Crossmatching products and methods

Abstract

The present disclosure relates to products and methods for crossmatching transplant donors and recipients. Products and methods provided are based on solid phase supports coated with transplant donor cell membrane fragments. The products and methods are useful in allotransplantation and xenotransplantation.

Description

CROSSMATCHING PRODUCTS AND METHODSCross Reference To Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 618,160, filed January 5, 2024 and U.S. Provisional Application No. 63 / 641 ,675, filed May 2, 2024, which are incorporated herein by reference in their entirety.Field

[0002] The present disclosure relates to products and methods for crossmatching transplant donors and recipients. Products and methods provided are based on solid phase supports coated with transplant donor cell membrane fragments. The products and methods are useful in allotransplantation and xenotransplantation.Background

[0003] 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 it.

[0004] Crossmatching assays are used to screen prospective recipient patients before receiving cells or tissues from a donor to try to avoid transplant rejection. Crossmatching assays are designed to detect when recipients make antibodies that 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 target cells for destruction by natural killer cells and macrophages among others. Organ transplants in which the recipient synthesizes pre-transplant anti-donor antibodies are at an increased risk of rapid graft failure.

[0005] Allotransplantation is the transplantation from one individual to another 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 and the transplant is called a xenograft.

[0006] In allotransplantation, there can be recipient antibodies that recognize three types of antigens on donor cells: (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). Pre-transplant diagnostic testing examines a potential recipient’s antibody reactivity towards molecules on cells fromprospective donors. The absence of any recipient antibodies towards these three classes of antigens indicates compatibility and marks ideal donor and recipient pairings.

[0007] 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 arrays have 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 also be used to help detect the de novo production of anti-HLA antibodies which indicate an active organ rejection process.

[0008] Xenotransplant crossmatching 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. As with allotransplant crossmatching, target cells which express xenoantigens are incubated with recipient serum. The target cells were then evaluated for binding of recipient antibodies.

[0009] There remains a need in the art for products and assay methods for screening and monitoring transplant patients.Summary

[0010] Humoral immunity can result in many of the same rejection problems in xenotransplantation as in allotransplantation. For allotransplantation or xenotransplantation, it is contemplated herein that pre-transplant crossmatching assay methods provided by the disclosure can determine which recipient patients may receive a transplant with reduced risk of rapid humoral rejection. For allotransplantation or xenotransplantation, it is contemplated herein that pre-transplant crossmatching assay methods provided herein can avoid rapid humoral rejection in recipients. For xenotransplantation, for example, pigs can be genetically modified to eliminate the expression of various xenoantigens recognized by human antibodies that exist prior to the transplant. It is contemplated herein that pre-xenotransplant crossmatching assay methods provided by the disclosure can determine which recipient patients may receive xenotransplants or such genetically modified xenotransplants withreduced risk of rapid humoral rejection. It is contemplated herein that pre-xenotransplant crossmatching assay methods provided herein can avoid rapid humoral rejection in recipients. For allotransplantation or xenotransplantation, it is also contemplated herein that methods provided herein can detect de novo antibodies arising in the recipient in the posttransplant period.

[0011] More is now understood about pig xenoantigens that are immunogenic in humans. Carbohydrates synthesized by the GGTA1 gene are recognized by IgG and IgM in all primates prior to transplantation. The B4GalNT2 gene produces glycans recognized by most humans pre-xenotransplant, and many non-human primates post xenotransplant. The pig CMAH gene produces a variant of sialic acid, Neu5Gc, that human antibodies recognize pre- xenotransplant. Though produced by and tolerated by most mammals, Neu5Gc is foreign to people because they carry a nonfunctional CMAH gene and fail to synthesize Neu5Gc. Gene editing of pigs, the preferred donors in xenotransplantation, eliminates these carbohydrate-based antigens creating ideal negative crossmatches for 30% of humans. However, 70% of people continue to exhibit pre-transplant anti-pig antibodies, many of which may target SLA. SLA have sufficient amino acid sequence and structural similarities to the homologous HLA that these swine proteins can also be targeted by human antibodies. Human antibodies that recognize SLA often, but not always, cross-react with HLA proteins.

