WO1995031209A1 - Prevention of hyperacute rejection in pig to primate organ transplant - Google Patents

Abstract

The invention provides a method to prevent or ameliorate a hyperacute rejection reaction which would normally occur after transplant of a pig organ to a primate recipient, including a human recipient. Normally, anti-pig antibodies in the blood of the recipient will bind to pig antigens on the endothelial cells of the grafted pig organ, and activate the complement cascade causing necrosis of the pig organ within minutes to hours. The invention method involves passing at least 2-3 plasma volumes of the primate recipient's plasma over a sterile and pyrogen-free column coupled to protein which binds to and thereby removes immunoglobulin from the recipient's plasma, and then transplanting a pig organ to the primate recipient. The column treatment is preferably repeated on several days before and after transplant, and thereby prevents or ameliorates the hyperacute rejection reaction by removing anti-pig antibodies from the recipient's plasma. The method can remove greater than 99 % of the primate recipient's total IgG and greater than 99 % of the recipient's total IgM. The method also effects a 50-500 fold reduction in anti-pig immunoglobulin, and a 15-60 % reduction in potential complement activity. This invention also provides immunoglobulin-depleted human plasma suitable for infusion to a human recipient of a pig organ transplant.

Description

PREVENTION OF HYPERACUTE REJECTION IN PIG TO PRIMATE ORGAN TRANSPLANT

Technical Field

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.

Background There is a great need world-wide for transplantable organs such as heart, kidney, liver, and pancreas. In the case of a single kidney or a partial pancreas transplant, it is sometimes possible to locate a living donor with immunological markers compatible with the transplant recipient, although organ donation by a living donor involves great risk and possible deleterious health effects for the donor. In all other cases, the organ donation must come from a high quality, heart-beating human cadaver, and again there must be a good immunological match between the donor and the recipient. Patients in need of an organ transplant often must be on a waiting list for longer than one year, and many patients die before a suitable organ becomes available.

If it were possible to use animals as organ donors for humans, the organ shortage could be solved. Moreover, 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.

The use of an animal organ for transplant to a human recipient is limited by 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.

In using an animal organ, 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. However, non-human primates of a suitable size, namely the larger apes, are mostly members of endangered species, and thus their use is limited. Moreover, 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, however, 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. These preformed antibodies in the recipient's blood are also known as "human heterophile antibodies", "natural antibodies" or "xenoreactive antibodies". When 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. It has been hypothesized that 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).

An analogy to the hyperacute rejection reaction can be seen in the transplant of 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. Usually, every attempt is made to locate a donor organ well-matched for both ABO blood type and HLA haplotype. However, in certain situations, an ABO- mismatched organ from an HLA-matched donor is the best or only organ available for transplant. In attempts to prevent hyperacute rejection when ABO-mismatched organs were transplanted, 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. Successful kidney and bone marrow transplants were reported using this procedure (Bannett, A.D. , et al., Transplant. Proc. 1987 XIX:4543-4546; Bensinger, W.I., et al.. Transplantation 1982 33:427-429; US Patent No: 4,137,401; European patent no: 89311540.2).

It was found that certain pre-formed antibodies in humans bind to carbohydrate residues on foreign antigens. In particular, the antigenic blood group substances A and B bear trisaccharides, which have been chemically synthesized. In a baboon/baboon model for ABO-mismatched heart transplant, the recipient was first administered intravenous A or B trisaccharide on the theory that the trisaccharide would form a "neutralizing" complex with the preformed anti-A or anti-B antibody, thereby preventing hyperacute rejection. When continuous A/B antigen treatment was combined with high-dose immunosuppression, hyperacute rejection was ameliorated in the majority of experiments (Cooper, D.K.C., et al., Transplant. Proc. 24:566-571, 1992).

Other types of carbohydrate antigens in xeno-grafts have been proposed as the targets of human pre-formed xenoreactive antibodies (Laus, R. , et al., Int. Archs.

