JP7159493B2 - Systems and methods for removing immune inhibitors from biological fluids - Google Patents

Description

[1. field]
[01] The present disclosure relates to systems and methods for removing immune inhibitors from plasma.

[2. Introduction]
[02] Harnessing the immune system to kill cancer has been the focus of oncologists and cancer researchers for over a century. The observation that patient tumors go into remission after immunostimulatory bacterial infection (eg Coley, WB (1991) Clin Orthop Relat Res, pp. 3-11; Hughes, WT and Smith, DR). (1973) Cancer 31, pp. 1008-1014;Yates, JW and Holland, JF (1973) Cancer 32, pp. 1490-1498; , Pathol Microbiol (Basel) 40, 297-304), and the correlation between cancer immune cell infiltration and survival (eg Lipponen, PK et al. (1992) Eur J Cancer 29A, 69-75). Ma, D. and Gu, MJ (1991) J Tongji Med Univ 11, 235-239; Pastrnak, A. and Jansa, P. (1989) Acta Univ Palacki Olomuc Fac Med 124, 7-71. (1992) Int Surg 77, 256-260); Di Giorgio, A.; (1992) Int Surg 77, 256-260) suggest the possibility of immunological control of neoplasia. However, it is only in the last decade that researchers in the field of cancer immunotherapy have been able to claim significant improvements in patient outcomes. A major breakthrough in this area has been the development of antibodies that inhibit negative regulators or checkpoints of T cell activation. These antibodies belong to a class of drugs termed "immune checkpoint inhibitors." The first FDA-approved, ipilimumab, an antagonistic antibody that targets cytotoxic T-cell-associated protein 4 (CTLA-4), improved overall survival in patients with metastatic melanoma in 2010 . A related study randomized to receive ipilimumab and glycoprotein 100 (gp100, also known as melanoma cell protein) (403 patients), ipilimumab alone (137), or gp100 alone (136). A total of 676 HLA-A ^* 0201 positive patients with unresectable stage III or IV melanoma were evaluated. The median overall survival was 10.0 months for patients receiving ipilimumab and gp100 compared to 6.4 months for patients receiving gp100 alone (hazard ratio for death 0.68, P< 0.001). The 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 with ipilimumab and gp100 1.04, P=0.76) (e.g. Hodi, FS et al. (2010) N Engl J Med 363 , pages 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 been shown to be effective in improving overall survival in multiple cancers. proven (eg 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) Topalian, SL et al (2014) J Clin Oncol 32, 1020-1030; Ribas, A. and Wolchok, JD (2018) Science 359, 1350-1355. reference). For example, in one study, 296 patients received anti-PD-1 ligand antibody treatment. Among 236 patients for whom response could be evaluated, objective responses (complete or partial responses) were observed in patients with non-small cell lung cancer, melanoma, or renal cell carcinoma. Cumulative response rates (all doses) were 18% (14/76 patients) in patients with non-small cell lung cancer, 28% (26/94 patients) in patients with melanoma, and 28% (26/94 patients) in patients with renal cell carcinoma. 27% (9 of 33 patients). Responses were durable, with 20 of 31 patients with a follow-up of ≥1 year having responses lasting ≥1 year (Topalian, SL et al. (2012) N Engl J Med 366, 2443- 2454). The encouraging results of these studies have stimulated interest from the field of cancer research and inspired further investigation into targeting alternative immune checkpoint molecules.

[03] Blockade of checkpoints represents a breakthrough in cancer therapy, but the majority of cancer patients do not respond to these treatments, and some tumor types appear to be inherently resistant. be. Treatments are designed to augment ongoing immune responses and are ineffective in cases lacking initial immune activation, including tumors that do not have infiltrating T cells. Therefore, the development of therapeutic strategies that enhance immune cell recruitment would increase the proportion of patients responding to immune checkpoint blockade. Limitations of checkpoint inhibitors include systemic exposure of the patient to the antibody used, as well as the inability to consistently elicit a response.

[04] TNFα (tumor necrosis factor-alpha, or TNF, referred to interchangeably herein) promotes anticancer activity and, as the name implies, was initially characterized as an antitumor agent. are potent cytokines (see, eg, Carswell, EA et al. (1975) Proc Natl Acad Sci USA 72, 3666-3670). Subsequently, TNF was shown to have both tumor-promoting and tumor-suppressive effects depending on its contextual activity within the tumor microenvironment (e.g. Wang, X. and Lin, Y. (2008) Acta Pharmacol Sin 29, pages 1275-1288). Within the tumor microenvironment, low levels of TNF expression contribute to angiogenesis, vascular permeability, and metastatic potential, whereas at high levels and during therapeutic delivery to tumors, TNF is mediated by apoptosis. demonstrated anti-tumor effects, including disruption of vascular integrity, direct tumor killing, and induction of anti-tumor immune responses (eg Berberoglu, U. et al. (2004) Int J Biol Markers 19, 130-134). (see Michalaki, V. et al. (2004) Br J Cancer 90, 2312-2316; Talmadge, JE et al. (1987) Cancer Res 47, 2563-2570). Beneficial effects of increased TNF in clinical settings have been reported. For example, a study of TNF expression in 61 patients with non-small cell lung cancer demonstrated TNF expression in 45.9% of cases directly correlated with more favorable clinical outcome (e.g. Boldrini, L. et al. G. (2000). ) Br J Cancer 83, pages 480-486). Administration of TNF has been approved for isolated extremity administration and has shown clinical benefit in isolated liver procedures for liver cancer.

[05] sTNF-R (a soluble receptor for TNF) inhibits anticancer immune responses and contributes to the control of TNF toxicity. The natural regulation or attenuation of the anti-tumor effects of TNF is attributed to the presence of inhibitory molecules present in the plasma that bind/neutralize TNF, including released soluble TNF receptors (e.g. Xanthoulea, (2004) J Exp Med 200, 367-376;Aderka, D. et al. (1998) J Clin Invest 101, 650-659;Aderka, D. et al. (see Selinsky, CL et al. (1998) Immunology 94, 88-93; Selinsky, CL and Howell, MD (2000) Cell Immunol 200, 81-87). The cancer-promoting activity of these soluble inhibitors was discovered after early observations of cancer regression that occurred in patients undergoing plasmapheresis (eg Israel, L. et al. (1976) Lancet 2, 642-643; Israel, L. et al. (1977) Cancer 40, 3146-3154). Continuing studies have shown that this observation can be attributed to the removal of sTNF-R. Molecular cloning of cDNAs and studies of recombinant proteins confirmed their anti-TNF activity and tumor-promoting function (eg Schall, TJ et al. (1990) Cell 61, 361-370; Engelmann, H. et al. (1990) J Biol Chem 265, 1531-1536).

[06] At low doses of TNF, normal concentrations of sTNF-R inhibitors are able to bind and inactivate small amounts of TNF administered. However, increased dose dosing or stimulation of higher TNF production to higher than normal levels induces release of sTNF-R that counteracts TNF's ability to reach therapeutic anti-tumor concentrations without toxic effects. there's a possibility that. Therefore, the ability to overcome TNF inhibition to achieve anticancer effects requires administration of amounts of TNF very close to the maximum tolerated dose (MTD). For this reason, systemic TNF therapy, although potentially effective, has shown toxicity in many human clinical trials. Because of this adverse risk/benefit consideration, systemic therapy with TNF has largely been abandoned. However, isolated extremity procedures that block systemic exposure to TNF have been performed in combination with chemotherapeutic agents (eg Deroose, JP et al. (2012) Ann Surg Oncol 19, 627-635; Verhoef, C (2007) Curr Treat Options Oncol 8, 417-427).

[07] There have been attempts to use medical devices to remove immune "blocking factors" produced by tumors outside the body. Unfortunately, to date, these devices have suffered from a) non-specific binding of other biological materials, b) "elution" of immunoadsorbent material from the device into the patient's circulatory system, and c) depletion of target proteins. There are many limitations such as ineffective removal from the circulatory system. The present invention overcomes these and other limitations of conventional systems.

[Overview]
[08] The following is a non-exhaustive list of some aspects of the present approach. Some and other aspects of this approach are described in the following disclosure.

[09] 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 sequestration chamber comprises a capture support configured to bind at least one target component of the bodily fluid and capture the at least one target component in the sequestration chamber in response to contact of the capture support with the bodily fluid. . The capture support is configured to bind at least one target component and reduce the amount of at least one target component in a bodily fluid. The isolation chamber comprises first and second access ports configured to provide access to the isolation chamber separate from the entrance. The first and second access ports are configured to facilitate insertion and/or removal of capture supports into and/or from the isolation chamber. The system places the body fluid having the reduced amount of the at least one target component in an isolation chamber for optional reintroduction of part or all of the body fluid having the reduced amount of the at least one target component back to the patient. and one or more filters configured to separate the capture support in the isolation chamber from the inlet and outlet. One or more filters are configured to retain the capture support within the isolation chamber.

[10] In one embodiment, (a) the capture support that binds the at least one target component has a capture efficiency of 80% or greater at a plasma flow rate of 45 mL/min or less; The binding affinity of the capture support to the component is at least about 10 ⁻⁷ K _D and/or (c) the elution rate of the capture support through the outlet is less than about 100 ng/mL/min.

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

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

[13] In one embodiment, the bodily fluid comprises plasma.

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

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

[16] In one embodiment, the at least one targeting moiety comprises one or more immunosuppressive agents.

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

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

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

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

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

[22] In one embodiment, the capture support is configured to bind two or more target components of the bodily fluid.

[23] In one embodiment, the capture support comprises a solid support to which an antibody, antibody fragment, binding peptide, aptamer, or avimer is immobilized.

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

[25] In one embodiment, the capture support comprises TNFα, multimers of TNFα, single-chain TNFα, fragments of TNFα, multimers of fragments of TNFα, or combinations thereof.

[26] In one embodiment, the TNFα multimer comprises TNFα monomers in which one or more monomers are in an amino-terminal to carboxy-terminal linkage.

[27] In certain embodiments, the TNFα multimer may or may not include spacers between the monomers.

[28] In certain embodiments, the spacer comprises one or more amino acid residues.

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

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

[31] In one embodiment, the capture support comprises a trimeric form of the TNFα ligand.

[32] 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.

[33] In one embodiment, the capture support comprises a ligand bound to a bead.

[34] In one embodiment, the ligands have a given density and orientation on a given bead. Density and/or orientation are configured to enhance binding of the ligand to at least one target component of the bodily fluid.

[35] In one embodiment, the size, number, density, and/or concentration of the beads facilitates laminar flow of bodily fluids past the beads to enhance binding of the ligand to at least one target component of the bodily fluid. is configured as

[36] In one embodiment, the beads are quenched in ethanolamine to enhance binding specificity.

[37] In one embodiment, the bodily fluid is whole blood.

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

[39] In one embodiment, the extracorporeal closed circuit column is configured to remain sterile during manipulation.

[40] In one embodiment, the system includes a target component outlet configured to facilitate sampling or removal of all or a portion of the captured at least one target component without compromising the extracorporeal closed circuit column. Equipped with ports.

