EP0759763A1 - Prevention du rejet hyperaigu dans les greffes d'organes porc-primate - Google Patents

Prevention du rejet hyperaigu dans les greffes d'organes porc-primate

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Publication number
EP0759763A1
EP0759763A1 EP95919185A EP95919185A EP0759763A1 EP 0759763 A1 EP0759763 A1 EP 0759763A1 EP 95919185 A EP95919185 A EP 95919185A EP 95919185 A EP95919185 A EP 95919185A EP 0759763 A1 EP0759763 A1 EP 0759763A1
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EP
European Patent Office
Prior art keywords
plasma
pig
human
column
antibodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95919185A
Other languages
German (de)
English (en)
Other versions
EP0759763A4 (fr
Inventor
Joseph R. Leventhal
Jutta MÜLLER-DERLICH
Wolfgang BÖHM
Franz A. Bieber
Marc Salit
Reiner Spaethe
R.Morton Bolman, Iii
Arthur J. Matas
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baxter International Inc
University of Minnesota
Original Assignee
Baxter International Inc
University of Minnesota
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Application filed by Baxter International Inc, University of Minnesota filed Critical Baxter International Inc
Publication of EP0759763A1 publication Critical patent/EP0759763A1/fr
Publication of EP0759763A4 publication Critical patent/EP0759763A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
    • A61M1/3489—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents by biological cells, e.g. bioreactor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2202/00—Special media to be introduced, removed or treated
    • A61M2202/04—Liquids
    • A61M2202/0413—Blood
    • A61M2202/0415—Plasma
    • A61M2202/0417—Immunoglobulin

Definitions

  • the present invention is in the general field of transplant of organs from discordant species to primate recipients, including humans. Specifically, the invention relates to methods for preventing the hyperacute rejection reaction by passing the plasma of the recipient over a column which removes anti-pig antibodies. The invention is also in the general field of processed human plasma.
  • organ shortage could be solved.
  • many more patients could receive organ transplants before their condition deteriorated to the extreme point that is presently required to receive a human organ transplant.
  • hyperacute rejection reaction the immunological reaction known as the "hyperacute rejection reaction" wherein the recipient's own immune system attacks and destroys the transplanted organ within minutes to hours, typically within 48 hours after transplant. Even when the recipient receives immunosuppressive therapy, hyperacute rejection is not ameliorated.
  • the hyperacute rejection reaction could be expected to be somewhat diminished if the organ is from a closely related "concordant" species, such as a non- human primate.
  • a closely related "concordant" species such as a non- human primate.
  • non-human primates of a suitable size namely the larger apes, are mostly members of endangered species, and thus their use is limited.
  • these animals are not well suited to breeding in captivity on a large scale.
  • a more distantly related "discordant" species such as the pig, would be ideal for organ availability.
  • the pig is widely bred for food.
  • Certain breeders have experience in breeding pathogen-free herds for current medical uses, particularly as a source for transplantable heart valves.
  • Pig heart valves are treated with fixatives and undergo other procedures which abolish their immunological reactivity. Such procedures would destroy the functionality of whole organs such as heart or kidney.
  • the use of the pig as an organ donor will not be possible unless a method is found to greatly reduce or prevent the hyperacute rejection reaction.
  • the hyperacute rejection reaction is thought to occur as a result of pre-formed antibodies in the blood of the recipient which recognize and bind to xeno-antigens in the tissue of the donor organ once the transplanted organ is in place and is perfused with the blood of the recipient.
  • preformed antibodies in the recipient's blood are also known as "human heterophile antibodies", “natural antibodies” or "xenoreactive antibodies”.
  • the xenoreactive antibodies bind to endothelial cells of the donor organ blood vessels, they stimulate the deposition of complement proteins, which also originate from the blood of the recipient.
  • Xenoreactive antibody/complement deposition is thought to initiate the "classical" pathway of complement action, which ultimately leads to disruption of the endothelial cell lining of the blood vessels of the donor organ (In: Immunology. Eds: Roitt, I.M., et al, J.B. Lippincott Co, Philadelphia, 1989, Chapter 13, pages 13.1- 13.16).
  • the hyperacute rejection reaction results in a necrotic donor organ within minutes to hours after xenotransplant.
  • necrosis of the donor organ results from "activation" of its endothelial cells, which in turn leads to interstitial hemorrhage, inflammation, edema, and small vessel thrombosis (Platt, J.L., et al., Immunology Today 11:450- 456, 1990).