Allergy appl. I mun. 85:201-207, 1988; Platt, J.L. , et al.,

Transplantation 50:817-822, 1990). Sandrin, et al. reported that most human xeno-reactive antibodies reacted with Gal(αl-3)Gal (Immunology 90:111391-111395, 1993;

Sandrin, et al., Tranplantation Proc. 25:2917-2918, 1993;

Vaughn, et al., Transplantation Proc. 25:2919-2920, 1993).

Sandrin, et al., also reported that, in their studies, the pig-reactive antibodies in human serum were of the IgM type, and clearly not IgG (see Sandrin, et al.,

Transplantation Proc. 1993 supra, p. 2917, 4th paragraph).

In a strategy specifically directed to the carbohydrate xeno -antigen Gal( l-3)Gal, it was proposed that transgenic pigs could be produced which are lacking in the enzyme which is rate-limiting for the synthesis of Gal(αl-3)Gal (Sandrin, et al. Immunology 1993 supra) . Of course, this strategy is predicated on the belief that Gal(αl-3)Gal is the major or only xeno-antigen responsible for hyperacute rejection of pig organs.

Several procedures have been proposed to remove xenoreactive antibodies from the blood of a recipient. For instance, the recipient's blood could be perfused through an organ of the proposed donor species prior to transplantation of a "fresh" organ. Alternatively, if a pig is to be the donor species, 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. (Bach, F.H.,, IN: XENOTRANSPLANTATION. Eds: Cooper, D.K.C., et al. Springer-Verlag, 1991, Chapter 6) .

It has also been proposed that antibodies be removed non- specifically from the recipient's blood prior to xeno- transplantation, in the hope that xenoreactive antibodies will be removed along with the rest.

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.

Repeated plasma exchange combined with splenectomy was used to prepare a baboon for transplantation of a pig kidney; one xenotransplant was reported to function for 22 days (Alexandre, G.P.J., et al., In: Xenograft 25, Ed: Hardy, M.A. , New York Elsevier Science Publishers 1989, p. 259).

There has been considerable interest in 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. has a high affinity for a portion of the Fc-region of sub-classes 1, 2, and 4 of irnmunoglobulin G (IgG.,, IgG₂, IgG₄) (Dantal, J. , et al., New England J. Med.550:7-14 , 1994; Nilsson, I.M., et al., Blood 58:38-44, 1981; Palmer, A., et al. , The Lancet January 7, 1989, pp.10-12). 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. The efficacy of the Protein A column treatment in several INS patients after kidney transplant was reported (Dantal,et al, supra; Palmer, et al., supra). The Protein A column treatment has also been reported in a pig-to-dog xenotransplantation (Shapiro, et al., J Invest Surg 3:39- 49, 1990).

Autoimmune diseases have also been treated by removal of a significant portion of the patient's immunoglobulins using a column coupled to antibodies directed against human irnmunoglobulin. Use of such columns in the treatment of auto-immune disease has been reported as follows: Muller- Derlich, J. , et al., Congress in Monte Carlo, April, 1992; Muller-Derlich, J. , et al., IX. Congress of the International Society for Artificial Organs July 1993; Muller-Derlich, J. , et al., Ninth Scientific Congress of the European Society for Haemapheresis in Association with the British Blood Transfusion Society. September 1993; Muller-Derlich, J. , et al. XXIV. Tagung der Gesellschaft fur Immunologie September/October 1993; Muller-Derlich, J. , et al. Conference on Immunoglobulins Intravenous IglV. Lisbon, November 1993.

Returning now to the subject of xenotransplant, it has been proposed that, even if xenoreactive antibodies were removed from the recipient's blood, it would still be necessary to inactivate or remove complement to prevent a hyperacute rejection reaction after a mismatched allograft or a xeno- graft from a discordant species such as pig. The administration of cobra venom factor can accomplish the depletion of complement activity by a massive activation of C3, which proceeds to exhaust all subsequent components in the complement cascade. However, the effectiveness of cobra venom factor is short-lived because the recipient rapidly forms neutralizing antibodies against the factor. The administration of cobra venom factor also carries the risk of anaphylactic reaction to the foreign substance. Other strategies to inactivate complement activity involve the production of transgenic donor animals which would express human complement inhibitors in their organ tissues.

If 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.

Summary of the Invention

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. Following the column procedure, 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.

When the coupled protein comprises 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.