[41] In one embodiment, the system comprises an eluent port configured to facilitate introduction of an eluent into the isolation chamber without compromising the extracorporeal closed circuit column.

[42] In an embodiment, the eluent port is further configured to receive a conditioning agent configured to prepare the system for reuse.

[43] In an embodiment, the system further comprises a pump configured to drive the reconditioning agent through the inlet, the isolation chamber, and the outlet.

[44] In an embodiment, the pump comprises a syringe pump, a peristaltic pump, a piston pump, a diaphragm pump, or a combination thereof.

[45] In one embodiment, the one or more filters have an average pore size of from about 3 microns to about 100 microns.

[46] In an embodiment, the system further comprises one or more additional isolation chambers comprising capture supports having the same function.

[47] In one embodiment, one or more additional sequestration chambers are combined with the sequestration chambers to form a multi-stage separation circuit configured to bind multiple different target components.

[48] In one embodiment, the patient is a human or veterinary subject. Veterinary subjects may include domestic animals such as dogs, cats, farm or ranch animals such as horses, pigs, cows, and/or other animals.

[49] According to another embodiment, there is provided a method of removing at least one or more target components of a bodily fluid by the system of any of the above embodiments. The method comprises the steps of: directing a bodily fluid from a patient through an inlet into an isolated chamber; binding at least one target component of the bodily fluid to capture the at least one target component in the sequestered chamber; and optionally passing some or all of the bodily fluid having the reduced amount of the at least one target component from the isolation chamber through the outlet for reintroduction back to the patient.

[50] In one embodiment, the method further comprises measuring a reduced amount of at least one target component in the bodily fluid reintroduced back into the patient.

[51] In one embodiment, the measuring step comprises liquid chromatography-mass spectroscopy (LC-MS), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (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, surface plasmon resonance (SPR), and/or bilayer interferometry (BLI).

[52] In one embodiment, the method further comprises measuring the elution rate of the capture support in the bodily fluid reintroduced back into the patient.

[53] In some embodiments, the methods are for human, veterinary, domestic/companion animal, ranch/farm animal, and/or other uses.

[54] The combination of these and other objects, features, and characteristics of the system or method disclosed herein, and the functions of the associated elements of the method and structure of operation, and the economies of parts and manufacturing The following description and appended claims, all of which form part of this specification, will become more apparent when considered with reference to the accompanying drawings. Here, like reference numerals in the various figures designate corresponding parts. It is expressly to be understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the invention. As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[55] The above aspects and other aspects of the present approach will be better understood upon reading this application in view of the following drawings. Like numbers in the drawings indicate similar or identical elements.

1 illustrates an exemplary embodiment of the system in accordance with one or more embodiments; FIG. 1 provides a more detailed overview of the system according to one or more embodiments; FIG. FIG. 2 illustrates a corresponding cross-sectional view of the system in accordance with one or more embodiments; According to one or more embodiments, a capture support (e.g., in this example Fig. 3 is a diagram for explaining ligand-coated beads). 5 is an enlarged view of the capture support shown in FIG. 4, according to one or more embodiments; FIG. An enlarged view shows a capture support containing beads and capture ligands. FIG. 4 illustrates an exemplary playback mechanism, in accordance with one or more embodiments; FIG. 4 illustrates an exemplary embodiment in which the system comprises two or more internal isolation chambers that can be configured in series or parallel for liquid flow from inlet to outlet, according to one or more embodiments; be. FIG. 2 illustrates an exemplary use in which multiple systems can be utilized in series and/or parallel configurations within a single plasmapheresis flow circuit, according to one or more embodiments. FIG. 10 illustrates multiple isolation chambers each having independently controllable flow valves within a single housing, according to one or more embodiments. FIG. 2 illustrates a method of removing target components of blood, plasma, and/or other bodily fluids with the present system, according to one or more embodiments. FIG. 4 illustrates representative system performance characteristics including reduction of sTNF-R1 and sTNF-R2 from a patient's blood pool as a function of procedure time and column capture efficiency, according to one or more embodiments. .

Detailed Description of Illustrative Embodiments
[67] While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and will herein be described in detail. Drawings may not be to scale. However, the drawings and detailed description thereof are not intended to limit the invention to the particular forms disclosed, but rather to within the spirit and scope of the invention as defined by the appended claims. should be understood to be intended to cover all modifications, equivalents, and alternatives contained therein.

[68] To alleviate the problems described herein, the inventors have invented solutions and, in some important cases, overlooked by others in the field ( or was not expected). Indeed, we emphasize the difficulty of recognizing these problems that are occurring now and will become more pronounced in the future if industry trends continue as we anticipate. Want to. Moreover, some embodiments are problem specific as many of them address concurrent problems, and not all embodiments address every problem in the conventional systems described herein. It should be understood that it does not provide all of the benefits described herein. Improvements that solve various permutations of these problems are then described below.

[69] The present systems and methods are useful for immunomodulation in cancer patients and may provide relatively useful immunomodulation for other diseases including, but not limited to, autoimmune and inflammatory disorders. In some embodiments, extracorporeal removal of immunosuppressive agents from a patient's blood using immunoadsorption 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 dependent on soluble TNF receptors (sTNF-R1 and sTNF-R2, eg Gatanaga, T. et al. (1990) Lymphokine Res 9, 225-229; Gatanaga, T. et al. (1990) Proc Natl Acad Sci USA 87, 8781-8784;Schall, TJ et al.(1990) Cell 61, 361-370;Berberoglu, U. et al. page) is blocked by the presence of inhibitory molecules in circulation known as These receptors, which block the therapeutic anti-tumor effects of endogenous TNF, have been shown to be elevated in cancer and correlate with disease stage (eg Aderka, D. et al. (1991) Cancer Res 51, 5602-5607). sTNF-R is also a prognostic indicator for breast cancer, melanoma, colorectal cancer, and osteosarcoma, and is negatively correlated with patient survival (eg Langkopf, F. and Atzpodien, J. (1994) Lancet 344 , 57-58; Viac, J. et al. (1996) Eur J Cancer 32A, 447-449). The selective removal of sTNF-R from a patient's blood by plasmapheresis, a process known as Immunopheresis™, demonstrates the antitumor effects of endogenous TNF by demonstrating its antitumor effect. It can enhance the patient's natural anti-tumor immune response, thereby facilitating reduction of tumor burden and improving patient survival.

[70] Figure 1 illustrates an exemplary embodiment of the system (item 10). In FIG. 1, system 10 is shown coupled to apheresis machine 12 . System 10 includes sTNF-R (eg, soluble tumor necrosis factor receptor 1 (sTNF-R1), also known as tumor necrosis factor receptor superfamily member 1A (TNFRSF1A and CD120a), and the tumor necrosis factor receptor superfamily Soluble tumor necrosis factor receptor 2 (sTNF-R2), also known as member 1B (TNFRSF1B and CD120b), is configured to be selectively removed from the blood of cancer patients 14 as an exemplary target component. The system 10 contains a highly selective binding matrix within a housing 16 having various ports to facilitate filling and plasma flow during use.Blood withdrawn from a patient 14 is processed to obtain plasma, Plasma is processed by placing the system 10 in the plasma flow path 18 of an apheresis machine for plasmapheresis procedures.

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

[72] As shown in FIG. 1, an apheresis device 12 performs intravenous removal of blood 20 from a patient 14 and then (eg, using centrifugal force, membrane filters, and/or other components) the blood. It may facilitate separation 22 into plasma and cellular fractions. The plasma fraction is then pumped into system 10, where the plasma captures sTNF-R using, for example, TNF ligands (described herein) (including binding matrices described herein). Pass through the support. The plasma is then pumped back out of the system 10 and some or all of the processed plasma is combined with the separated cells of the patient 14 and reintroduced 24 back into the circulatory system of the patient 14 .

[73] In some embodiments, the processed plasma is discarded and replaced by fresh plasma. For example, plasmapheresis may be performed concurrently and the exchanged plasma is further processed to remove inhibitors. For example, plasmapheresis may be followed by immunopheresis (eg, as described herein).

[74] Although blood, plasma, sTNF-Rs, and TNF ligands are specifically mentioned throughout this application, the components and/or principles described herein are applicable to other body fluids, other target components of body fluids, and/or other capture or binding elements.

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

[76] The closed circuit column may be configured to remain sterile during operation. In some embodiments, the components of system 10 are cleaned with, for example, 70% isopropyl alcohol prior to assembly to remove particles. Filters 212 and 214 may be attached to end caps 250 and 252 and pressed onto the distal end of a barrel or (for example) tube to form housing 16 . Caps 254 and 256 may be threaded and/or otherwise connected to inlet 200 and outlet 210 . This intermediate assembly is packaged and sterilized using, for example, ethylene oxide (EtO). Residual EtO is dissipated before continuing the manufacturing process. The EtO-sterilized intermediate assembly is aseptically filled with capture support 204 through access ports 206 and 208, and ports 206 and 208 are then tightly capped with (for example) polycarbonate luer (for example as described above) caps. do. The assembled devices are then individually packaged and terminally sterilized with (for example) 17.5-30 kGy electron beam irradiation. In some embodiments, other sterilization means that may be utilized include gamma irradiation, ethylene oxide, hydrogen peroxide, bleaching, heat sterilization, steam sterilization, ozone, and/or the stability of capture support 204 and/or other Other sterilization procedures are included depending on factors.

[77] Housing 16, inlet 200, outlet 210, and/or other components of system 10 are configured to interface with a flow path (eg, plasma) of an apheresis device (eg, device 12 shown in FIG. 1). good too. Housing 16, inlet 200, outlet 210, and/or other components of system 10 are present after the patient's blood has been separated, for example, into a cellular fraction and a plasma fraction (eg, as described herein). It may be configured to connect with the flow path of the apheresis device at one point of the flow path. Housing 16 may form a fluid channel or flow path that directs patient bodily fluids between inlet 200 and outlet 210 .

[78] Housing 16 may provide structural support for capture support 204 and/or other components of system 10 . Housing 16 may form an elongate tubular body having a circular cross-sectional shape and/or other cross-sectional shapes. Housing 16 may contain isolation chamber 202 with 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.

[79] Housing 16 houses 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 in isolation chamber 202 from inlet 200 and outlet 210 . Filters 212, 214 may be configured to retain capture support 204 within isolation chamber 202 and/or perform other functions.

[80] In some embodiments, filters 212 and/or 214 may form a porous barrier that is mounted substantially perpendicular to the direction of fluid flow through housing 16 . Filter 212 may be positioned proximate inlet 200 and filter 214 proximate outlet 210 , thereby forming an isolation chamber 202 within housing 16 . Filters 212 and/or 214 allow a portion (up to and including all) of capture support 204 (e.g., one or more beads described herein) to escape system 10, e.g. may be configured to prevent it from flowing into the In some embodiments, filters 212 and/or 214 can include, for example, porous frits. In some embodiments, filters 212, 214 can have an average pore size of, for example, about 3 microns to about 100 microns, and/or other average pore sizes. In some embodiments, filters 212 and 214 have a diameter greater than the inner diameter of housing 16 such that filters 212 and 214 fit snugly within housing 16 and displace as bodily fluids flow through system 10 . do not do. In some embodiments, filters 212 and 214 are provided with end caps 250 and 252 (described below) that press filters 212 and 214 against the rims of housing 16 (eg, at either end of the tube formed by housing 16). ) is held in place by pressure from the In some embodiments, filters 212 and 214 are held in place within housing 16 by adhesives, washers, gaskets, stitching, overmolding, ultrasonic welding, and/or other components and/or processes. there is In some embodiments, filters 212 and 214 may be made from polyethylene and/or other materials.

