Products and methods relating to transplant recipient antibodies bound to donor cell membrane fragments

Abstract

The present disclosure relates to products and methods relating to transplant recipient antibodies bound to donor cell membrane fragments. Products and methods provided are used to separate and collect transplant recipient antibodies from donor cell membrane fragments, as well as to characterize the transplant recipient antibodies. The products and methods are useful for allotransplant or xenotransplant recipients.

Description

PRODUCTS AND METHODS RELATING TO TRANSPLANT RECIPIENT ANTIBODIES BOUND TO DONOR CELL MEMBRANE FRAGMENTSField

[0001] The present disclosure relates to products and methods relating to transplant recipient antibodies bound to donor cell membrane fragments. Products and methods provided are used to separate and collect transplant recipient antibodies from donor cell membrane fragments, as well as to characterize the transplant recipient antibodies. The products and methods are useful for allotransplant or xenotransplant recipients.Background

[0002] Transplant rejection occurs when the immune system of the recipient of a transplant, particularly antibody produced by the recipient, attacks the donor cells. The recipient's immune system recognizes the transplant as foreign tissue and attempts to destroy the transplant.

[0003] Recipient antibodies bind to donor cells. Because of this binding, the recipient antibodies initiate damaging immune responses in the recipient by activating the complement cascade or by marking donor cells for destruction by natural killer cells and macrophages among others.

[0004] Allotransplantation is the transplantation from one individual to another individual of the same species but a different genotype. The transplant in allotransplantation is called an allograft. Xenotransplantation is the transplantation from one species to another species and the transplant is called a xenograft.

[0005] In allotransplantation, there can be recipient antibodies that recognize different types of antigens on donor cells such as: (i) carbohydrate-based blood group antigens from the ABO blood groups, (ii) various protein-based endothelial antigens, and (iii) the highly polymorphic proteins known as human leukocyte antigens (HLA).

[0006] Antibody interaction with blood groups has been detected by agglutination of donor red blood cells after mixing donor red blood cells with recipient serum, or monoclonal antibodies known to recognize ABO carbohydrates. Antibodies towards endothelial antigens have been detected by incubating recipient serum with solid phase supports (e.g., beads or ELISA plates) coated with potentially antigenic recombinant proteins. Alternatively, endothelial cells have been used as cell targets of recipient immunoglobulins. Traditionally, donor lymphocytes, which synthesize high levels of HLA molecules, have been incubated with recipient serum to probe for HLA-specific immunoglobulin. More recently, bead arrayshave been developed that display numerous unique HLA alleles to allow probing of recipient serum for IgG towards HLA proteins found in most of the available donor pools. These assays can be used to help detect the de novo production of anti-HLA antibodies which indicate an active organ rejection process.

[0007] Xenotransplant assays have used donor cells (e.g., red blood cells, peripheral blood mononuclear cells, and endothelial cells) as targets to detect recipient antibodies. Molecules from donors, such as swine leukocyte antigens (SLA, the homologs of HLA) have also been expressed in various cell types, including cells that are from the same species as the recipient, as a platform to display these xenoantigens. Donor cells which express xenoantigens are incubated with recipient serum. The donor cells are then evaluated for binding of recipient antibodies.

[0008] There remains a need in the art for products and methods for isolating and characterizing recipient antibodies arising from an immune response to a transplant.Summary

[0009] Humoral immunity can result in many of the same rejection issues in xenotransplantation as in allotransplantation. Monitoring of recipient immune responses after transplant is important for both types. It is contemplated herein that, after allotransplantation and xenotransplantation, donor cell membrane fragments in the transplant recipient bind (“soak up”) transplant recipient antibodies and that the donor cell membrane fragments “hide” the transplant recipient antibodies from assays previously used in the field to monitor the transplant recipient immune response to the transplant. Products and methods provided herein address this problem.

[0010] Products and methods provided herein isolate antibodies from a transplant recipient in the post-transplant period that react with the transplant cell membrane fragments. In particular, methods provided herein isolate recipient antibodies from donor cell membrane fragments obtained from the recipient and separate transplant recipient antibodies from the donor cell membrane fragments. Further, the transplant recipient antibodies may be characterized in the methods.