It is an object of this invention to provide a method, suitable for use in a human subject, to prevent or ameliorate the hyperacute rejection reaction which normally occurs upon transplantation of a pig organ to a primate subject.

It is a further object of this invention to provide immunoglobulin-depleted human plasma, suitable for use in human subjects in need of such plasma.

Brief Description of the Figure 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.

Detailed Description 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.

Surprisingly, this procedure works to prevent or ameliorate the hyperacute rejection reaction, even without intentional inhibition or removal of complement, per se. Before this discovery, it was thought that even if most of the xeno¬ reactive antibodies could be removed, the residual xeno¬ reactive antibodies in plasma would activate the complement cascade in geometric fashion to cause hyperacute rejection. However, using the present invention, no direct intervention in the complement system is necessary. For instance, it was found that perfusion over a human immunoglobulin-binding column according to the present invention also reduced potential complement activity in the processed plasma by approximately 15-60%. The mechanism by which potential complement activity is reduced in the column procedure is not known. It is also not certain whether the reduction of complement effected by the column procedure is necessary for the efficacy of the procedure in preventing hyperacute rejection. However, the importance of the discovery is not diminished by the unknown nature of its mechanism of efficacy. 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.

Suitably, 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.

Since 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) . Preferably, the column procedure results in a 50 to 500 fold or greater reduction in total anti-pig immunoglobulin in the subject's plasma.

Suitably, the column procedure results in no significant reduction in plasma fibrinogen, Factor V or Factor VII levels.

Preferably, the protein-coupled column binds both human IgG and IgM. It has been proposed that 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). However, 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) . Moreover, after immunoglobulins have been removed, and after xenotransplant, it is very likely that the resurgent anti- pig antibodies will be predominantly of the IgG type. It is expected that removal of predominantly IgM prior to xenotransplant could prevent or ameliorate the hyperacute rejection reaction immediately post-transplant. However, it is desirable to remove also xeno-reactive IgG in the days following xenotransplant. Therefore, it is most practical to have one type of immunoglobulin-binding column which can remove both IgG and IgM from the plasma of the primate recipient. Fortunately, it was discovered that a primate recipient can tolerate very well the removal of the majority of both IgG and IgM antibodies over several days pre- and post-transplant.

Therefore, 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. Preferably, 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. Alternatively, the coupled antibodies may be monoclonal or recombinant antibodies which bind to human immunoglobulins.

In an alternative embodiment of the invention, 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. However, it is generally believed that neither Protein A nor Protein G will bind human IgM. 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.

Methods and compositions for the production of sterile and pyrogen-free protein-coupled columns are provided in U.S. patent application serial number 08/242,215, filed May 13, 1994, entitled STERILE AND PYROGEN-FREE COLUMNS COUPLED TO PROTEIN FOR BINDING AND REMOVAL OF SUBSTANCES FROM BLOOD. This patent application is herein incorporated by reference, with specific reference to the enabling information contained in the pages of application number 08/242,215 as follows:

For production of antibodies and virus inactivation, Example 1, pages 15-19.

For description of pre-columns and working columns. Example 2, pages 19-20.

For sterile purification of antibodies/protein destined to be coupled to the therapeutic column, Example 3, pages 20-25.

For preparation of sterile and pyrogen-free column matrix, Example 4, pages 26-27.

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.

Preferably, 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. When anti-pig antibody titers begin to rise after about post-operative day 5, without other signs of organ rejection, that is an indication that "accommodation" of the recipient's immune system may be occurring, and that the danger of hyperacute xenograft rejection is probably past. At this point, the column procedure does not have to be repeated, and the subject may be maintained on conventional immunosuppressive therapy.

In case a transplant subject is in need of plasma replacement, 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. Alternatively, 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. Most preferably, 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.

Thus, it has been determined that 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. It is understood that the term "primate subject" includes human subjects in need of organ transplant. Moreover, it has been discovered that 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.

It is expected that 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 Prednisone™, anti-proliferative drugs such as Azothioprine™, and/or anti-T-lymphocytic drugs such as cyclosporine. It is also possible that 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. However, 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.