[81] In some embodiments, the housing 16 includes end caps 250 and 252 at either end of the housing 16 that define and/or include an inlet 200 and an outlet 210 . End caps 250 and 252 may be threaded onto housing 16 and/or otherwise coupled to housing 16 (eg, by clips, clamps, adhesives, ultrasonic welding, fitting, etc.). In some embodiments, end caps 250 and/or 252 may be secured to housing 16 to ensure system 10 is substantially airtight. In other words, 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 terminate at inlet 200 and outlet 210, respectively. In some embodiments, inlet 200 and/or outlet 210 may include caps 254,256. 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 from, for example, high density polyethylene and/or other materials.

[82] In some embodiments, the housing 16 may be formed from plastic and/or other materials. For example, housing 16 may be made of ECTFE (ethylene-chlorotrifluoroethylene copolymer, halarECTFE, ETFE (ethylene-tetrafluoroethylene), tefzel ethylenetetrafluoroethylene (ETFE), FEP (fluorinated ethylene polypropylene), HDPE (high density polyethylene) , LDPE (low density polyethylene), PC (polycarbonate), Makrolon polycarbonate, PEI (polyetherimide), PET (polyethylene terephthalate), PETG (polyethylene terephthalate copolymer), PFA (polyfluoroalkoxy), Teflon PFA, PMMA (polymethyl methacrylate), PMP (polymethylpentene), polypropylene, PPCO (polypropylene copolymer), polystyrene, PSF (polysulfone), PTFE (polytetrafluoroethylene), SAN (styrene acrylonitrile), TFE (tetrafluoroethylene), Teflon TFE, TMX (Thermanox), PMX (Permanox), and/or other materials.In some embodiments, the housing 16 is made of a metallic material (e.g., iron, iron alloys, steel, stainless steel, aluminum, aluminum alloys), glass, and/or other materials.

[83] Portal 200 may be configured to receive blood, plasma, and/or other bodily fluids from a patient (eg, patient 14 shown in FIG. 1). Outlet 210 provides blood, plasma, and/or other bodily fluids having a reduced amount of one or more target components for reintroduction back into the isolation chamber 202 of the patient (eg, patient 14 shown in FIG. 1). It may be configured to pass from 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 are commonly used in apheresis machine tubing sets, intravenous tubing extension sets, fluid tubing adapters, filters, stopcocks, and/or closed-loop patient fluid line assemblies. configured to be fluidly coupled to other elements. In some embodiments, inlet 200 and/or outlet 210 allow blood, plasma, and/or other bodily fluids from the patient to flow through system 10 at between about 5 mL/min and about 300 mL/min, and/or other flow rates. It may be configured to flow. In some embodiments, the flow rate may be from about 10 mL/min to about 100 mL/min. In some embodiments, the flow rate may be from about 25 mL/min to about 75 mL/min. In some embodiments, the flow rate may be from about 35 mL/min to about 70 mL/min. In some embodiments, the flow rate may be from about 40 mL/min to about 60 mL/min. These exemplary flow rates are within the 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 in conjunction with an apheresis device. However, it is expected that a flow rate of 300 mL/min is possible. Inlet 200 and/or outlet 210 may have a particular size diameter and/or other features to facilitate such flow.

[84] In some embodiments, inlet 200 and/or outlet 210 may be configured to allow human plasma containing (for example) up to about 200 μg of sTNF-R protein to flow through system 10 . This example is based on the patient's expected total plasma level of sTNF-R. The concentration range of sTNF-R (combination of sTNF-R1 and sTNF-R2) in human plasma is approximately 3-10 ng/mL, and an exemplary patient plasma volume is in the range of 50 cc/kg body weight (W). deaf. The total amount of sTNF-R is approximately (W (kg) x 50 mL/kg x (3-10 ng/mL)/1000 ng/μg). Thus, for a 70 kg individual (for example), the amount of sTNF-R would range from 10.5 to 35 μg. In some embodiments, the excess capture capacity of system 10 is based on laboratory bench testing of excess sTNF-R for plasma flowing through system 10 at flow rates up to (for example) about 45 mL/min. , can create sufficient efficiency margins. In some embodiments, inlet 200 and/or outlet 210 may be formed from polypropylene, polycarbonate, Makrolon(TM), and/or other materials.

[85] The isolation chamber 202 may be connected to the inlet 200. FIG. Isolation chamber 202 may be configured to receive blood (eg, whole blood), plasma, and/or other bodily fluids through inlet 200 . The isolation chamber 202 may include a capture support 204, access ports 206, 208, and/or other components.

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

[87] In some embodiments, at least one target component may comprise a complex, assembly, or cell. In some embodiments, at least one target component may comprise one or more blood products such as plasma or serum components that function to inhibit an anti-cancer immune response in a patient. In some embodiments, at least one targeting component may include one or more immunosuppressive agents. For example, at least one targeting component may target soluble TNFα receptors, sTNF-R1 receptors, sTNF-R2 receptors, sTNF-R1 and sTNF-R2 receptors, and/or other receptors and combinations of receptors. can contain.

[88] In some embodiments, the capture moiety may be selected to bind and capture other specific molecules in plasma. Examples of these other molecules or targets include, but are not limited to, acetylcholine receptors, adenosine receptors, adrenoceptors, GABA receptors, angiotensin receptors, cannabinoid receptors, cholecystokinin receptors, dopamine receptors. , glucagon receptor, glucocorticoid receptor, glutamate receptor, histamine receptor, mineralocorticoid receptor, orfactory receptor, opioid receptor, purinergic receptor, secretin receptor, serotonin receptor, somatostatin receptor , steroid hormone receptors, calcium-sensing receptors, hormone receptors, erythropoietin receptors, and natriuretic peptide receptors, or ligands thereof. Other examples include, but are not limited to, type I cytokine receptors such as type I interleukin receptors, erythropoietin receptors, GM-CSF receptors, G-CSF receptors, growth hormone receptors, oncostatin M receptors. body, myostatin receptor, leukemia inhibitory factor receptor, type II cytokine receptor such as type II interleukin receptor, interferon alpha/beta receptor, interferon gamma receptor, or ligands thereof; interleukin-1 receptor, members of the immunoglobulin superfamily such as CSF1, ckit receptor, interleukin-18 receptor or ligands thereof; CD27, CD40 and lymphotoxin receptors or their ligands; serpentine CCR and CXCR such as CCR1 and CXCR4 and chemokine receptors, including interleukin 8 receptors or ligands thereof; TGFβ receptors, including TGFβ receptor 1 and TGFβ receptor 2, or their ligands; galectins; and/or other structures. (See Ozaki and Leonard, J. Biol. Chem 277:29355-29353, 2002).

[89] In some embodiments, the capture support 204 can include a solid support and/or other components. Solid supports can be affinity chromatography support materials, hollow fiber membranes, sheet membranes, membrane cassettes, rolled sheet membranes, and/or other materials. In embodiments where capture support 204 comprises an affinity chromatography support material, the affinity chromatography support material comprises carbohydrates or polysaccharides such as sugars, sepharose, agarose, or polymers such as acrylamide, and/or other materials. obtain.

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

[91] In some embodiments, the capture support 204 is an affinity chromatography matrix that includes different capture moieties including, but not limited to, support-bound affinity agents (e.g., ligands as described herein). may be Affinity chromatography matrices can alternatively be cyanogen bromide, tresyl, triazines, vinyl sulfones, aldehydes, epoxides, or activated carboxylic acids to facilitate linkage of affinity agents (eg, ligands) to solid supports. such as, but not limited to, linking groups. The chromatographic matrix links the methyl-lysine affinity agent with the linking group to the solid support by chemically activating the solid support if necessary to covalently bind the affinity agent to the solid support. may be prepared by contacting a solid support with a methyl-lysine affinity agent. Additionally, the affinity agent may be linked to the solid support through a linker such that the affinity agent is more accessible for binding to methylated proteins and peptides.

[92] In some embodiments, capture support 204 may comprise a solid support to which antibodies, antibody fragments, binding peptides, aptamers, avimers, and/or other components are immobilized. 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.

[93] In some embodiments, the capture support 204 comprises TNFα (as noted above, TNF and TNFα are used interchangeably herein), multimers of TNF, single chain (sc) TNF. , fragments of TNF, multimers of fragments of TNF, or combinations thereof. In some embodiments, capture support 204 may be and/or include a TNF ligand bound to one or more solid supports. In some embodiments, a bond may be, for example, a covalent bond and/or other bond.

