identified in the table below may be used.

Table 1. Total blood volume calculation formula

Table 2. Formula for calculating patient plasma volume

Table 3. Treatment time calculation formula

INTENDED CLINICAL PERFORMANCE—Immunopheresis columns of (e.g., system 10) use centrifugation or membrane separation techniques and secondary plasma processing (e.g., by system 10) It is a device designed to efficiently remove sTNF-R from a patient's plasma by successfully integrating with a commercially available apheresis machine that allows The system disclosed herein meets the clinical performance requirements described below. Certain processing times and flow rates were found to be within the capabilities of modern apheresis systems using centrifugation techniques.

System Performance Specifications - 1. Biosafety: Proven to be biosafe for use as an extracorporeal device supporting prolonged exposure to circulating blood. 2. Binding target: sTNF-R (including sTNF-R1 and sTNF-R2). 3. Coupling efficiency: >80% after 30 minutes of treatment at clinically relevant flow rates. 4. Binding capacity: > 230 μg of sTNF-R. 5. Flow (plasma): ≤ 60 mL/min. 6. Treatment time: about 2 hours. 7. Target plasma exchange volume: 2 plasma volumes. 8. Leach rate (TNFα): < 50 ng/min. 9. Shelf life: > 6 months @ 2-8℃. 10. Pressure tolerance: 776 mmHg.

Example 3 - Efficacy of system 10 based on in vitro parameters

Preclinical Testing - In vitro testing demonstrated that the system 10 met the performance and safety specifications defined in "Intended Clinical Performance" (previous section). The TNF-coupled binding matrix effectively captured >98% of sTNF-R in both spiked buffer and human plasma in standard in vitro tests, while achieving a calculated sc-TNFα leaching rate of <50 ng/min (2 h). equal to a maximum of 6 μg), which is well below the accepted safety limit since the maximum tolerated dose (MTD) per day of TNFα is approximately 200 μg (see, e.g., Goossens, V., et al. (1995) Proc. Natl Acad. Sci. USA, 92, 8115-8119). These performance and safety measurements were repeated after the beads were sterilized by E-beam irradiation (15-30 kGy). Additionally, in this embodiment, system 10 is designed for sustained flow rates of <300 mL/min, 10 to 44 mL/min, and/or 45 to 100 mL/min. The housing (eg, 16 shown in FIGS. 2 and 3 ) can withstand internal pressures of >776 mmHg, well above the typical back pressure cut-off threshold for commonly used apheresis machines.

Characterization of the binding matrix (e.g. capture scaffold 204)—identity, purity, and integrity of the TNF ligand—sTNF- Captured R. TNFα was characterized by assessing purity by high performance liquid chromatography (HPLC), integrity and identity by mass spectrometry, and functional strength by flow test binding assay. HPLC was performed using a column with phosphate buffer as mobile phase at a flow rate of 1 mL/min. Completeness was determined by mass spectrometry, which confirmed a calculated molecular weight of about 54 kD.

Binding Efficiency of Binding Matrix in sTNF-R Spiked Buffer - For the standard flow test, an amount of 2 mL of bead bed was obtained from the irradiated column of the present invention, transferred to a small column and spiked with sTNF-R1 or sTNF-R2. Capture efficiency from phosphate buffer was tested. TNF-R1 spiked buffer (20 ng/mL) was passed through the column at 10 mL/min, 5 mL/min/, 2.5 mL/min, 5 mL/min and 10 mL/min, respectively. Samples were collected at 0.5 minute intervals and assayed for sTNF-R. The result was a capture efficiency of greater than 98% for all samples. Assay for TNFα showed release to be less than 7 ng/min.

Capture Capacity of Binding Matrix in sTNF-R Spiked Human Plasma - To test the capacity of system 10, a column of the present invention was run by running 1.8 liters of spiked human plasma through the device at a flow rate of 45 mL/min. tested. Human plasma used for each run was spiked with high concentrations of each receptor (9.74 ng/mL for sTNF-R1 and 127.5 ng/mL for sTNF-R2). Samples of post-column plasma were collected every 10 minutes and assayed for sTNF-R concentration using a commercially available high-precision sTNF-R1/sTNF-R2 diagnostic kit. The amount of sTNF-R retained on the column was calculated to determine the capture efficiency and total capacity.