The following experimental examples are offered by way of illustrating the invention and are not intended to limit the scope of the claims in the invention.

EXAMPLE 1 Removal of immunoglobulin, including anti-pig antibodies. from human and baboon plasma in vitro.

Two different column were used. 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

lgG(mg/dL) lgM(mg/dl_) Transferrin Fibrinogen Factor 5 Fac

Experiment #1

Ig-column Pre-Rx 349 34 124 0.11

Human FFP Pass #1 <6 <9 93 0.08

500cc total Pass #2 <6 10 101 0.1 volume

Fixperiment #2

Ig-column Pre-Rx 371 33 159 0.19 31 46

Human FFP Pass #1 <6 <4 151 0.18 29 37

500cc total Pass #2 <6 <4 133 0.16 25 37 volume

Experiment #3

Ig-column Pre-Rx 420 38 161 0.2 35 49

Human FFP Pass #1 <6 <4 154 0.18 32 40

500cc total Pass #2 <6 <4 148 0.17 30 41 volume

Experiment #4

Ig-column Pre-Rx 705 81 238 0.24 67 77

Human FFP Pass #1 57 32 235 0.26 65 74

500cc total Pass #2 37 28 225 0.24 61 77 volume

Experiment #5

Ig-column Pre-Rx 483 53 57 0.12 82 66

Baboon plasma Pass #1 <6 10 53 0.14 _{* *}

250cc total Pass #2 <6 10 39 0.15 _{* *} volume

Experiment #6

Ig-column Pre-Rx 510 48 80 0.15 _{* *}

Baboon plasma Pass #1 <6 11 72 0.14 _{* *}

300cc total Pass #2 <6 10 68 0.14 _{* *} volume

Experiment #7

IgM column Pre-Rx 711 76 262 _{* * *}

Human FFP Pass #1 549 17 215 _{* * *}

300 cc total Pass #2 540 12 210 _{* * *} volume

Experiment #8

IgM column Pre-Rx 800 85 280 _{* * *}

Human FFP Pass #1 700 15 240 _{* * *}

350 cc total Pass #2 690 14 238 _{* * *} volume

Experiment #9

IgM column Pre-Rx 361 86 59

Baboon 505 Pass #1 312 17 55 plasma

400cc total Pass #2 310 15 54 volume Other than the dilutional effect, no significant reduction in plasma fibrinogen, factor V or Factor VII levels occurred.

Anti-pig antibodies in the plasma were measured in a 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 (Nunc™). Cells in the wells were rinsed in PBS and fixed in 200 μl of cold glutaraldehyde solution (0.1%) at 4°C for 5 minutes, followed by washing in Hank's balanced salt solution (HH) . Non-specific binding sites on the cells were blocked by incubating the cells in HH containing 1% bovine serum albumin (BSA) for 45 - 60 minutes at room temperature. Positive control sera were pooled human serum (PHS) and unmodified baboon serum. Fifty μl of the control and test sera were added to the wells and incubated for 1 hour at 4°C (for the IgM ELISA) or at 37°C (for the IgG ELISA) , followed by 3 rinses in HH. Fifty μl of the secondary antibody was added to each well and incubated at room temperature for 1 hour. The secondary antibody for the IgM ELISA was goat anti-human IgM (μ; Zymed) conjugated to alkaline phosphatase; the secondary antibody for the IgG ELISA was goat anti-human IgG similarly conjugated (Sigma) . After washing in HH, the marker reaction was developed in diethanolamine (0.1M with 0.5 X 10^" M MgCl₂) with phosphatase substrate (lmg/ml p- nitrophenyl phosphate) . The developer/substrate was added at lOOμl/well, and the plates were incubated in the dark at room temperature for about 30 minutes, or until the positive control read 1-1.5 absorbance at 405 nm. The plates were read at 405 nm on a standard ELISA plate reader. Results are shown in Table 2 below. Anti-pig IgG was reduced by 54 to 486 fold. Anti-pig IgM was reduced by 9 to 54 fold.