[0011] Every cell type releases a variety of cell membrane fragments that retain molecules from the cell surface. Several websites exist which contain databases of the protein, nucleic acid, and lipid content of these subcellular particles (microvesicles. org / # and exocarta.org / are examples). These cell membrane fragments are variously referred to as exosomes, microvesicles, ectosomes, and micromeres (plus even more names have been used with each representing a unique characteristic such as size). The cell membranefragments may be obtained from cells in culture as well as from bodily fluids such as urine and serum.

[0012] The disclosure provides methods of collecting transplant recipient antibodies bound to transplant donor cell membrane fragments comprising: a) applying transplant recipient bodily fluid to a chromatography column, b) separating in the chromatography column, donor cell membrane fragments in the bodily fluid from transplant recipient antibodies in the bodily fluid, c) removing the donor cell membrane fragments from the chromatography column, and d) collecting the transplant recipient antibodies remaining in the chromatography column.The separation of step b) may employ a pH gradient, a high salt concentration, a chelating agent or a chaotropic chemical.

[0013] The separation of step b) may employ a pH gradient. The separation of step b) may employ a pH gradient, wherein the pH in the column of steps a) and b) is about pH 2.5 to about pH 5.0. The separation of step b) may employ a pH gradient, wherein the pH in the column of steps a) and b) is about pH 9.0 to about pH 11 .5. The pH may be adjusted in step c) to about pH 6.0 to about pH 8.5.

[0014] The separation of step b) may employ a high salt concentration. The salt concentration may be about 0.5 M to about 2.5 M. The salt may be sodium chloride.

[0015] The separation of step b) may employ a chelating agent. The chelating agent may be ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(P-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA). The chelating agent added may be about 5 mM to about 10 M.

[0016] The separation of step b) may employ a chaotropic chemical. The chaotropic chemical may be urea, guanidinium hydrochloride or arginine. The chaotropic chemical added may be about 10 mM to about 10 M.

[0017] Methods provided herein may further comprise characterizing the antibodies collected in step d) by an assay for binding to at least one antigen. The antigen or antigens may be coated on a solid phase support. Antibody binding to antigen may be detected by fluorescence. The solid phase support may comprise microparticle beads.

[0018] In methods provided herein, the recipient may have received an allotransplant. The allotransplant may be from a human.

[0019] In methods provided herein, the recipient received a xenotransplant. The xenotransplant may be from a pig.

[0020] Kits are provided herein. Kits may comprise one or more of: a chromatography column packed with a porous stationary phase, a solution or solutions for separating in thechromatography column transplant recipient antibodies from donor cell membrane fragments, and an antigen-coated solid phase support.Brief Description of the Drawings

[0021] Fig. 1 shows a schematic of illustrative steps of methods provided herein for collecting transplant recipient antibodies from donor cell membrane fragments and characterizing the transplant recipient antibodies.

[0022] Fig. 2 shows the median fluorescence intensity (MFI) resulting from detection of antibodies collected from cell membrane fragments.Detailed Description

[0023] As referred to in the Background section above, “allotransplantation” is the transplantation from one individual to another of the same species but a different genotype. Allotransplantation may be from a human donor to a human recipient. “Xenotransplantation” is the transplantation from one species to another. Xenotransplantation may be from a pig donor to a human recipient.

[0024] A “transplant” herein may be a cell, fluid, tissue or organ. The transplant may be a temporary or permanent replacement in a recipient in need of the transplant. A transplant in allotransplantation is called an “allograft.” A transplant in xenotransplantation is called an “xenograft.” A transplant may be a human cell, fluid, tissue or organ. A transplant may be a pig cell, fluid, tissue or organ. A transplant may be a genetically engineered pig cell, fluid, tissue or organ. An illustrative cell is a blood cell (e.g., lymphocyte), stem cell, endothelial cell or islet cell. Illustrative fluids are plasma and lymph. Illustrative tissues are blood, cartilage, tendon, ligament. Illustrative organs are a kidney, liver, lung, heart, brain, lung, eye, stomach, pancreas, kidney, uterus, bladder, skin, hair follicle, gland (such as salivary, hypothalamus, pituitary, pylorus, adrenal, mammary, thyroid or thymus), nose, mouth, lip, spleen, teeth, tongue, tonsil, pharynx, esophagus, large intestine, small intestine, small bowel, rectum, anus, bone, cartilage, tendon, ligament, suprarenal capsule, skeletal muscle, smooth muscle, blood vessel, spinal cord, trachea, ureter, urethra, ovary, oviduct, uterus, vagina, testes, seminal vesicles, penis, lymph nodes and lymph vessels. A “transplant antigen” is an antigen of a transplant that is antigenic and immunogenic to a transplant recipient.

