JP2002530093A - Stabilizing milk in transgenic animals - Google Patents

Abstract

(57) Abstract: The present invention relates to stabilization of milk of a transgenic animal. In particular, the invention provides for the expression of a protein (eg, fibrinogen) expressed in the milk of a transgenic animal by co-expressing a serine proteinase inhibitor (eg, α ₁ -antitrypsin) in the milk of the transgenic animal. Regarding protection.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the stabilization of milk of transgenic animals. In particular, the invention relates to the protection of fibrinogen expressed in milk of transgenic animals, together with the expression of serine proteinase inhibitors.

[0002]

[Prior art]

During the last decade, recombinant DNA technology has been increasingly used to produce commercially important biological materials. For this purpose, DNA sequences encoding various human proteins of medical importance have been cloned.

[0003] Expressing DNA sequences in bacteria to produce the desired medically important protein appears to be an attractive proposition, but in fact, in bacterial cells, exogenous proteins are not. Bacteria are often not suitable as hosts because of their stability and poor processing.

In view of this problem, attempts have been made to express cloned genes in mammalian tissue culture, which in some cases has proven to be a viable strategy. However, batch fermentation of animal cells is an expensive and technically difficult process.

[0005] It has been clarified that the use of transgenic animals as hosts can be a solution to the above problems. WO-A-8800329 discloses transgenic animals that secrete useful pharmaceutical proteins into their milk. To date, it has been successful to produce transgenic proteins in milk of transgenic animals without causing proteolytic damage to the protein by other components of the milk. Until now, practical precautions taken against the degradation of foreign proteins in milk have been to treat the milk quickly after collection or to add known chemical inhibitors to the milk after collection. Met.

[0006] Milk is a complex mixture of proteins, lipids, and carbohydrates. The protein component basically consists of three classes of proteins, casein, serum proteins,
And acidic whey protein. Casein assembles into large molecular weight complexes called micelles that can precipitate by low speed centrifugation or under mildly acidic conditions.
Serum proteins consist primarily of albumin and immunoglobulins, but there are small amounts of other proteins, possibly including the pro-protease plasminogen and prothrombin. Whey proteins are generally small, stable to acids and tend not to precipitate under acidic conditions.

[0007] Proteolytic activity can be measured in certain circumstances, especially when the natural composition changes.
That is, it can be detected in milk after a drastic change in environmental conditions, such as heat treatment or prolonged storage. The most prominent of these is plasmin, a protease active under alkaline conditions that cleaves proteins behind the basic amino acids lysine and arginine. Other proteases in milk have thrombin-like activity and are more active under acidic conditions.

[0008] Plasmin in milk exists in two forms, the predominantly inactive precursor plasminogen and smaller amounts of the active form. Some of them do leak from the plasma into the glandular lumens, but the source of this protease is uncertain. Leakage from plasma is due to the observation that the level of plasmin / plasminogen in colostrum is high when the barrier between blood and milk becomes more leaky and there may be high protein transport to milk. Supported. However,
It has also been observed, at least in the most studied cows, that plasmin / plasminogen levels increase during lactation, with a sharp rise at the end. The last increase in activity is associated with a proteolytic step involved in mammary gland regression when this tissue begins to enter the process of stopping milk production. This is a reasonable assumption since plasmin activity is involved in the remodeling process. In addition to being present in milk in two forms, plasminogen is primarily associated with casein micelles, which form part of the natural mechanism for preventing activation. May be.

[0009] There is always the theoretical possibility that proteases known to be present in milk can act to damage the expressed heterologous protein to provide a source of therapeutic products. Although possible, the significant degree of damage that prevents economical production of the target protein is not widespread. As a test system for assessing expression levels, many proteins are expressed in mouse milk, and mouse milk is often expressed in blood due to trauma induced during the mechanical process of collecting milk However, there are few reports on the profound degradation of target proteins due to proteolysis. For example, a modified tissue plasminogen activator, a protein designed to be activated by proteolysis and also a strong activator of plasminogen, is present in both mouse and goat milk. It has been successfully expressed. This is very important for the general stability of milk, as it is expected that the presence of active plasminogen activator could activate all plasmin activity in milk and cause the milk itself to break down. Good evidence. This was not observed in the test system described.

However, the expression of human fibrinogen in milk of transgenic livestock presents a surprising degree of proteolytic damage,
It became clear. This is human plasma sp. IX spiked in sheep's milk.
As previously reported in 1989 when the factor was found not to be degraded (Clark et al, 1989), contrary to the general observation that sheep's milk is not high in protease activity, and a great deal of Contrary to the successful expression of a number of heterologous proteins including AAT (α-1-antitrypsin), Factor IX, Factor VII, and bile salt-stimulated lipase without identification of proteolytic disruption . This experience in sheep milk is repeated by proteins expressed in other ruminant milk. For example, in goats, both AAT and antithrombin III, atypical tissue plasminogen activator, and at least two monoclonal antibodies are expressed, but proteolytic damage causes problems with economic recovery of the product. There is no report on.