(See Table 2 next page)

Table 2

Effect of Ig-column on anti-pig IgM titers inverse serial dilutions

2 6 18 54 162 486 1458

HS 1.72 0.93 0.42 0.24 0.18 0.16 0.129

Pre-column FFP 0.62 0.36 0.215 0.16 0.14 0.14 0.129

1 volume 0.114 0.13 0.129 0.135 0.139 0.138 0.127

2 volume 0.118 0.12 0.128 0.14 0.13 0.134 0.137 inverse serial dilutions

2 6 18 54 162 486 1458

HS 1.72 0.93 0.42 0.24 0.16 0.16 0.129

Pre-column FFP 0.34 0.21 0.16 0.134 0.137 0.122 0.118

1 volume 0.12 0.12 0.15 0.13 0.135 0.132 0.134

2 volume 0.115 0.12 0.13 0.14 0.135 0.132 0.112 inverse serial dilutions

2 6 18 54 162 486 1458

HS 1.78 0.88 0.48 0.28 0.18 0.168 0.147

Pre-column FFP 0.908 0.6 0.335 0.245 0.192 0.177 0.13

1 volume 0.48 0.28 0.2 0.185 0.17 0.155 0.145

2 volume 0.38 0.22 0.18 0.165 0.14 0.14 0.146 inverse serial dilutions

2 6 18 54 162 486 1458

HS 1.56 0.93 0.45 0.272 0.205 0.167 0.146

Pre-column baboon plasma 0.59 0.38 0.32 0.227 0.195 0.176 0.174

1 volume 0.306 0.26 0.23 0.215 0.14 0.16 0.14

2 volume 0.32 0.25 0.23 0.184 0.17 0.162 0.17

Effect of Ig-column on anti-pig IgG titers* inverse serial dilutions

2 6 18 54 162 486 1458

HS 1.07 0.61 0.58 0.48 0.35 0.24 0.17

Pre-column FFP 0.285 0.27 0.2 0.17 0.14 0.13 0.122

1 volume 0.118 0.11 0.11 0.112 0.114 0.11 0.11

2 volume 0.118 0.1 1 0.11 0.114 0.112 0.112 0.112 inverse serial dilutions

2 6 18 54 162 486 1458

HS 1.07 0.61 0.58 0.48 0.35 0.24 0.17

Pre-column FFP 0.3 0.27 0.22 0.18 0.152 0.14 0.118

1 volume 0.113 0.12 0.13 0.128 0.122 0.115 0.122

2 volume 0.112 0.12 0.112 0.115 0.12 0.12 0.117 inverse serial dilutions

2 6 18 54 162 486 1458

HS 1.15 0.82 0.64 0.46 0.34 0.26 0.177 pre-column FFP 0.92 0.61 0.52 0.35 0.22 0.17 0.15

1 volume 0.263 0.17 0.143 0.138 0.13 0.118 0.116

2 volume 0.26 0.15 0.145 0.125 0.128 0.118 0.11 inverse serial dilutions

2 6 18 54 162 486 1458

HS 1.1 0.69 0.58 0.425 0.325 0.23 0.17

Pre-column baboon plasma 0.257 0.2 0.19 0.154 0.138 0.13 0.122

1 volume 0.1 0.1 0.119 0.118 0.125 0.118 0.118

2 volume 0.1 0.1 0.118 0.11 0.12 0.113 0.115

*NOTE: antibody levels measured with pig aortic endothelial cell ELISA EXAMPLE 2 Removal of immunoglobulin. including anti-pig antibodies, from primate subjects in vivo.

Baboon subjects were housed and cared for under the federally approved guidelines for research institutions where experiments involving laboratory animals are conducted. These guidelines are published as U.S. National Institutes of Health (NIH) Publication # 86-23 entitled The Guide for the Care and Use of Laboratory Animals.

Each baboon subject was prepared for plasma treatment or surgery by initial induction of anesthesia with ketamine HC1 (Ketalar™) and pentathol; the subject was then maintained during all procedures under inhalation anesthesia using Forane™ 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) . From the plasmapheresis machine, 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, Munich) . The plasma effluent from the column was collected in a sterile 125 ml Viaflex™ 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.