The binding efficiency at each time point was calculated by comparing the pre- and post-column concentrations of sTNF-R. The binding matrix captured more than 85% of sTNF-R1 and sTNF-R2. The binding efficiency was overall greater than 95% for sTNF-R1 and lower efficiency was observed for sTNF-R2, but the capture still remained greater than 85%. The slight linear decrease in binding efficiency to sTNF-R2 is probably due to the lower binding affinity of the soluble receptor for the TNFα ligand used.

The total amount of sTNF-R captured on the column was determined by comparing the concentration of sTNF-R before and after treatment. The column captured 230 μg of sTNF-R at 15 mL/min run and 149.4 μg at 45 mL/min run. All of these values exceed the amount of about 30 μg of sTNF-R normally present in cancer patients. The total TNF-R capture for the 15 mL/min run at 80 min, at which point the column efficiency began to decline, was 99.9 μg, still far exceeding the typical amount present in cancer patients.

Rate of Leaching of TNFα from Devices of the Present Invention—System 10 is used to prevent infusion of potentially pharmacologically significant amounts into a patient without unintended release (i.e., 'leaching') of TNFα from system 10. ) is configured to prevent TNF release from the beads was determined during flow testing. Phosphate buffered saline was spiked with approximately 20 ng/mL of sTNF-R1 and run through a 2 mL bead bed from a System 10 (FIGS. 1-3) column. Flow rates were sequentially performed at 10 mL/min, 5 mL/min, 2.5 mL/min, 5 mL/min and 10 mL/min for 30 seconds at each flow rate. Samples were analyzed according to a commercially available high-precision TNF diagnostic kit. The TNF leaching rate in ng/min was calculated by multiplying the TNF concentration in the sample (ng/mL) by the flow rate (mL/min). The release of the final fraction collected at 10 mL/min was either 0.65 ng/min or 0.325 ng/min/mL per 2 mL of beads (0.65 ng/min/2 mL). In this example, the upper limit of the bead bed volume, which is within the specification of 50 ng/min, is (50 ng/min)/(0.325 ng/min/mL) or 154 mL of beads.

Device Housing Integrity—Tests were performed on empty housings of the present invention (eg, 16) to ensure that the integrity of the system 10 is maintained at increased internal pressure. The side ports (eg, 206, 208) of system 10 were attached to the compressor using tubing and connectors. The capped housing was immersed in a water bath, pressurized to 776 mmHg, and blistering was observed. No device failure (presence of air bubbles leaking from the device submerged in water) was observed. This test pressure exceeds the maximum pressure of the Optia unit (500 mmHg).

Example 4 - Efficacy Demonstration in Dog Model

The effect of in vitro clearance of sTNF-R using system 10 from dogs with spontaneously occurring solid malignancies or melanomas (stage 4) was evaluated in a proof-of-concept comparative oncology study. This study used a system (10) with a sc-TNFα peptide-bead matrix. System 10 was used with a Terumo BCT Spectra Optia apheresis system for secondary plasma processing through system 10. Dual lumen catheters were used in most dogs for vascular access, and extracorporeal anticoagulation was achieved with acid-citrate dextrose anticoagulant (ACDA) solution and with calcium gluconate infusion as recommended per the Spectra Optia user's operating manual. reversed A total of 20 dogs were treated. Dog patients received 12-24 apheresis treatments over the course of the 4-8 week treatment phase of the study. More than 300 immunopheresis treatments were performed during this study.

Device Performance—To confirm the in vivo safety and performance of the device, TNF and sTNF-R were measured every 30 minutes in pre- and post-column plasma (extracted from the inlet and outlet ports of the device, respectively) during treatment; And systemic (from blood) TNF and sTNF-R were measured before each treatment, every 30 minutes during treatment, and every 30 minutes after treatment (for 1.5 hours immediately after treatment).

During each treatment, analysis of the plasma before and after the column (System 10) showed that sTNF-R was efficiently and consistently removed over the course of each individual treatment without a measurable increase in TNF levels in the blood. Analysis of plasma before and after treatment generally showed nearly 50% reduction in systemic sTNF-R levels.