[0011] The proteolysis of fibrinogen takes many forms and occurs to varying degrees. The α chain, in its undamaged form, has a relatively long carboxy-terminal region that is somewhat protected from proteolysis, probably by associating with the fibrinogen molecule in its entirety. The α-chain clipping is
Presumably, the carboxy region tends to become more exposed once in the solution phase and, once it occurs, to get closer to the protease and tends to progress. In fact, fibrinogen isolated from human plasma already has some α-chain proteolysis in at least one of the two subunits present, and the entire fibrinogen molecule contains F1 (parent molecule) and Expands as a double line known as F2 with partial alpha chain degradation. Further proteolysis results in the removal of two α-chains per molecule, but there is also a progressive clipping back resulting in the appearance of “fragment X” with relatively large damage to both chains. here,
Further proteolysis may result in smaller ladders of molecular weight, such as fragments Y, D, etc.
Occurs for damaged molecules that result in The clotting activity functionality begins to decrease significantly at fragment X and beyond. Therefore, the presence of this fragment is a useful indicator of the percentage of functional fibrinogen present in the mixture, and it is desirable to keep the content of fragment X to a minimum when it comes to recovering useful products.

The site of damage to the α-chain found in the F2 component of human plasma is inconsistent with the known specificity of plasmin and does not occur at a single site. despite this,
One of the biological functions of plasmin is to break down the fibrin component of the clot, and fibrinogen is damaged in the alpha chain by plasmin before activation by thrombin and uptake into the clot. It is known that this can be done. Therefore,
Plasmin in milk causes a partial degradation of fibrinogen, resulting in F2
Which can then be broken down to X.

[0013] In general, the higher the expression level of a heterologous protein in milk, the lower the degree of proteolytic damage to that protein. However, there is a clear and substantial proportion of fragment X in sheep's milk that can be observed by gel analysis, even at a total expression level of about 5 g per liter. This has the effect of reducing the levels of recoverable fibrinogen and requiring the removal of fragments below F2 by any step designed to recover the functional product. The degree of fibrinogen damage in milk is much higher than expected based on experience with other proteins. Thus, fragment X is observed at all stages of lactation, starting with high levels in colostrum, decreasing during the first 30 days of lactation, and increasing steadily for the next 30 days. A further complication with fibrinogen is that it removes fibrinopeptides A and B, at least under non-denaturing conditions such as those used in processes designed to produce biologically active substances, allowing fibrinogen to become autologous. The effect of a second protease activity present in milk that has a thrombin-like effect of increasing the tendency to aggregate and become insoluble.

[0014] Both plasmin and thrombin activity in milk, which previously did not seem to interfere with the recovery of heterologous proteins expressed in ruminant milk, both significantly reduce the process yield of fibrinogen. And requires a more complex and therefore more expensive recovery process, reducing the shelf life of usable milk. This is a combination of irreversible material lost by precipitation and degraded material that must be removed to obtain functional fibrinogen. The overall effect is that the yield of purification is low during the first 15 days of lactation,
It increases at day 0, stays high, and then falls off rapidly as breastfeeding progresses. This reduces the supply of normal, useful milk from lactation, possibly beyond four months, to almost one third, and thus increases the cost of milk production by at least a factor of three. In addition, the presence of a significant proportion of fragments X requires larger column matrix volumes and basically more complicated steps. From the standpoint of economic recovery of recombinant fibrinogen obtained from milk of transgenic sheep or other mammals, the expression of the recombinant fibrinogen during milk expression, during milk storage, and during subsequent processing It would clearly be beneficial to find a way to substantially reduce proteolytic damage to the molecule.

[0015] Controlling proteolysis in stored milk and during processing can theoretically be accommodated by adding protease inhibitors immediately after milk collection. However, this product is intended to be used for therapy in humans, and thus, as an essential property of being an inhibitor, adds a protease inhibitor that is often toxic in nature. It should be noted that the risks of monitoring and the costs of monitoring and removal must be realistically considered. An additional complication about fibrinogen is
It appears that there are two different proteases that jointly contribute to the loss of yield. Many inhibitors that are acceptable from a therapeutic processing standpoint (
(Except for the active site serine alkylating agent such as diisopropylfluorophosphate), which does not appear to work effectively for both activity and safety issues that further exacerbate the process.