Potential complement activity in the plasma was measured using a modification of the technique of Kabat and Mayer (Experimental Immunoche istry, 2nd edition, Springfield, IL: Charles C. Thomas, 1961:149) . The assay determines the concentration of serum which will cause lysis of 50% of a standardized preparation of antibody-sensitized erythrocytes. It is to be noted that this assay measures the potential complement activity in the plasma.

Results: Initial use of the anti-human Ig column procedure for 2 plasma volumes resulted in a mean reduction of total IgG and IgM of 97.5% and 78.4% respectively (see Table 3 below) . Use of the column procedure over 3 additional days maintained the lowered levels of IgG and IgM. A reduction of about 15% up to about 60% in potential complement activity in the plasma was observed (Table 3) . There was no subject morbidity or mortality from the column procedure. Specifically, there was no hypoxia or hypotension. There was no reduction in total plasma volume, nor was there any evidence of edema. It was not necessary to replace plasma proteins because the subject's albumin and transferrin were not removed by the column procedure.

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. At three and four days after the second IgM column treatment, the titer of baboon anti- pig IgM had risen to about 30% of the original value. Table 3

IgG igM TF ςso

Expt #10:IN VIVO

Ig-column

Baboon 505 on-line

1200 cc plasma passsed over column pre-Rx 291 57 56

1 PV 10 18 55

2 PV 10 15 50

Expt #11:IN VIVO Ig-column

Baboon 505 on-line 1200 cc plasma passed over column pre-Rx 350 55 80

1 PV 10 18 75

2 PV 10 15 70

Expt #12: IN VIVO pre-Rx 291 40 54 47

2 PV 35 13 46 27

Expt #13: IN VIVO pre-Rx 261 35 62 39

2 PV 32 9 43 22

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.

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-Therasorb™ , Baxter, Immunotherapy Division, Europe) were connected with the primary separation system as shown (Figure 1) . All tubing connections were made under aseptic conditions.

To remove the preservative solution from the columns, 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.

Before start of the procedure, the entire system was tested for absence of air bubbles and leaks, correct connections of the solutions, including the anticoagulants, correct installation of the programming of the device, functionality of the automatic clamps, and the safety system.

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.

After the venous puncture and the connection of the tubing system to the patient, the blood cell separator was filled with the patient's blood. The blood flow rate was kept between 50-90 ml/min. When a column with a volume of 100 ml was used, the liquid level was maintained at about 0.8 cm over the Sepharose™ in the column. After the stabilization of the separation process, 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 Sepharose™ 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.

Using a plasma flow rate of up to 40 ml/min, 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.

During the time of filling of the second column, 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.

Also during filling of the second column, 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.

Then, 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.

Blood samples were taken for analysis of the success of the procedure.

After each procedure, the columns assigned to each patient were cleaned and stored under aseptic conditions at 2-8°C until the next use for the same patient.

Results: Preliminary results showed that the IgG concentration in the subjects' blood was reduced by at least 70% to over 99% compared to starting concentrations. IgA and IgM levels were reduced by 70% to 90%. An example of the results from one patient are shown in Table 4 below: Table 4 after Circulating Immunoglobulins [%] column treatment IgG IgM IgA

0 100 100 100

1 23 52 51 2 7 23 24

3 8 10 8

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.

There was no morbidity or mortality associated with the use of the column procedure. Plasma loss was typically low (4- 8%) , and no plasma replacement was required.

Conclusions: 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.

At least five days prior to transplant, 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. Also administered on day -4 were cyclophosphamid (Cytoxan™) at 20 mg/kg i.v., cefuroxime (Zinacef™, an antibiotic) at 8cc i.v. gl2 hrs, and nystatin popsicles (anti-fungal) at ql2. On day -3, in addition to DSPG, cefuroxime, and nystatin, an antihista ine (Benadryl™) was administered at 50 mg i.v. Thirty minutes after infusion of Benadryl™ , horse anti-baboon lymphocyte globulin (bALG) at 20 mg/kg was administered over 3-4 hours. On day -2, nystatin, cefuroxime, and DSPG were administered, but DSPG was decreased if the white blood cell count fell below 1500. Also on day -2, the baboon subjects underwent plasmapheresis, which resulted in an 80% reduction in antibodies and a great reduction in clotting factors. We have previously reported that plasmapheresis is followed by a rebound in total and in anti-pig antibodies within 72-96 hours in baboons who do not receive any column treatment (Transplantation 57:974-983, 1994).