  • ABO-mismatched organs from human donor to human recipient.
  • a recipient with type O blood for instance, is expected to have preformed anti-A and anti-B antibodies in his blood.
  • every attempt is made to locate a donor organ well-matched for both ABO blood type and HLA haplotype.
  • an ABO- mismatched organ from an HLA-matched donor is the best or only organ available for transplant.
  • pre-formed anti-A/anti-B antibodies were removed from the recipients' blood using extracorporeal perfusion of the recipients' plasma over synthetic A/B blood group antigens covalently linked to silica.
  • carbohydrate antigens in xeno-grafts have been proposed as the targets of human pre-formed xenoreactive antibodies (Laus, R. , et al., Int. Archs.
  • the recipient's blood could be perfused through an organ of the proposed donor species prior to transplantation of a "fresh" organ.
  • a "column” could be constructed of isolated pig endothelial cells, for instance. The recipient's plasma could be perfused over this column to remove anti-pig antibodies prior to transplantation.
  • Irnmunoglobulin can be removed non-specifically by plasmapheresis.
  • Conventional plasmapheresis, or plasma exchange results in loss of blood volume, recipient sensitization, and activation of the complement and clotting systems.
  • These side effects of plasmapheresis are somewhat alleviated by volume replacement with pooled preparations of fresh frozen plasma, human albumin, irnmunoglobulin, and/or a type of bulking agent such as starch.
  • Coagulation factors, platelets, and anti- thrombotic factors must also be replaced. This treatment carries the risk of virus transfer from the pooled human preparations, as well as the risk of anaphylactic reaction to foreign substances.
  • Plasmapheresis does not appear to be either practical or safe for immediate pre-transplant or post-transplant use because of the risk of excessive bleeding.
  • Non-specific antibody removal for indications other than organ transplant, mainly for the treatment of autoimmune disease.
  • One method for non-specific antibody removal involves perfusing the autoimmune subject's plasma over a column coupled with Protein A from Staphylococcus aureus.
  • Protein A a major component of the cell wall of S. aureus.
  • the Protein-A coupled columns have also been used for the non-specific removal of anti-HLA antibodies from hypersensitized patients who are in need of a kidney transplant. These patients typically suffer from idiopathic nephrotic syndrome (INS) . They commonly suffer a relapse of INS soon after transplantation of even the most well-matched donor kidney, thus practically excluding them from the possibility of having any kind of currently available kidney transplant.
  • INS idiopathic nephrotic syndrome
  • the hyperacute rejection phenomenon could be prevented in the early days following transplant, there is a good chance that the patient's immune system would undergo a process of "accommodation" which would diminish or eliminate the reaction between the xenoreactive antibodies and complement of the patient with the endothelial cells of the donor organ (Bach, F.H. et al., supra) . Then, a conventional immunosuppressive regimen could maintain the compatiblity of the patient and the xeno-graft to allow long-term survival of the graft even after the return of antibodies in the recipient's blood. What is needed is a method to prevent or ameliorate the severity of the hyperacute rejection reaction in the transplantation to a human recipient of an immunologically mismatched allograft or an organ from a discordant species such as the pig.
  • the invention provides a method for preventing or ameliorating the hyperacute rejection reaction which would normally occur upon transplant of a pig organ to a primate subject.
  • the method of the invention is suitable for use in human subjects, because all materials used are sterile and pyrogen-free.
  • the method requires the use of a sterile and pyrogen-free column coupled to protein which binds to human immunoglobulin, perfusion of the recipient's plasma over the column, thereby removing immunoglobulin, and replacement of the immunoglobulin-depleted plasma to the primate recipient.
  • the primate subject receives a pig organ transplant. The method can be repeated several times pre- and post- transplant.
  • the invention also provides immunoglobulin-depleted human plasma which is useful for plasma replacement to recipients of pig organ transplants.
  • the immunoglobulin-binding protein coupled to the column is selected from a group consisting of Staphylococcus aureus protein A, Streptococcus protein G, and anti-human immunoglobulin antibodies.
  • the coupled antibodies can bind specifically to human IgG, to human IgM, or to a mix of human immunoglobulin classes and sub-classes.
  • the coupled antibodies can be polyclonal or monoclonal.
  • the coupled antibodies may also be recombinantly produced as double-chain or single-chain antibodies which bind to human immunoglobulin.