One of the inhibitors tested from a group of potential candidates that is effective against plasmin, namely tranexamic acid, was found to be effective in stabilizing milk during storage. But this did not affect this initial level of fragment X,
Or does not inhibit thrombin damage. A similar experiment that observed the level of fragment X produced when incubating transgenic milk at higher temperatures revealed that 1 g of AAT per liter was not effective per se.

A second, previously unexplored approach to reduce or reduce the further loss of useful products due to proteolysis is to combine protease inhibitors with said products in the milk of the producing species. Expression. The problem of proteolytic damage of heterologous expressed proteins present in the milk of transgenic animals has not been recognized at a level that requires prevention in addition to the means already identified in the art, Until now, there was no motivation to do this. However, there is a problem in selecting inhibitors without accurate identification of the proteases that cause damage and their relative contribution to such damage. For example, an inhibitor of plasmin that is highly active against plasmin, such as serpin α2-antiplasmin, can be selected but has little effect on thrombin. This can be co-expressed with fibrinogen, but may still cause significant precipitation-related loss or may not provide protection against thrombin, which can damage the end product and adversely affect its stability. The expression of potent plasmin inhibitors in the mammary gland may also have unpredictable effects on morphological development or milk production, as plasmin may have a possible role in gland biological phenomena Absent.

[0018] Conversely, it could express antithrombin III and suppress thrombin damage to fibrinogen, but this could be due to plasmin and possibly other proteases that contribute to the overall damage of the target molecule. Will not be valid. For this reason, none of the above protease inhibitors will be recognized as suitable “protective proteins” in the production of therapeutically useful proteins present in milk of transgenic animals.

The protease inhibitor AAT has little effect as an inhibitor on both plasmin and thrombin due to its narrow protease specificity (see Example 1 and FIG. 1). The natural target protease for which AAT acts as an inhibitor is elastase. Except during infections where neutrophils are capable of releasing large amounts of elastase, there is no report that the protease, elastase, appears in milk. Since any animal affected with mastitis or other mammary gland infections is weaned from the milking herd, it is unlikely that this protease is responsible for proteolytic damage to fibrinogen made in sheep milk. Few. further,
AAT has previously been shown by the present inventors to add to transgenic milk after collection, but to provide no provision for the stability of transgenic fibrinogen. In contrast to the stabilizing effect of the plasmin inhibitor tranexamic acid when added to transgenic milk after collection. Thus, along with the other protease inhibitors described above, AAT would not have been expected to provide any protective effect when produced in the milk of transgenic animals.

It has been established that the serine protease inhibitors described above are less likely to be effective candidates for inhibiting fibrinogen proteolysis when co-expressed in the mammary gland of transgenic animals. The dramatic, and therefore surprising, effect of such co-expression is the basis of the present invention. In this example, crossing two separate lines promoted co-expression of AAT and fibrinogen. Analysis of the induced lactation revealed that the resulting fibrinogen was A
It was significantly different from fibrinogen expressed in the absence of AT. The main difference is that the fibrinogen α-chain co-expressed with AAT had no or little detectable proteolytic processing.
This is indicated by the presence of predominantly F1 species on the non-reducing gel, which observation indicates that after partial purification, after precipitation, F1 and F2 can be separated into separate components. Predominantly a single α-chain species is present by sex interaction chromatography and instead of the normal doublet on reducing gel analysis (Example 4).
c and FIG. 10). The second evidence that the proteolysis is clearly reduced is the non-reducing gel analysis, in which the derived milk of the fibrinogen-only line and the absence of the fragment X normally found in natural milk. is there.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION

According to a first aspect of the invention, in the milk of a non-human transgenic animal,
There is provided the use of a expressed serine proteinase inhibitor to stabilize the milk. The present invention provides for such use to extend the storage of milk, which is particularly useful when said milk contains therapeutically valuable proteins. In particular, milk stabilization involves reducing proteolytic damage of proteins present in the milk.

As used herein, “milk” is understood to be a fluid secreted from the mammary glands of an animal. Milk stabilization is pronounced with reduced proteolytic damage. The level of serine proteinase inhibitor required to achieve a stabilizing effect will vary somewhat but can be readily determined by one of ordinary skill in the art without undue burden by standard procedures. Factors to consider include serine proteinase inhibitors and the type of animal in which the serine proteinase is expressed. The level of serine proteinase inhibitor in the milk may be elevated above any background or endogenous level, for example, from an introduced transgene or from an endogenous gene that has been modified to increase levels. Increased expression levels.

The serine proteinase inhibitor is preferably of the serpin or Kunitz family, especially antithrombin III, heparin cofactor II, α-2-antiplasmin, protease nexin-1, preferably α-1- Antichymotrypsin, most preferably α-1-antitrypsin.