[0025] A “transplant donor” is the individual providing a transplant. A transplant donor may be, for example, a human. A transplant donor may be, for example, a pig.

[0026] The term "pig" refers to any pig known to the art including, but not limited to, a wild pig, domestic pig, mini pigs, a Sus scrofa pig, a Sus scrofa domesticus pig, as well as an in-bred pigs or a genetically engineered pig.

[0027] A “genetically engineered” cell or animal is a cell or animal the genome of which has been engineered by genetic engineering techniques, for example, to change the expression level of a gene, to knock out a gene or to add a gene, or combinations thereof.

[0028] The term "knockout" herein relates to an animal or cell in which a given gene has been altered, removed or disrupted such that no active gene transcript is produced and / or no active gene product is produced.

[0029] The terms "non-naturally occurring," “synthetic” or "engineered" as used herein are interchangeable and refer to material that has been created or modified by the hand of man (e.g., a genetically modified animal or cell having one or more predetermined engineered genetic modifications in its genome) or is derived using such material (e.g., a tissue or organ obtained from such genetically modified animal). For example, cells comprising one or more synthetic or engineered nucleic acids are considered herein to be engineered or non-naturally occurring cells. As used herein, the term "engineered tissue" refers to aggregates of engineered / non-naturally occurring modified cells.

[0030] Gene expression may be analyzed by any means known in the art including, but not limited to, RT-PCR, Western blots, Northern blots, microarray analysis, immunoprecipitation, radiological assays, polypeptide purification, spectrophotometric analysis, Coomassie staining of acrylamide gels, ELISAs, 2-D gel electrophoresis, in situ hybridization, chemiluminescence, silver staining, enzymatic assays, ponceau S staining, multiplex RT-PCR, immunohistochemical assays, radioimmunoassay, colorimetric analysis, immunoradiometric assays, positron emission tomography, fluorometric assays, fluorescence activated cell sorting, radioimmunosorbent assays, real-time PCR, hybridization assays, sandwich immunoassays, flow cytometry, SAGE, differential amplification, or electronic analysis.

[0031] A “transplant recipient” is the individual receiving a transplant. In methods provided herein, a transplant recipient may be a human.

[0032] Transplant donor cell membrane fragments are used in methods provided herein as the source of transplant recipient antibodies. Cell membrane fragments from transplant recipients provided herein comprise transplant recipient antibodies bound to their surface. The cell membrane fragments, include, but are not limited to, exosomes, microvesicles, ectosomes, and micromeres. Transplant donor cell membrane fragments may be obtained from, for example, from any transplant recipient bodily fluid that has been in direct orindirect contact with any transplant donor cell, fluid, tissue or organ. As another non-limiting example, transplant donor cell membrane fragments may be isolated from a ground up cell debris from a transplant biopsy.

[0033] In provided methods, a transplant may be an allotransplant. The allotransplant may be from a human.

[0034] In provided methods, a transplant may be a xenotransplant. The xenotransplant may be from a pig.

[0035] Methods of collecting transplant recipient antibodies bound to donor cell membrane fragments from a transplant comprise, for example, a) applying transplant recipient bodily fluid to a chromatography column, b) separating in the chromatography column, donor cell membrane fragments in the bodily fluid from transplant recipient antibodies in the bodily fluid, c) removing the donor cell membrane fragments from the chromatography column, and d) collecting the transplant recipient antibodies remaining in the chromatography column.

[0036] Transplant recipient bodily fluids containing donor cell membrane fragments are the starting material in methods provided herein. Such fluids include, but are not limited to, serum, plasma, urine and blood. Such bodily fluids are obtained from a subject by conventional medical procedures.

[0037] Serum, plasma, urine or blood from a transplant recipient may be used in methods provided herein for detecting transplant recipient antibodies to a transplant that pre-existed in the recipient before the transplantation and / or transplant recipient antibodies that developed post-transplant (e.g., real-time methods).

[0038] Cell membrane fragments from a body fluid may be concentrated before being applied to a chromatography column. Concentration may be by, for example, centrifugation (taking advantage of the physical properties of size or density) or affinity isolation e.g., using an antibody).

