RU2252533C2 - Method for obtaining transgenic animal - Google Patents

Abstract

FIELD: gene engineering.

SUBSTANCE: one should introduce DNA into animal genetic material. Moreover, DNA codes a complement's homologous restriction factor of discordant type and a sequence providing expression of coding DNA in the cells of transgenic animal. Cells isolated in such transgenic animal could be transplanted to animal of the mentioned discordance type without acute detachment. The innovation could be applied to obtain materials for transplantation, as well.

EFFECT: higher efficiency.

22 dwg, 14 ex

Description

The present invention relates to a biocompatible material for use in transplants, as well as to the preparation and use of such material.

The transplantation of missing or damaged animal (especially human) tissue, including organs, over the past four decades has acquired the character of conventional therapy in clinical medicine. These transplant methods range, for example, from the use of polyethylene terephthalate sold under the DACRON trademark by DuPont to replace damaged blood vessels, to use the saphenous vein as an autograft to bypass arteries, and to transplant a heart from one person to another.

Organ transplantation has undergone significant development with the receipt of immunosuppressants, allowing to achieve high results at a relatively moderate cost. The need for an organ transplant has increased rapidly. Currently, more than 20,000 organ transplants are performed worldwide per year, which, however, make up only about 15% of the required quantity, as estimated by current criteria. The supply / demand relationship of all types of donor organs cannot be decided on the basis of existing sources. Perhaps this is best illustrated by meeting the need for a heart transplant. The first heart transplant performed by Dr. Barnard in 1967 aroused great interest around the world. During the year, in 22 countries, 101 heart transplants were performed with 64 different surgical teams. The disappointment following the poor results led to less than 30 transplants per year by the beginning of the 1970s. However, the introduction of cyclosporins into immunosuppression has revolutionized heart transplantation so that most surgical centers now expect successful cardiac transplant results from more than 80% of transplants per year (and patient survival). According to experts, this percentage of survival can be increased further. The success of this technique undoubtedly predetermined the fact that professional physicians and the public generally became more confident that heart transplantation was a real alternative to death, and an increasing number of patients undergo this technique. Currently, over 2,000 heart transplants are performed annually in the world.

Today, the greatest likelihood of death during a heart transplant falls on the waiting period of a suitable donor organ. If an artificial heart makes it possible to obtain short-term care devices for such patients, then long-term use devices suggest the presence of a greater number of heart transplant centers and donors. The potential number of people who would have been able to survive a heart transplant was never determined by scientific methods, however, published estimates of the need for a heart transplant vary widely from 50 to 250 million people per year depending on the criteria chosen, the age of the recipient, the disease, and so on. Whatever the actual figure, it is already clear that the current number of donors is not able to solve this need. Similar remarks can be made with regard to kidney and liver transplants, and it is very likely that soon, as a result of the now widely used therapeutic technique for pancreatic transplantation or Lagerhans islet for diabetes, the reduction of this tissue will be the first concern.

There are other disadvantages associated with the current state of transplant therapy. Of course, donor organs are not always suitable for use in transplantation, since many donors themselves are victims of some accident (for example, a road accident) that caused death as a result of damage to a certain organ other than the one that is being transplanted; the transplanted organ may be damaged as a result of another case or related difficulties.

In addition, due to the unpredictability of donor organ suitability, transplant therapy can often not be planned as a routine operation, and too often, surgical teams and hospital administrators must respond at the time the donor organ is identified and engage in non-social activities, which adds to administrative and personal difficulties.

For heart, liver, and lung transplants, if tissue rejection occurs, transplantation usually cannot be done until another suitable donor appears in a short period of time.

In addition to the above medical difficulties, the existing practice of transplantation in some cases is associated with social difficulties. Firstly, there may be religious objections to organ transplants from potential donors, in particular among peoples who believe in reincarnation. Undoubtedly, there are a number of other ethical and social difficulties encountered in organ transplants from deceased people, in particular in some countries where consent for such an operation is required. Finally, the emergence of commercial trade in living kidney donors, especially in some regions of the Third World, is of concern, and it would be desirable to prohibit or weaken such trade.

As emphasized above, conventional transplant surgery with all its drawbacks consists in transplanting organs from one animal of a particular species (usually a human) to another animal of the same species. Such transplants are called allografts. Due to difficulties in obtaining a conventional allograft, as noted above, attention should be paid to the possibility of using xenografts for transplantation. Xenotransplantation is a general term used to implant tissues, including cells and organs, through species barriers.

There are already several examples of the successful use of xenografts in a therapeutic transplant. For example, recent years have shown the use of pig tissue for replacing the aortic valve, pig skin for treating patients with severe burns, and the umbilical vein of a cow as a graft for replacing veins. All these xenografts have one common feature: they provide only mechanical tissue replacement. However, the tissue used is biologically non-functional. The reason for this is that the immune processes that take place in a person immediately (within minutes or hours) destroy the cellular integrity of tissues from most species. Such xenografts are known as discordant xenografts.

The ferocity of such destruction is phylogenetically associated. So, tissue from chimpanzees, a primate who is a close relative of a person, can be preserved in a person in the same way as an allograft; such a xenograft is known as a concordant xenograft.

Although one might think that concordant xenografts might answer the question regarding difficulties with allografts, in practice this is not entirely true. Chimpanzees are much smaller than humans, and chimpanzees, as a rule, do not function in humans. With regard to the kidneys, this problem can be solved by transplantation of the kidneys of other chimpanzees in order to replace damaged human kidneys, but this is not possible with respect to the liver and heart. In addition, chimpanzees breed slowly in nature and are poorly caught, and the need for chimpanzees as experimental animals (especially at present, to study the acquired immune deficiency syndrome (AIDS)) means that demand again outstrips supply. In addition, there may be some social difficulty associated with public opinion regarding the use of other primates as xenograft donors.

Therefore, attention is again drawn to discordant xenografts. It is generally believed that the reason why discordant xenografts fail so quickly is the existence of reciprocal "naturally occurring" antibodies against undetermined antigens from donor species (Shons et al., (Europ.Surg.Res., 5, 26 -36 1973)). Antibodies are called "naturally occurring" because they are present in living things that do not have any immunological evidence from a donor species.

Rapid rejection, known as hyper-acute antibody-mediated organ transplant rejection, is well documented. In the early 60s, when kidney transplantation became the usual treatment, it was noticed that the transplanted kidneys were rejected by the recipient from time to time, although the operation was still ongoing. During a transplant operation, the kidney usually becomes red and solid in consistency shortly after the vessels of the recipient and donor are stitched. Such transplants often produce urine almost immediately. In the form of rejection, when the transplant is destroyed while the patient is still on the table (hyper-acute rejection), destructive processes begin in the first few minutes or shortly after transplantation. When this happens, the kidney becomes bluish and spotty, and then stagnant. The consistency of the organ also changes. As a rule, the transplant becomes swollen, urine production does not occur, and the newly transplanted kidney is then rejected immediately. It became clear that a humoral-mediated immunological reaction between preformed circulating antibodies in the recipient and antigens in the donor kidney is involved in this process. The only way to resolve this problem in allograft is to verify before transplantation that no antibodies exist in the recipient against donor cells. With the increasing knowledge of testing for such antibodies (known as crossover), it became clear that this generalization regarding the fact that the antibody in the recipient interacts with the antigens in the donor is not correct, and that hyper-acute graft rejection when it involves grafts between individual patients of the same species, limited to the existence of specific types of antibodies, known as positive cross-tests with thermal lymphocytotoxins (lymphocytotoxic v); and almost certainly, these antibodies belong to a subclass of immunoglobulin G (IgG). In addition, the presence of these antibodies is always due to the pre-existing sensitization technique, either as a result of early blood transfusions, or as a result of pregnancy, or, most often, as a result of previous transplant damage.