[0024] The serine proteinase inhibitors of the present invention are numbered well known in the art (number
), Many of which have already been cloned and expressed by gene transfer. Contains milk, especially heterologous proteins, whose role is to isolate as a therapeutically useful component, rather than expressing them primarily to isolate them as useful proteins per se Their use in the present invention is unique in that it is expressed as a protective protein in the stabilization of milk. Thus, the expressed serine proteinase can be in any form that confers protective capacity. Serine proteinases according to the first aspect of the present invention include all native proteins as well as truncated proteins, amino acid sequence variants (mutant proteins or polymorphic variants), and added residues. And other related species, including natural variants thereof.

The serine proteinase inhibitor may be endogenous to the animal or exogenous to the animal. If endogenous, there would be some modification to the animal to "turn on" its expression, including expression in the animal's milk. If the serine proteinase is exogenous to the animal (ie, the transgene), it can be obtained from any source. Preferably, said serine proteinase inhibitor is of bovine, ovine or human origin.

Of particular interest in the present invention is the proteinase inhibitor α-1-antitri
This is Psin. α-1-antitrypsin (AAT) is a mature peptide
And contains 394 amino acids. First of all, with a 418 amino acid preprotein
Is expressed. The mRNA encoding the preprotein is 1.4 kb long
Which is the length of the cDNA encoding AAT used in this application (about 1).
. 3 kb). Structural gene coding for AAT (liver, P
erlino et al, the EMBO Journal Volume
6 p. 2767-2771 (1987)) contains four introns;
It is 10.2 kb long. As described above, the AAT of the present invention is in its natural form.
Need not be. Examples include mutants that are resistant to oxidation and serine pros such as AAT.
Other analogs of the protease inhibitors are included. These analogs include α-1-anti
Includes a novel protease inhibitor created by modification of the active site of trypsin
You. For example, if Met-358 of AAT is modified to Val, it is in the active center.
By substituting an oxidation-sensitive residue with an inert valine, the molecule becomes oxidized.
Resistance to inactivation by

The animal according to the first aspect of the present invention may be a sheep, cow, goat, rabbit, mouse, camel, buffalo, pig, or horse.

The stabilization according to the first aspect of the invention can be extended to the stabilization of heterologous proteins that are also expressed in milk of non-human animals.

In order to obtain the desired expression in the milk of the transgenic animal, it may be necessary to alter the genomic configuration of said animal. Under the control of a milk protein gene promoter, it may be necessary to introduce a serine proteinase inhibitor (and any other heterologous protein for expression also in milk). The milk protein gene promoter includes long or short whey acidic protein promoter, α-lactalbumin promoter, including those known to those skilled in the art,
The β-lactoglobulin gene is particularly preferred, although it can be anything such as a short or long α, β, or κ casein promoter. The promoter is selected based on a number of factors, such as the composition of the various milks of the various animals. For example, the sheep BLG promoter is particularly useful for expressing proteins in the sheep mammary gland. Details of the genes and their promoters can be found in "Clark
et al, TIBTECH 5:20 (1987) and Henning.
housen, Protein Expression and Purifi
41: 3 (1990).

The 5 ′ flanking sequence (as part of the milk gene promoter) will generally include a milk protein, eg, β-lactoglobulin (BLG) transcription start site. For BLG, it preferably includes about 800 base pairs upstream of the transcription start site of BLG (eg, 799 base pairs). In a particularly preferred embodiment, at least 4.2k base pairs upstream are included.

[0031] Suitable 3 'flanking sequences may be present. However, the presence of such a sequence may not be essential, especially if the DNA encoding the protein contains its own polyadenylation signal sequence. However, in some embodiments of the present invention, it may be necessary or convenient to provide a 3 'sequence. The 3 'untranslated region apparently stabilizes the RNA transcript of the expression system, thus increasing the yield of the desired protein. Such sequences may be derived from the casein 3 'untranslated region, the SV40 small and antigen, 3' untranslated regions of other milk protein genes, especially the BLG gene 3 'untranslated region.

If necessary or desired, suitable signal and / or secretion sequences may be present. In this regard, both homogeneous and heterologous control sequences can be used in the present invention. In addition to the above promoters, useful sequences that control transcription include enhancers, splice signals, transcription termination signals, and polyadenylation sites, many of which are known in the art.

The animal species selected for expression is not particularly critical and will be selected by one of skill in the art to suit their needs. Mammals are preferred,
It may be essential. Laboratory mammals suitable for the experimental ease of manipulation include mice and rats. More yield may be obtained from livestock such as cattle, pigs, buffaloes, camels, goats, and sheep. Intermediate between experimental and domestic animals are animals such as rabbits, which are equally suitable.