  • Figure 1 illustrates the aseptic connections from the subject's bloodstream, to the plasmapheresis machine, over the column, and back to the subject.
  • Figures 2A and 2B show the reduction in anti-pig immunoglobulins in baboon subjects after treatment with the method of the invention.
  • Transplantation of a pig organ to a non-human or human primate recipient will normally lead to hyperacute rejection of the organ within a few minutes to 48 hours.
  • the principle clinical features of hyperacute rejection are a sudden drop in urine output accompanied by a sharp increase in serum creatinine levels.
  • the hyperacute rejection reaction is thought to be largely due to preformed antibodies in the blood of the primate recipient which bind to xeno-antigens on the endothelial cells of the blood vessels of the transplanted organ, thereby activating complement, disrupting the endothelial cell lining, and causing necrosis of the donor organ.
  • the present invention provides a method to prevent or ameliorate the hyperacute rejection reaction by separating the primate recipient's plasma from cells and platelets, perfusing the recipient's plasma over a sterile and pyrogen-free column coupled to protein which binds to human immunoglobulin, thereby removing immunoglobulin from the recipient's plasma.
  • the recipient's immunoglobulin- depleted plasma is remixed with his blood cells and platelets, and the reconstituted blood is returned to the recipient. This procedure may be repeated several times before and after transplant of the organ.
  • the present invention provides a method to prevent or ameliorate the hyperacute rejection reaction without the need for remedies specifically directed to the complement system such as the administration of cobra venom factor or the use of transgenic pig organs.
  • the protein coupled to the sterile and pyrogen-free column is selected from the group consisting of Staphylococcus aureus Protein A, Streptococcus Protein G, and antibodies which bind human immunoglobulin.
  • the primate recipient's separated plasma is continuously passaged over the sterile and pyrogen-free protein-coupled column, and the column effluent is returned to the subject for at least 2-3 plasma volumes.
  • This treatment preferably results in a significant reduction of total IgG of at least about 75% to about 99%, more preferably at least about 85% to about 99% or greater.
  • This treatment also preferably results in a significant reduction of total IgM of at least about 50% to about 99%, more preferably at least about 60% to about 99% or greater. It is expected that the treatment can reduce total IgM and/or IgG below the sensitivity level of a standard immunoglobulin assay, thus the assay results may indicate no detectable IgM and/or IgG remaining after treatment.
  • the intended donor organ is of pig origin, it is also important to monitor the reduction in anti-pig antibodies in the recipient's plasma.
  • the reduction in anti-pig specific immunoglobulin is measured in a porcine endothelial cell enzyme-linked immunosorbent assay (ELISA) .
  • ELISA porcine endothelial cell enzyme-linked immunosorbent assay
  • the column procedure results in a 50 to 500 fold or greater reduction in total anti-pig immunoglobulin in the subject's plasma.
  • the column procedure results in no significant reduction in plasma fibrinogen, Factor V or Factor VII levels.
  • the protein-coupled column binds both human IgG and IgM.
  • the xeno-reactive immunoglobulins which mediate the hyperacute rejection reaction are primarily, if not solely, of the IgM class (Platt, J.L., et al., Immunology Today 11:450-457, 1990).
  • the present inventors consider it likely that a significant proportion of anti-pig antibodies are of the IgG class, particularly in human subjects, because of evidence of human IgG binding to pig endothelial cells in an ELISA assay (see Table 2 in Example 1 below) .
  • the sterile and pyrogen-free column is preferably coupled to antibodies which bind to both human IgG and human IgM.
  • the coupled antibodies can be pooled polyclonal antibodies raised in animals such as sheep immunized with pooled human immunoglobulins plus adjuvant.
  • the coupled antibodies bind to human light chains such as lambda and kappa light chains, and thereby recognize and bind to both human IgG and IgM. It is preferable that the coupled antibodies also bind to IgG heavy chain.
  • the coupled antibodies may be monoclonal or recombinant antibodies which bind to human immunoglobulins.
  • the column matrix material can be coupled to Staphylococcus aureus protein A, which binds certain subclasses of human IgG.
  • the column matrix material can also be coupled to Streptococcus Protein G, which binds a different set of human IgG subclasses.
  • Streptococcus Protein G which binds a different set of human IgG subclasses.
  • a mixture of Proteins A and G could be coupled to a single column to effect adequate binding to and removal of human IgG. It is expected that Protein A/G columns could be used in conjunction with columns that also bind human IgM in order to prevent or ameliorate the hyperacute rejection reaction.