The mechanism of hyper-acute xenograft rejection is believed to be similar to the mechanism of hyper-acute allograft rejection, as emphasized above. Extensive literature on the mechanism of xenograft rejection, it covers about 83 years. During this time period, only three publications dealt with the hypothesis of a xenograft rejection mechanism that does not involve antibodies. The assumption was based on the involvement of an alternative metabolic pathway for complement activation in xenograft rejection. The assumption first appeared in 1976 by Schilling et al., (Surgery, Gynaecology and Obstetrics 142; 29-32 (1976)). The assumption was again put forward in 1988 and 1989 (the same data were published twice) Miyagawa et al., (Transplantation, 46 (6) 825-830 (1988) and Transplantation Proceedings, 21 (1), 520-521 (1989) ) However, the results were not convincing, since both experiments experienced basically the same error. The model selected by the applicant was a xenograft model in which cross-species antibodies did not exist. However, it now seems that the analyzes used to detect cross-species antibodies were incorrect, and that the conclusions drawn in these works were based on inaccurate data.

Most of the steps currently being taken experimentally to eliminate or reduce xenograft rejection include chemotherapeutic intervention in the recipient’s immune system, largely on a nonspecific basis, such as cyclosporin A and other immunosuppressants, by plasmapheresis, by treatment of cobra venom factor, and antibody absorption Staphylococcus protein A and so on. This approach naturally follows from chemotherapy, which supports allografts.

The present invention involves a completely different approach: instead of nonspecific interference with the recipient’s immune system, the invention allows for the co-administration of a substance that affects the donor transplant, which is considered “own”, through certain components of the recipient’s immune system. In particularly preferred embodiments of the invention, the donor tissue itself is modified until immunological to the recipient, which in some respects should be "himself."

It has also been found that hyper-acute xenograft rejection is not necessarily mediated by antibodies. This conclusion is based on two observations. Firstly, in the absence of an antibody, but in the presence of complement, hyper-acute rejection is observed; secondly, in the presence of an antibody, but in the absence of complement, no hyper-acute rejection is observed.

The invention is based on the discovery that complement activation plays a major role in hyper-acute xenograft rejection, regardless of whether or not such activation is accompanied by the binding of molecules of the corresponding antibodies. Activation of the alternative complement metabolic pathway can be induced by a variety of cellular products. These products are not limited to foreign cells, such as bacteria or xenografts, but exist on many cells. So, in principle, many human cells can activate an alternative metabolic pathway of complement, causing extensive autoimmune rejection. If this does not happen, then the fact that there are a number of proteins that negate complement present in the natural state in serum and on the surface of the cells. These molecules (referred to as “homologous complement restriction factors”) prevent the full activation of self-complement either by the classical or alternative metabolic pathway under the action of the products of the cells themselves, thereby preventing autoimmune rejection of “their own”. The functioning of such molecules is illustrated with nocturnal paroxysmal hemoglobinuria. With such a disease, the “membrane anchor” of at least one of these molecules (decay factor) is absent. Thus, the protein is not retained in the erythrocyte cell membrane and is disconnected from the cell, activating the alternative metabolic pathway of complement, after which it is lysed, thereby causing hemoglobinuria.

In accordance with a first embodiment of the present invention, there is provided a method of transplanting animal tissue into a recipient, wherein the tissue originates from a donor of a species different from the recipient, the donor species is a discordant species with respect to the recipient, characterized in that the tissue is transplanted into the recipient and obtained in combination with the transplanted tissue one or more homologous complement restriction factors, active in recipient form, to prevent complete complement activation.

The word “tissue” as used herein means any biological material to be transplanted and includes organs (especially internal vital organs such as the heart, lung, liver and kidney, pancreas and thyroid gland), cornea, skin, blood vessels and other connective tissue, cells, including blood and hematopoietic cells, islets of Langerhans, brain cells and cells from endocrine and other organs, as well as general body water (for example, a plasma-protein preservative), and they can all be cand idatami for transplantation from one species to another.

A “discordant species” is a species (usually vascularized) whose xenograft to the recipient usually gives rise to hyperostroma rejection, that is, rejection within minutes or hours rather than days (Calne, Transplant. Proc., 2: 550, 1970) . Such hyper-acute rejection should be well known to those skilled in the art and may occur within 24 hours, within 6 hours, or even within one hour after transplantation.

Complement and its activation are now well known and described in Roitt, Essential Immunology (Fifth Edition, 1984) Blackwell Scientific Publications, Oxford. The activity attributed to complement (C ’) depends on the functioning of nine protein components (C1 to C9), acting sequentially, of which the first consists of three main sub-fractions called C1q, C1r and C1s. Complement can be activated by the classical or alternative metabolic pathway, both pathways being described briefly.

In the classical metabolic pathway, the antibody binds to C1, the C1s subunit of which acquires esterase activity and causes activation and transfer to sites on the membrane or immune complex, first C4 and then C2. This complex has the activity of “C3 convertase” and cleaves C3 in solution to produce a small peptide fragment of C3a and a residual C3b molecule, which have completely different functions. C3a has anaphylatoxin activity and does not play any other role in the complement amplification cascade. C3b is membrane-bound and can induce immune adhesion of the antigen-antibody-C3b complex, thereby facilitating subsequent phagocytosis.

In an alternative metabolic pathway, the activity of C3 convertase is represented by C3bB, the activation of which can be triggered by external agents, in particular microbial polysaccharides, such as endotoxin, acting independently of the antibody. The convertase is formed under the influence of Factor I on the complex of C3b and Factor B. This complex forms a loop with positive feedback in which the decomposition product of C3 (C3b) helps to form more than one cleavage enzyme.

Both in the classical and in the alternative metabolic pathway, the C3b level is maintained under the influence of the C3b inactivator (Factor I). C3b easily combines with Factor H to form a complex that is destroyed by Factor I and loses its hemolytic and immune-adhesive properties.

The classic and alternative paths are common after the implementation of stage C3. C5 is cleaved to give fragments C5a and C5b. C5a has anaphylatoxin activity and gives rise to chemotaxis of polymorphonuclear neutrophils. C5b binds as a complex to C6 and C7, forming a heat-resistant site on the membrane, which replenishes the final components of C8 and C9 with the formation of a membrane-attacking complex (MAC). It is an annular structure inserted into and extending from the membrane, which forms a transmembrane channel that is completely permeable to electrolytes and water. Due to the high internal colloid osmotic pressure, the resulting influx of sodium and water ions occurs, which leads to cell lysis.

Homologous complement restriction factors (HCRF _S ) useful in the present invention can generally interfere with any part of the complement activation cascade. HCRF _S can interfere exclusively with the part that makes up the classical metabolic pathway, or exclusively with the part that makes up the alternative metabolic pathway, or, more usually, can interfere with the part that is common to both the classic and alternative metabolic pathway ways. Preferably, the HCRF _S regulator interferes with a common part of the metabolic pathway. HCRF _S may be identical to natural HCRF or may simply have a corresponding function. Synthetic and semi-synthetic HCRF _S , including those obtained by recombinant DNA methods, as well as their variants, are included in the concept of the term HCRF _S.

As mentioned above, homologous complement restriction factors are substances that regulate the action of the complement cascade in such a way as to reduce or prevent its lytic activity; they are used by the animal body to label tissue as "their own" so that an autoimmune reaction can be avoided. In the present invention, in principle, it is possible for HCRF _{S to} be either membrane-bound or serum free, although in practice it would be preferable to have HCRF _S membrane-bound on xenograft tissue cells. In this way, it is easier for this factor to be “in association” with the graft tissue. Preferred HCRF _s include putative cell membrane factors including receptor C3b / C4b (CR1), S3.dg receptor (CR2), stimulant hemolysis (decay factor, DAF), C3b inactivator and membrane cofactor protein (MCP). Routine serum HCRFs include Factor H, a hemolysis stimulant (DAF), and a C4 binding protein (C4bp). These HCRF _s all regulate downward complement activity by interference in step C3. Homologous restriction factor (HRF), which blocks in C8, is also a putative membrane factor.

Many, but not all, genes for suitable HCRF _S are located in RCA (complement activation regulator), the locus of which maps chromosome 1 with the q32 disk (Rey-Campos et al., J.Exp Med. 167: 664-669 (1988)) .