Throughout this specification, we carry, at some stage, any kind of genetic modification (eg, gene deletion, mutation, substitution) resulting from in vitro genetic manipulation. The term "transgenic" is used in a broader sense to include animals. The genetic manipulation may be performed on the animal itself during a certain stage, or may be performed on an animal involved in the production of the animal (eg, a parent animal). Thus, not only is the first generation of the animal protected by the present invention, but also its progeny with the necessary genetic modifications described above. The term "transgenic" refers to whether the endogenous or exogenous gene is "switched on", usually by the introduction or rearrangement of a promoter sequence into the genome of the animal,
Or, an animal whose expression level has been manipulated to be "switched up".

According to a second aspect of the present invention there is provided use of a non-human transgenic animal capable of expressing a serine proteinase inhibitor in its mammary gland in the manufacture of stabilized milk.

According to a third aspect of the invention, the use of a non-human transgenic animal, in which the exogenous DNA sequence encoding a serine proteinase inhibitor has been stably integrated into its genome, in the production of stabilized milk Is provided.

In the second and third aspects of the present invention, it is desirable that the non-human transgenic animal secretes and produces milk. All preferred features of the first aspect of the invention also apply to the second and third aspects.

According to all aspects of the invention, the transgenic animal is preferably capable of transmitting the construct to its progeny.

According to a fourth aspect of the present invention there is provided a non-human transgenic animal capable of expressing a serine proteinase inhibitor and fibrinogen in its milk.

According to the fifth aspect of the present invention, a non-human transgenic gene whose exogenous DNA sequence encoding a serine proteinase inhibitor and an exogenous DNA sequence encoding fibrinogen are stably introduced into its genome. Animals are provided.

According to the fourth and fifth aspects of the present invention, the non-human transgenic animal comprises:
It is preferable that a serine proteinase inhibitor and fibrinogen can be co-expressed in the milk. Alternatively, the serine proteinase inhibitor may be expressed earlier than fibrinogen, but should also be expressed at the same time fibrinogen is expressed. Preferably, in order to express serine proteinase inhibitors and fibrinogen in the mammary gland of the animal, the coding sequence is under the control of one or more milk gene promoters.

[0042] Fibrinogen has now been identified as a suitable protein of the present invention as it has now been shown that it is susceptible to proteolysis when expressed as a transgenic protein in the milk of transgenic animals.

[0043] Fibrinogen, the major structural protein in the blood essential for clot formation, exists as a dimer of three polypeptide chains. That is, Aα (66.5
kD), Bβ (52 kD) and γ (46.5 kD) are linked by 29 disulfide bonds. Addition of asparagine-linked carbohydrate to the Bβ and γ chains results in a molecule with a molecular weight of 340 kD. Fibrinogen has a three-bar structure. The central node, called the E domain, contains the amino termini of all six chains, including the fibrinopeptide (Fp), while the two distally located nodes, called the D domains, are Aα , Bβ and γ chains. The amino termini of the Aα and Bβ chains of fibrinogen are proteolytically cleaved by thrombin (a serine protease converted from its inactive form by factor Xa), releasing fibrinopeptides A and B (FpA &FpB);
Converted to fibrin monomer. The resulting fibrin monomers non-covalently assemble into profibrils by DE contact with adjacent fibrin molecules. This builds fibrin polymer chains in a half-shifted and overlapping manner. Contact also occurs longitudinally between adjacent D domains (DD contact), resulting in lateral aggregation. Factor XIII, another serine protease, is cleaved by proteolysis by thrombin in the presence of Ca ²⁺ to become active. This activated factor XIII (factor XIIIa) catalyzes the cross-linking of polymerized fibrin by creating an isopeptide bond between lysine and glutamine side chains. The first glutamine-lysine bond formed occurs at the C-terminus of the gamma chain making the DD bridge. Thereafter, numerous crosslinks are formed between adjacent Aα chains.
The cross-linking step gives the clot both biological stability (resistance to fibrinolysis) and mechanical stability [Sienbenlist and Mosesson, P.
Progressive Cross-Linking of Fibrin γ
chains Increases Resistance to Fibr
inolisis, Journal of Biological Chemi
stry, 269 : 28414-28419, 1994].

Fibrinogen and XII as the first component catalyzing the polymerization process
By having factor I with thrombin and Ca ²⁺ as second components, the coagulation process can be easily processed in vitro into a self-sustaining adhesive system. These adhesive systems known in the art as "Fibrin Sealants" or "Fibrin Tissue Adhesives" include surgical techniques and as a delivery device for a wide range of pharmaceutically active compounds. Numerous applications have been found [Sierra, Fibrin Se
alt Adhesive Systems: A Review of T
heir Chemistry, material Properties a
nd Clinical Applications, Journal of
Biomaterials Applications, 7 : 309-352,
1993].