One day before transplant, and immediately prior to transplant (day 0) , the anti-human IgG column procedure described in Example 2 above was performed. Also on day -1 and day 0, DSPG, cefuroxime, and nystatin were administered. On day 0, cyclosporine was administered as a continuous i.v. infusion at 4 mg/kg, adjusted to keep the level at 250-300. Also on day 0, an i.v. steroid (Solumedrol™) was administered at 500 mg Q12 hours.

The subjects were prepared for standard kidney transplant as described in Manual of Vascular Access and Organ Transplant, Eds: Najarian, Simmons, Asher; Springer Verlag, 1983.

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.

During the first three days following transplant, DSPG, cyclosporine, Solumedrol™, cefuroxime, and nystatin administration was continued. However, the white blood count was monitored each day before administration of DSPG and cyclosporine, and the doses were adjusted accordingly.

During the 13 days following transplantation, one subject underwent the 2-plasma volume anti-human Ig column procedure on post-operative days 2 and 5. Another subject only received the post-operative day 5 treatment because its antibody titers were still low on post-operative day 2.

Results: Total plasma IgG and IgM levels were maintained at a reduction of 70-90% and 70-90% respectively. Potential complement activity in plasma ranged from 25-85% of pre-treatment levels in the post-transplant period, with reductions most closely associated with column absorption on post-transplant days.

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.

There was no clinical evidence of hyperacute rejection of the pig kidney graft. Animals died on post-operative days 11 and 13 respectively of non-transplant-related causes with functioning xenografts. Post-mortem histological analysis of

the grafted kidneys showed no evidence of hyperacute rejection.

Conclusion: 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. Moreover, 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.

In contrast, human subjects are not anesthetized for the column treatment, and they are generally able to lie quietly for several hours, if necessary, while their plasma is being passed through the column system (Figure 1; see Example 3 above) . It is expected that the initial plasmapheresis treatment used in these experiments will be replaced by the column treatment in human subjects as in Example 3 above, given that sufficient numbers of anti- human Ig columns will be available for clinical use. It is expected that the anti-human Ig column treatment can prevent hyperacute rejection of transplanted pig organs in human recipients.

EXAMPLE 5

Attenuation of hyperacute rejection reaction in pig heart perfused with primate blood.

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.

Whole blood (450ml) was obtained from healthy human volunteers, and separated into plasma and cellular components by high speed centrifugation. When the cells and plasma were recombined to form whole human blood, and when this human blood was perfused through the model pig heart, there was a swift hyperacute rejection reaction in the pig heart (mean=25.2 minutes; n=5) . Plasma obtained from additional volunteers (n=5) was passed twice over a column containing polyclonal anti-human IgG (gamma and light chain specific) conjugated to Sepharose^w (Ig-Therasorb™, Baxter), resulting in a 98.1% reduction in total IgG and 88.4% reduction in total IgM. This correlated with a 78.0% reduction in human anti-pig IgG, and a 76.2% reduction in anti-pig IgM, as measured by a porcine endothelial cell ELISA. When the cellular and plasma components were recombined, the reduction was somewhat less, presumably due to a small amount of retained plasma in the cellular fraction.

Total potential complement activity as measured by C_H50 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.

Results: When perfused with autologous blood, the pig heart functioned relatively normally for 6 hours (n=4) . When the column-treated, recombined human blood was perfused through the model pig heart, the heart functioned for a mean of 4 hours and 18 minutes. In comparison to the swift hyperacute rejection reaction (25 minutes) , this duration of more than 4 hours of function was considered comparable to control.

Histological analysis demonstrated deposition of IgG, IgM, C3, C4, and fibrinogen on pig heart perfused with untreated human blood. No antibody or complement component was found on pig hearts perfused with autologous pig blood or column- treated human blood.

It was concluded that Ig-Therasorb™ column treatment of human blood prevented the hyperacute rejection reaction which normally occurs when a pig heart is perfused with human blood.