Although there is some confusion with the nomenclature and location of HCRFs, factors C4BP, CRI, DAF and factor H were identified by Rey-Campos et al., (Above), as well as in their earlier work (J.Exp.Med, 166: 246-252 (1987)). Membrane cofactor protein (MCP) is processed as a synonym for C4 binding protein (C4bp), and it may be that these two factors are either related or identical. Pother and Till ("The Complement System", Springer-Verlag, Berlin (1988)) provide regulatory factors for C3 convertase in section 1.2.3.2; they established the equality between the C4-binding protein (C4bp) and the hemolysis stimulator, as well as between the factor H, B _I H-protein and the accelerator of the inactivator C3b. There is no doubt that the nomenclature, location and characterization of HCRF _S will continue to evolve, however, it should be understood that the present invention contemplates the use of all HCRF _S in accordance with the requirement of suitability and preference.

Other references to HCRF _S include the following:

Factor I (also known previously as C3b inactivator or KAF): Tamura and Nelson (J. Immmunol., 99: 582-589 (1967);

Factor H: Pangburn et al., (J. Exp. Med., 146: 257-270 (1977);

C4 binding protein: Fujita et al., (J. Exp. Med., 148: 1044-1051 (1978));

DAF (also known as CD55): Nicholson-Weller et al., (J. Immunol., 129: 184 (1982));

Membrane cofactor protein (MCP; also known as CD46 and first described as qp45-70 and hereinafter known as qp66 / 56): Seya et al./ (J.Exp.Med., 163: 837-855 (1986));

CRI (also known as CD35): Medof et al., (J. Exp. Med., 156: 1739-1754 (1982)) and Ross et al., (J. Immmunol., 129: 2051-2060 (1982) );

CR2 (also known as CD21, 3d / EBV receptor and p140: Iida et al., (J.Exp.Med., 158: 1021-1033 (1983)) and Weis et al., (PNAS 81: 881-885 ( 1984)).

The following is the tissue distribution of some RCA proteins:

CR1: Membrane (limited): red blood cells; monocytes; most B cells and some T cells; polymorphonuclear leukocytes; follicular dendritic cells; podocytes;

CR2: Membrane (limited): most B cells; follicular dendritic cells; some epithelial cells and several T cell lines;

MCP: Membrane (wide): all peripheral blood cells (except red blood cells); epithelial, endothelial and fibroblast cell lines; trophoblast and sperm;

DAF: Membrane (wide): all peripheral blood cells; epithelial, endothelial and fibroblast cell lines; trophoblast and sperm;

C4bp: Plasma: hepatic synthesis;

N: Plasma: hepatic synthesis; fibroblast and monocytic cell lines.

As for the proteins involved in homologous restriction at the level of the membrane-attacking complex, the use of which is also provided for by the present invention, there is general agreement (however, not yet evidence) in the form of a protein sequence that the following proteins weighing 65 kDa (or so) are identical: C8-binding proteins (Schonermark et al., J. Immunol., 136: 1772 (1986)); homologous restriction factor (HRF) (Zaiman et al., Immunology, 83: 6975 (1986)), as well as MAC-inhibiting protein (MIP) (Watts et al., (1988)).

The C8bp / HRF / MIP protein is attached to the cell surface using a glycolipid anchor, like CD59 and DAF: it is known that these proteins are functionally absent in nocturnal paroxysmal hemoglobinuria.

A protein of 18-20 kDa is also involved at the MAC level. The following proteins are believed to be identical (or maybe not):

P-18 (Sudita et al., (J. Biochem. 104: 633 (1988));

HRF - 20 (Okada et al., (Intl. Immmunol., 1 (1989));

CD59 (Davies et al., (J. Exp. Med., (September 1989)));

and membrane reactive lysis inhibitor (MIRL) (Hologuin et al., (J. Clin. Invest., 84: 7 (1989))).

Evidence of the putative identity of these proteins is that the protein and / or cDNA sequences for CD59 and HRF-20 are shown to be identical: it is likely that they are the same as P-18 / MIRL. It should be noted that there is some homology to the CD59 / HRF-20 sequence with the murine LY-6 antigen sequence that is involved in T-cell activation (Gronx et al., (J. Immmunol., 142: 3013 (1989))).

SP-40.40 is also involved in the regulation of MAC (Kivszbaum et al., EMBO 8, 711 (1989)).

Preferably, HCRF _S interferes with complement activation in step C3. Both MCP and DAF block the positive feedback loop in the alternative metabolic pathway of C3 activation, and they constitute the preferred HCRF _S.

HCRF _{S is} offered in association with transplanted tissue. This means that HCRF _S is administered in such a way that the graft tissue is labeled as “native,” however, other foreign material, such as invading bacteria, is not labeled. One or more HCRF _S can be introduced into the graft tissue parenterally but locally. However, in practice this may not be preferred due to the difficulty in ensuring adequate localization of HCRF _S on the graft tissue, and also because of the other difficulty that HCRF _S can be administered to the recipient repeatedly after the graft is received, and this difficulty can be eliminated through the use of special pharmaceutical delivery systems.

In general, it would be more convenient to obtain HCRF _S so that it integrates with the cell membrane on the donor tissue. Although there may be some mild infections of the transplanted tissue causing suitable expression, by far the most preferred way to achieve this goal is the transgenic pathway for donor tissue, consisting in the fact that the tissue contains and expresses a nucleic acid encoding one or more HCRF _S , active in recipient form when it is transplanted into the recipient. Such transgenic tissue may continue to express HCRF _S indefinitely. Genetically, HCRF _S can be derived from a recipient species or, less preferably, from a closely related species for which concordant xenografts are possible.

Although, in principle, the transgenic donor tissue can be derived from cell culture, it is preferred that the donor tissue is derived from a transgenic animal. The transgenic animal must express (or be able to express) HCRF _S at least in the transplanted tissue. However, even this is unimportant, since it is possible to bind HCRF _S to the cell membranes of donor tissue using a specific binder (such as a hybrid monoclonal antibody (Milstein and Cuello, Nature, 305: 537 (1983)) or a receptor.

The recipient species is mainly human, but not exclusively. Other primates may also be suitable recipients, like many other species, if considerations of economics and ethics permit.

The donor species may be any suitable species that is different from the recipient species and which, taking into account the physiology of the recipient species, is capable of producing tissue suitable for transplantation. Pork donors are suitable for human recipients, although other species are also suitable.

According to a second embodiment of the present invention, there are provided transplantable animal cells or tissue of a donor species, wherein the cells or tissue are associated with one or more homologous complement restriction factors active in the desired recipient form to prevent complete complement activation, while the donor species is a discordant species with respect to recipient species.

In accordance with a third embodiment of the present invention, there is provided a transgenic animal having transplantable tissue that does not allow xenograft rejection to occur during transplantation into the immune system of at least one discordant species. A discordant species is one that is usually hyper-constructive and rejects a xenograft from an animal.

Therefore, the present invention provides for the use of animal tissue obtained from a donor species and one or more homologous complement restriction factors that are active in a recipient form, the donor species being a discordant species relative to the recipient species, in the preparation of tissue transplanted into the recipient species.

According to a fourth embodiment of the present invention, there is provided a transgenic animal having cells capable of expressing a homologous complement restriction factor of another species. A homologous restriction factor is typically active in a form that is discordant with respect to the type of transgenic animal. Cells can be from one specific tissue, preferably described with reference to the first embodiment of the invention, or from more than one, or from all tissues, in which case the animal can become a donor of more than one tissue. Such a transgenic animal can be considered as a collection of non-transformed (in the sense of non-proliferative) cells.

According to a fifth embodiment of the present invention, there is provided an untransformed animal cell capable of expressing one or more homologous complement restriction factors active in a form that is discordant with respect to the animal cell.

According to a sixth embodiment of the present invention, there is provided recombinant DNA comprising a DNA encoding at least one homologous complement restriction factor and one or more sequences so that the encoding DNA is expressed by an untransformed animal cell. An animal cell may be a cell of a transgenic animal genetically incorporating the construct. Alternatively, the cell may be a cultured organ along with other tissue, such as a Lagerhans islet.