The annual demand for US clinical fibrin sealant is 300 Kg / kg, which can be recovered using the current cryoprecipitation method used by plasma sorters.
It is estimated to be much more than a year. Therefore, other sources of fibrinogen, the major component of fibrin sealants, have been sought (recombinant sources are preferred). [Butler et al, Curre
nt Progress in the Production of Rec
ombinant Human Fibrinogen in the Mil
k of Transgenic animals, Thrombosis a
nd Haemostasis, 78 : 537-542, 1997]. Since mammals have been shown to be able to produce transgenic human fibrinogen up to a level of 5.0 g / L in their milk, this constitutes a commercially viable method of producing human fibrinogen [Prunkard et al, High
-Level expression of recombinant hum
an fibrinogen in the milk of transg
nic mice, Nature Biotechnology, 14 : 867.
-871, 1996; Cottingham et al. , Human Fi
brinogen from the milk of transgenic
sheep. In: Tissue Sealants: Current P
ractice, Future Uses. Cambridge Institute
ute, Newton Upper Falls, MA, March 30A
pril2 1996 (abstract)].

As used herein, the term “fibrinogen” refers to the major structural proteins required to form a clot, not only the entire glycoprotein form of fibrinogen, but also truncated fibrinogen, the amino acids of fibrinogen. Sequence variants (mutant proteins or polymorphic variants), fibrinogen species containing added residues, and other related fibrinogen species, including any naturally occurring variants thereof, are included. The same mutations described above apply to other fibrinogen-like proteins that can be isolated from milk according to the invention. The present invention is useful for the production and isolation of proteins that have been modified in some way to promote transgenic expression, such as by fusion to individual proteins themselves or to other proteins.

[0047] Fibrinogen may be obtained from any source, but is preferably of bovine or human origin.

As used herein, “fibrin adhesive”
The term ")" or "fibrin sealant" refers to a substance containing fibrinogen that can form a biodegradable adhesive or seal by the formation of polymerized fibrin. Such adhesive / sealant systems are alternatively referred to as "fibrin tissue adhesives".
adhesive "or" fibrin tissue glue "
glow) ". The adhesive or seal may act as a hemostat, a barrier to fluids, a substrate that fills gaps, or a drug delivery agent, among others. Above all, neurosurgery, ophthalmology,
It can be used in orthopedic or cardiothoracic surgery, skin grafting, and various other types of surgery.

[0049] The fibrin glue or sealant may be, besides fibrinogen, thrombin,
Ca ⁺⁺ , may include substances that promote the formation of a fibrin glue / seal, such as Factor XIII (also including Factor XIIIa herein). Although it is recognized that thrombin may be the preferred enzyme to be incorporated into any system where formation of a fibrin clot is desired, there are other enzymes capable of proteolytic cleavage of fibrinogen that result in the formation of a fibrin clot. You have to understand. An example of this is the snake venom enzyme Batroxobin.
[Weisel and Cederholm-Williams, F.
ibrinogen and Fibrin: Characteriazatio
n, Processing and Applications, Handbo
ok of Biodegradable Polymers (Series:
Drug targeting and Delivery) 7: 347-36.
5, 1997]. If desired, albumin, fibronectin, solubilizers, bulking agents and / or suitable carriers or diluents (dil)
other components, such as ent).

One advantage of fibrin sealant as a biodegradable polymer is that fibrin sealant is a temporary drug for hemostasis or wound healing because there are natural mechanisms in the body to effectively remove blood clots. The point is that it can be a stopper. Although various proteolytic enzymes and cells can, under certain conditions, lyse fibrin, the most specific mechanisms include the fibrinolytic mechanism. The dissolution of fibrin clots under physiological conditions involves the binding of circulating plasminogen to fibrin and the active protease plasmin (by plasminogen activator).
(Also capable of binding to fibrin). Subsequently, plasmin cleaves specific sites of fibrin.

In some cases, after applying fibrin glue or sealant to a site,
It may be advantageous to have a natural fibrin disruption step. In fact, this disintegration can be promoted, for example, by including plasminogen. Or
In some cases, for example, delaying the process by including an anti-fibrinolytic compound that can block the conversion of plasminogen to plasmin or that binds directly to the active site of plasmin and inhibits fibrinolysis May be advantageous. Such anti-fibrinolytic agent, a major physiological inhibitor alpha ₂ plasmin - contained antiplasmin, and ε- aminocaproic acid - macroglobulin, aprotinin, alpha _2.