[0039] A chromatography column is a column that contains a matrix which can separate biological molecules by a variety of properties including, for example, by size and / or by interactions at the molecular level involving unstable or permanent bonds between solutes, solvents and stationary phases. Illustrative chromatography columns contemplated herein for gel filtration contain, for example, a porous stationary phase that lets cell membrane fragments (larger in size) pass through the chromatography column quickly while temporarily retaining antibodies (smaller in size) for subsequent collection.

[0040] In an illustrative method, donor cell membrane fragment / recipient antibody complexes are separated in a chromatography column using a combination of a pH gradient and gel filtration. See, for example, the schematic shown in Fig. 1 .

[0041] A pH gradient, for example, may be used in a chromatography column herein to dissociate recipient antibodies from donor cell membrane fragments. For example, the pH in the column of steps a) and b) of paragraph

[0027] above may be a low pH of about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0 or about 5.5. The pH in the column of steps a) and b) may be about 2.5 to about 6.0. The pH in the column of steps a) and b) may be about 2.5 to about 5.0. The pH in the column of steps a) and b) may be about 3.0 to about 4.0. The pH in the column of steps a) and b) may be about 3.0. As another example, the pH in the column of steps a) and b) may be a high pH of about 8.5 to about 1 1 .5. The pH in the column of steps a) and b) may be a high pH of about 9.0 to about 1 1 .5. The pH in the column of steps a) and b) may be a high pH of about 9.0, about 9.5, about 10.0, about 10.5, about 1 1 .00 or about 11 .5. The pH in the column may be adjusted in step c) of paragraph

[0027] above to a more neutral pH of about 6.0 to about 8.5. The pH may be adjusted in step c) to about 6.0, about 6.5, about 7.0, about 7.5, about 8.0 or about 8.5. The pH may be adjusted in step c) to about pH 7.5. The pH may be adjusted in step c) to about pH 7.4.

[0042] The present disclosure contemplates that cell membrane fragment / antibody complexes may be separated by varying one or more chromatography column conditions including, but not limited to, pH, salt concentration and presence of other chemicals. Thus, other examples of dissociating transplant recipient antibodies from donor cell membrane fragments in methods provided herein include adding high salt concentrations (such as about 0.5 M to about 2.5 M; such as about 0.5 M, about 1 .0 M, about 1 .5 M, about 2.0 M, or about 2.5 M salt), chelating agents (some antibodies need divalent cations to bind so a chelator of the cations such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol- bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) can dissociate antibodies) or chaotropic chemicals such as urea, guanidinium hydrochloride and arginine. The chelating agents and chaotropic chemicals may typically be used in concentrations ranging from millimolar to molar. The chelating agents may be used in concentrations ranging from about 5 mM to about 10 M. The chaotropic chemicals may be used in concentrations ranging from about 10 mM to about 10 M.

[0043] Various salts may be used for dissociating transplant recipient antibodies from donor cell membrane fragments including, but not limited to, sodium chloride (NaCI). Other particular salts may be chosen, for example, in view of the Hofmeister series of ions which ranks ions in decreasing order of their effect on proteins: for anions, COs2-> SO42-> S20s2“r cations, N(CHs)4+> NH4+>

[0044] Gel filtration utilizes a chromatography column packed with a porous stationary phase. Mixtures of materials (e.g., salts, antibodies, and membranes) are passed through the column in a liquid mobile phase. The stationary phase may be composed of crosslinked agarose, dextrans and acrylamides or other molecules. The size of the pores in the stationary phase preferentially retards the flow of some, but not all components. Appropriate selection of the stationary phase separates donor cell membrane fragments from recipient antibodies which may be further separated, for example, from ions that make up the pH gradient. Examples of stationary phase material are Sepharose-CL4B (Cytiva; separation range 7 x 104to 2 x 107daltons) and Sephacryl-S500 (Cytiva; separation range 4 x 104to 2 x 107daltons).

[0045] Additives such as polyethylene glycol (or other agents) may be used to aid the concentration or separation of cell membrane fragment / antibody complexes.

[0046] The antibodies collected in step d) may be characterized by determining binding to at least one antigen of interest. The at least one antigen may be coated on a solid phase support.

[0047] A “solid phase support” herein is an insoluble support material coated with chemically reactive functional groups. The chemically reactive functional groups are used to covalently or non-covalently link (or “coat”) the solid phase support with at least one antigen.