According to a seventh embodiment of the present invention, there is provided a genetic construct suitable for incorporation into an animal’s genetic material to produce a transgenic animal, the construct comprising DNA encoding at least one homologous complement restriction factor and one or more sequences so that the encoding DNA is expressed in at least some cells of a transgenic animal genetically incorporating the construct. Such a genetic construct may be in the form of a mini-chromosome known as YAC. As indicated above, the homologous complement restriction factor is active, usually in a form that is discordant with respect to the type of transgenic animal.

According to an eighth embodiment of the present invention, there is provided a method for producing a transgenic animal, comprising incorporating into the genetic material of an animal DNA encoding at least one homologous complement restriction factor and one or more sequences so that the encoding DNA is expressed in at least some cells of the transgenic animal .

Methods for producing transgenic animals are becoming more common, and detailed steps can be taken from known conventional sources. For example, PCT application WO-A-8800239 discloses the steps necessary, in principle, for constructing a transgenic animal.

The actual method of incorporating the construct into the cells of a transgenic animal can be accomplished by microinjection, sperm-mediated administration, or any other suitable method. Preliminary genetic manipulations can be carried out in a prokaryote, as is generally preferred.

DNA encoding HCRF _S is either obtained in the form of cDNA or is derived using traditional cloning techniques. DNA encoding a hemolysis promoter (DAF) is probably best characterized and described by Medof et al., (PNAS, 84: 2007-2011 (1987)).

A physical map of the RCA gene cluster is given by Rey-Campos et al., (1988) (above). Options DAF and their preparation by recombinant DNA methods are disclosed in the application for Europatent A-0244267; such options can be used in the present invention.

Due to the better understanding of DAF genetics and the known cDNA sequence encoding DAF, this factor is the preferred homologous complement restriction factor.

Other preferred features of 2-7 embodiments of the present invention are similar to those of the first embodiment, mutatis mutandis.

The invention will now be illustrated by the following examples. In the examples, the reference is made to the drawings, in which:

figa-1E show sequential ECG recordings for the heart of a rabbit transplanted to newborn piglets in accordance with Example 1;

figure 2 shows the result of radio-immunoassay, which suggests that the pigs used in Example 1 do not have significant amounts of anti-species antibodies;

figure 3 shows some stages of protein electrophoresis used in Example 4;

figure 4 shows certain stages of two-dimensional cross electrophoresis used in Example 4;

figure 5 shows the "20-Rockets" obtained by carrying out Example 4;

6 shows the result of the analysis of cell lysis with radioactive chromium in Example 5;

7 illustrates titers of lytic anti-hamster antibodies from a rat hamster heart transplant recipient before transplantation (day 0) and 5, 7, and 9 days after transplantation, as described in Example 6;

Fig. 8 shows graphically optical densities (OD) by fraction G200; the histogram illustrates the titers in each fraction of lytic anti-hamster antibodies from a rat hamster heart recipient, as described in Example 6;

Fig.9 shows Southern blotting of DNA extracted from T5, b10 and DB3 cell lines, as described in Example 7;

10 shows ⁵¹ Cr release values indicative of a human T5 cell line lysing a rabbit complement, but not a human complement, in the presence of porcine anti-human antibodies, as described in Example 7;

11 shows release values indicative of the ineffectiveness of human antibodies to lyse a human T5 cell line with either human or rabbit complement, as described in Example 7;

12 shows ⁵¹ Cr release values that demonstrate that human antibodies can lyse a mouse-mouse hybridoma (DB3) in the presence of both rabbit complement and human complement, as described in Example 7;

13 shows ⁵¹ Cr release, showing that the B10 human-mouse hybrid cell line is lysed with human antibodies in the presence of rabbit complement, but not lysed with human antibodies in the presence of human complement, as described in Example 7;

Fig.14 shows the consumption of 3H-adenine (in pulses per minute) by CHO cells, which indicates that these cells are destroyed by immune rat serum in the presence of human complement or rabbit complement, as described in Example 8;

Fig. 15 shows consumption of 3H-adenine (in pulses per minute) by CHO cells transfected with the human membrane cofactor protein (MCP), which indicates that these cells are destroyed by immune rat serum in the presence of rabbit complement, but not destroyed by this immune rat serum in the presence of human complement, as described in Example 8;

FIG. 16 shows “2D-rockets” suggesting that under the circumstances described with respect to FIG. 15, the human complement component C3 does not cleave to form C3b, as described in Example 8;

Fig. 17 shows ⁵¹ Cr release values indicative of 3T3 murine fibroblasts lysed by naturally occurring antibodies in the presence of human complement and the protective effect of human MCP expression by mouse cells;

Fig. 18 shows a slot blot of DNA analysis of transgenic second generation mice using labeled MCP cDNA (upper) or labeled DAF cDNA as a probe.

EXAMPLE 1. Xenograft rejection occurs in the absence of any anti-species antibodies.

In general, animals cannot survive without circulating immunoglobulins. They are produced by lymphocytes in response to antigenic stimuli. In the early neonatal period, however, passively tolerated maternal immunoglobulin acts as a temporary replacement for this self-produced antibody. This passively tolerated immunoglobulin confers protection to the young, while an early immune experience is acquired. In mammals, this passive transfer of maternal immunolobulin usually occurs both transplacentally and through colostrum. In several species, however, the structure of the placenta is such that no maternal antibody can be carried this way. A pig is one such species. The entire maternal antibody is derived from colostrum. Thus, newborns breast-fed piglets are in principle non-immunoglobulin.

Large white pigs selected at birth are placed in wooden pens heated by hot water bottles, and they do not gain access to the chest. Two pigs from each farrow are selected for each experiment. These animals weigh approximately 1 kg after birth.

Young New Zealand white rabbits weighing approximately 300 g are used as donors. These donors are anesthetized with hypnol and diazepam, the chest is opened and the vena cava is catheterized with a 19 gauge needle. Cold (+ 4 ° C) cardioplegia (Thomas No. 2) is infused until the heart stops beating and perfusion with cardioplegia begins. Cooling is also applied externally by cold cardioplegia directly from the syringe. Then the rabbit heart is removed using surgical techniques and stored in a cardioplegia solution at a temperature of + 4 ° C. These precautions are necessary because it is proven that the rabbit’s heart is very sensitive to ischemic disturbance.

Recipient piglets are anesthetized initially by aspiration of Halotan / 02. Then, an intravenous needle (23 gauge) is inserted into the vein of the mammary gland, anesthesia is maintained by intravenous ketamine. Piglets are simultaneously hydrated with intravenous saline. Serum and EDTA blood samples are taken after transplantation.

The rabbit’s heart is transplanted into the neck of the piglets according to the Heron method (Acta Pathol. Microbiol. Scand., 79: 366-372 (1971)). The aorta is anastomized end to side (6-0 proline) to the carotid artery, and the pulmonary artery is anastomized to the jugular vein. All other cardiac vessels are ligated. Hearts begin to beat within a few minutes after removing the clamps. The heart rate is recorded using a diascope / ECG monitor. The neck of the piglet is not closed during the experiments, the hearts are kept moist, covering with a sticky film.

The ECG results are shown in figa-1E. The curve shown in FIG. 1A shows normal heartbeat immediately after transplantation. Violation occurs after about twenty minutes (pigv) and within an hour (pigv), when it is not possible to detect heartbeat, which indicates a hyper-acute transplant rejection.

Therefore, this example demonstrates that hyper-acute rejection of a discordant xenograft occurs even in the absence of antibodies.

EXAMPLE 2. Newborn piglets used in Example 1 do not have any anti-species antibodies.

Rabbit anti-pork immunoglobulin G (IgG) is radioiodinated by the method of Greenwood et al., Biochemical Journal 89: 114-123 (1963), modified by Davies and Howard (not published).

The following ingredients are added to polystyrene tubes (L P2, 6 cm × 1 cm) in quick succession: 25-50 μl of protein (at a concentration of 1 mg / ml); 3-4 μl Na ¹²⁵ I (100 μcub.inch / ml); 10 μl of chloramine-T (* 4 mg / ml; 0.5 M); sodium phosphate buffer (pH 7.5).