Device Safety and Clinical Efficacy - To evaluate the clinical safety and efficacy of the device, safety, tolerability and impact on tumor progression were measured throughout the study.

During a course of more than 300 immunopheresis treatments across a total of 20 dogs, few serious side effects were reported, and there were no adverse events resulting from treatment with a specific apheresis procedure or system (10) or removal of sTNF-R. there was nothing Tolerability to treatment is best described when examining QoL data in canine patients. Changes in QoL were scored as 'neutral' for most scores across the active treatment phase of the study, which generally included 12-24 treatments per patient. Although some scores got worse, most showed stable parameters or improvement over the entire course of treatment, indicating a favorable outcome for the study's Phase 4 patient population.

Treatment with the device of the present invention had an overall beneficial effect on tumor progression. Most dogs (12/17 evaluable cases) were scored as "stable disease" (SD) at some point during treatment (data not shown) and 7 of 17 evaluable dogs were favorable at the end of treatment. Treatment-related effects were seen, with one case showing complete remission (CR).

CONCLUSIONS—The dog's overall condition generally improved during the study, with observable stabilization or reduction in tumor burden and improvement in quality of life. In addition, the absence of clinically significant safety concerns is consistent with the established relative safety of common apheresis procedures, suggesting the potential to provide clinically meaningful benefits without the typical and significant side effects associated with conventional chemotherapy and radiation. It is persuasive because This canine companion animal study indicates that in vitro removal of sTNF-R using an apheresis immunosorbent affinity column of the present invention containing a sc-TNF peptide-bead matrix can be therapeutically effective in dogs with cancer. and provided strong clinical evidence that the use of such devices could be used in human subjects.

Example 5 - Biocompatibility

System 10 was evaluated for biosafety according to the Food and Drug Administration (FDA) biocompatibility test matrix and international standard ISO 10993-1 (2009). System 10 can be classified as an external communication device that has an extended contact period and circulates blood. This is the same test class used for several other commercially available in vitro immunosorbent columns.

Biocompatibility testing was performed according to 21 CFR Part 58 (Good Laboratory Practice for Nonclinical Laboratory Studies). All testing was performed by an accredited independent testing laboratory on sterile and finished devices. Based on the tests performed, the device complies with the ISO 10993-1 (2012) Consensus Standard, "Biological Evaluation of Medical Devices - Part 1: Evaluation and Testing within Risk Management Processes" and FDA's June 16, 2016 publication. Follow the recommendations and principles contained in relevant guidance documents.

Example 6 - Demonstration of Safety in Human Subjects

A first human compassionate use clinical study was conducted to collect pilot safety and performance data for the system 10. The device was used in combination with the Terumo BCT Spectra Optia system with secondary plasma processing via system 10 as was done in the canine comparative oncology study (described above). A double lumen catheter was used for vascular access based on the experience of the canine study, in vitro anticoagulation (ACDA solution) and reversal (with calcium gluconate infusion) were similar to those used in the canine study and as described in the Spectra Optia user's operating manual. as recommended according to

Apheresis Procedure Performance—The system 10 has been successfully used with Terumo BCT Spectra Optia's apheresis equipment. For each treatment performed, system 10 could be properly integrated into the extracorporeal plasma circuit. There were no reports of equipment-induced failures in secondary plasma processing circuits (eg, circuits coupled to system 10). System circuit integrity remained consistent (eg, no leaching/fluid loss was reported) and all treatments could be successfully completed. Based on these aggregate data, the device of the present invention appears suitable for use with the Terumo BCT Spectra Optia.

Safety and Tolerability Results - A total of 14 patients were enrolled in the study. All patients failed current treatment but otherwise had stable, advanced cancer (baseline Eastern Cooperative Oncology Group (ECOG) score 0-2). The range of treatments each patient received varied (eg, one patient received up to 16 treatments). A total of 93 individual treatments were completed and no unexpected apheresis-related adverse events (AEs) or device adverse events (ADEs) were reported. Based on the data collected in this study, immunopheresis using the present approach and system (10) appears to be generally safe and well tolerated.