[0048] The solid phase supports provided herein include, but are not limited to, beads. Beads may be, for example, microbeads, magnetic beads, ion torrent beads or flow cytometry beads. Beads may comprise silica, gold, latex, polymers (such as polystyrene, polysulfone and polyethyl), or hydrogel. An illustrative type of bead, when chemically activated, crosslinks to primary amines of other molecules. Suitable beads are commercially available.to name a few examples, Aldehyde Sulfate, 4% w / v, 4um latex beads (Invitrogen, part of ThermoFisher Scientific, Carlsbad California, Catalog No. A37304); Microplex Microspheres (Luminex, now part of Diasorin, Austin Texas, Catalog Nos. LC10001 -YY through LC10100-YY); Streptavidin Polystyrene, 5uM (Bangs Laboratories, Fishers, Indiana, Catalog No. CP01006); EV Isolation Kit (Miltenyi Biotech, Waltham, MA, Catalog No. 130-1 11 -572); and Polylysine Magnetic Beads, PLYSMB-20 (Carolina Biosystems, Prague, Chezk Republic).

[0049] Beads may have a diameter ranging from about 1 pm to about 15 pm, inclusive of each endpoint of the range. Microbeads having a diameter of about 1 pm, 2 pm, 3 pm, 4Hm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm or 15 pm are also contemplated.

[0050] Other illustrative solid phase supports provided herein include, but are not limited to, dishes, such as those used in ELISA assays, coated with: (i) polylysine, or (ii) antibodies against known microvesicle proteins such as tetraspanin molecules or (iii) other chemicals enabling microvesicles to adhere to them. Other illustrative solid phase supports include those that can interact with microvesicles (e.g., larger agaraose or sephacryl beads than the sizes listed in

[0038] ) that have biochemical or biophysical characteristics that drive association with microvesicles). These may include supports with negatively charge surfaces (Capto-S- Agarose), positively charged surfaces (DEAE Sepharose), or hydrophobic surfaces (phenyl sepharose. Such supports or equivalents thereof may be obtained from, for example, Cytiva (Marlborough, MA). Solid phase supports provided herein may comprise a detectable label or other identifying characteristic. For example, the solid phase supports may comprise a single fluorescent dye or multiple fluorescent dyes. For example, the beads may be labeled with a dye including, but not limited to, acryloylmethylphenanthrene, rhodamine B, rhodamine 6G. methylene blue, acryflavin, acridine orange, janus green B, rose Bengal, luciferol CH and aminohydroquinones. For example, the solid phase supports may comprise bar codes as an identifiable characteristic. The solid phase supports may be unlabeled.

[0051] Antigens may be coated on / linked to the solid phase supports by a variety of techniques conventional in the art. One illustrative technique is physical crosslinking of molecules on the solid phase support to molecules in the antigen. Another illustrative technique would be using specific affinity interactions to couple a solid phase support and antigen. An example of this would be to coat the solid phase support with a monoclonal antibody that recognizes molecules found in the antigen. Yet another illustrative technique would rely on non-specific charge-charge interactions or hydrophobic interactions to trap antigens on solid supports.

[0052] Detection of recipient antibodies bound to antigens coated on solid phase supports may be by labeled reagents that interact with the bound recipient antibodies. For example, detection may be by using fluorescently tagged goat-antihuman serum that binds to human recipient antibodies. The level of retained fluorescence is used as a marker of human recipient antibodies trapped on the antigens. Other methods of detecting recipient antibodies bound to antigens coated on solid phase supports include, but are not limited to, using reagents that become active when recipient antibodies are present. An example of this would be to examine whether the antibody-dependent complement system activates when incubated with recipient antibody-exposed antigens. Activation may be detected by:(i) observation of increased proteolytic activity of the activated complement components, (ii) observation of activated complement components adhered to the solid support, or (iii) observation of donor cell membrane fragments lysed by activated complement.

[0053] In an illustrative system, fluorescent anti-human antibody is used to detect human recipient antibody which was collected from transplant donor cell membrane fragments and bound to antigen on a solid phase support.