* must be prepared again before use.

These components are mixed for 30 seconds, constantly mixing. Then, the following components are quickly added: 50 μl of DL-tyrosine (saline in 0.5 ml of sodium phosphate buffer, pH 7.5); 300 μl of 2% bovine serum albumin (BSA) / phosphate buffered saline (PBS) / azide.

The labeled protein is then isolated from unreacted iodine by using a small column (8 cm × 1.0 cm) of Sephadex G-25, the average gradient of which is made in PBS / azide. The mixture for the iodination reaction was quantitatively transferred to the obtained G-25 column and eluted with PBS / azide. Six-drop fractions are collected in polystyrene tubes (L P2). The column is eluted until both peaks of the protein and ( ¹²⁵ I) iodine become eluted, and the radioactivity in all fractions is measured. The radioactivity incorporated into the protein can be calculated as follows:

the amount of radioactivity in the protein = initial total amount - the amount in iodide peaks.

The radioactively labeled IgG (now referred to as the “isotope”) is then used for analysis of (pig) antibodies in newborn piglets as follows:

Materials

PBS + 0.01% azide - oxoid;

PBS / BSA 1% - BSA-Sigma;

isotope is a complete molecule of the rabbit anti-pork IgG with 12-18 × 10 ³ cycles / min;

heat inactivated serum (56 ° C for 30 minutes);

anticoagulated blood tests.

Method

1. Receive a 1% suspension of rabbit red blood cells in PBS, and 100 μl of the amount of suspension is added to the tubes. The cells unwind to the supernatant,

2. Serial dilutions of inactivated sera are obtained in PBS / BSA from an adult pig (positive control), a newborn piglet (test sample) or rabbit (negative control). 0.025 ml of the amount is added to red cells with doubling. The tubes are incubated at 4 ° C for 4 hours.

3. After incubation, the tubes are washed three times in PBS / BSA. Then 0.05 ml of the isotope is added to each tube and incubation is continued overnight at 4 ° C.

4. The tubes are washed repeatedly three times and the calculation of the number for 1 minute is carried out on a gamma-ray counter.

5. Results are postponed as the number of cycles per minute depending on the titer.

The results are shown in figure 2. Rabbit serum is used as a negative control value, and adult (i.e. breast-fed) pig serum is used as a positive control value. You can see that the level of pig antibody in the body of breast-fed pig 2 is comparable to the level in the negative control sample.

EXAMPLE 3. Demonstration of the relevance of complement C3 to the destruction of xenograft

Complementary deficient guinea pigs, derived from the line described by Burger et al., (Eur.J. Iimnunol., 16: 7-11 (1986)), are used as donors for heart transplantation using basically the same technique which is described for rabbit-to-pig xenografts in Example 1. Rats are anesthetized with ether aspiration and the hearts are cooled with cardioplegia and cut off as described above. Guinea pig donors are anesthetized with valium and intramuscular hypnol. Hearts are implanted in the neck as described above. For control guinea pigs, that is, those that have normal complement levels, graft rejection usually occurs within a few minutes, which eliminates the need to close the neck. In experimental animals, the neck is closed and the hearts are monitored by two daily palpations. Normal ECGs are observed for several hours after surgery, which indicates the absence of hyper-acute rejection.

EXAMPLE 4

A. Swine lymphocytes and kidney cells activate human complement in an alternative way of metabolism

In accordance with the methodology of Grabar and Williams (Biochem. Biophys.Acta, 10: 193 (1953)) agarose gels I pour into 8 × 8 cm glass cups (figure 3). A 10 ml gel mixture is required, and it consists of 5 ml of 2% agarose and 5 ml of Veronal buffer (VB), (VB is 75 mM sodium barbitone, 10 mM EDTA, 10 mM NaN ₃ , pH 8.5). Agarose and VB are mixed at a temperature of 60 ° C immediately before use. The gels are poured and cooled on a leveling platform. After sedimentation, the gel consists of 1% agarose and has a depth of about 1.5 mm.

Wells 3 with a diameter of 3 mm are cut at a distance of about 1 cm from the end of the gel. Each well may contain about 8 μl of sample. The sample has no special preparation, except for the addition of bromophenol blue for staining the sample. After application of the sample, the gel is carefully placed on the platform of the electrophoresis vessel. Then, cotton swabs soaked in VB (sample buffer) are carefully applied along the edge of the gel closest to the wells, and another swab is applied to the opposite edge of the agarose. (It is important to ensure that the ends of the swabs are immersed in tanks with a buffered mixture). Then a current of 25-30 mA is passed through the gel until albumin (visualized with the aid of bound bromphenol) reaches a positive (anode) swab. The process takes approximately 2.5 hours to 3 hours. If two or more gels need to be processed simultaneously and in parallel, then the applied current should be increased in power accordingly so that two gels consume a current of 50 mA, and three gels consume a current of 75 mA, and so on.

When the electrophoresis is complete, as indicated by the movement of the marker 9 based on the egg protein, which is visualized with bromphenol blue, the gel is removed from the electrophoresis vessel.

B. Two-dimensional cross-hair immunoelectrophoresis (2-D Rockets)

Strips 11 (FIG. 4) containing the electrophoresis proteins from step (A) are cut and stacked on one end of a new glass cup 13. Then a 1: 1 mixture of 15 2% agarose: HC containing about 1% antiserum for the protein to be visualized , poured onto a cup and defend. The antiserum is added to a mixture of agarose and VB when the latter is cooled to a temperature of about 50 ° C.

The "rocket" cup is then subjected to electrophoresis as described above, with the end of the gel containing strips of the first size connected through a cotton swab to the negative electrode (cathode) 17, and the opposite end connected to the anode 19. The gels are electrophoresed overnight with an applied current , the value of which depends on the size of the gels; 10 mA is needed for each 8 cm of gel length so that the 16 cm gel consumes 20 mA, and so on.

Proteins are separated by electrophoresis in the first size, and counted and visualized by electrophoresis in the second size; the following describes the staining technique for visualization purposes.

C. Softening and staining of gels

This technique is similar for both traditional immunoelectrophoresis and missiles. The gel to be coated is coated with 1 layer of POSTL IP (trademark) non-fibrous paper manufactured by Adiard Evans and Co., which is pre-wetted with water. The gel is then coated with 5 layers of absorbent paper towel. The structure is softened for 1 hour, after which all the paper pulp is removed, and the process is repeated.

After secondary softening, the gel is dried in a stream of warm air and then impregnated with PBS for at least 1 hour in order to remove the non-precipitated protein. The gel is dried again and stained for 10 minutes in a solution of 0.5% (in terms of mass to volume) of coomassie brilliant blue G250, 45% water, 45% methanol and 10% acetic acid.

Then the gel is freed from staining by continuous washing in 20% methanol, 6% acetic acid until the background is clear. After that, the gel is dried in warm air.

Figure 5 reproduces the dried gel. Rocket 1 is a negative control sample containing 50 μl of normal human serum (HHS) and 25 μl of VBS, including EGTA. EGTA is a chelator that cleaves calcium; calcium is an essential component for complement activation in the classical metabolic way, and therefore the presence of EGTA ensures that complement can only be activated by an alternative metabolic pathway. The left-side (larger) peak represents C3, and the right-side (smaller) peak represents C3bi, the decay product of activated C3. Therefore, in the control sample, a smaller amount of C3bi indicates only a small amount of complement activation.

In rocket 2, 75% porcine red blood cells (volume ratio) are added to the buffered mixture. An insignificant, but probably insignificant increase in the level of C3bi is observed, suggesting that porcine red blood cells only marginally activate human complement in an alternative metabolic way. The reason for this poor answer is not clear.

In rockets 3 and 4, 75% of pork lymphocytes (volume ratio) or 75% of pig kidney cells (volume ratio) are respectively added to the buffered mixture. In each case, there is a significant increase in the level of C3bi, indicating the activation of human complement by porcine lymphocytes.

EXAMPLE 5. Porcine lymphocytes are not lysed by human antibodies in the presence of porcine complement, but are lysed in the presence of rabbit or human complement.