11 illustrates representative system 10 (FIGS. 1-3) performance characteristics, including column capture efficiency and sTNF-R1 and sTNF-R2 reduction from a human patient's blood pool as a function of procedure time. 11 illustrates typical system 10 performance results from a single human patient treatment. At baseline (T=0 min), at 30 min, 60 min, 90 min, and at the end of treatment (i.e., completion of 2 plasma volumes circulated through the system) (which in this example occurred at 175 min from the start time of the procedure) Samples of whole blood and plasma were taken. At each time point, whole blood is drawn from the patient's central line catheter, and the inlet (eg, 200 shown in FIGS. 2-3) and outlet (eg, 210 shown in FIGS. 2-3) of system 10 Plasma samples were taken from Additionally, whole blood samples were taken at 30 and 60 minutes post treatment. The sTNF-R1 and sTNF-R2 concentrations of the samples were analyzed using commercially available high-precision diagnostic assays. 11 shows a significant difference between the inlet 200 and outlet 210 sTNF-R1 and sTNF-R2 concentrations corresponding to each time point, indicating that system 10 maintains sTNF-R1 and sTNF-R1 and sTNF-R2 concentrations throughout the course of treatment. It is demonstrated that sTNF-R2 is effectively captured. Moreover, a steady time-based decrease in sTNF-R1 and sTNF-R2 concentrations observed in the patient's whole circulatory system (i.e., measured in central line whole blood) followed by a rebound at 30 and 60 minutes post-treatment, with levels of sTNF-R1 and sTNF-R2 over the treatment period. It indicates that the therapeutic goal of reducing endogenous levels of sTNF-R2 was effectively achieved.

SEQUENCE LISTING <110> IMMUNICOM, INC. JOSEPHS, Steven ONG, Matthew JAFRI, Amir SEGAL, Robert Prince, Stephen <120> SYSTEM AND METHOD FOR REMOVAL OF IMMUNE INHIBITORS FROM BIOLOGICAL FLUIDS <130> 022947-0508680 <140> PCT/US2019/062177 <141> 2019-11-19 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 155 <212> PRT <213> Homo sapiens <400> 1 Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala 1 5 10 15 Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn 20 25 30 Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val 35 40 45 Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly 50 55 60 Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg 65 70 75 80 Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys 85 90 95 Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp 100 105 110 Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp 115 120 125 Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu 130 135 140 Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 2 <211> 509 <212> PRT <213> artificial sequence <220> <223> trimeric peptide <400> 2 Met Cys Gly Ser His His His His His His Gly Ser Ala Ser Ser Ser 1 5 10 15 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 20 25 30 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 35 40 45 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 50 55 60 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 65 70 75 80 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 85 90 95 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 100 105 110 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 115 120 125 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 130 135 140 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 145 150 155 160 Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Ser Gly Gly Gly 165 170 175 Ser Gly Gly Gly Ser Gly Gly Gly Ser Ser Ser Arg Thr Pro Ser Asp 180 185 190 Lys Pro Val Ala His Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu 195 200 205 Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu 210 215 220 Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile 225 230 235 240 Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val 245 250 255 Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys 260 265 270 Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro 275 280 285 Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly 290 295 300 Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg 305 310 315 320 Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile 325 330 335 Ile Ala Leu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 340 345 350 Gly Gly Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 355 360 365 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 370 375 380 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 385 390 395 400 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 405 410 415 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 420 425 430 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 435 440 445 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 450 455 460 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 465 470 475 480 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 485 490 495 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 500 505 <210> 3 <211> 499 <212> PRT <213> artificial sequence <220> <223> trimeric peptide <400> 3 Gly Ser Ala Ser Ser Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala 1 5 10 15 His Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn 20 25 30 Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn 35 40 45 Gln Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val 50 55 60 Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His 65 70 75 80 Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu 85 90 95 Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu 100 105 110 Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu 115 120 125 Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu 130 135 140 Asp Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly 145 150 155 160 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ser 165 170 175 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 180 185 190 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 195 200 205 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 210 215 220 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 225 230 235 240 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 245 250 255 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 260 265 270 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 275 280 285 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 290 295 300 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 305 310 315 320 Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Ser Gly Gly Gly 325 330 335 Ser Gly Gly Gly Ser Gly Gly Gly Ser Ser Ser Arg Thr Pro Ser Asp 340 345 350 Lys Pro Val Ala His Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu 355 360 365 Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu 370 375 380 Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile 385 390 395 400 Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val 405 410 415 Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys 420 425 430 Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro 435 440 445 Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly 450 455 460 Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg 465 470 475 480 Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile 485 490 495 Ile Ala Leu