[0052] The fibrin / sealant may comprise two components, one containing fibrinogen and factor XIII (and / or factor XIIIa) and the other containing thrombin and Ca ⁺⁺ . If desired, the other materials may contain one or both of the components.

As mentioned above, the primary use of fibrinogen is thought to be in the preparation of adhesives or sealants, but fibrinogen has been described, for example, in US Pat. No. 5,272.
There is another use in the medical arts as a coating for polymeric articles as disclosed in U.S. Pat. The lyophilized fibrinogen according to the invention is, in particular, (
Preferably used in or as part of a gauze or bandage (made of a polylactic acid compound used in surgical suturing). If desired, such wound dressings may be in the form of a package or kit for direct application to the skin or internal organs (including other components necessary to form the clot described above). Can be supplied.

All of the preferred features of the first to third aspects also apply to the fourth and fifth aspects.

According to a sixth aspect of the present invention there is provided use of a non-human transgenic animal according to the fourth or fifth aspect of the present invention in the production of recombinant fibrinogen. All of the preferred features of the first to fifth aspects also apply to the sixth aspect.

According to a seventh aspect of the present invention, there is provided a method for producing a non-human transgenic animal according to the fourth or fifth aspect of the present invention, comprising: (a) a first method encoding a serine proteinase inhibitor; Obtaining a DNA sequence or sequence family of: (b) in a cell, zygote or embryo of an animal capable of producing a transgenic animal having said first DNA sequence stably integrated into its genome; (C) obtaining a second DNA sequence or sequence family encoding fibrinogen; and (d) transgenic animals having the second DNA sequence stably integrated into their genome. Introducing said second DNA sequence or sequences into a cell, zygote or embryo of an animal that can be produced; and (e) transforming said cell, zygote or embryo into an animal. In growing; a method of producing a non-human transgenic animals having a are provided.

In this aspect of the invention, the “first” and “second” DNA sequences need not actually be separate, discrete sequences. For example, fibrinogen is often introduced in the above steps on one or more sequences (family of sequences), in this case on three separate constructs encoding the three subunit chains of fibrinogen. The same may be true for the coding sequence of a serine protease inhibitor.

The first and second sequences can be introduced separately, simultaneously or sequentially into a cell, zygote or embryo. The first and / or second DNA sequences can be operably linked to a milk gene promoter.

In the method according to the seventh aspect of the invention, the animal is preferably induced to secrete milk.

The method for producing a transgenic animal according to the present invention is not limited. They were first described in pronuclear microinjection (1980. Gordo.
n JW et al, Proc. Natl. Acad. Sci. USA 77
: 7380-7384) and the introduction of genes into livestock (Eb).
ert KM and Schinder, JES, Therigenol
ogy 39, 121-135, 1993); in vitro maturation and fertilization (K
Rimpenfort, P .; et al, Biotechnology 9,
844-847, 1991); Retrovirus-mediated gene transfer (Weiss
R. et al, RNA tumour viruses. Cold Spr
ing Harbour Laboratory, New York 1985
); DNA transfer via sperm (Bachiller D. et al, Mo.)
l. Reprod. Dev. 30, 194-200, 1991); transgenesis via embryonic stem cells; embryonic germ cells (Stew)
art L. C. , Dev. Bio. 161 626-628, 1994); and the introduction of nuclei as described in WO 97/07669 and WO 97/07668. Microinjection and nucleus transfer are preferred methods for producing transgenic animals according to the present invention.

The preferred features of the first to sixth aspects also apply to the seventh aspect.

[0062] An eighth aspect of the present invention provides a transgenic animal produced by a method according to the seventh aspect of the present invention. All of the preferred features of the first to seventh aspects also apply to the eighth aspect.

A ninth aspect of the present invention is a method for producing recombinant fibrinogen, comprising: (a) inducing a transgenic animal according to the fourth, fifth or eighth aspect of the present invention to induce lactation. (B) milking the animal; (c) collecting the milk; and (d) isolating the fibrinogen (including a purification step if necessary). I do.

The isolation and subsequent purification of fibrinogen may be by any method.

The preferred features of the first to eighth aspects also apply to the ninth aspect.

A tenth aspect of the present invention provides a recombinant fibrinogen produced in milk of an animal according to any one of the fourth, fifth, eighth or ninth aspects. Preferred features of any of the first to ninth aspects also apply to the tenth aspect.

[0067] An eleventh aspect of the present invention provides a fibrin sealant comprising the recombinant fibrinogen according to the tenth aspect of the present invention. Details regarding fibrin sealants are described herein above. All of the preferred features of the first to tenth aspects also apply to the eleventh aspect.