The chromium release method is used to monitor lysis of cells mediated by human serum in the presence of either porcine complement, or complement of a newborn rabbit, or human complement.

Materials

Medium for the isolation of lymphocytes - Flowlabs;

RPMI 1640 + 10% inactivated fetal calf serum;

SFR (without azide) - Oxoid;

Tablets with V-shaped holes - Sterilin;

Newborn Rabbit Complement Lymphocytes - Serums;

Labels - either human or pig complement (dilutions 1 + 7 in RPMI);

Heat Inactivated Serums (56 ° C, 30 minutes)

Method

1. Defibrinated whole pig blood, diluted 1: 1 in PBS, is applied in layers to an equal volume of medium to isolate Ficoll Hypaque lymphocytes. The tubes are centrifuged at 1200 g for 80 minutes at a temperature of 20 ° C.

2. The obtained porcine lymphocytes at the interface are removed and washed once in PBS. The supernatant was resuspended in RPMI 1640, and the number of cells was adjusted to 2 × 10 ⁷ / ml.

3.200 mccub. an inch of ⁵¹ Cr is added to a 2 × 10 ⁷ cell pellet and incubated at room temperature for 1.5 hours.

4. Labeled cells are washed twice at 900 g for 5 minutes and adjusted to obtain a final number of cells of 1 × 10 ⁶ / ml in RPMI.

5. 0.05 ml of the amount of inactivated sera to be tested as serial dilutions with doubling, together with control samples, is plated on plates. The diluted complement is added to the relevant wells in an amount of 0.05 ml followed by the addition of 0.05 ml of labeled cells. The cups are incubated for 1 hour at a temperature of 30 ° C in a furnace with CO ₂ .

6. After incubation, the plates are centrifuged for 15 minutes at 900 g, 20 ° C so as to pellet the cells. 100 μl of the supernatant is removed into labeled tubes and counts are carried out for 1 minute on a gamma-ray counter.

7. Results are plotted in% of the number of initially labeled cells depending on the titer.

Control samples

Full Release Control (FRC) - 50 μl of cells + 100 μl of water + 0.1% Tween;

Negative control - 50 μl of cells + 100 μl of RPMI;

Complement control (CC) - 50 μl of cells + 50 μl of diluted complement + 50 μl of RPMI.

results

The test results are shown in Fig.6. It can be seen that porcine lymphocytes are lysed by human serum only in the presence of non-guinea (i.e., rabbit or human) complement, but not in the presence of porcine complement.

The conclusion is that one or more homologous complement restriction factors present on pig cells are successfully regulated to decrease the effect of pork complement, but not the action of human or rabbit complement.

EXAMPLE 6

The purpose of this example is to show that an antibody can cause hyper-acute transplant rejection. The concept on which this invention is based has arisen as a result of the discovery that hyper-acute rejection can occur in the absence of anti-transplant antibodies, but requires functional complement. Since this discovery is new, there is no description in the literature of experiments that formally show that an antibody can cause xenograft rejection. Since it is difficult to determine whether these antibodies play a role or not in the presence of naturally occurring antibodies, such an experiment is not easy to carry out. In this example, the role of the antibody is demonstrated by reversing the concordant xenograft into a discordant xenograft by introducing an antibody of appropriate specificity. The recipients used in this study are male rats of the PVG (RTIC) line (Banting and Kingdom, Bicester, Oxon, UK) aged 3 to 6 months and weighing 250 to 300 g. Syrian hamsters, also obtained from Banting and Kingdom and weighing from 100 to 150 g. Heart transplantation is carried out in accordance with the Heron method (see the link in Example 1). Hamster hearts are transplanted into the neck of the rats, connecting the aorta with the carotid artery, and the pulmonary artery with the jugular vein using the cuff technique. All other vessels are ligated. Hearts begin to beat a few minutes after being released from vascular clamps and are monitored by external palpation. All operations are carried out on animals anesthetized by aspiration of halotane and oxygen.

Anti-hamster lytic antibody levels are measured as follows: 50 μl of a 1% hamster erythrocyte solution is added to 50 μl of test serum, which is subsequently diluted. 50 μl of 1 out of 7 dilution of the complement of a newborn rabbit (Sera Lab., Crawley Down, Sussex) is added to the solution and incubated for 1 hour at a temperature of 37 ° C. 750 μl of diluent was added to fix complement and centrifuged (Beckman MICROFUGE, 13000 rpm, 4 minutes), after which OD 415 was measured in the supernatant. (The word MICROFUGE is a trademark). Positive and negative control samples are CFD and distilled water added to a 1% cell solution, respectively. The measurement results of OD 415 are set as a function of serum titration on the X axis. As can be seen from FIG. 7, transplantation of a hamster’s heart into a rat results in a rat producing very high titers of lytic anti-hamster antibodies. Serum from some of these rats was separated into its component protein fractions by column chromatography on a SEPHADEX G200 column (Pharmacia GB Ltd, London) using a standard column chromatography technique ("Using SEPHADEX in the Separation, Purification and Characterization of Biological Materials", Curling in Exp.Physiol . and Biochem., 3 (1970) 417-484 (GA Kerkut Ed.) Academic Press, London and New York, 1970). (The word SEPHADEX is a trademark). Each of the 7 ml fractions collected from the column was assayed for lytic anti-hamster activity as described above. Fig. 8 shows that, despite the fact that these antibodies were induced as a result of heart transplantation, anti-species activity is maintained almost exclusively in the immunoglobulin M fraction. After the activity test, the fractions are concentrated using CX10 ultrafilters (Pharmacia) to a concentration of 0.5 mg ml and stored at a temperature of -70 ° C until use.

In order to study their ability to reject a xenograft as opposed to lysing red cells, hamster hearts are transplanted into the neck of inactive rats. Shortly after the hearts began to beat, 2 ml of pure serum or 0.5 mg of purified immunoglobulin containing lytic anti-hamster antibodies was administered intravenously to rats. Unseparated serum and 0.5 mg of immunoglobulin M cause rejection of a hamster heart transplant for 15 minutes. The results from the introduction of immunoglobulin G are not consistent with some drugs that cause transplant rejection, while in other cases the transplant continues to function. When albumin from the G200 column was introduced as a control sample, heart grafts always survive and reject during the normal time period for this model, which is 3 days. This experiment shows that binding of this antibody to a transplant can induce its hyper-acute rejection.

EXAMPLE 7

The data cited so far in this application show that xenograft rejection may involve complement activation either by the alternative metabolic pathway or by antibody-mediated complement activation (classical pathway of metabolism). In addition, complement regulators on the surface of a xenograft target can protect it from rejection by a homologous but not heterologous complement. The decisive activation stage common to both metabolic pathways of complement activation is the cleavage of the complement component C3. This cleavage is carried out by C3-convertase, C4b2a (C3-convertase of the classical metabolic pathway) or C3bBb convertase (C3-convertase of the alternative pathway). These enzymes cleave C3 to form C3b, which in turn can use an alternative metabolic pathway to form more C3 convertases (feedback loop). As a result, the complement system is able to amplify C3b deposition on a “foreign” target. However, a plurality of C3b does not successfully interact with a foreign target and remains in the liquid phase and can therefore randomly bind to host cells. In order to protect these cells from the effects of random binding of complement, control proteins are involved in the inactivation of complement components either in the liquid phase or in the state of binding to their own tissue. Those glycoproteins that are involved in the control of C3 are all genetically associated within the same region of the human chromosome 1, called the RCA locus (complement activation regulators). In this example, the applicant shows that murine cells that have acquired the human chromosome I through expression techniques and express RCA locus proteins on their surface behave in an in vitro analysis of xenograft rejection as if they were human cells, not mouse cells.