[0012] As mentioned above, despite genetic engineering, 70% of humans contain antibodies to the least antigenic engineered pig which lacks GGTA1 , B4GalNT2, and CMAH genes. Though SLA proteins have also been identified as xenoantigens, other currently undefined antigens likely also exist. Typically, swine peripheral blood mononuclear cells (PBMC) are used as targets in xenotransplant crossmatching. While these cells display SLA molecules, it remains unclear whether they display all xenoantigens that may be expressed by a transplanted organ. Consequently, using PBMC as the crossmatching target could miss anti-pig antibodies against a transplanted organ when evaluating various donor-recipient combinations. An alternative target source capable of displaying the full spectrum of xenoantigens may be needed. Though endothelial cells have been evaluated as crossmatching targets, they too may fail to represent all xenoantigens produced in an organ. In addition, cell culture conditions have not yet been identified where endothelial cells can be cultured without fetal bovine serum. Unfortunately, fetal bovine serum contains Neu5Gc which transfers to the target cells in culture which increases the background signal and diminishes the ability to detect recipient antibodies recognizing less abundant antigens.

[0013] Every cell type examined releases a variety of cell membrane fragments that contain 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 membrane fragments can be obtained from cells in culture as well as from bodily fluids such as urine and serum and are known in humans to be enriched in HLA molecules. As noted above, cell membrane fragments carry many molecules from the cells and are contemplated by the disclosure to be useful as crossmatching targets. As illustrated in the Examples below, when cell membrane fragments are linked to beads, specific antigenic molecules have been detected in these membrane fragments by monoclonal antibodies and glycan-specific lectins. In detection methods, membrane-coated beads that have been incubated with fluorescent antibodies are examined using flow cytometric analyses.

[0014] Cell membrane fragments exhibit one or more characteristics which are contemplated herein to be valuable in crossmatching assays. As one example, serum- derived cell membrane fragments originate from all tissues offering the potential to represent all antigens that may be found in a donor (e.g., xenoantigens that may be found in a pig). This is contemplated herein to alleviate the issues associated with using only one or two cell types, such as PBMC, as a proxy for the antigens found in an organ. As another example, cell membrane fragments released from cells engineered to express single HLA or SLA molecules can be linked to beads. As another example, by using isolated membranes devoid of intracell organelles e.g., nucleus, mitochondria or endoplasmic reticulum), the assay background is diminished thus increasing assay sensitivity. As another example, perfusate from donor organs are contemplated to contain the cell membrane fragments useful as organ-specific crossmatching targets.

[0015] The disclosure thus provides products and methods for crossmatching transplant donors and recipients.

[0016] The disclosure provides products such as crossmatching solid phase supports coated with transplant donor cell membrane fragments.

[0017] The disclosure provides methods of preparing a crossmatching solid phase support comprising coating a solid phase support with transplant donor cell membrane fragments.

[0018] The disclosure provides products such as kits comprising the crossmatching solid phase supports.

[0019] The disclosure provides methods of detecting transplant recipient antibody binding or transplant recipient immune cell binding to transplant donor cell membrane fragments comprising: for detecting antibody binding contacting a crossmatching solid phase supportcoated with the transplant donor cell membrane fragments with transplant recipient serum, plasma, urine, or blood, or for detecting immune cell binding contacting a crossmatching solid phase support coated with the transplant donor cell membrane fragments with transplant recipient blood or urine, and detecting transplant recipient antibody binding or transplant recipient immune cell binding to the crossmatching solid phase support.

[0020] The disclosure provides crossmatching methods for determining the compatibility of a transplant donor and a transplant recipient comprising: for detecting antibody binding contacting a crossmatching solid phase support coated with the transplant donor cell membrane fragments with transplant recipient serum, plasma, urine, or blood, or for detecting immune cell binding contacting a crossmatching solid phase support coated with the transplant donor cell membrane fragments with transplant recipient blood or urine, and detecting transplant recipient antibody binding or transplant recipient immune cell binding to the crossmatching solid phase support. In the methods, binding indicates a lack of compatibility between a transplant donor and a transplant recipient while a lack of binding indicates compatibility.

[0021] The disclosure provides methods of detecting a transplant recipient immune reaction to a transplant from a transplant donor comprising: contacting a crossmatching solid phase support coated with transplant donor cell membrane fragments with transplant recipient serum, plasma, urine, or blood, or for detecting immune cell binding contacting a crossmatching solid phase support coated with the transplant donor cell membrane fragments with transplant recipient blood or urine, and detecting transplant recipient serum antibody binding or transplant recipient immune cell binding to the crossmatching solid phase support. In the methods, binding indicates a transplant recipient immune reaction to the transplant.