[94] Types of TNF include mammalian TNF, such as primate TNF and human TNF. Exemplary human TNF sequences include:
1. （ＳＳＳＲＴＰＳＤＫＰＶＡＨＶＶＡＮＰＱＡＥＧＱＬＱＷＬＮＲＲＡＮＡＬＬＡＮＧＶＥＬＲＤＮＱＬＶＶＰＳＥＧＬＹＬＩＹＳＱＶＬＦＫＧＱＧＣＰＳＴＨＶＬＬＴＨＴＩＳＲＩＡＶＳＹＱＴＫＶＮＬＬＳＡＩＫＳＰＣＱＲＥＴＰＥＧＡＥＡＫＰＷＹＥＰＩＹＬＧＧＶＦＱＬＥＫＧＤＲＬＳＡＥＩＮＲＰＤＹＬＤＦＡＥＳＧＱＶＹＦＧＩＩＡＬ、配列番号１）［処理されたＴＮＦモノマー、Ｇｅｎｂａｎｋ受託番号ＡＱＹ７７１５０．１］；
2. Trimmer type:
（ＭＣＧＳＨＨＨＨＨＨＧＳＡＳＳＳＳＲＴＰＳＤＫＰＶＡＨＶＶＡＮＰＱＡＥＧＱＬＱＷＬＮＲＲＡＮＡＬＬＡＮＧＶＥＬＲＤＮＱＬＶＶＰＳＥＧＬＹＬＩＹＳＱＶＬＦＫＧＱＧＣＰＳＴＨＶＬＬＴＨＴＩＳＲＩＡＶＳＹＱＴＫＶＮＬＬＳＡＩＫＳＰＣＱＲＥＴＰＥＧＡＥＡＫＰＷＹＥＰＩＹＬＧＧＶＦＱＬＥＫＧＤＲＬＳＡＥＩＮＲＰＤＹＬＤＦＡＥＳＧＱＶＹＦＧＩＩＡＬＧＧＧＳＧＧＧＳＧＧＧＳＧＧＧＳＳＳＲＴＰＳＤＫＰＶＡＨＶＶＡＮＰＱＡＥＧＱＬＱＷＬＮＲＲＡＮＡＬＬＡＮＧＶＥＬＲＤＮＱＬＶＶＰＳＥＧＬＹＬＩＹＳＱＶＬＦＫＧＱＧＣＰＳＴＨＶＬＬＴＨＴＩＳＲＩＡＶＳＹＱＴＫＶＮＬＬＳＡＩＫＳＰＣＱＲＥＴＰＥＧＡＥＡＫＰＷＹＥＰＩＹＬＧＧＶＦＱＬＥＫＧＤＲＬＳＡＥＩＮＲＰＤＹＬＤＦＡＥＳＧＱＶＹＦＧＩＩＡＬＧＧＧＳＧＧＧＳＧＧＧＳＧＧＧＳＳＳＲＴＰＳＤＫＰＶＡＨＶＶＡＮＰＱＡＥＧＱＬＱＷＬＮＲＲＡＮＡＬＬＡＮＧＶＥＬＲＤＮＱＬＶＶＰＳＥＧＬＹＬＩＹＳＱＶＬＦＫＧＱＧＣＰＳＴＨＶＬＬＴＨＴＩＳＲＩＡＶＳＹＱＴＫＶＮＬＬＳＡＩＫＳＰＣＱＲＥＴＰＥＧＡＥＡＫＰＷＹＥＰＩＹＬＧＧＶＦＱＬＥＫＧＤＲＬＳＡＥＩＮＲＰＤＹＬＤＦＡＥＳＧＱＶＹＦＧＩＩＡＬ、配列番号２）；及び３． Trimmer type:
（ＧＳＡＳＳＳＳＲＴＰＳＤＫＰＶＡＨＶＶＡＮＰＱＡＥＧＱＬＱＷＬＮＲＲＡＮＡＬＬＡＮＧＶＥＬＲＤＮＱＬＶＶＰＳＥＧＬＹＬＩＹＳＱＶＬＦＫＧＱＧＣＰＳＴＨＶＬＬＴＨＴＩＳＲＩＡＶＳＹＱＴＫＶＮＬＬＳＡＩＫＳＰＣＱＲＥＴＰＥＧＡＥＡＫＰＷＹＥＰＩＹＬＧＧＶＦＱＬＥＫＧＤＲＬＳＡＥＩＮＲＰＤＹＬＤＦＡＥＳＧＱＶＹＦＧＩＩＡＬＧＧＧＳＧＧＧＳＧＧＧＳＧＧＧＳＳＳＲＴＰＳＤＫＰＶＡＨＶＶＡＮＰＱＡＥＧＱＬＱＷＬＮＲＲＡＮＡＬＬＡＮＧＶＥＬＲＤＮＱＬＶＶＰＳＥＧＬＹＬＩＹＳＱＶＬＦＫＧＱＧＣＰＳＴＨＶＬＬＴＨＴＩＳＲＩＡＶＳＹＱＴＫＶＮＬＬＳＡＩＫＳＰＣＱＲＥＴＰＥＧＡＥＡＫＰＷＹＥＰＩＹＬＧＧＶＦＱＬＥＫＧＤＲＬＳＡＥＩＲＰＤＹＬＤＦＡＥＳＧＱＶＹＦＧＩＩＡＬＧＧＧＳＧＧＧＳＧＧＳＧＧＧＳＳＳＲＴＰＳＤＫＰＶＡＨＶＶＡＮＰＱＡＥＧＱＬＱＷＬＮＲＲＡＮＡＬＬＡＮＧＶＥＬＲＤＮＱＬＶＶＰＳＥＧＬＹＬＩＹＳＱＶＬＦＫＧＱＧＣＰＳＴＨＶＬＬＴＨＴＩＳＲＩＡＶＳＹＱＴＫＶＮＬＬＳＡＩＫＳＰＣＱＲＥＴＰＥＧＡＥＡＫＰＷＹＥＰＩＹＬＧＧＶＦＱＬＥＫＧＤＲＬＳＡＥＩＮＲＰＤＹＬＤＦＡＥＳＧＱＶＹＦＧＩＩＡＬ、配列番号３）
is included.

[95] Exemplary TNFs are monomers of the sequence of SEQ ID NO: 1, dimers of the sequence of SEQ ID NO: 1, trimers of the sequence of SEQ ID NO: 1, or trimeric forms of SEQ ID NO: 2 or SEQ ID NO: 3, or subsequences thereof can include Monomers comprising SEQ ID NO:2 or SEQ ID NO:3 may optionally be covalently linked by a glycine or serine spacer sequence 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 forms.

[96] Naturally occurring or non-naturally occurring variants of TNF are included. Such variants include variants with gain or loss of function.

[97] Non-limiting examples of TNF variants include substitutions of one or more amino acids of a reference TNF sequence (eg, 1-3, 3-5, 5-10, 10-15, 15-20, 20- 25, 25-30, 30-40, 40-50, 50-100 or more residues), additions (eg 1-3, 3-5, 5-10, 10-15, 15-20, 20 insertions of ˜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 a portion of the function or activity of the unmodified sequence, such as the ability to bind sTNF-R (eg, sTNF-R1 receptor and/or sTNF-R2 receptor). is doing.

[98] A variant may have one or more non-conservative or conservative amino acid sequence differences or alterations, or both. A "conservative substitution" is the replacement of one amino acid by a biologically, chemically, or structurally similar residue. "Biologically similar" means that the substitutions do not destroy biological activity. "Structurally similar" means that amino acids have side chains of similar length or similar size, such as alanine, glycine, and serine. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Particular examples include substitution of one hydrophobic residue for another, such as isoleucine, valine, leucine, or methionine, or substitution of one polar residue for another, such as lysine for arginine. to, glutamic acid to aspartic acid, or glutamine to asparagine, serine to threonine, and the like. Particular examples of conservative substitutions include substitution of a hydrophobic residue such as isoleucine, valine, leucine, or methionine for another residue, substitution of a polar residue for another residue, e.g. arginine for lysine. , glutamic acid to aspartic acid, or glutamine to asparagine, and the like. For example, conservative amino acid substitutions typically include those of the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; Includes substitutions in A "conservative substitution" also includes substituting a substituted amino acid for the unsubstituted parent amino acid.

[99] Such variants include proteins or polypeptides that have been or can be modified using recombinant DNA technology, e.g., such that the protein or polypeptide has been modified or has additional properties. . A variant may differ from a reference sequence, such as a naturally occurring protein or peptide.

[100] At the amino acid sequence level, a naturally occurring or non-naturally occurring variant protein is at least about 70% identical, more typically about 80% identical, and even more typically about 90% or less identical to the reference protein. Although identical above, non-identity in substantial regions (eg, less than 70% identity, eg, less than 60%, 50%, or even less than 40% identity) is permitted in non-conserved regions. In other embodiments, the sequence is at least 60%, 70%, 75%, or more identical to the reference sequence (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity). Techniques for introducing amino acid changes into proteins or polypeptides are known to those of skill in the art (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual (2007)).

[101] The term "identity" and grammatical variations thereof means that two or more reference entities are identical when they are in an "aligned" sequence. Thus, by way of example, where two polypeptide sequences are identical, they both have identical amino acid sequences, at least within the reference region or portion. Identity can span defined segments (regions or domains) of the sequences. A "zone" or "region" of identity refers to two or more reference entities that are the same. That is, if two protein sequences are identical over one or more segments or regions of the sequence, they share identity within that region. An "aligned" sequence refers to multiple protein (amino acid) sequences often containing corrections for missing amino acids (gaps) as compared to a reference sequence.

[102] Identity can extend over the entire sequence length or over a portion of the sequence. For example, the length of the sequences sharing the percent identity may be 2, 3, 4, 5 or more contiguous amino acids, or more, such as 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 sharing identity is 20 or more contiguous amino acids, or more, e.g. , 31, 32, 33, 34, 35, etc. In further non-limiting examples, the sequences sharing identity are 35 or more contiguous amino acids in length, e.g. 48, 49, 50, etc. contiguous amino acids. In more specific non-limiting examples, the length of sequences sharing identity is 50 amino acids or more, such as 50-55, 55-60, 60-65, 65-70, 70-75, 75-80 amino acids. , 80-85, 85-90, 90-95, 95-100, 100-110, etc., are contiguous amino acids.

[103] The degree of identity between two sequences can be determined using computer programs and mathematical algorithms. Such algorithms that calculate percent sequence identity generally account for sequence gaps or mismatches over the compared regions or sections. For example, a BLAST (eg, BLAST 2.0) search algorithm (see, eg, Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI) has the following exemplary search parameters: ing. Mismatch: -2, gap open: 5, gap extension: 2. For protein or polypeptide sequence comparisons, the BLASTP algorithm is typically used in conjunction with a scoring matrix such as PAM100, PAM250, BLOSUM62, or BLOSUM50. FASTA (eg, FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantify degrees 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 has also been developed to quantify protein structural similarity using Delaunay-based topological mapping (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[104] Ligands and proteins such as TNF include additions and insertions, eg, heterologous domains. An attachment (eg, a heterologous domain) can be a covalent or non-covalent attachment of any type of molecule. Additions and insertions (eg, heterologous domains) typically confer complementary or unique functions or activities.

[105] Non-limiting examples of additions or insertions include 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 spacer amino acids and amino acids that function as multimers include glycine, serine, and alanine.

[106] Additions and insertions include chimeric and fusion sequences, which are protein sequences that have one or more molecules not normally present in the native (wild-type) reference sequence covalently attached to the sequence. be. The terms "fusion" or "chimera", and grammatical variations thereof, mean that a portion or portions of the molecule are distinct (heterologous) from the molecule because they typically do not coexist in nature. means to contain an entity. Thus, for example, one portion, a fusion or chimera, comprises or consists of portions that are not naturally coexistent and structurally distinct.

[107] In some embodiments, methods for covalently linking a TNF ligand to a solid support(s) include amine reduction chemistry, cyanogen bromide (CNBr), N-hydroxysuccinimide esters, Carbonyldiimidazole, reductive amination, 2-fluoro-1-methylpyridinium (FMP) activation, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) mediated amide bond formation, organic sulfonyl chlorides, tosyl chloride and tresyl chloride, divinyl sulfone, azlactone, cyanuric chloride (trichloro-s-triazine), sulfhydryl-reactive chemistry, iodoacetyl and bromoacetyl activation, maleimide, pyridyl disulfide, divinyl sulfone, epoxy or bisoxirane, TNF-thiol , carbonyl-reactive chemistry, hydrazide, reductive amination, hydroxyl-reactive chemistry, cyanuric chloride, active hydrogen-reactive chemistry, diazonium, Mannich condensation, photoreactive cross-linking, CNBr activation and immobilization with periodate activation Serum albumin and/or other methods are included.

[108] In some embodiments, the binding can be, for example, ionic binding, electrostatic binding, van der Waals binding, hydrophobic binding, and/or other binding. In some embodiments, electrostatic binding involves binding compatible molecules such as immobilized avidin conjugated to biotin, streptavidin and monomeric avidin, antibody-antigen complexes, ligand-receptor complexes, and/or other linking molecules. may be used to form between the TNF ligand and one or more solid supports.