Cell lines

T5 is an tonsil B-cell line transformed by the Epstein-Barr virus obtained by the method of Bird et al., (Nature, 289: 300-301 (1981)). B10 is a human hybridoma producing an anti-tetanus monoclonal antibody, which is obtained by synthesizing the human B-lymphoblastoid line (BLL) with the mouse myeloma cell line X63-AG8.653 (Kierney et al., (J. Immmunol., 123: I548- I550 (1979) .T5 and B10 cell lines are obtained from M.C. Carter and Dr. N.K. Hughes-Johnson from MRC MITI Group in Babraham, Cambridge. DB3 is a mouse hybridoma cell line that produces an anti-progesterone monoclonal antibody ( Wright et al .. Nature, 295: 415-417 (1982)). oligonucleotide primers specific for human chromosome I: (5'-CCACAGGTGTAACATTGTGT-3 ') (SEQ ID NO: 1) and (5'-GAGATAGTGTGATCTGAGGC-3') SEQ ID NO: 2; they are, respectively, located higher and lower in the sequence of the primers of the human gene for antithrombin 2 (AT3), which is known to be on human chromosome I (Wu et al., Nucl. Acids Res., 17: 6433 (1989)). Oligonucleotides can be synthesized by methods well known in the art.

High molecular weight genomic DNA is obtained using the method of Herrmann and Frischauf (Methods Enzymol., 152: 180-183 (1987)). Briefly, 100 × 10 ⁶ cells from each cultured cell line are lysed with 5 ml TNE (100 mM Tris, pH 7.5, 100 mM Nad, 10 mM EDTA 1% Sarkozil) and treated with fresh proteinase K (100 μg / ml ) The drug is extracted with phenol (saturated water and balanced against 0.1 M Tris, pH 8), phenol chloroform (1: 1, vol.) And then isoamyl alcohol: chloroform (1:24, vol.). DNA is obtained by ethanol precipitation and dialyzed against TE (10 mm Tris, pH 8.0, 1 mM EDTA), adjusted to 100 mM in NaCl, as well as only TE at 4 ° C. The isolated DNA was analyzed on a 0.5% agarose gel and the concentration was determined by optical density at 260 nm. The polymerase chain reaction (PCR) for each cell line is carried out as described by Saiki et al., (Science, 239: 487-491 (1989)). A volume of 100 μl contains 500 ng of genomic DNA, 1.2 ng of each primer and 2.5 units of Taq DNA polymerase (type 3 Thermos acquaticus) (Cambio Ltd., Cambridge, UK) using a buffered solution supplied with the enzyme. Nucleotides (dNTP _s ) (Boehringer Mannheim Diagnostics and Biochemicals Ltd, Lewis / East Sussex, UK) have a concentration of 2 mM each. DNA was amplified for 30 cycles using a programmable thermal controller (Genetic Research Instrumentation Ltd, Dunmow, Essex, UK): denaturation at 93 ° C for 1 minute: annealing at 55 ° C for 1 minute: and insert at temperature 72 ° C for 2 minutes. 10 μl of the reaction product is analyzed directly on a 2% agarose gel in a buffered solution of Tris, boric acid, EDTA. Product size is determined by comparison with HincII-digested phage X-174-rf DNA (Pharmacia LKB Biotechnology, Upsala, Sweden).

Cultured T5, B10 and DB3 cells are treated with anti-DAF (hemolysis promoter) with a monoclonal antibody (Kinoshita et al., (J.Exp.Med., 162: 75-92 (1985)) and fluorescent-labeled “second” antibody. Cells (1 × 10 ⁶ ) are reacted with the mouse anti-DAF monoclonal antibody IAIO (IgG2a 10 μg / ml in 100 μl of 10% fetal fetal serum, 0.1% azide). IAIO (Kinoshita et al., (J. Immunol., 136: .3390 -3395 (1986)) is obtained from Dr. M. Devitz of the University of New York Medical Center; New York, USA. Only buffers serve as “blind” control samples. After incubation for 2 hours, the cells are washed with ice 3 times, resuspended and incubated in 100 μl of buffer containing 1 in 100 FITC conjugated goat F (ab ') 2 anti-mouse immunoglobulin (with purified heavy and light chains by affinity and absorbed in human immunoglobulin) (Tago Immunochemicals Inc. , Burlingame, California, USA) for one hour on ice Some cells are incubated with the second antibody only as staining control samples. Because DB3 is a mouse IgG1 secreting cell line, a FITC-conjugated sheep anti-mouse IgG2A (I in 40, The Binding Site Ltd, Birmingham, UK) or goat anti-mouse IG pre-absorbed by equal volumes of DB3 cells are also used to eliminate anti-IgG1 reactivity that occurs when staining DB3 cells. All cells are thoroughly washed and resuspended in 200 μl of buffered solution. DAF-positive cells are detected using a Beckton Dickinson FACS-STAR apparatus for analysis of a cell sorter with excitation of fluorescence (FACS). (The expression FACS-STAR is a trademark).

The polymerase chain reaction (PCR) method is used to detect the presence of human chromosome 1 in three different lines - cultured cells: T5 (human), B10 (human-mouse) and DB3 (mouse-mouse). Fig. 9 shows that after amplification, T5 and B10 have a strip size of 495 base pairs, while DB3 (i.e., mouse-mouse hybrid) does not have a strip at all. It was stated that PCR products using ATZ primers consist of 2 alleles of 572 base pairs (allele 1) and 496 base pairs (allele 2) (Wu et al., Above). The bands found in the genomic DNA of T5 and B10 correspond to allele 2. This proves that the B10 cell line of the human-mouse hybrid contains the human chromosome I.

FACS analysis for the presence of DAF shows that most human T5 cells (85.7%) are stained positively with anti-DAF monoclonal antibody. A similar level (83.1%) of positive cells was found in B10 cells of the mouse-human hybrid. Mouse-mouse hybrid DB3 cells exhibit identical staining patterns for both anti-DAF-treated and untreated drugs. However, this reactivity of the anti-mouse IgG1 disappears if

(1) use a FITC-conjugated anti-mouse IgG2a or

(2) the above goat anti-mouse IgG is pre-absorbed by DB3 cells. The results show that the human-mouse hybrid B10 cell line expresses human DAF on the surface of the cell membrane as detected by specific anti-DAF monoclonal antibodies. The expression level is similar to the expression level relative to the human T5 cell line. The mouse-mouse hybridoma cell line does not express human DAF.

Cytotoxic studies of the activity of killer cells with radioactive chromium are carried out in relation to these cell lines, as described in Example 5 above. Figure 10 shows that when the pork anti-human antibodies are incubated with the human T5 cell line, the addition of rabbit complement causes lysis, whereas lysis does not occur when the human complement is added, since, of course, the T5 cell line has human HCRF _S. This is a confirmation of the results of Example 5. When human antibodies are used on a human cell line, lysis does not occur with both human complement and rabbit complement, indicating the absence of autoantibodies. The technique with radioactive chromium does not allow a long incubation to detect any significant levels of activation by the alternative metabolic pathway of rabbit complement in human cells (Fig. 11). However, when human antibodies are incubated with the DB3 mouse-mouse hybridoma cell line (FIG. 12), cell killing (lysis) is achieved by both rabbit complement and human complement, which demonstrates the possibility of the existence and functioning of human complement in such a trial. When using a B10 human-mouse hybrid having a human chromosome I and expressing at least DAF, the rabbit complement causes cell line lysis, while the human complement is not able to cause cell line lysis (Fig. 13). The explanation for this phenomenon is that human HCRF _S expressed through the possession of chromosome I on a mouse-human hybrid inhibit the activity of human complement.

EXAMPLE 8

The previous example shows that having chromosome I can prevent xenograft cell rejection. While strong indirect evidence exists that it is the CRA locus that protects the mouse cell from xenograft rejection, this example provides formal evidence. This example demonstrates the effect of transfection of non-human cell lines using human MCP and their interaction with human or rabbit complement.

cDNA is obtained for MCP (human membrane cofactor protein) in accordance with the detailed description in Lublin et al., (J.Exp.Med., 168: 181-194 (1988)). The construction of transfected cell lines was carried out using the expression plasmid SFFV.neo in accordance with the procedure described by Uhlbrigge et al., (Proc.Natl. Acad.Sci., 85: 5649-5653 (1988)). It contains a long terminal repeat of the 5 'virus that forms the focus of avian leukemia (Freund virus) (SFFV.LTR) (Clark and Mak (Nucl.Acids.Res., 10: 3315-3330 (1982)) and (Proc.Natl.Acad .Sci, 80: 5037-5041 (1983))). Cell lines are obtained from the American Type Culture Collection, 12301, Parklawn Drive, Rockville, Maryland, USA. The cell lines used are CHO-KI (ATCC CCL 61) and NIH / 3T3 (ATCC CPL 1658). Gene expression is confirmed using a monoclonal antibody to MCP (Andrews et al., Ann.Hum.Genet., 49: 31-39 (1985) and FACS analysis, which is already described above. In some cases, cell lines are selected according to the principle of high expression level MCP by cell sorting on FACS using a standard methodology.