[0022] In the provided methods, binding can be detected by, for example, fluorescence. In the provided methods, binding can be detected by, for example, the activation of pathways where antibody interaction with the coated crossmatching solid phase support is the initiating event (e.g., complement pathway activation).

[0023] In the provided products and methods, the solid phase support can comprise, for example, microparticle beads.

[0024] In the provided products and methods, the transplant donor can be an allotransplant donor. In the provided products and methods, the allotransplant donor can be human.

[0025] In the provided products and methods, the transplant donor can be a xenotransplant donor. In the provided products and methods, the xenotransplant donor can be a pig.

[0026] In the provided products and methods, the transplant donor cell membrane fragments can be isolated from one or more of a transplant donor bodily fluid, a transplant donor cell or a perfusate of the transplant. The transplant donor cell membrane fragments can be isolated from a transplant donor bodily fluid. The transplant donor bodily fluid can be one or more of serum, plasma or urine. The transplant donor bodily fluid can be serum. The transplant donor bodily fluid can be plasma. The transplant donor bodily fluid can be urine. The transplant donor cell membrane fragments can be isolated from a transplant donor cell. The transplant donor cell can be a cell of the transplant from the transplant donor. When the transplant donor is, for example, a genetically engineered animal such as a genetically engineered pig, the transplant donor cell membrane fragments may be isolated from the same type of genetically engineered pig but not from the donor pig itself. The transplant donor cell membrane fragments can be isolated from a perfusate of the transplant from the transplant donor.

[0027] The disclosure also provides methods of detecting one or more viral, bacterial and / or fungal infections of a transplant donor or transplant therefrom comprising contacting a solid phase support coated with transplant donor cell membranes with a viral-, bacterial- or fungal-specific reagent and detecting binding of the reagent.

[0028] The methods provided herein may include negative, blocking cell membrane fragments along with the transplant donor cell membrane fragments.Brief Description of the Drawings

[0029] Figure 1 shows the levels of IB4 antibody binding to glycan antigens in cell membrane fragments coated on beads after the cell membrane fragments were released from a wild-type pig lymphoma (WT) and from a lymphoma variant edited to eliminate GGTA1 , B4GalNT2, and class I SLA genes (KO).

[0030] Figure 2 shows binding of beads coated with WT and KO lymphoma cell membrane fragments by antibodies against the class II SLA-DR proteins. A negative control (-) indicated background fluorescence.

[0031] Figure 3 shows beads coated with cell membrane fragments can be used as targets in crossmatching assays that evaluate recipient antibody binding.

[0032] Figure 4 shows binding of IgG and IgM from human serum [Serum (+)] to pig cells. No serum controls [Serum(-)] reveal signals obtained in the absence of antibody binding. IgG and IgM exhibited high binding in the absence of antigen competition (-). Serum was also pretreated with membranes collected from unedited cells (W) or from edited cells (D) that lack functional xenoantigen-producing genes (GGTA1 , B4GalNT2, and SLA-1 , SLA-2, and SLA-3). These membranes provide a source of xenoantigens to compete with binding of antibodies to either unedited (top panel) or edited (bottom panel) cells.Detailed Description

[0033] A “crossmatching” method, test or assay are terms used interchangeably herein to refer to a test that determines the immunologic risk for a recipient associated with a transplant from a potential donor. The test determines whether there are transplant-reactive circulating antibodies or immune cells in the recipient directed against transplant antigens. "Compatibility” of the donor and recipient is another term associated with immunologic risk. A lower immunologic risk signifies higher compatibility between the donor and recipient. A higher immunologic risk signifies lower compatibility between the donor and recipient.

[0034] 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. “Xenotransplantation” is the transplantation from one species to another.

[0035] A “transplant” herein can 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 can be a human cell, fluid, tissue or organ. A transplant can be a pig cell, fluid, tissue or organ. A transplant can 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 a antigen of a transplant that is antigenic and immunogenic to a transplant recipient.

[0036] A “transplant donor” is the individual providing a transplant. A transplant donor can be, for example, a human. A transplant donor can be, for example, a pig.

[0037] 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.

[0038] 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.

[0039] The term "knockout" herein relates to a 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.

[0040] 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 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.

[0041] 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.

[0042] A “transplant recipient” is the individual receiving a transplant. A transplant recipient can be a human.