[0054] Antibodies useful for detection herein may be polyclonal antibodies, monoclonal antibodies, antibody fragments which retain their ability to bind their unique epitope [e.g., Fc, Fab and F(ab)2 fragments], single chain antibodies and humanized antibodies. Antibodies may be generated by techniques standard in the art. Antibodies classes include, for example, IgG (including subtypes lgG1 , lgG2a, lgG2b, lgG2, lgG3 and lgG4), IgM, IgA, IgD and IgE.

[0055] Antibodies used for detection may be labeled for detection of antigen binding. The antibodies may be labeled with a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, phycoerythrin, rhodamine, or luciferin. The antibodies may comprise a radioactive label such as 3H, 14C, 32P, 35S, or 1251. The antibodies may be labeled with heavy metal labels. The antibodies may be labeled with enzymes such as alkaline phosphatase, p-galactosidase, biotin ligase or horseradish peroxidase. The antibodies may be labeled with biotin and avidin.

[0056] Illustrative assays for characterization of recipient antibodies to allotransplant antigens of interest include, but are not limited to, solid phase single HLA-antigen bead (SAB) assays of Immucor and of One Lambda / ThermoFisher which are described in Bertrand et al., Transplantation, 103: 597-603 (2019) (Luminex assays) and Clerkin et al., Transplantation Direct, 3: e218 (2017). Another such illustrative assay is a single-antigen bead-based LABScreen assay (Labscreen assays 1 , 2 and 3) of One Lambda (Canoga Park, CA, USA) in which the antigens listed in Table 1 are linked to beads.

[0057] Allotransplant antigens of interest contemplated herein include, but are not limited to, HLA antigens as well as the antigens listed in Table 1 .Table 1

[0058] Kits are provided for carrying out methods provided herein. Kits provided herein comprise one or more of: a chromatography column packed with a matrix such as a porous stationary phase, a solution or solutions (e.g., acid solution, salt solution, solution of a chelating agent, or solution of a chaotropic chemical) for separating transplant recipient antibodies from donor cell membrane fragments, and an antigen-coated solid phase support.

[0059] Other terminology and disclosure

[0060] As used herein and in the appended claims, the singular forms "a," "and" and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any element, e.g., any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusiveterminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0061] When a range of values is provided herein, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present disclosure.

[0063] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method may be carried out in the order of events recited or in any other order which is logically possible. This disclosure is intended to provide support for all such combinations.

[0064] As used herein, “contemplated,” “may,” “may comprise,” “may be,” “can,” “can comprise” and “can be” all indicate something envisaged by the inventors that is functional and available as part of the subject matter provided.

[0065] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials for the purpose for which the publications are cited.Examples

[0066] While the following examples describe specific embodiments, variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be placed on the invention.Example 1

[0067] An experiment was performed to demonstrate the release of human antibodies from pig lymphocyte cell membrane fragments.

[0068] The pig cell membrane fragments were prepared and exposed to human serum as follows. Ten million pig lymphocytes were incubated in 5mls of serum free media (Aim V, ThermoFisher) containing 500ul of heat inactivated (56“C for 30 minutes) human serum. After 30 minutes at 37“C, the cells were pelleted (400g, 5 minutes, 18“C-25“C) and the supernatant discarded. The cell pellet was resuspended in 5mls of serum free media (Aim V) and centrifuged again (400g, 5 minutes, 18“C-25“C). Again, the supernatant was discarded, and the pellet resuspended in 5mls of fresh serum free media (Aim V) and incubated at 37“C in 5%CO2 for 12-16 hours. The cells were pelleted from the media by centrifugation (400g, 5 minutes, 18“C-25“C), and then the cell pellet was resuspended in 1 ml of fresh media (Aim V) and stored at 4“C for later use. Another preparation of the same pig lymphocytes was treated identically, except the preparation did not get incubated with human serum, to serve as a negative control. To concentrate cell membrane fragments, the supernatants from the resuspended pig lymphocyte pellets were centrifuged at 1000g for 10 minutes to remove residual cells. The supernatants of this centrifugation step were collected diluted with an equal volume of phosphate buffered saline and centrifuged for 1 hour and 15 minutes at 100,000g at 19“C-25“C. The supernatant was discarded and the 100,000g cell membrane fragment pellets were resuspended in 10Oul of media (Aim V).