  • Example 1 For production of antibodies and virus inactivation, Example 1, pages 15-19.
  • Example 4 For preparation of sterile and pyrogen-free column matrix, Example 4, pages 26-27.
  • Example 5 For activation of column matrix material and coupling of protein thereto, Example 5, pages 27-30. For finishing of final column product, Example 6, page 30.
  • the passage of at least 2-3 volumes of the subject's plasma over the column is repeated once per day on at least 3 different days prior to transplantation.
  • the column procedure can also be repeated on the day of, and just prior to, transplantation.
  • Post-transplantation in the case of a human recipient, the column procedure is preferably repeated on at least 2 separate days following the day of transplant. This procedural preference is based on the observation that immunoglobulin titers rise rapidly in humans due to replenishment of the vascular titer from the interstitial space (see Example 3 below) .
  • the decision to repeat the column procedure post-transplant can be supported by monitoring of kidney function, anti-pig antibody titers, serum creatinine, and biopsy of the grafted organ.
  • the column procedure should be repeated if there is a rise in xeno-reactive (anti-pig) antibody titer in conjunction with a decrease in urine output, an unexplained rise in serum creatinine (i.e. not a drug side- effect) , and/or a graft biopsy showing signs of hyperacute rejection.
  • the present invention also provides human immunoglobulin-depleted plasma prepared by passing normal donated human plasma over the immunoglobulin-binding columns according to the method of the invention.
  • Normal donated pooled fresh-frozen and thawed human plasma could be passed many times over the presently used therapeutic columns, thereby removing more than 99% of its total IgG and IgM content.
  • a large column can be constructed, which can remove more than 99% of the total IgG and IgM in one pass. Normal ranges of total IgG (640 - 1700 mg/dL) and total IgM (50 - 3500 mg/dL) have been reported for normal, pooled, fresh-frozen human plasma.
  • the column treatment can provide a normal, pooled human plasma product containing preferably no more than 65 mg/dL total IgG, no more than 6 mg/dL total IgM, at least 50 fold less anti-pig IgG, and at least 10 fold less anti-pig IgM, as compared with a normal human plasma pool.
  • the immunoglobulin-depleted plasma contains at least 200 fold less anti-pig IgG and at least 25 fold less anti-pig IgM.
  • the immunoglobulin-depleted plasma can then be fresh-frozen and stored for future use for the treatment of organ transplant patients or any other patients in need of immunoglobulin-depleted plasma.
  • a primate subject can be effectively and safely treated using the column procedure to remove xeno-reactive and anti-pig antibodies, both before and after transplant.
  • the term "primate subject” includes human subjects in need of organ transplant.
  • use of the column procedure of the invention can prevent or ameliorate symptoms of hyperacute rejection of a transplanted pig organ such as a kidney or heart. Prolonged survival of a pig kidney transplanted to a primate subject was observed, and histological analysis of the grafted kidney showed no signs of hyperacute rejection.
  • follow-up treatment of a human recipient of a pig organ will include standard long-term immunosuppressive therapy, such as administration of a steroid such as PrednisoneTM, anti-proliferative drugs such as AzothioprineTM, and/or anti-T-lymphocytic drugs such as cyclosporine.
  • a steroid such as PrednisoneTM
  • anti-proliferative drugs such as AzothioprineTM
  • anti-T-lymphocytic drugs such as cyclosporine.
  • the column treatment of the present invention could be used in conjunction with transplant of a transgenic pig organ which has been genetically altered to eliminate certain xeno- antigens or to express human complement inhibitors.
  • the column procedure of the present invention will allow a pig organ transplant to survive the initial post ⁇ operative period during which hyperacute rejection would otherwise occur, and thereby make pig to human organ transplantation feasible.
  • EXAMPLE 1 Removal of immunoglobulin, including anti-pig antibodies. from human and baboon plasma in vitro.
  • the first column was a sterile and pyrogen-free column coupled to anti-human Ig polyclonal antibodies raised in sheep by immunization with pooled human IgG and adjuvant according to the methods described in Example 1 of U.S. patent application serial number 08/242,215 (supra) .
  • the second column was coupled to anti-human IgM polyclonal antibodies raised in sheep immunized with a pooled human IgM fraction plus adjuvant.