[0043] Transplant donor cell membrane fragments are used in methods provided herein as crossmatching targets. Cell membrane fragments provided herein comprise antigens from the surface of transplant donor cells, including cells of the transplant. The cell membranefragments, include, but are not limited to, exosomes, microvesicles, ectosomes, and micromeres. Transplant donor cell membrane fragments can be isolated from any fluid that has been in direct or indirect contact with any transplant donor cell, fluid, tissue or organ. For example, isolation can be from donor blood, plasma, serum or urine. As other non-limiting examples, isolation can be from media collected from cell culture of donor isolated primary cells, continuous or immortalized cells, or organoid cultures. As yet another non-limiting example, isolation can be from the perfusate of any donor organ (e.g., preservative solution that has been passed through an isolated donor organ.) As another non-limiting example, transplant donor cell membrane fragments can be isolated from ground up cell debris.

[0044] Cell membrane fragment isolation begins with a step that eliminates unwanted large debris from the starting material. Various methods for cell membrane fragment isolation are known in the art. For example, cell membrane fragments can be isolated by centrifugation. As described in Example 1 , this can be achieved by, for example, centrifugation at 10,000g for 30 minutes. A second step can include concentration by, for example, centrifugation at 100,000g for an hour.

[0045] As a non-limiting alternative, solutions containing cell membrane fragments can be passed over solid phase supports that can interact with beads. These include other types of solid phase supports described herein. Yet other alternatives include, but are not limited to, the following.

[0046] Cell membrane fragments may also be isolated by aggregation after incubating them with short peptides that bind proteins on the cell membrane fragment surface. The membrane fragments aggregate and can be isolated at much lower centrifugation speeds. Once collected the membranes can be dissociated and fixed to solid supports. See, e.g., Microvesicle vs Vesicle - ME™ Kit (Biosynth, UK).

[0047] Cell membrane fragments may also be isolated by size exclusion chromatography, the use of non-specific precipitating reagents such as polyethylene glycol, and ultrafiltration (Sidhom etal., Int J Mol Sci. 2020 Sep 4;21 (18):6466. doi: 10.3390 / ijms21186466. PMID: 32899828; PMCID: PMC7556044).

[0048] Cell membrane fragments may also be isolated as described in Cohen etal., J Cell Biol. 1977 Oct 1 ;75(1 ):119-34.

[0049] 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 transplant donor cell membrane fragments provided herein.

[0050] The solid phase supports provided herein include, but are not limited to, beads. Beads can be, for example, microbeads, magnetic beads, ion torrent beads or flow cytometry beads. Beads can 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- 111-572); and Polylysine Magnetic Beads, PLYSMB-20 (Carolina Biosystems, Prague, Chezk Republic).

[0051] Beads can 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, 4 pm, 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.

[0052] 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 can 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.

[0053] Cell membrane fragments can be linked to the solid phase supports by a variety of techniques. One illustrative technique is physical crosslinking of molecules on the solid phase support to molecules in the cell membrane fragments. An example of this would be a primary amine on a cell membrane fragment crosslinking to an aldehyde group that is part ofthe solid phase support. Another illustrative technique would be using specific affinity interactions to couple a solid phase support and a cell membrane fragment. An example of this would be to coat the solid phase support with a monoclonal antibody that recognizes molecules found in the cell membrane fragments. Yet another illustrative technique would rely on non-specific charge-charge interactions or hydrophobic interactions to trap cell membrane fragment on solid supports. Examples of this would be to make a solid phase support highly negatively charged so that it may attract positive charges on the cell membrane fragment, or vice versa.

[0054] Transplant recipient fluids containing transplant recipient antibodies or transplant recipient immune cells are used in methods provided herein. Such fluids include, but are not limited to, serum, plasma, urine and blood.

[0055] Serum, plasma, urine or blood from a transplant recipient can be used in methods provided herein for detecting pre-transplant ( / .e., pre-existing) transplant recipient antibodies that recognize ( / .e., bind to) transplant antigens and / or blood or urine from a transplant recipient can be used in methods provided herein for detecting pre-transplant ( / .e., preexisting) transplant recipient immune cells that recognize transplant antigens. In addition, methods provided herein can be used for detecting transplant recipient humoral / antibody and cellular immune responses to the transplant that arise post-transplant (e.g., real-time methods). Detection may be by any detection system that detects binding between the transplant recipient antibodies and transplant antigens.

[0056] Transplant recipient immune cells include, but are not limited to, macrophages, neutrophils, T cells and natural killer (NK) cells.