[0069] To separate human serum antibodies bound to the pig cell membrane fragments, a size exclusion chromatography column containing approximately 4mls of Sephacryl-S500 (Cytiva) was prepared by passing 10mls of media (Aim V) through the column. Next 10Oul of 0.1 M citric acid pH 3.0 was added to the top of the column. The resuspended membranes with bound antibodies from the 100,000g pellet were then loaded onto the column. 5mls of fresh media (Aim V) was added to the column. The first 1 .2 mis that eluted from the column were discarded. Then three 850ul fractions were collected. The third 850ul fraction was expected to (and did) contain the human antibodies separated from the pig cell membrane fragments.

[0070] The human antibodies in the third 850ul fraction were confirmed by their binding to 200,000 pig lymphocytes as test antigens. As a negative control, the corresponding column fraction collected from pig cells that were not incubated with human serum was also used. This “mock” treated fraction was also incubated with 200,000 pig lymphocytes. After 30 minutes at 4“C the lymphocytes were pelleted (400g, 5 minutes, 18“C-25“C), resupended in 1 ml phosphate buffered saline and pelleted again 400g, 5 minutes, 18“C- 25“C). The cell pellet was then resuspended in 10Oul of a 1 :200 dilution of anti-human IgG labeled with the fluorescent molecule Alexa Fluor 488 (AF488-donkey antihuman IgG 709- 546-098, Jackson Immunoresearch). After 30 minutes at 4“C the 1 ml of phosphate buffered saline was added to the cells and centrifuged (400g, 5 minutes, 18“C-25“C). This PBS-centrif ugation step was repeated once. The pelleted cells were resuspended in 50ul of PBS and binding of antibodies was analyzed on a flow cytometer.Figure 2 shows the median fluorescence intensity (MFI) resulting from the human antibodies collected from cell membrane fragments originating from the pig cells that had been incubated with human serum (Ab+).

Claims

Claims1 . A method of collecting transplant recipient antibodies bound to transplant donor cell membrane fragments comprising: a) applying transplant recipient bodily fluid to a chromatography column, b) separating in the chromatography column, donor cell membrane fragments in the bodily fluid from transplant recipient antibodies in the bodily fluid, c) removing the donor cell membrane fragments from the chromatography column, and d) collecting the transplant recipient antibodies remaining in the chromatography column.

2. The method of claim 1 , wherein the separation of step b) employs a pH gradient, a high salt concentration, a chelating agent or a chaotropic chemical.

3. The method of claim 2 employing a pH gradient.

4. The method of claim 3 employing a pH gradient, wherein the pH in the column of steps a) and b) is about pH 2.5 to about pH 5.0.

5. The method of claim 3 employing a pH gradient, wherein the pH in the column of steps a) and b) is about pH 9.0 to about pH 11 .5.

6. The method of claim 3, 4 or 5 employing a pH gradient, wherein the pH is adjusted in step c) to about pH 6.0 to about pH 8.5.

7. The method of claim 2 employing a high salt concentration.

8. The method of claim 7, wherein the salt is sodium chloride.

9. The method of claim 7 or 8, wherein the salt concentration is about 0.5 M to about 2.5 M.

10. The method of claim 2 employing a chelating agent.11 . The method of claim 10, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'- tetraacetic acid (EGTA).

12. The method of claim 10 or 11 , wherein the chelating agent added is about 5 mM to about 10 M.

13. The method of claim 2 employing a chaotropic chemical.

14. The method of claim 13, wherein the chaotropic chemical is urea, guanidinium hydrochloride or arginine.

15. The method of claim 13 or 14, wherein the chaotropic chemical added is about 10 mM to about 10 M.

16. The method of any preceding claim, wherein the antibodies collected in step d) are characterized by an assay for binding to at least one antigen.

17. The method of claim 16, wherein the at least one antigen is coated on a solid phase support.

18. The method of claim 16 or 17, wherein antibody binding to antigen is detected by fluorescence.

19. The method of claim 17 or 18, wherein the solid phase support comprises microparticle beads.

20. The method of any preceding claim, wherein the recipient received an allotransplant.21 . The method of any preceding claim, wherein the allotransplant was from a human.

22. The method of any preceding claim, wherein the recipient received a xenotransplant.

23. The method of any preceding claim, wherein the xenotransplant was from a pig.

24. A kit comprising one or more of: a chromatography column packed with a porous stationary phase, a solution or solutions for separating in the chromatography column transplant recipient antibodies from donor cell membrane fragments, and an antigen-coated solid phase support.

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