[109] In some embodiments, the solid support is agarose, sepharose, cellulose, porous glass, silica, acrylamide-derivative polyacrylamide beads, Trisacryl, Sephacryl, Ultrogel® AcA chromatography adsorption. Pall Corporation, azlactone beads, methacrylate derivatives, TSKgel (registered trademark) chromatography gel (Tosoh Corporation), TOYOPEARL (registered trademark) HW polymer gel (Tosoh Corporation), HEMA (2- hydroxymethacrylate), poly(2-hydroxymethacrylate), Eupergit, polystyrene and its derivatives, Poros, polyethersulfone, polysaccharides, polytetrafluoroethylene, polysulfone, polyester, polyvinylidene fluoride, polypropylene, poly(tetrafluoroethylene-co - perfluoro(alkyl vinyl ether)), polycarbonate, polyethylene, glass, polyacrylate, polyacrylamide, poly(azolactone), polystyrene, polylactide, ceramic, nylon, metal, and/or other materials. . In some embodiments, solid supports may be formed by plates, membranes, beads, ceramics, and/or other components.

[110] In some embodiments, the solid support can be, for example, or include beads. In some embodiments, capture support 204 comprises ligands bound to beads. As noted above, in some embodiments, the ligand may be or include an sc (single chain)-TNFα ligand. In some embodiments, the ligand may be or include a dimeric or trimeric form of the sc-TNF ligand. In some embodiments, the ligand is, for example, a single-chain polypeptide (sc-TNF) ligand (monomeric, dimeric, or trimeric) that binds to and efficiently captures sTNF-R from patient plasma. It may be and/or contain a TNF ligand.

[111] Ligands with altered amino acid sequences or conformational changes due to protein purity are believed to cause substantial changes, such as increased or decreased binding affinities. Such mutations in the sequence may improve or reduce the binding efficiency of the polypeptide to sTNF-R, respectively. Impurities in the TNF formulation can lower the amount of TNF used for ligation by reducing the amount of TNF ligated in proportion to the total amount of protein used. These ligands may have a high target affinity or binding affinity for this target portion of the patient's biological material. Such binding affinities are expressed as _KDs (note that lower _KD values indicate higher binding affinities). Typical target affinities of ligands are, for example, greater than about 10 ⁻⁶ K _D , or greater than about 10 ⁻⁷ K _D , or greater than about 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. The affinity of TNF for sTNF-R1 may be, for example, approximately 10 ⁻¹¹ K _D. The affinity of TNF for sTNF-R2 may be, for example, approximately 10 ⁻¹⁰ K _D.

[112] In some embodiments, the sc-TNF ligand comprises an sc-TNFα molecule. In some embodiments, the sc-TNF ligand comprises a TNFα monomer, one or more complexes of TNFα protein, and/or other components. In some embodiments, complexes include dimers, trimers, multimers, muteins, and/or fragments thereof. In some embodiments, the capture support 204 is a sc-TNF protein ligand conjugated to a plurality of agarose beads (eg, in plasma circulating through the system 10, such as shown in FIGS. 1-3). selectively binds to existing sTNF-R).

[113] In some embodiments, production of sc-TNF (and/or other ligands) may be accomplished through various means of genetic engineering and protein expression (eg, Muller, R. et al. (1986) FEBS Lett 197, 99-104; Mori, T. et al. (1994) Gene 144, 289-293; Horwitz, AH et al. (2019) World J Microbiol Biotechnol 35, 27; Ashman, K. et al. (1989) Protein Eng 2, 387-391; Su, X. 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. (see Tang, P. et al. (1996) Biochemistry 35, 8216-8225).

[114] A ligand may have a given density and orientation on a given support (eg, a bead, etc.). Ligands can be covalently linked to beads (and/or other supports), for example, through amine or thiol moieties. Density and orientation may be configured to enhance binding between ligands and target components of bodily fluids. In some embodiments, the ligand may be configured to extend out of the support matrix (eg, a given bead) at the amino (N) or carboxyl (C) terminus for binding accessibility. . In some embodiments, a linker is used (for example) between the bead surface and the ligand to extend the ligand into passing body fluids as a means of reducing steric hindrance that may interfere with binding. may be provided.

[115] Density can be expressed as mg of ligand per mg of support. In some embodiments, the ligand may be a 54K _D protein. By way of example and not limitation, the support may contain, for example, at least about 0.1 mg (1.8×10 ⁻⁶ mmol) ligand/mg support (eg, beads), at least about 1 mg (18×10 ⁻⁶ mmol) ligand/ mg support (eg, beads), at least about 5 mg ligand/mg support (eg, beads), at least about 7 mg (1.3×10 ⁻⁴ mmol) ligand/mg support (eg, beads), at least about 10 mg ligand/mg Support (eg, beads), may have a ligand density of at least about 15 mg ligand/mg support (eg, beads), or at least about 20 mg ligand/mg support (eg, beads), or higher.

[116] The size, number, density, and/or concentration of supports, such as beads (e.g., in combination with the shape and size of housing 16), may be selected to enhance binding of ligands to target components in bodily fluids. It may be configured to facilitate laminar flow of blood, plasma, and/or other bodily fluids through the beads. An increase in bead size will accommodate proportionally higher flow rates. Increasing the density of the linked ligands can increase the capture capacity while avoiding concentrations that can contribute to steric hindrance that can interfere with effective binding of the target molecule. The size, number, density, orientation, and/or concentration of supports (e.g., beads) can effectively balance the trade-off between, for example, capture rate and clinically practical procedure time. can facilitate the flow rate of For example, for a plasmapheresis procedure involving one embodiment of system 10, a patient plasma volume is passed through isolation chamber 202 to achieve a specific target concentration reduction of sTNF-R1/R2 from the patient plasma. may need to be cycled twice. On the other hand, an alternative embodiment with twice the capture rate efficiency of the first embodiment achieves a similar reduction in sTNF-R1/R2 concentrations, thereby nearly halving the clinical procedure time. To reduce, it is only necessary to circulate one patient plasma volume through the isolation chamber. In some embodiments, beads may comprise one or more different bead materials, such as commercially available agarose or polyacrylamide compositions.

[117] In some embodiments, the beads and/or other solid supports have a size that prevents them from passing through filters 212 and 214 (see discussion of filter pore size herein). You may In some embodiments, the beads may be quenched with ethanolamine or ethylenediamine (if the remaining binding sites after ligation of TNF are saturated in their occupancy) to enhance binding specificity. . Ethanolamine, for example, is used as a quenching agent because of its biocompatibility profile. In some embodiments, to improve control and maximize recovery (or for other reasons), the beads may be pretreated with agents such as Immuron, polystyrene, or polyethylene, and/or the beads may be It may be pretreated with a commercially available cross-linking agent. A cross-linking agent can be any chemical used to facilitate binding of a molecule that captures one or more circulating immune complexes to a solid phase. Non-limiting examples of commercially available cross-linking agents include, for example, poly-L-lysine, glutaraldehyde, and/or cyanogen bromide.

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

[119] The beads may be formed from materials that are biocompatible and to which various ligands are covalently linked (eg, as described above) or electrostatically attached. Coupling of the ligands can be accomplished using amine reduction chemistry, cyanogen bromide (CNBr), N-hydroxysuccinimide esters, carbonyldiimidazole, reductive amination, FMP activation, EDC-mediated amide bond formation, organic sulfonyl chlorides, tosyl chloride and tresyl. chloride, divinyl sulfone, azlactone, cyanuric chloride (trichloro-s-triazine), sulfhydryl-reactive chemistry, iodoacetyl and bromoacetyl activation methods, maleimide, pyridyl disulfide, divinyl sulfone, epoxy or bisoxirane, TNF-thiol, carbonyl reaction chemistry, hydrazides, reductive amination, hydroxyl-reactive chemistry, cyanuric chloride, active hydrogen-reactive chemistry, diazonium, Mannich condensation, photoreactive cross-linking, and/or other manipulations. good too. Ligation can also be performed using immobilized serum albumin with CNBr activation or periodate activation. In some embodiments, the binding involves non-covalent interactions such as a) immobilized avidin, streptavidin and monomeric avidin conjugated to biotin, b) antibody-antigen complexes, and/or c) ligand-receptor complexes. may be implemented using

[120] In some embodiments, the capture support 204 that binds to the target component is [1−(plasma concentration of sTNF-R at outlet 210/plasma concentration of sTNF-R at inlet 200)]×100 , the capture efficiency, expressed as a percentage, may be at least 10% or more of sTNF-R1 and/or sTNF-R2. In some embodiments, the capture efficiency may be at least 50% or greater. In some embodiments, the capture efficiency may be at least 80% or greater. In some embodiments, the capture efficiency may be at least 90%, 95%, 96%, 97%, 98%, 99%, or greater.

[121] The capture efficiency value takes into account the time-based component as more target agent accumulates and is captured within the column (system 10) and the available binding sites in the capture matrix of the system decrease. (eg capture efficiency over the first 5, 10, 15, 30, 45, 60, 90, 120 minutes or longer of treatment). By way of some non-limiting examples, in some embodiments, at least 80% or more of the sTNF-R protein (sTNF-R1 and/or sTNF-R2) in, for example, a flowing biological sample is about 30 It can become bound to the TNF ligand within the isolation chamber 202 within minutes. In some embodiments, at least 90% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flowing biological sample are induced by TNF ligands in the isolation chamber 202 within about 30 minutes. can become bound to In some embodiments, at least 95% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flowing biological sample are exposed to TNF ligands in the isolation chamber 202 within about 30 minutes. can become bound to In some embodiments, at least 96% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flowing biological sample are soluble in TNF ligand within the isolation chamber 202 within about 30 minutes. can become bound to In some embodiments, at least 97% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flowing biological sample are soluble in TNF ligand within the isolation chamber 202 within about 30 minutes. can become bound to In some embodiments, at least 98% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flowing biological sample are soluble in TNF ligand within the isolation chamber 202 within about 30 minutes. can become bound to In some embodiments, at least 99% or more of the sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in the flowing biological sample are soluble in TNF ligand within the isolation chamber 202 within about 30 minutes. can become bound to

[122] While not wishing to be bound by any theory, the capture efficiency values described herein are consistent with the use of sc-TNF ligands (eg, with extremely high target affinity) as described above, the trimeric form of sc - use of TNF ligand, purity of ligand, density of ligand on beads, binding orientation of ligand on beads, size of beads, number, density and concentration of beads in system 10, capture efficiency and clinical procedure time. the physical size and configuration of the housing 16 that provides laminar flow through the beads, and is used to couple the ligands to the beads (e.g., chemically and/or electrostatically). It may be due to process, sterilization technique and radiation dose, and/or other factors. In other words (again, without wishing to be bound by any theory), one reason for the high efficiency of, for example, the column (system 10) is the high binding efficiency of the ligand coupled with the capture ligand on the bead matrix. It may be due to the large quantity.