[0057] In post-transplant recipient humoral / antibody and cellular immune response detection methods, donor cell membrane fragments can be used that are obtained from a transplant donor like the donor cell membrane fragments obtained for use in pre-transplant methods.

[0058] Detection of recipient antibodies bound to donor cell membrane fragments coated on solid phase supports can be by labeled reagents that interact with the bound antibodies. For example, detection can be by using fluorescently tagged goat-antihuman serum that binds to human antibodies. The level of retained fluorescence is used as a marker of human antibodies trapped on the cell membrane fragments. Other methods of detecting recipient antibodies bound to donor cell membrane fragments include, but are not limited to, using reagents that become active when antibodies are present. An example of this would be to examine whether the antibody dependent complement system activates when incubated with antibody-exposed donor cell membrane fragments. Activation can 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.

[0059] In an illustrative system, fluorescent anti-human antibody is used to detect human recipient serum antibody bound to transplant donor cell membrane fragments coated on crossmatching solid phase supports.

[0060] 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.

[0061] Antibodies used for detection may be labeled for detection of 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.

[0062] Specific binding of an antibody to an antigen described herein within a biological sample may be carried out using: Western blot analysis with immunoblotting, immunocytochemistry, immunohistochemistry, dot blot analysis, flow cytometry, ELISA assays or RIA assays. These techniques and other approaches are conventional in the art.

[0063] A parallel use of donor cell membrane fragments contemplated herein is to detect one or more viral, bacterial or fungal infections of the transplant donor and any transplant therefrom. The transplant donor may be a human. The transplant donor may be a nonhuman animal such as a pig. Detection methods provided include detecting one or more viral, bacterial or fungal components such as nucleic acids (including RNA or DNA), lipids, carbohydrates or proteins (including fragments thereof) present in transplant donor cell membrane fragments from a transplant donor or present in transplant donor cell membrane fragments from a transplant recipient. In the methods, the transplant donor cell membrane fragments may be coated on solid phase supports as described herein. Detection of the viral, bacterial and / or fungal components in the methods may be accomplished with any detection system / reagent known in the art. For example, a viral nucleic acid may be specifically detected by sequencing or PCR using a primer specific for the viral nucleic acid.For example, a viral protein may be detected by specific binding by an antibody targeting the viral protein.

[0064] Methods are provided for detecting infection of pig xenotransplant donors by one or more viruses, bacteria and / or fungi. Methods are provided for detecting infection of pig xenotransplant donors by viruses, bacteria and / or fungi such as the following.Table 1Methods are provided for detecting infection of xenotransplant donors by viruses such as simian T-lymphotropic virus (STLV), simian foamy virus (SFV), simian virus 40 (SV40), baboon endogenous retrovirus (BaEV), porcine endogenous retroviruses (PERVs), herpesviruses [including porcine cytomegalovirus (PCMV) and porcine lymphotropic herpesvirus (PLHV)], pseudorabies virus (PRV), hepatitis E virus (HEV), and porcine circoviruses (PCVs).