Claims (41)

A system for removing at least one target component of a bodily fluid, the system comprising:
an inlet configured to receive bodily fluid from a patient;
An isolation chamber coupled to an inlet and configured to receive bodily fluid from the inlet, the isolation chamber comprising a capture support and first and second access ports, wherein the capture support is responsive to contact between the capture support and the bodily fluid to form an isolation chamber configured to bind to at least one target component of a bodily fluid to capture the at least one target component in the body fluid and to bind to the at least one target component to reduce the amount of the at least one target component in the bodily fluid; an isolation chamber, wherein the first and second access ports are configured to provide access to the isolation chamber separate from the inlet and to facilitate insertion and/or removal of a capture support into and/or from the isolation chamber;
an outlet configured to pass the bodily fluid having the reduced amount of the at least one target component from the isolation chamber to selectively reintroduce at least a portion of the bodily fluid having the reduced amount of the at least one target component back to the patient; and
A system comprising one or more filters configured to separate a capture support within the isolation chamber from an inlet and an outlet and configured to retain the capture support within the isolation chamber. 2. The method of claim 1, wherein (a) the entrapment efficiency of the entrapment scaffold that binds to the at least one target component is greater than or equal to 80% at a plasma flow rate of less than or equal to 45 mL per minute, and optionally
(b) the binding affinity of the capture support for the at least one target component is at least about 10 ⁻⁷ K _D and/or
(c) a system wherein the leaching rate of the capture support through the outlet is less than about 100 ng/mL/min. 2. The method of claim 1, wherein (b) the binding affinity of the capture support for at least one target moiety is greater than 10 ^-7 K _D , and optionally
(a) the capture efficiency of the capture support that binds to the at least one target component is 80% or greater at a flow rate of 45 mL/min or less; and/or
(c) a system wherein the leaching rate of the capture support through the outlet is less than about 100 ng/mL/min. 2. The method of claim 1, wherein (c) the leaching rate of the capture support through the outlet is less than about 100 ng/mL/min, and optionally
(a) the capture efficiency of the capture support that binds to the at least one target component is 80% or greater at a flow rate of 45 mL/min or less; and/or
(b) a system wherein the binding affinity of the capture support to the at least one target component is at least about 10 ⁻⁷ K _D . 5. The system according to any one of claims 1 to 4, wherein the bodily fluid comprises plasma. 6. The system according to any one of claims 1 to 5, wherein the at least one target component comprises a protein, complex, assembly or cell. 7. The system according to any one of claims 1 to 6, wherein the at least one target component comprises one or more plasma components that function to suppress an anti-cancer immune response in the patient. 8. The system of any one of claims 1-7, wherein the at least one target component comprises one or more immunosuppressive agents. 9. The system of any one of claims 1-8, wherein the at least one targeting component comprises a soluble TNFa receptor. 10. The system according to any one of claims 1 to 9, wherein the at least one target component comprises the sTNF-R1 receptor and/or the sTNF-R2 receptor. 11 . The system according to claim 1 , wherein the capture support comprises an affinity chromatography support material, a hollow fiber membrane, a sheet membrane, a membrane cassette, a bead or a rolled sheet membrane. 12. The system of claim 11, wherein the capture support comprises an affinity chromatography support material and the affinity chromatography support material comprises sepharose, agarose or acrylamide. 13. The system according to any one of claims 1 to 12, wherein the entrapment support comprises a porous or non-porous matrix material. 14. The system of any one of claims 1-13, wherein the capture support is configured to bind to more than one target component of a bodily fluid. 15. The system according to any one of claims 1 to 14, wherein the capture support comprises a solid support on which an antibody, antibody fragment, conjugated peptide, aptamer or avimer is immobilized. 16. The system of claim 15, wherein the antibody is selected from the group consisting of IgA, IgD, IgE, IgG, IgM and combinations thereof. 