[123] In some embodiments, the high binding specificity and/or affinity of the capture support 204 for the target component is such that the only known interaction of the capture ligand is sTNF-R1 and/or sTNF-R1 in plasma. This is probably because it is limited to R2. This binding specificity is determined by the use of the sc-TNF ligands described above, the trimeric form of the sc-TNF ligand, the materials used to form (eg, the chemical composition of) the beads and/or the particular bead matrix, the quenching of the beads. may be due to the use of ethanolamine (eg, used to reduce non-specific binding), optimization of target protein binding versus non-specific binding, pore size used for filters 212 and 214, and/or other factors.

[124] In some embodiments, the binding affinity of the capture support for at least one target component 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 component is at least about 10 ⁻⁶ K _D. In some embodiments, the binding affinity of the capture support for at least one target component is at least about 10 ⁻⁷ K _D. In some embodiments, the binding affinity of the capture support for at least one target component is at least about ^{10-8 KD} _. In some embodiments, the binding affinity of the capture support for at least one target component is at least about 10 ⁻⁹ K _D. In some embodiments, the binding affinity of the capture support for at least one target component is at least about ^{10-10 KD} _. In some embodiments, the binding affinity of the capture support for at least one target component is at least about ^{10-11 KD} _. In some embodiments, the binding affinity of the capture support for at least one target component is at least about ^{10-12 KD} _. In some embodiments, the binding affinity of the capture support for at least one target component is at least about ^{10-13 KD} _. In some embodiments, the binding affinity of the capture support for at least one target component is at least about ^{10-14 KD} _.

[125] In some embodiments, the system 10 may be configured to have a TNF elution rate that is less than 1/1000 of the maximum tolerated daily dose (MTD) limit (e.g. Goossens, V. et al. (1995)). ) Proc. Natl Acad. Sci. USA, 92, pages 8115-8119). In some embodiments, system 10 may be configured to have a dissolution rate less than 10000 times the MTD dose limit. In some embodiments, system 10 may be configured to have a dissolution rate that is less than 500 times less than the daily limit MTD. In some embodiments, system 10 may be configured to have a dissolution rate less than 100 times less than the daily limit MTD. This is not biased by clinical effects and/or side reactions due to escape (i.e., elution) of some of the capture support 204 (e.g., ligands described herein) into the patient's circulatory system, Clinical efficacy of system 10 can be assured, including successful, efficient and specific capture of one or more target components.

[126] The elution rate is the total volume of capture supports 204 contained within the isolated chamber 202 of the system 10 after production (e.g., in units of ng/mL/min) of capture supports 204 escaping from system 10. ) can be defined as a percentage. For example, in some embodiments, system 10 can have an elution rate of less than about 150 ng/mL/min. In some embodiments, system 10 can have an elution rate of less than about 100 ng/mL/min. In some embodiments, system 10 can have an elution rate of less than about 80 ng/mL/min. In some embodiments, system 10 can have an elution rate of less than about 50 ng/mL/min. In some embodiments, system 10 can have an elution rate of less than about 40 ng/mL/min. In some embodiments, system 10 can have an elution rate of less than about 30 ng/mL/min. In some embodiments, system 10 can have an elution rate of less than about 20 ng/mL/min. In some embodiments, system 10 can have an elution rate of less than about 10 ng/mL/min.

[127] The elution rate is determined by the strength and integrity of the binding (e.g., chemical and/or electrostatic) between the sc-TNF ligand and the bead, the use of reductive amination chemistry with certain ligands, trimer form of sc-TNF ligand, the process (e.g., chemical and/or electrostatic) used to link the ligand to the beads, clinical pretreatment approaches to prepare the system 10 for patient treatment ( flush volumes and flow rates prior to use), clinically practical flow rates through the system 10 to balance capture efficiency and clinical procedure time, cleaning of the housing 16 during manufacture, sterilization techniques and radiation used in production. amount, and/or other factors. In some embodiments, clinical pretreatment preparation (eg, pre-use flush volume and flow rate) of system 10 includes flushing with 1 liter of normal saline at a flow rate of about 100 mL/min.

[128] By way of non-limiting example, Figures 4 and 5 show target components 402 (sTNF-R1) and or housing 16 that binds to a second target component 403 sTNF-R2 (these are merely examples, more target components are possible) to capture target components 402 and 403 in isolation chamber 202; capture support 204 (eg, bead 400 in this example) in isolation chamber 202 of FIG. FIG. 5 is an enlarged view of the bead 400 (eg, part of the capture support) shown in FIG. FIG. 5 shows the direction 500 of body fluid (eg plasma 404) passing through system 10 (FIGS. 1-3) from inlet 200 to outlet 210 (FIGS. 1-3). In some embodiments, the system 10 may be symmetrical, with either end of the device being bi-directional so that the clinical user can choose to use either end of the device to act as an upflow inlet or a downflow outlet. direction may be used.

[129] As shown in Figures 4 and 5, capture occurs when there is contact between the capture support 204 and the bodily fluid (plasma 404). Specifically, the capture is between sTNF-R1 and sTNF-R2 molecules 402, 403 in plasma 404 and ligands (eg, the sc-TNF ligands described above) 410 bound (linked) to beads 400. Occurs in response to close and/or direct contact. Binding 412 between ligand 410 and bead 400 is also shown. A capture support 204 (eg, a combination of beads 400 and ligands 410) binds one or more target moieties 402, 403 (sTNF-R1 and sTNF-R2 molecules) to one in a body fluid (eg, plasma 404). Or it may be configured to reduce the amount of multiple target components 402 , 403 .

[130] In some embodiments, once the processed sTNF-R deficient plasma passes through outlet 210 (FIGS. 2 and 3), some or all of the processed sTNF-R depleted sample (eg, FIG. 1) can be reinfused into the patient. Depletion of sTNF-R reduces the pool of unconjugated sTNF-R in the patient's blood and increases the availability of TNF to promote anti-cancer effects at the tumor site(s). A shift in the concentration equilibrium towards an increase results.

[131] As described herein, the system 10 (FIGS. 1-3) may be configured to selectively remove immunosuppressive agents associated with immunosuppressive conditions. 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 body fluids results in increased availability of endogenous TNF, which has anticancer effects at the tumor site(s). promote

[132] Returning to FIGS. This access may be segregated from access through entrances 200, exits 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 out of isolation chamber 202 . This may be done, for example, at the time system 10 is manufactured and/or at other times.

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

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

[135] FIG. 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, pump 606, waste line 608, and/or other components. In some embodiments, the cleaning solution 602 and regeneration solution 604 may be alternately pumped through the system 10 by a pump 606 to a waste line 608 .

[136] In some embodiments, the system 10 facilitates sampling or removal of all or a portion of the captured target component (e.g., without damaging the extracorporeal closed circuit column formed by the system 10). A target component exit port configured to.

[137] In some embodiments, the system 10 may be configured to facilitate reuse by interchanging the capture and dissociation steps. By dissociating the target prior to further capture, the column can be regenerated to near original specifications and function. To accomplish this dissociation, the captured complex between the bead-bound protein and its ligand is disrupted, resulting in dissociation of the complex. As such dissociation stages may be application dependent, specific dissociation conditions may depend on the particular subtype of circulating protein or the complexing moiety captured. Agents such as hydrophilizing agents or low pH that induce high salt concentrations can be effective dissociation agents. High salt concentrations of hydrophilizing agents such as sodium chloride (300 mM-1.5 M) or guanidine hydrochloride (3-8 M) are used to dissociate the capture complex. In some embodiments, the salt solution has a pH of approximately 7.2 and is 500 mM NaOH, 2 mM EDTA, and 50 mM Tris buffer or 500 mM NaOH, 2 mM EDTA, and 50 mM phosphate. May contain sodium. Alternatively, captured immune complexes can be dissociated with a low pH solution. An effective pH for the dissociation stage may be, for example, approximately 2.8. An exemplary pH range may be 1.5-2.5. This can be accomplished with pH adjusted citrate or glycine solutions. In some embodiments, the pH range may be 2.0-2.5. In some embodiments, the pH range may be about 2.5-3.5. However, it should be understood that a lower pH will require less dissociation time. Acids such as, for example, acetic acid, citric acid, and hydrochloric acid may be used to lower the pH. In addition to increasing salt concentration or decreasing pH, other methods of dissociating immune complexes are possible. In addition to increasing salt concentration or decreasing pH, the dissociation conditions are configured to occur for a short period of time and to contain bovine serum albumin (BSA) and/or other components that compete with the ligand or receptor. may

[138] In some embodiments, the system 10 includes an elution agent configured to facilitate introduction of an eluant into the sequestration chamber (eg, without compromising the extracorporeal closed circuit column formed by the system 10). A drug port may be provided. The eluent port may further be configured to receive a conditioning agent configured to prepare system 10 for reuse. In some embodiments, the eluent port(s) may be identical to one or both of access ports 206 and/or 208 .

[139] In some embodiments, the pump 606 is configured to drive the elution/regenerate agent through the inlet 200 (Fig. 2), the isolation chamber 202 (Fig. 2), and the outlet 210 (Fig. 2). You may Pump 606 may include syringe pumps, peristaltic pumps, piston pumps, diaphragm pumps, combinations thereof, and/or other pumps.

[140] In some embodiments, the system 10 may include one or more additional isolation chambers 202, as shown in FIG. FIG. 7 illustrates two exemplary embodiments 700 and 750 of system 10 with additional isolation chambers 202 . In exemplary embodiment 700 , isolation chambers 202 A and 202 B are located in series within housing 16 . In exemplary embodiment 750 , chambers 202 C, 202 D, and 202 E are positioned side-by-side within housing 16 . Different chambers (eg, 202A and B, and 202C-E) may be separated by filters 212, 214 (and/or other filters), separation membrane 710, and/or other components. In some embodiments, separation membrane 710 may be, for example, filters 212 and/or 214 and vice versa.

[141] These additional sequestration chambers 202A-E may comprise capture supports similar and/or identical to capture support 204 (FIG. 2) described above, or different capture supports. Additional isolation chambers 202 (AE) 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) may be combined to form a multi-stage separation circuit configured to bind multiple different target components ( eg 202A and B, and 202C, D and E). In some embodiments, multiple isolation chambers may be configured in a series configuration (e.g., embodiment 700), where the total volume of bodily fluid (e.g., plasma) flowing through system 10 is divided into multiple isolation chambers. Each of the chambers is passed sequentially (eg 202A then 202B). In some embodiments, multiple isolation chambers may be configured in a parallel configuration (e.g., embodiment 750), where the volume of bodily fluid (e.g., plasma) flowing through system 10 is evenly or unequally fractionated. , whereby these fractionated sub-volumes of fluid pass in parallel through each of a plurality of isolated chambers (eg, 202C, 202D, and 202E).

[142] Figure 8 illustrates additional embodiments 800 and 850 in which multiple systems (eg, multiple systems 10) are configured into a single plasmapheresis flow circuit 802 or 852 simultaneously during a treatment procedure. do. In one embodiment (eg 800), multiple systems (10) are connected in series, eg plasma flows through one system (10) to the next. In another embodiment (e.g., 850), two or more systems (10) are configured in parallel and the volume of bodily fluid (e.g., plasma) flowing through the plasmapheresis flow circuit is divided into equal or unequal fractions, Fractionated sub-volumes of these fluids may thereby pass through each of the multiple systems in parallel (eg, two systems 10 are shown in embodiment 850, but this is intended to be limiting). not). In one embodiment involving such a parallel system configuration, the entire volume of fluid in the plasmapheresis flow circuit is first directed through one or more of the parallel system isolation chambers (eg, 202 above). and then at subsequent times during the same treatment procedure, the fluid in the circuit is redirected through different systems, or through different combinations of parallel systems coupled in the flow circuit. can be subdivided. In any of the above configurations, each individual system may contain the same or different capture matrices targeting the same or different target agents in bodily fluids.