  • Anti-human Ig column Human fresh frozen plasma (FFP, 500ml) or baboon plasma (300ml) were passed 2x over this column. Total IgG and total IgM in the plasma samples were measured by standard immunoglobulin nephelometry (University of Minnesota, clinical laboratory) . This column treatment reduced total IgG in the plasma by 95-99% (see Table 1 below) . The anti-Ig column treatment also reduced total plasma IgM by 62-85%. The effect on IgM was explained by the column-coupled antibodies binding to lambda and kappa light chains, thus binding both IgG and IgM classes.
  • Anti-human IgM column Human FFP (300ml) or baboon plasma (400 ml) were passed 2x over an anti-IgM column. This column lowered total plasma IgM by 80 - 83%. No specific removal of IgG occurred. Table 1
  • porcine endothelial cell ELISA as described in Platt, J.L., et al. Transplantation 49:1000-1001, 1990. Briefly, porcine aortic endothelial cells were isolated and cultured in Dulbecco's modified Eagle's medium containing 20% fetal calf serum (Ryan, U.S., et al., J Tissue Cult Methods 1986; 3) . The cells were grown to confluence in 96-well microtiter plates (NuncTM).
  • the secondary antibody for the IgM ELISA was goat anti-human IgM ( ⁇ ; Zymed) conjugated to alkaline phosphatase; the secondary antibody for the IgG ELISA was goat anti-human IgG similarly conjugated (Sigma) .
  • the marker reaction was developed in diethanolamine (0.1M with 0.5 X 10 " M MgCl 2 ) with phosphatase substrate (lmg/ml p- nitrophenyl phosphate) .
  • Each baboon subject was prepared for plasma treatment or surgery by initial induction of anesthesia with ketamine HC1 (KetalarTM) and pentathol; the subject was then maintained during all procedures under inhalation anesthesia using ForaneTM gas.
  • the subject's bloodstream was aseptically connected via intravenous puncture to a plasmapheresis machine (Baxter Autopheresis C ⁇ ) which separated the plasma from the cells and platelets ( Figure 2) .
  • the plasma was aseptically conducted to and passed over a sterile and pyrogen-free column coupled to anti-human Ig (binds to lambda and kappa light chains) (Ig-Therasorb ⁇ , Baxter Immunotherapy, Kunststoff) .
  • the plasma effluent from the column was collected in a sterile 125 ml ViaflexTM bag (Baxter Travenol) from which it was pumped and remixed on ⁇ line with the subject's blood cells and platelets, and reinfused to the subject. This procedure was carried out continuously for 2 plasma volumes. A maximum plasma flow of 40cc/min was well tolerated by the subject.
  • the entire column procedure lasted 1.5-2.5 hours. The column procedure was repeated on 3 additional days.
  • Total plasma IgG and IgM were measured as described above.
  • the anti-human IgM column was used in one in vivo procedure.
  • Total serum IgM ws reduced by 91% in one baboon by the IgM column procedure when used for two consecutive days following plasmapheresis.
  • Total IgG was not significantly reduced and indeed rose during this period; anti-pig IgG also was not reduced by the IgM column procedure.
  • the use of the IgM column reduced baboon anti- pig IgM antibodies to less than 1% of the original value after each column treatment.
  • the titer of baboon anti- pig IgM had risen to about 30% of the original value. Table 3
  • EXAMPLE 3 Removal of immunoglobulin from the blood of human subjects.
  • Anti-human immunoglobulin coupled columns were used for the removal of immunoglobulin from the blood of human patients suffering from idiopathic thrombocytopenic purpura (ITP) , systemic lupus erythematosus (SLE) , and vasculitis. These procedures were part of controlled clinical trials carried out in Europe for the treatment of ITP patients whose conditions were refractory to conventional treatments.
  • ITP idiopathic thrombocytopenic purpura
  • SLE systemic lupus erythematosus
  • FIG. 1 The overall system for immunoglobulin depletion is shown in Figure 1. Briefly, the tubing system of the primary separation system was first filled with sterile 0.9% NaCl. Two anti-human Ig columns (Ig-TherasorbTM , Baxter, Immunotherapy Division, Europe) were connected with the primary separation system as shown ( Figure 1) . All tubing connections were made under aseptic conditions.
  • each column was rinsed before its first use with 5 liters sterile 0.9% NaCl solution, at a flow rate of 90-100 ml/min. For each subsequent use, it was sufficient to rinse each column with 2 liters of the sterile solution, at the same flow rate.