[0065] Methods are provided for detecting infection of human allotransplant donors by one or more viruses. Methods are provided for detecting infection of human allotransplant donors by viruses such as the following.Table 2Family Name Representative VirusesAdenoviridae Human adenovirus types 1 to 57 in seven species (human adenovirus species A to G)Anelloviridae Torque teno virus 1 (TTV1 ), Torque teno mini virus 1 , Torque teno midi virus 1 (type species for numerous viruses in 3 genera)Arenaviridae Lassa virus, lymphocytic choriomeningitis virus, Junin virus, Machupo virus, Guanarito virus, Sabia virus, Whitewater Arroyo virus, Chapare virus, Lujo virusAstroviridae Human astroviruses (eight serotypes)Bornaviridae Mammalian 1 bornavirus (formerly Borna disease virus [BDV])Bunyaviridae California encephalitis virus, Sin Nombre virus, La Crosse virus, Hantaan virus, Muerto Canyon virus, Crimean-Congo hemorrhagic fever virus, Sandfly fever viruses, Rift Valley fever virus, Heartland virusCaliciviridae Noroviruses, sapovirusesCoronaviridae SARS coronavirus; MERS coronavirus; human coronaviruses OC43, 229E, NL63, and HKLI1 ; human torovirus and other human enteric coronavirusesFiloviridae Ebola viruses (e.g., Zaire ebolavirus, Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus), Marburg virusFlaviviridae Genus Alphavirus dengue virus, yellow fever virus, Japanese encephalitis virus, West Nile virus, Murray Valley encephalitisvirus, Kyasanur encephalitis virus, tick-borne encephalitis virus, Zika virus, and others; Genus Hepacivirus: hepatitis C virus (HCV); Genus Pegivirus: GB virus-Cc (GBV-C) (formerly hepatitis G virus [HGV])Hepadnaviridae Hepatitis B virus (HBV)Hepeviridae Hepatitis E virus (HEV)Herpesviridae Herpes simplex virus type 1 , herpes simplex virus type 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8 (i.e., Kaposi sarcoma-associated herpesvirus), herpes simian B virusOrthomyxoviridae Influenza A virus (e.g. subtype H1 N1 ), influenza B virus, influenza C virus, Thogoto virus, Dhori virus, Bourbon virusPapillomaviridae Human papilloma virus (>150 serotypes)Paramyxoviridae Measles (rubeola) virus, mumps virus, parainfluenza viruses, Hendra virus, Nipah virus, Menangle virusParvoviridae Human parvovirus B19, human bocavirus, adeno-associated virusesPicobirnaviridae Human picobirnavirusPicornaviridae Genus Enterovirus: human rhinoviruses (>100 serotypes), enteroviruses (>100 serotypes, including poliovirus 1-3, coxsackievirus A and B, echoviruses, and other human enteroviruses); Genus Hepatovirus: hepatitis A virus (HAV); Genus Parechovirus: human parechoviruses; Genus Kobuvirus: Aichi virus; Genus Cosavirus: human cosaviruses; Genus Cardiovirus: Vilyuisk human encephalomyelitis virus, Saffold viruses; Genus Salivirus: human klassevirus, salivirus A; Genus Senecavirus: Seneca Valley virus; Unassigned: Syr- Darya Valley fever virusPneumoviridae Respiratory syncytial virus, human metapneumovirusesPolyomaviridae JC virus, BK virus, KI virus, WU virus, Merkel cell polyomavirus, lymphotropic polyomavirus, human polyomavirus 6, human polyomavirus 7, trichodysplasia spinulosa-associated polyomavirus, human polyomavirus 944, Poxviridae Molluscum contagiosum virus, variola (smallpox) virus, monkeypox virus, vaccinia virus, orf virus, pseudocowpox virus, Tanapox virus, Yaba monkey tumor virusReoviridae Human rotavirus, Colorado tick fever virus, human reovirus,c Kemerovo virusRetroviridae Human immunodeficiency viruses types 1 and 2, human T- lymphocyte lymphotropic viruses, 47 xenotropic murine leukemia virus-related virus, human endogenous retroviruses (HERVs), simian foamy virusRhabdoviridae Rabies virus, vesicular stomatitis virus, Australian bat lyssavirus, Duvenhage virus, Mokola virusTogaviridae Rubella virus; Chikungunya virus; eastern equine, western equine, and Venezuelan equine encephalitis viruses; Ross River, Sindbis, and Semliki Forest virusesMethods are provided for detecting infection of human allotransplant donors by one or more viruses such as Adenoviridae, Anelloviridae, Arenaviridae, Astroviridae, Bornaviridaea, Bunyaviridae, Caliciviridae, Coronaviridae, Deltae, Filoviridaea, Flaviviridae,Hepadnaviridae, Hepeviridae, Herpesviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridaea, Parvoviridae, Picobirna, Picobirnaviridae, Picornaviridaed, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridaea, and / or Togaviridae.

[0066] To increase sensitivity of methods provided herein, “negative, blocking” cell membrane fragments can be used in the methods to block non-specific reactivity of donor cell membrane fragments with transplant recipient antibodies or cells. Negative, blocking cell membrane fragments may be prepared from human cell lines manipulated (by genetic engineering or other methods) such that they lack epitopes of interest to ( / .e., intended to be detected in) the assay.

[0067] The disclosure provides kits to carry out methods disclosed herein. The kits comprise crossmatching solid supports provided herein. The kits provided herein may further comprise any components necessary to carry out the detection assays that are conventional in the art. The kits may comprise one or more of the following. The kits may comprise antibodies. The kits may comprise buffers, loading dyes, gels such as polyacrylamide gels and molecular weight markers for preparing SDS-PAGE gels to carry out Western blots.The kits may comprise filters, membranes, blocking buffers, control buffers, isotype control antibodies, wash buffers or buffers and reagents for detection to carry out immunoblotting or dot blotting analysis such as labeled secondary antibodies. The kit may comprise fixing reagents, blocking buffers, control buffers, wash buffers, staining dyes and detection reagents including anti-isotype antibodies. The kits may comprise the necessary reagents and tools to carryout flow cytometry, ELISA assays, RIA assays or cytotoxicity assays. The kits may comprise instructions for use of the crossmatching solid supports.