This example illustrates the effect of transfection of CHO cells using MCP. Since these cell lines grow as monolayers, cell killing is assessed by the analysis of terminal adenine uptake, which is described by Voeno et al., (Immunology 32: 221-226 (1977)). Briefly, this assay involves incubating cell cultures in flat-bottomed sterile 96-well complement and antibody plates. By the end of the period of experimental incubation, cell viability is assessed by the ability of the culture to take radioactive adenine. Viable cells take adenine, dead cells do not; therefore, viable cells have high numbers, dead cells have low numbers.

By analogy with many transformed cells, CHOs are insensitive to naturally occurring antibodies and to the effects of the alternative complement metabolic pathway. However, these cells are sensitive to those antibodies that have been shown to cause hamster heart xenograft rejection, as described in Example 6. Since CHO cells are obtained from the hamster, these antibodies kill CHO cells with both human and rabbit complement (Fig. 14). When CHO cells are transfected with human MCP, cells can only be lysed in the presence of rabbit complement. The human complement is inhibited due to the presence of human MCP on the surface of the hamster cell line (Fig. 15). The proof that the decay of the cells to be destroyed is really due to the decay of C3 convertase is the analysis of the decay of human C3 after incubation of CHO cells under the influence of missile immunoelectrophoresis, as described in Example 4 above. As you can see, there is no decay above the control levels of complement (Fig.16).

These data confirm that the genetically engineered proteins that regulate downward complement on the surface of non-human cells protect those cells from the mechanisms of hyper-acute xenograft rejection that, as a common hallmark, need to cleave the C3 component of complement.

EXAMPLE 9

In accordance with the procedure of Example 8, mouse fibroblast cells ZTZ transfect cDNA encoding MCP (MCP clone K5.23). ⁵¹ Cr is added to the cells as described in Example 5. One volume of cells is then incubated with one volume of human complement inactivated by heat and one volume of human complement previously absorbed at 4 ° C with mouse spleen cells in order to remove the anti-mouse antibody from human complement. The mixture and subsequent dilutions of complement are plated on plates. The main conditions and distinguishing features of the analysis with radioactive chromium are similar to those described in Example 5. The results regarding clone K5.23 are shown in Fig. 17, which also shows, as a control experiment, the effect of the MCP cDNA introduced in the reverse orientation (in this case, it is not transcribed). Correctly transcribed MCP cDNA gives protection against killing of cells, as can be seen from the relatively low ⁵¹ Cr release level, whereas untranscribed cDNA does not give protection, as indicated by relatively high ⁵¹ Cr release levels.

EXAMPLE 10

Results similar to those described in Example 8 above can be obtained using LI / 210 cells (murine leukemic cell line) transfected with cDNA for DAF. cDNA obtained for DAF in accordance with the description in the work of Lublin and Atkinson (Ann. Rev. Immunol., 7: 35-58 (1989)).

EXAMPLE 11

cDNA for MCP is obtained and ligated into the plasmid SFFV.neo, as in Example 8 above.

Using this preparation, DNA transgenic mice are obtained as described in the "Manipulation of Mouse Embryos. Laboratory Manual" by B. Nogan et al., Cold Spring Harbor Laboratory (1986). From ten to fifteen female mice (CBAxBlO) fI aged 3-4 weeks are induced to superovulate by intraperitoneal injection of 5 units of serum gonadotropin from pregnant mares (commercially available as Folligon) followed by an intraperitoneal injection of 5 units of urinary chorionic gonadotropin from urine from pregnant women (supplied commercially as Chorulon). Females are left for mating, on the day of Chorulon injection, with males (CBAxB10) FI, and the next day, females with vaginal plugs are sacrificed by cervical (cervical) mixing and fertilized eggs are isolated from their fallopian tubes.

Three to four hundred eggs, isolated in this way, contain two pronuclei, clearly visible using Nomarski differential interference contrast optics at 400x magnification. One of the two pronuclei is injected with approximately 2000 copies of the DNA preparation containing the MCP cDNA transgene at concentrations from 0.5 to 2 ng / μl.

The eggs that survived the microinjection are reimplanted into the fallopian tubes of the (CBAxB10) FI females, which were mated the previous night with vasectomized males and therefore were pseudopregnant (that is, they are ovulated and their hormonal state is a state of pregnancy, however, their own oocytes are not fertilized). About 30 microinjected eggs are transferred into the fallopian tubes of each pseudo-pregnant animal, under anesthesia, either on the day of microinjection or the next day, when the eggs are in the second cell stage. A normal pregnancy occurs and seventeen mice are born from ten mothers. Progeny is screened by slot blotting and / or southern blotting (see Example 8), as well as by polymerase chain reaction, DNA analysis from tail skin cells using ³² P-labeled probes and primers that recognize the transgene. One offspring animal, a male, is transgenic for the MCP DNA sequence.

EXAMPLE 12

The procedure of Example 11 is repeated except that cDNA for DAF, as described in Example 10, is used instead of cDNA for MCP. Twenty-three mice are born from ten mothers. Three of them (two females, one male) transgenic for DAF are obtained using the Southern blotting method.

EXAMPLE 13

The male mouse obtained in Example 11, which contains the human MCP cDNA transgene, is allowed to grow to maturity and mated with the female (CBAxB10) FI. Offspring of eleven animals are obtained. The DNA of tail cells from each member of the offspring is screened by slot blot analysis using labeled human MCP cDNA as a probe to determine whether the transgene is inherited or not. The analysis results are shown in the upper part of Fig. 18. You can see that the descendants 0, 1, 5, 7, 8, and 10 inherited. (Four control analyzes are undertaken: with human DNA (H), mouse DNA (M), mouse DNA mixed with 10 pg of human MCP-labeled cDNA, and mouse DNA mixed with 100 pg of human MCP-labeled cDNA).

EXAMPLE 14

The male mouse obtained in Example 12, which contains the human DAF cDNA transgene, is allowed to grow to maturity and mated with a female (CBAxB10) FI. For each descendant obtained, tail DNA was screened by slot blotting using labeled human DAF cDNA as a probe to determine if the transgene is inherited or not. The analysis results are shown in the lower part of Fig. 18. It can be seen that offspring 13.3 (female) inherited. (Four control samples are performed: human DNA (H), murine DNA (M), murine DNA mixed with 10 pg of human labeled DAF cDNA, and murine DNA mixed with 100 pg of labeled human DAF cDNA (hemolysis stimulator)).

Sequence List

SEQ ID NO: 1

Sequence Type: Nucleotide

Sequence Length: 20

Properties: The upstream primer of the human antithrombin 2 gene (AT3)

Sequence: CCACAGGTGT AACATTGTGT 20

SEQ ID NO: 2

Sequence Type: Nucleotide

Sequence Length: 20

Properties: Below the primer of the human antithrombin 2 gene (ATZ)

Sequence: GAGATAGTGT GATCTGAGGC 20

Claims (2)

1. A method for producing a transgenic animal, said method comprising introducing DNA into the animal’s genetic material, characterized in that the DNA encodes a homologous restriction factor of complement of a discordant species and a sequence that provides expression of the coding DNA in at least some cells of the transgenic animal, cells can be removed from the transgenic animal and transplanted to the animal of the indicated discordant species without hyper-acute rejection due to the presence of homol limiting complement restriction factor, preventing complete complement activation. 2. The method according to claim 1, characterized in that in the specified method, microinjection or semen is used to introduce the DNA into the animal’s genetic material.

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