  • the appropriate canulae were connected to the left and right cubital veins of the patient. Blood samples were taken. The connection to the blood cell separator was put in place. Anticoagulation was accomplished with either heparin or citrate (ACD-A or ACD-B) . When citrate was the anti ⁇ coagulant, during the first half of the procedure, the citrate was used at a dilution of 1:22 to 1:18. In the second therapy phase, the dilution utilized was 1:12 to 1:8. Symptoms of hypocalcemia were monitored (paraesthesia in fingers or lips) , and the administration of citrate was diminished accordingly. Calcium tablets were kept at hand in case of frank hypocalcemia.
  • the blood cell separator was filled with the patient's blood.
  • the blood flow rate was kept between 50-90 ml/min.
  • the liquid level was maintained at about 0.8 cm over the SepharoseTM in the column.
  • the cell-free plasma was directed through the tubing system over the first column. It was important to keep the flow rate even and to monitor the plasma level over the SepharoseTM in the column. A higher plasma level was undesirable, because it would have led to a higher volume burden for the patient, and plasma loss due to plasma retention in the column.
  • the column was loaded with as much plasma as possible during 15 minutes. Thereafter, the plasma flow was switched to the second column, which was likewise filled with as much plasma as possible in 15 minutes.
  • the plasma in the first column was flushed out using sterile 0.9% NaCl at the plasma flow rate.
  • One column volume of plasma was returned to the patient together with the blood cells which had been removed.
  • the first column was regenerated as follows: (1) A further rinse with 50 ml 0.9 % NaCl at a flow rate of 100 ml/min; (2) Desorption of the bound immunoglobulin with one column volume of sterile 0.2 M glycine/HCl buffer, pH 2.8. The controller of the device prevented contact between this solution and the patient. The desorbed immunoglobulin was discarded. (3) Neutralization with one column volume of sterile PBS, pH 7.4. Testing of the neutralization using lack us paper. (4) Rinsing out of the PBS with at least one column volume of sterile 0.9% Nacl. The column was then ready for the next round of adsorption.
  • the filling of the columns was again automatically switched. This procedure was repeated as many times as necessary to process the desired volume of plasma.
  • the number of cycles used was chosen by the attending physician, according to the condition and needs of the patient. So far, within the inventors' clinical experience, it has been possible to process up to 3.5 times the extracorporeal volume of a given patient during one column procedure. Moreover, the number of cycles used was not limited by the binding capacity of the columns, but rather by the needs of the individual patient.
  • the patient in Table 4 received the above described immunoglobulin-depleting column treatment every second day for about 3.5 hours, thereby processing about 8.3 liters of plasma per treatment.
  • the results in Table 4 illustrate the rationale for repeated column treatments. According to literature reports, immunoglobulin is redistributed from the extravascular compartment to the intravascular compartment within 24 hours after removal of plasma immunoglobulin. Therefore, successive column treatments repeated after 24-48 hours are most effective in removing the human subject's body stores of immunoglobulin.
  • Plasma loss was typically low (4- 8%) , and no plasma replacement was required.
  • the column procedure of the present invention can be used safely for the removal of immunoglobulin from the blood of human patients.
  • EXAMPLE 4 Transplant of pig kidney to primate recipient; prevention of hyperacute reaction.
  • the baboons underwent splenectomy and placement of intravenous lines.
  • Four days prior to transplant (day -4) , the subjects were administered deoxyspergualine (DSPG) , an anti-B cell humoral immunosuppressant, at 4 mg/kg i.v. over 3 hours.
  • DSPG deoxyspergualine
  • an anti-B cell humoral immunosuppressant at 4 mg/kg i.v. over 3 hours.
  • Also administered on day -4 were cyclophosphamid (CytoxanTM) at 20 mg/kg i.v., cefuroxime (ZinacefTM, an antibiotic) at 8cc i.v. gl2 hrs, and nystatin popsicles (anti-fungal) at ql2.
  • Kidneys were harvested from normal pigs which were size- matched for the baboon recipient; the harvested kidneys were flushed with sterile preservation solution and used immediately.
  • Figures 2A and 2B show the reduction in baboon anti-pig titers after the indicated treatments. Both baboon subjects had low starting levels of anti-pig IgG, and much greater levels of anti-pig IgM. (It has been observed that human plasma, in general, has a higher titer of anti-pig IgG than does baboon plasma.) The Ig-coupled column treatments maintained a dramatically reduced titer of anti- pig IgM antibodies for the duration of the experiments.