Claims

What is claimed is:

1. A method to prevent or ameliorate a hyperacute rejection reaction which would normally occur after transplantation of a pig organ to a primate subject, said method comprising the steps of;

(a) providing a sterile and pyrogen-free column coupled to protein which binds human immunoglobulin,

(b) passing plasma of a primate subject in need of an organ transplant over the column under conditions which effect the binding of said protein to immunoglobulin in the subject's plasma, thereby removing a significant portion of the immunoglobulin from said plasma,

(c) reinfusing said plasma to the subject, and

(d) transplanting a pig organ to said subject.

2. The method of claim 1 wherein said coupled protein is selected from the group consisting of Staphylococcus aureus Protein A, Streptococcus Protein G, and anti-human immunoglobulin antibodies.

3. The method of claim 2 wherein the coupled protein comprises anti-human immunoglobulin antibodies selected from the group consisting of polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

4. The method of claim 3 wherein the antibodies are recombinant double-chain or recombinant single-chain antibodies.

5. The method of claim 3 wherein the antibodies coupled to the column specifically bind to epitopes on a member of the group consisting of human lambda-type light chain, human kappa-type light chain, human gamma-type heavy chain, and human mu-type heavy chain.

6. The method of claim 1 wherein said immunoglobulin in the subject's plasma which is bound by the antibodies coupled to the column is selected from the group consisting of IgG and IgM.

7. The method of claim 1 wherein said step (b) further comprises the passing of at least two plasma volumes of the subject over the column.

8. The method of claim 7 wherein said step (b) is conducted on at least one day prior to step (d) .

9. The method of claim 8 further comprising conducting step (b) on at least one additional day prior to step (d) .

10. The method of claim 8 further comprising repeating step (b) on the day of and immediately prior to step (d) .

11. The method of claim 1 further comprising repeating step (b) on at least one of the days following the day of step (d) .

12. The method of claim 1 further comprising conducting step (b) on at least three separate days prior to step (d) and on at least 2 separate days after step (d) .

13. The method of claim 7 wherein said step (b) effects the removal of at least about 75% of the total IgG from the plasma.

14. The method of any of claims 7-12 wherein said step (b) effects the removal of at least about 90% of the total IgG from the plasma.

15. The method of claim 7 wherein said step (b) effects the removal of at least about 50% of the total IgM from the plasma.

16. The method of any of claims 7-12 wherein said step (b) effects the removal of at least about 90% of the total IgM from the plasma.

17. The method of claim 7 wherein said step (b) effects the reduction of potential complement activity in the plasma by at least about 15%, as measured by a standard assay based on the amount of plasma required to lyse 50% of a preparation of antibody-sensitized erythrocytes.

18. The method of claim 7 wherein said step (b) effects the reduction of anti-pig antibodies in the plasma by at least about 50 fold, as measured by an anti-pig antibody ELISA based on the binding of immunoglobulins in the plasma to pig endothelial cells in test wells.

19. Human plasma suitable for infusion to a human recipient of a pig organ transplant, said plasma being prepared by passing plasma from at least one normal healthy human donor over a sterile and pyrogen-free column coupled to protein which binds to and thereby removes human immunoglobulin from human plasma.

20. Human plasma containing no more than about 65 mg/dL total IgG, no more than about 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 plasma from a normal healthy human donor.

21. Human plasma containing no more than 10% of the total IgG, no more than 10% of the total IgM, at least 50 fold less anti-pig IgG, and at least 10 fold less anti-pig IgM, as compared with plasma from a normal healthy human donor.

22. Human plasma containing less than about 1.0% of the total IgG, less than about 1.0% of the total IgM, at least 250 fold less anti-pig IgG, and at least 25 fold less anti- pig IgM, as compared with plasma from a normal healthy human donor.

23. The use of a protein which binds to human immunoglobulin in the manufacture of a column for the diminution of a hyperacute rejection reaction which would normally occur after transplantation of a pig organ to a primate subject, said diminution being accomplished by a method comprising passing plasma of the primate subject over the column prior to transplantation of the pig organ.

24. The use of a protein according to claim 23 wherein said protein comprises anti-human immunoglobulin antibodies.