[143] FIG. 9 illustrates an inlet 200 for introducing fluid into the system 10, each independent flow path from the inlet 200 of the system 10 to an outlet 210 (which may be formed by male luer ports, for example). 903-909), each with its own flow control valves 910, 912, 914, 916 (eg, stopcocks and/or other components) that can individually turn the isolation chamber "on" or "off." 9 illustrates an embodiment 900 of system 10 comprising an isolation chamber 902 of . In this embodiment, fluid can be directed through any one chamber or any combination of multiple chambers simultaneously. An exemplary flow path 950 is shown at the bottom of FIG. Fluids exiting one or more chambers used at a time are recombined at system fluid outlet 210 for return to the apheresis machine. In this embodiment, each of the isolation chambers 902-908 is configured to target one or more different target moieties in bodily fluids (eg, included in capture support 204 described above). of different capture molecules. As shown in FIG. 10, multiple isolation chambers 902-908 and their corresponding flow control valves 910-916 may be contained within a single unified outer housing (eg, 16 described above). .

[144] Figure 10 illustrates a method 1000 for removing target components in blood, plasma, and/or other bodily fluids by the system 10 (Figures 1-3). The operations of method 1000 presented below are intended to be illustrative. In some embodiments, method 1000 may be accomplished with one or more additional operations not described and/or without one or more of the discussed operations. Additionally, the order in which the operations of method 1000 are illustrated in FIG. 10 and described below is not intended to be limiting.

[145] In operation 1002, blood, plasma, and/or other bodily fluids are passed from a patient (eg, patient 14 shown in FIG. 1) through portal 200 (FIGS. 2-3) into isolation chamber 202 (FIGS. 2-3). led to.

[146] At operation 1004, the target component in the blood, plasma, and/or other body fluid is captured in the isolated chamber 202 (FIGS. 2-3) to cause the target component in the blood, plasma, and/or other body fluid to can be conjugated to reduce the amount of the target component of

[147] At operation 1006, the bodily fluid having the reduced amount of the target component passes from the isolation chamber 202 (FIGS. 2-3) through the outlet 210 (FIGS. 2-3) for reintroduction back to the patient. .

[148] In operation 1008, the reduced amount of the target component in the bodily fluid is reintroduced back into the patient, and this amount may or may not be measured quantitatively (e.g., operation 1008 is optional may be). In some embodiments where quantitative measurements are used, the measurements include liquid chromatography-mass spectroscopy (LC-MS), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), resistance measurements, 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 computer One or more of performing tomography (SPECT), enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) measurements, and/or other manipulations may be included.

[149] Operation 1010 may or may not measure the elution rate of the capture support in the bodily fluid that is reintroduced back into the patient (eg, operation 1010 may be optional). Concentrations of capture agents (eg, TNF) may be determined from fluid samples taken from inlet 200 and outlet 210 and/or from fluid connectors attached thereto. The difference between the two measured concentration values may be used to determine the rate and amount of scavenger escaping from the isolation chamber. In some embodiments, the flow rate in mL/min is used to determine the total amount of sequestrant that elutes from the system into the patient's circulation over a period of time (e.g., the duration of a single treatment procedure). good too.

[150] For purposes of explanation, the system(s) or method(s) of the present disclosure will be described in detail based on what is presently believed to be the most practical and preferred implementation. Although described, such details are for that purpose only, and the disclosure is not limited to the disclosed implementations, but rather may include modifications and equivalent arrangements that fall within the spirit and scope of the appended claims. It should be understood that it is intended to be inclusive. For example, it is to be understood that this disclosure can combine one or more features of any implementation with one or more features of any other implementation to the extent possible.

[151] The examples attached below provide various demonstrations of the successful use of various embodiments of the systems and methods described herein.

Example 1 - Materials and Assembly of the System
[152] Materials - The immobilized solid support matrix (capture support 204) utilized in this embodiment of the system was agarose-based beads (ie, strongly cross-linked support resin).

[153] Chemistry—Sodium metaperiodate was used to activate the solid support matrix (capture support 204), which was then coupled to single-chain TNF ligands by reductive amination. TNF was ligated in an amount of 1 mg per mL of solid support matrix.

[154] The assembly-binding matrix (capture support 204) comprises single chain TNF (SC-TNF) proteins covalently linked to agarose beads. The device housing consists of a polycarbonate tube capped with a non-vented luer cap with two side ports for filling. The end caps have male luer ports and are rigidly attached to the tubing to create a fluidly sealed housing terminating at each end by end cap luer ports. Inside the housing at the junction between each endcap and the tube is a filter frit with an average pore size of 15-50 μm to hold the TNF ligand-linked beads, the minimum diameter of the filter frit being (e.g. ) larger than the pore size of the filter.

Example 2 - Parameters for Utilizing the System and Method
[155]1. In configurations where pre-column as well as post-column plasma concentration measurements are made to determine capture efficiency and/or elution, which are not necessarily routine clinical applications, a 3-way stop is used to allow periodic plasma sampling. Attach cock valves to the inlet and outlet of the column end caps. 2. The system fitted with a stopcock is connected to the plasma circuit of the apheresis machine. 3. To complete steps 4, 6, and 10 below, configure the apheresis unit with procedure-independent and/or procedure-specific operating parameters according to the apheresis machine's instructions. 4. Prior to connecting the patient to the apheresis machine, flush the system with 1 L of normal saline. 5. Connect the patient to the apheresis machine. 6. A patient is treated with the system. 7. Blood and plasma samples are collected according to the study protocol and/or clinical treatment protocol, if applicable. 8. The patient's vital signs are monitored continuously according to typical apheresis procedure practice guidelines. 9. Patients are continuously monitored for adverse events before, during, and after treatment. 10. Flush/wash the device with normal saline after treatment and recap the column with the retained end cap to ensure proper storage and/or disposal of used devices.

[156] The columns of the present invention are intended for use with an apheresis device, such as the Terumo BCT or Spectra Optia System, which is designed to be compatible with a plasma processing column such as system 10. Such systems automate calculations based on patient data and device parameters configured by an operator. If the apheresis machine does not automate such calculations, the formulas identified in the table below may be used.

[157]

[158]

[159]

[160] Intended Clinical Performance—An immunopheresis column (eg, System 10) is used for efficient removal of sTNF-R from patient plasma (eg, such as by System 10) using centrifugation or membrane separation techniques. A device designed to be successfully integrated with commercial apheresis machines that allow for secondary plasma processing. The system disclosed herein meets the clinical performance requirements described below. The specified treatment times and flow rates have been found to be within the capabilities of modern apheresis systems using centrifugation techniques.

[161] Standards for system performance-1. Biological Safety: Demonstrated to be biologically safe for use as an extracorporeal device supporting long-term exposure to circulating blood. 2. Binding target: sTNF-R (including sTNF-R1 and sTNF-R2). 3. Binding efficiency: greater than 80% after 30 minutes of treatment at clinically relevant flow rates. 4. Binding capacity: greater than 230 μg sTNF-R. 5. Flow rate (plasma): 60 mL/min or less. 6. Processing time: approximately 2 hours. 7. Target plasmapheresis volume: twice the plasma volume. 8. Elution rate (TNFα): less than 50 ng/min. 9. Shelf life: >6 months at 2-8°C. 10. Pressure resistance: 776mmHg.

Example 3 - Efficacy of system 10 based on in vitro parameters
[162] Preclinical Testing - In vitro testing has demonstrated that the system 10 meets the performance and safety specifications defined in "Intended Clinical Performance" (previous section). The TNF-linked binding matrix effectively captured over 98% of sTNF-R in both spiked buffer solutions and human plasma in standard in vitro assays, while the calculated elution rate of sc-TNFα was Maintained below 50 ng/min (equivalent to a maximum of 6 μg for 2 hours). Since the maximum tolerated dose (MTD) of TNFα per day is approximately 200 μg, this is well below acceptable safety limits (eg Goossens, V. et al. (1995) Proc. Natl Acad. Sci. See page 8119). These performance and safety measures were reproduced after the beads were sterilized by electron beam irradiation (15-30 kGy). Further, in this embodiment, system 10 is designed for sustained flow rates of less than 300 mL/min, 10-44 mL/min, and/or 45-100 mL/min. The housing (eg, 16 shown in FIGS. 2 and 3) can withstand internal pressures in excess of 776 mmHg, well above the typical back pressure shutoff threshold for commonly used apheresis devices.

[163] Binding matrix (e.g., capture support 204) characterization - TNF ligand identification, purity, and integrity - Recombinant single-chain TNFα ligand (sc-TNFα ligand, containing three TNFα monomers) was used. TNFα was characterized by assessing purity by high performance liquid chromatography (HPLC), integrity and identity by mass spectroscopy, and potency of functionality by flow test binding assay. HPLC was performed using a column with a phosphate buffer as a mobile phase and a flow rate of 1 mL/min. Integrity was determined by mass spectroscopy, which confirmed the calculated molecular weight of approximately 54 kD.

[164] Binding Efficiency of Binding Matrix in sTNF-R Supplemented Buffer - For a standard flow test, a bead bed volume of 2 mL was obtained from an irradiated column of the invention and transferred to a small column, and sTNF-R1 Alternatively, the capture efficiency from phosphate buffer supplemented with sTNF-R2 was tested. Buffer (20 ng/mL) spiked with TNF-R1 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 taken at 0.5 minute intervals and assayed for sTNF-R. The result was a capture efficiency of over 98% for all samples. Assays for TNFα showed its release to be less than 7 ng/min.

[165] Binding Matrix Capture Capacity in Human Plasma Spiked with sTNF-R—To test the capacity of system 10, the present invention was tested by flowing 1.8 liters of spiked human plasma through the device at a flow rate of 45 mL/min. Inventive columns were tested. Human plasma used in each run was spiked with high concentrations of each receptor (sTNF-R1 9.74 ng/mL and sTNF-R2 127.5 ng/mL). Samples of post-column plasma were taken every 10 minutes and analyzed for sTNF-R concentration using a commercial high accuracy sTNF-R1/sTNF-R2 diagnostic kit. The amount of sTNF-R retained on the column was calculated to confirm capture efficiency and total capacity.

[166] The binding efficiency at each time point was calculated by comparing the pre- and post-column concentrations of sTNF-R. The binding matrix captured over 85% of sTNF-R1 and sTNF-R2. The binding efficiency was over 95% for sTNF-R1 by the end and a lower efficiency was observed for sTNF-R2, although its capture remained over 85%. The slight linear decline in binding efficiency for sTNF-R2 is probably due to the low binding affinity of this soluble receptor for the TNFα ligands used.