  • the column treatment of the present invention successfully prevented the hyperacute rejection reaction which would normally follow within minutes to 48 hours after transplant of a pig kidney to a primate subject.
  • the initial plasmapheresis treatment used in these experiments functioned to spare the limited binding capacity of the few columns available at the time for experimental use.
  • the initial plasmapheresis treatment diminished the number of times a baboon subject would have to be anesthetized and subjected to the column treatment; the duration of an individual column treatment was also diminished thereby.
  • a model for pig heart transplant was developed in which a fresh, beating pig heart was perfused with either autologous pig blood or human blood.
  • Plasma obtained from healthy human volunteers was passed twice over a column containing polyclonal anti-human IgG (gamma and light chain specific) conjugated to Sepharose w (Ig-TherasorbTM, Baxter), resulting in a 98.1% reduction in total IgG and 88.4% reduction in total IgM.
  • Total potential complement activity as measured by C H 50 assay revealed a 70% reduction resulting from column treatment, as compared to a 40% reduction in untreated controls from the cardiopulmonary bypass circuit in each group taken immediately prior to reperfusion.
  • Ig-TherasorbTM column treatment of human blood prevented the hyperacute rejection reaction which normally occurs when a pig heart is perfused with human blood.

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Abstract

Procédé permettant de prévenir ou de limiter une réaction de rejet hyperaigu qui se produirait normalement après une greffe sur un récepteur primate, y compris humain, d'un organe prélevé sur un porc. Normalement, les anticorps anti-porc dans le sang du récepteur se lient aux antigènes de porc présents sur les cellules endothéliales de l'organe greffé, et déclenchent une série de réactions complémentaires en cascade qui provoquent la nécrose de l'organe dans les minutes ou les heures qui suivent. Le procédé décrit consiste à faire passer au moins 2 à 3 volumes de plasma du récepteur primate sur une colonne stérile et exempte de pyrogène couplée à une protéine qui se lie à l'immunoglobuline du plasma du récepteur et l'élimine, et à procéder ensuite à la greffe de l'organe de porc dans le récepteur primate. Ce traitement, que l'on répète de préférence pendant plusieurs jours avant et après la greffe, prévient ou limite la réaction de rejet hyperaigu, en éliminant les anticorps anti-porc du plasma du récepteur. Ce procédé permet d'éliminer plus de 99 % de l'IgG totale et plus de 99 % de l'IgM totale du récepteur. Il réduit également de 50 à 500 fois l'immunoglobuline anti-porc, ainsi que de 15 à 60 % l'activité complémentaire potentielle. Cette invention permet aussi d'obtenir du plasma humain dépourvu d'immunoglobuline pouvant être perfusé dans le récepteur humain d'une greffe d'organe de porc.
EP95919185A 1994-05-13 1995-05-12 Prevention du rejet hyperaigu dans les greffes d'organes porc-primate Withdrawn EP0759763A4 (fr)

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US24220894A 1994-05-13 1994-05-13
US242208 1994-05-13
PCT/US1995/006028 WO1995031209A1 (fr) 1994-05-13 1995-05-12 Prevention du rejet hyperaigu dans les greffes d'organes porc-primate

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WO1995031727A1 (fr) * 1994-05-13 1995-11-23 Therasorb Medizinische Systeme Gmbh Colonne sterile et apyrogene couplee a une proteine en vue de la fixation et de l'extraction de substances donnees du sang
EP0775493A3 (fr) * 1995-11-22 1998-08-12 Baxter International Inc. Perfusion extracorporelle d'organe xénogénique après déplétion d'anticorps par immunoaphérèse
US6464976B1 (en) * 1999-09-07 2002-10-15 Canji, Inc. Methods and compositions for reducing immune response
AU2013204469B2 (en) * 2008-12-19 2015-04-09 Baxalta GmbH Systems and methods for obtaining immunoglobulin from blood
US20100158893A1 (en) * 2008-12-19 2010-06-24 Baxter International Inc. Systems and methods for obtaining immunoglobulin from blood
US20120219633A1 (en) * 2011-02-28 2012-08-30 Pall Corporation Removal of immunoglobulins and leukocytes from biological fluids

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WO1995031209A1 (fr) 1995-11-23
AU2514595A (en) 1995-12-05

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