[0068] A non-limiting, illustrative example of a crossmatching kit relating to xenotransplantation of a transplant from a genetically engineered pig, is a kit including: crossmatching solid phase supports coated with transplant donor cell membrane fragments and positive control solid phase supports coated with wild-type pig cell membrane fragments. Such kits may further include negative, blocking cell membrane fragments. Such kits can be used to test patient fluid samples by methods described herein.

[0069] Other terminology and disclosure

[0070] 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 exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0071] 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.

[0072] 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 can also be used in the practice or testing of the present disclosure.

[0073] 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 can 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.

[0074] 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.

[0075] 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

[0076] 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

[0077] An experiment was performed to demonstrate transplant donor cell membrane fragments contain glycan antigens.

[0078] Cell culture supernatants taken from a wild type (WT) pig lymphoma and from a lymphoma variant edited to eliminate GGTA1 , B4GalNT2, and class I SLA genes (KO) were used as the source of released cell membrane fragments. The cell membrane fragments were isolated by preparative ultracentrifugation to first clarify the media of large cell debris, and finally to isolate small membrane fragments using a 100,000g and 90-minute-long centrifugation step. The cell membrane fragments were resuspended using a dounce homogenizer and linked to chemically activated beads [Aldehyde Sulfate, 4% w / v, 4um latex beads (Invitrogen, Carlsbad, California) (part of ThermoFisher Scientific) (Catalog No. A37304)] which crosslink to primary amines. After covalently linking the membrane fragments to the beads, residual active sites were blocked with bovine serum albumin. Beads coated with WT membrane fragments and KO membrane fragments were incubated with fluorescently labeled IB4 antibody which recognizes glycans modified by the GGTA1 gene.

[0079] As shown in Figure 1 , the WT beads but not the KO beads exhibit high levels of IB4 antibody binding. Thus, the membrane fragments released from the WT pig cells contained glycan antigens recognized by IB4 antibody.Example 2

[0080] Experiments were performed to demonstrate transplant donor cell membrane fragments contain class I SLA proteins and / or class II SLA proteins.

[0081] Beads coated with the WT membrane fragments and KO membrane fragments as described in Example 1 were incubated with fluorescently labeled antibodies against the class II SLA-DR proteins. WT beads incubated with a fluorescent antibody that did not target the beads were used as a as a negative control (-, in Figure 2).

[0082] As shown in Figure 2, the WT and KO membrane fragments bound high levels of antibodies against the class II SLA-DR proteins. Thus, the membrane fragments released from the WT and KO pig cells contained class II SLA proteins.

[0083] Lastly, cell membrane fragments isolated from cells edited to lack class I SLA and WT cell membrane fragments were linked to beads and incubated with a fluorescent antibody specific for class I SLA proteins. Class I SLA-positive WT beads exhibited a greater signal than the SLA-deficient KO beads.Example 3

[0084] An experiment was performed to demonstrate cell membrane fragment-coated beads can be used as targets in crossmatching assays that measure recipient antibody binding.

[0085] Beads as described in Example 1 were incubated with human serum. A fluorescent anti-human IgG or IgM antibody was used to detect human immunoglobulin that bound to the beads. Beads were incubated with a fluorescent secondary antibody only to define background signal.

[0086] As shown in Figure 3, WT beads incubated with human serum showed high levels of IgG and IgM binding. KO beads also bound human IgG and IgM but to a lesser extent than the WT beads.Example 4

[0087] Experiments were performed to demonstrate that cell membrane fragments comprise antigens useful for crossmatching assays.

[0088] In the experiments, human serum was pre-incubated with cell membrane fragments produced from WT pig cells or KO pig cells (deficient in GGTA1 , B4GalNT2, and SLA-1 , SLA-2, and SLA-3 genes). Then, those sera were incubated with either WT or KO pig cells and a standard crossmatch was performed where fluorescent anti-human IgG or IgM were used to detect antibody binding to the cells. As a control, the human serum was also prepared without any pre-incubation cell membrane fragment addition. The IgG and IgM detection reagents were also incubated with the pig cells that had not received any human serum as a negative control.