[167] The total amount of sTNF-R trapped on the column was determined by comparing the concentrations 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. Both of these values exceed the amount of sTNF-R of approximately 30 μg typically present in cancer patients. Running at 15 ml/min, the total amount of TNF-R captured was 99.9 μg at 80 minutes, at which point column efficiency began to decline. This is well above the typical amount still present in cancer patients.

[168] Elution of TNFα from the Devices of the Present Invention—To avoid the possibility of injecting a pharmacologically significant amount into the patient, the system 10 is designed to prevent the unintentional release of TNFα from the system 10 during its use (i.e., , “elution”). The release of TNF from the beads was determined during the flow test run. Approximately 20 ng/mL of sTNF-R1 was added to phosphate buffered saline and passed through a 2 mL bead bed obtained from the column of System 10 (FIGS. 1-3). Flow rates were performed sequentially as 10 mL/min, 5 mL/min, 2.5 ml/min, 5 mL/min, and 10 mL/min for 30 seconds for each flow rate. Samples were analyzed using a commercially available high precision TNF diagnostic kit. The elution rate of TNF in ng/min was calculated by multiplying the concentration of TNF in the sample (ng/mL) by the flow rate (mL/min). The amount released in the final fraction taken at 10 mL/min was 0.65 ng/min per 2 mL of beads, or 0.325 ng/min/mL (0.65 ng/min/2 mL). In this example, the upper limit of bead bed volume within the 50 ng/min specification is (50 ng/min)/(0.325 ng/min/mL), or 154 mL of beads.

[169] Device Housing Integrity—Tests were performed on empty housings (eg, 16) of the present invention to confirm that system 10 maintains integrity at elevated internal pressures. 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 to apply a pressure of 776 mmHg and observed for bubble generation. No device failure (presence of bubbles leaking from the submerged device) was observed. This test pressure exceeds the maximum pressure of the Optia unit (500 mmHg).

Example 4 - Demonstration of Efficacy in a Canine Model
[170] The effect of ex vivo ablation of sTNF-R using System 10 from dogs with naturally occurring solid malignancies or melanoma (stage 4) was evaluated in a proof-of-concept comparative oncology study. The study used system 10 with the sc-TNFα peptide-bead matrix. System 10 was used in conjunction with the Terumo BCT Spectra Optia Apheresis System for secondary plasma processing through system 10 . Dual lumen catheters are employed in most dogs for vascular access, and extracorporeal anticoagulation is achieved with citrate dextrose anticoagulant (ACDA) fluids and calcium supplementation, as recommended by the Spectra Optia user operating manual. It was retrograded by injection of gluconate. A total of 20 dogs were treated. Patient dogs received 12-24 apheresis treatments over the course of the 4-8 week treatment phase of the study. Over 300 immunopheresis procedures were performed during the study.

[171] Device performance - Pre- and post-column plasma (collected from the inlet and outlet ports of the device, respectively) every 30 minutes during the procedure to confirm the in vivo safety and performance of the device. TNF and sTNF-R were measured, and systemic (from blood) TNF and sTNF-R was measured.

[172] Pre- and post-column plasma analysis (of system 10) during each treatment resulted in efficient and consistent removal of sTNF-R over the course of each individual treatment, It was shown that there was no measurable increase in TNF levels. Overall, pre- and post-treatment plasma analysis showed an almost 50% reduction in systemic sTNF-R levels.

[173] Device Safety and Clinical Efficacy—To assess the clinical safety and efficacy of the device, safety, tolerability, and impact on tumor progression measurements were taken throughout the study.

[174] During the course of more than 300 immunopheresis procedures performed in a total of 20 dogs, there were few reports of serious adverse events, attributed to the specific apheresis procedure or by System 10. There were no adverse events as a result of treatment or removal of sTNF-R. Tolerability of the treatment is best described by looking at patient dog QoL data. Change in QoL was scored as 'neutral' overall for most scores throughout the active treatment phase of the study, which included 12-24 treatments per patient dog. Although some scores worsened, most scores showed stable parameters or improvement throughout the course of treatment, representing a favorable outcome for the Stage 4 patient dog population in this study.

[175] Treatment with the device of the present invention had an overall beneficial effect on tumor progression. Most dogs (12/17 evaluable) were scored with 'stable disease' (SD) at some point during treatment (data not shown), and of 17 evaluable dogs Seven dogs showed favorable treatment-related effects at the end of the treatment phase and one had a complete response (CR).

[176] Conclusions - The dog's overall condition improved during the study with an observable stabilization or reduction in tumor burden and improvement in quality of life. In addition, the lack of clinically significant safety issues presents the potential to provide clinically meaningful benefits without the typical and significant side effects associated with conventional chemotherapy and irradiation, and is therefore generally Consistent with and convincing with the established relative safety of therapeutic apheresis procedures. This canine companion animal study demonstrates that in vitro depletion of sTNF-R utilizing the Apheresis Immunoadsorption Affinity Column of the present invention comprising a sc-TNF peptide-bead matrix is therapeutically effective in dogs with cancer, We have provided compelling clinical evidence that the use of such devices can be employed in human subjects.

Example 5 - Biocompatibility
[177] The system 10 was evaluated for biological safety according to the Food & Drug Administration's (FDA) Biocompatibility Testing Matrix and International Standard ISO 10993-1 (2009). The system 10 can be classified as a circulating extracorporeal conduction device with a long contact time. This is the same test category utilized by many other commercially available extracorporeal immunoadsorption columns.

[178] Biocompatibility studies were performed in accordance with 21 CFR Part 58 (Good Laboratory Practice for Nonclinical Laboratory Studies). All testing was performed on sterilized final devices by an accredited independent laboratory. Based on the tests performed, the device meets the recommendations and principles contained within the ISO 10993-1 (2012) consensus standard "Biological evaluation of medical devices-Part 1: Evaluation and testing within a risk management process" and 6 Conforms to the FDA's accompanying guidance document issued May 16.

Example 6 - Demonstration of Safety in Human Subjects
[179] To collect experimental safety and performance data for system 10, a first-in-human ad hoc clinical study was conducted. The device was used in combination with the Terumo BCT Spectra Optia System with secondary plasma processing through the system 10 as done in the companion canine comparative oncology study (described above). A dual lumen catheter was employed for vascular access based on experience from studies in dogs. Extracorporeal anticoagulation (ACDA solution) and retrograde (by infusion of calcium gluconate) were similar to those employed in canine studies and were as recommended in the Spectra Optia user operating manual.

[180] Apheresis Procedure Performance - System 10 has been successfully utilized with a Terumo BCT Spectra Optia apheresis machine. For each procedure performed, the system 10 was successfully integrated into the extracorporeal plasma circuit. No device-attributable interference in secondary plasma processing circuits (eg, circuits coupled to system 10) was reported. System circuit integrity was consistently maintained (eg, no leaks/fluid loss reported) and all procedures could be completed successfully. Based on this overall data, the device of the present invention appears suitable for use with the Terumo BCT Spectra Optia.

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

[182] Figure 11 depicts performance characteristics of a representative system 10 (Figures 1-3), including reduction of sTNF-R1 and sTNF-R2 from a human patient blood pool as a function of procedure time and column capture efficiency. is illustrated. FIG. 11 is an illustration of typical system 10 performance results from the treatment of a single human patient. Whole blood and plasma samples were collected at baseline (T=0 min), 30 min, 60 min, 90 min, and at the end of treatment (i.e., when twice the plasma volume had completed circulation of the system), which was In this example, it occurred 175 minutes after the start time of the procedure). At each time point, whole blood was drawn from the patient's midline catheter and plasma samples were drawn from the inlet (eg, 200 shown in FIGS. 2-3) and the outlet (eg, 210 shown in FIGS. 2-3) of the system 10 . In addition, whole blood samples were taken at 30 and 60 minutes after treatment. The concentrations of sTNF-R1 and sTNF-R2 in the samples were analyzed using commercially available high-precision diagnostic assays. FIG. 11 shows the concentrations of sTNF-R1 and sTNF-R2 at the corresponding inlet 200 and outlet 210 at each time point, indicating that system 10 effectively captured sTNF-R1 and sTNF-R2 throughout the course of treatment. have demonstrated that Furthermore, a steady decrease in sTNF-R1 and sTNF-R2 concentrations over time observed in the patient's entire circulatory system (i.e., midline whole blood measurements) and subsequent rebound at 30 and 60 minutes post-treatment The levels indicate that the clinical goal of reducing endogenous levels of sTNF-R1 and sTNF-R2 during the treatment period was effectively achieved.

Claims (15)

A system for removing a target component from a patient's bodily fluid, comprising:
housing,
an inlet coupled to the housing and configured to receive the bodily fluid from a patient;
an isolation chamber disposed within the housing and configured to receive the bodily fluid from the inlet;
a capture support disposed within the isolation chamber and configured to bind to the target component;
an access port coupled to the housing and configured to insert and/or remove the capture support into and /or from the isolation chamber;
an outlet coupled to the housing and configured to pass the bodily fluid from the isolation chamber for reintroduction of some or all of the bodily fluid back to the patient; A system comprising a filter configured to separate said capture support in a chamber from said inlet and said outlet. 2. The system of claim 1, wherein the capture efficiency of said capture support that binds said target component is at least 80%. 3. The system of claim 2, wherein said capture efficiency is provided at a flow rate of 45 ml/min of said bodily fluid. 2. The system of claim 1, wherein the binding affinity of said capture support for said target component is greater than ^{10-7 KD} _. 2. The system of claim 1, wherein the elution rate of said capture support through said outlet is less than 100 ng/mL/min. 3. The system of claim 1, further comprising an eluant port configured to introduce eluant into the isolation chamber. 2. The system of claim 1, wherein said targeting component comprises a tumor necrosis factor (TNF) receptor. 2. The system of claim 1, wherein said capture support comprises fragments of TNF. 2. The system of claim 1, wherein the capture support comprises TNF[alpha], multimers of TNF[alpha], single-chain TNF[alpha], fragments of TNF[alpha], multimers of fragments of TNF[alpha], or combinations thereof. said capture support comprising ligands bound to beads, said ligands having a given density and orientation on a given bead ;
said given density is at least 0.1 mg ligand/mg support; and
2. The system of claim 1 , wherein the orientation is such that the ligand binds to the bead at the amino (N) or carboxyl (C) terminus of the ligand and the ligand extends out of the bead. . said capture support comprising ligands bound to beads, said ligands having a given density and orientation on a given bead;
the given density is at least 1.8×10 ^-6 millimoles ligand/mg support, and
2. The system of claim 1, wherein the orientation is such that the ligand binds to the bead at the amino (N) or carboxyl (C) terminus of the ligand and the ligand extends out of the bead. . The system according to claim 10 or 11 , wherein the size of said beads is 20-1,000 μm in diameter . The system of any one of claims 10-12, wherein said beads are quenched in ethanolamine to enhance binding specificity. 3. The system of claim 1, further comprising a target component exit port coupled to said housing configured to sample or remove all or a portion of captured target components without damaging said housing. . 2. The system of claim 1, further comprising an eluant port coupled to the housing configured to introduce eluant into the sequestration chamber without damaging the housing.

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