17. The system according to any one of claims 1 to 16, wherein the capture support comprises TNFα, multimers of TNFα, single-chain TNFα, fragments of TNFα, multimers of fragments of TNFα, or combinations thereof. 18. The system according to any one of claims 1 to 17, wherein the capture support comprises a sc-TNFα ligand, optionally a sequence or partial sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 . 19. The system of claim 18, wherein the capture scaffold comprises a trimeric form of an sc-TNFα ligand. 20. The system of claim 19, wherein the trimeric form of the sc-TNFα ligand comprises the sequence or partial sequence of SEQ ID NO:2 or SEQ ID NO:3. 21. The system of any one of claims 1-20, wherein the capture support comprises a ligand coupled to a bead. 22. The system of claim 21, wherein the ligand has a given density and orientation in a given bead, the density and orientation configured to enhance binding between the ligand and at least one target component of the bodily fluid. 23. The system according to claim 21 or 22, wherein the size, number, density and/or concentration of the beads is configured to promote laminar flow of bodily fluid through the beads to enhance binding between the ligand and at least one target component of the bodily fluid. . 24. The system of any one of claims 21-23, wherein the beads are quenched in ethanolamine to enhance binding specificity. 25. The system of any preceding claim, wherein the bodily fluid is whole blood. 26. The system of any preceding claim, wherein the inlet, isolation chamber and outlet form an extracorporeal closed circuit column. 27. The system of claim 26, wherein the extracorporeal closed circuit column is configured to remain sterile during operation. 28. The system of claim 26 or 27, further comprising a target component exit port configured to facilitate sampling or removal of all or a portion of the entrapped at least one target component without damaging the extracorporeal closed circuit column. 29. The system of any one of claims 26-28, further comprising an elution reagent port configured to facilitate introduction of an elution reagent into the isolation chamber without damaging the extracorporeal closed circuit column. 30. The system of claim 29, wherein the elution reagent port is further configured to receive a conditioning agent configured to prepare the system for reuse. 31. The system of any one of claims 1-30, further comprising a pump configured to drive the regenerant through the inlet, the isolation chamber and the outlet. 32. The system of claim 31, wherein the pump comprises a syringe pump, a peristaltic pump, a piston pump, a diaphragm pump, or a combination thereof. 33. The system of any one of claims 1-32, wherein the one or more filters have an average pore diameter of 3 microns to 100 microns. 34. The system of any one of claims 1 to 33, further comprising one or more additional isolation chambers comprising a capture support having the same function. 35. The system of claim 34, wherein one or more additional isolation chambers are combined with the isolation chambers to form a multi-stage isolation circuit configured to engage a plurality of different target components. 36. The system of any preceding claim, wherein the patient is a human or veterinary subject. 37. A method for removing at least one target component of a bodily fluid with the system of any one of claims 1-36, the method comprising:
passing bodily fluid from the patient through the inlet into the isolation chamber;
binding the at least one target component of the bodily fluid to capture the at least one target component in the isolation chamber to reduce the amount of the at least one target component in the bodily fluid; and optionally
passing some or all of the bodily fluid having the reduced amount of the at least one target component through the outlet for reintroduction from the isolation chamber back to the patient. 38. The method of claim 37, further comprising measuring the reduced amount of the at least one target component in the bodily fluid reintroduced to the patient. 39. The method of claim 38, wherein the measurement is LC-MS, HPLC, UHPLC, resistance measurement, luminescence measurement, chemiluminescence, electroluminescence, electrochemiluminescence, chromatographic monitoring, positron emission tomography (PET), x-ray computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, gamma camera, single photon emission computed tomography (SPECT), ELISA, SPR, or BLI. 40. The method of any one of claims 37-39, further comprising measuring the rate of leaching of the capture scaffold in bodily fluid reintroduced to the patient. 41. The method of any one of claims 37-40, wherein the method is for human or veterinary use.

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