[0089] As shown in Figure 4, when WT cells were used as targets, WT cell membrane fragments competed for binding very well ( / .e., they reduced antibody binding to the cells) but the KO cell membrane fragments did not compete as well. This was expected since KO cell membrane fragments lack xenoantigens that are found on WT cell membrane fragments. When KO cells were the targets, the KO cell membrane fragments blocked antibody binding to the KO cells better than WT. This indicates that WT cell membrane fragments have the same xenoantigens as the KO cell membrane fragments, but the abundance of xenoantigens varies because some xenoantigens have been removed in the KO.

Claims

Claims1 . A crossmatching solid phase support coated with transplant donor cell membrane fragments.

2. A method of preparing a crossmatching solid phase support comprising coating a solid phase support with transplant donor cell membrane fragments.

3. A kit comprising the crossmatching solid phase support of claim 1 .

4. A method of detecting transplant recipient antibody binding or transplant recipient immune cell binding to transplant donor cell membrane fragments comprising: for detecting antibody binding contacting a crossmatching solid phase support coated with the transplant donor cell membrane fragments with transplant recipient serum, plasma, urine or blood, or for detecting immune cell binding contacting a crossmatching solid phase support coated with the transplant donor cell membrane fragments with transplant recipient blood or urine, and detecting transplant recipient antibody binding or transplant recipient immune cell binding to the crossmatching solid phase support.

5. A crossmatching method for determining the compatibility between a transplant donor and a transplant recipient comprising: for detecting transplant recipient antibody binding contacting a crossmatching solid phase support coated with the transplant donor cell membrane fragments with transplant recipient serum, plasma, urine or blood, or for detecting transplant recipient immune cell binding contacting a crossmatching solid phase support coated with the transplant donor cell membrane fragments with transplant recipient blood or urine, and detecting transplant recipient antibody binding or transplant recipient immune cell binding to the crossmatching solid phase support, wherein binding indicates a lack of compatibility and wherein a lack of binding indicates compatibility.

6. A method of detecting a transplant recipient immune reaction to a transplant from a transplant donor comprising: for detecting transplant recipient antibody binding contacting a crossmatching solid phase support coated with transplant donor cell membrane fragments with transplant recipient serum, plasma, urine, or blood, or for detecting transplant recipient immune cellbinding contacting a crossmatching solid phase support coated with the transplant donor cell membrane fragments with transplant recipient blood or urine, and detecting transplant recipient antibody binding or transplant recipient immune cell binding to the crossmatching solid phase support, wherein binding indicates a transplant recipient immune reaction to the transplant.

7. The method of any of claims 4-6, wherein transplant recipient serum antibody binding is detected by fluorescence.

8. The support, kit or method of any preceding claim, wherein the solid phase support comprises microparticle beads.

9. The support, kit or method of any preceding claim, wherein the transplant donor is an allotransplant donor.

10. The support, kit or method of any preceding claim, wherein the allotransplant donor is human.11 . The support, kit or method of any preceding claim, wherein the transplant donor is a xenotransplant donor.

12. The support, kit or method of any preceding claim, wherein the xenotransplant donor is a pig.

13. The support, kit or method of any preceding claim, wherein the transplant donor cell membrane fragments are isolated from one or more of a transplant donor bodily fluid, a transplant donor cell or a perfusate of the transplant.

14. The support, kit or method of claim 13, wherein the transplant donor cell membrane fragments are isolated from a transplant donor bodily fluid.

15. The support, kit or method of claim 14, wherein the transplant donor bodily fluid is one or more of serum, plasma or urine.

16. The support, kit or method of claim 13, wherein the transplant donor cell membrane fragments are isolated from a transplant donor cell.

17. The support, kit or method of claim 16, wherein the transplant donor cell is a cell of the transplant.

18. The support, kit or method of claim 13, wherein the transplant donor cell membrane fragments are isolated from a perfusate of the transplant.

19. A method of detecting one or more viral, bacterial or fungal infections of a transplant donor or transplant therefrom comprising: contacting a solid phase support coated with transplant donor cell membranes with a viral-, bacterial- or fungal-specific reagent and detecting binding of the reagent.

20. The method of any of claims 4-18 wherein negative, blocking cell membrane fragments are included with the transplant donor cell membrane fragments.

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