For simultaneous expression of fibrinogen and AAT, the same tissue,
That is, it is preferable to create an expression construct that can induce expression at a sufficient level in mammary epithelial cells. This may include promoter regions, means for inducing tissue-specific expression (may be, but not necessarily, activated by prolactin), cDNA or genomic DNA encoding the protein of interest, and preferably Can be achieved by a number of methods using constructs that contain some means of stabilizing the mRNA transcript, which could be the polyadenosine 3 'region. It contains a gDNA sequence preceded by a truncated β-lactoglobulin promoter and followed by a 3 ′ poly A terminator region.
This can be achieved by inducing fibrinogen expression using one (one for each of the three subunits) separate constructs. Details of the construction of these constructs are described in PCT / US95 / 02648. One skilled in the art would use natural breast-specific regulatory regions, such as those for the alpha lactalbumin promoter, WAP promoter or casein promoter, or artificial promoters with appropriate regulatory elements, to induce expression in breast tissue. You will know many other promoters that can be.

The expression construct for AAT has been described by Clark et al.
Clark et al, 1989. Bio / Technology 7:48
7-492) BLG promoter and AAT modified gDNA (2 of exons)
A similar design was used. As described above for fibrinogen, designing constructs with other promoters is standard practice in the art. For example, one can envision altering the genetic composition of an animal so that its endogenous AAT is highly expressed in the mammary gland by "switching on" genes in normally inactive mammary cells.

For both fibrinogen and / or AAT according to the present invention, the homologous recombination of gDNA or cDNA (which may be) inserted into the appropriate active region of the genome of the transgenic animal behind the promoter present is Can be used to manipulate expression.

[0071] Transgenic animals can be produced using a variety of methods, including pronuclear microinjection, introducing nuclei into oocytes obtained from transformed cell lines, recombinant techniques, and the like. For example, an animal into which both human fibrinogen and human AAT have been transfected, after excision and purification from an E. coli vector, all four BLG constructs (described above) (one for each fibrinogen subunit, one for AAT) The equimolar ratio is mixed and injected into the pronucleus of a fully fertilized animal, e.g., an oocyte of sheep, in order to inject the mixture so produced to ensure proper expression levels. It can be made by creating a number of transgenic offspring. Useful in this case means sufficient AAT to inhibit fibrinogen and proteolysis at levels sufficient for commercial collection.

Another method for producing an animal in which both fibrinogen and AAT have been transfected is
Crossing a stable and separate line that expresses each protein individually. For example, a G1 female from a strain into which fibrinogen has been introduced is mated with a male into which AAT has been introduced. If heterozygous AAT males are used, approximately one out of eight offspring is a double transgenic female. Thus, a more efficient method is to breed a fibrinogen transgenic female with a homozygous AAT male, which results in approximately one in four double transgenic females. Alternatively, the AAT transgenic animal can be a heterozygous female crossed with a heterozygous or homozygous male fibrinogen. Again, this would result in about one in eight or about one in four females being double transgenic females, respectively. There are advantages. The first of these is the stability and shelf life of the product in milk. It has been demonstrated that the product remains stable in milk stored under warming, whereas fibrinogen expressed alone in milk is further damaged by proteolysis. The concentration in milk in which AAT is co-expressed is the same as that in milk with only fibrinogen, but utilizes all of this expression level, as well as the undamaged substance present therein, for processing. This is advantageous in the availability of fibrinogen.
Similarly, there is no longer any need to remove fibrinogen fragments from the product, which can be considered as closely related contaminants, which can reduce process complexity and allow the use of smaller chromatography columns, thus reducing total There is considerable room to reduce costs.

Co-expression of fibrinogen also has product-related advantages. The first advantage is that the resulting product, which can contain a high proportion of intact α-chain, is highly defined and therefore (with the addition of thrombin and factor XIII components, if necessary) It can be accurately formulated to give the fibrin sealant or glue preparation optimal properties. For example, it has been known that increasing the percentage of fragment X adversely affects the desired physical properties of such compositions. Increasing the proportion of F1 is useful in making stronger, more flexible fibrin sealant compositions, and at lower fibrinogen concentrations, creates a sealant with predetermined selected properties, increasing the economics of manufacturing It is anticipated that it may be possible to do so.

A further product-related advantage arises from the observation that fibrinogen has a type of progressiveness in proteolytic susceptibility. Therefore, F2 becomes F
Fragment X is more easily degraded than F1, and fragment X is more easily and more degraded than the ladder of F2 and smaller fragments. Therefore, the most major F1 fibrinogen is more stable to the action of proteases and therefore has a longer shelf life,
It is expected that it will be more suitable for liquid composition formulations. A second factor that will extend the shelf life is an effectively protease-free source of AAT co-expressed fibrinogen. The sensitivity of fibrinogen to proteases will generally limit the detrimental effects of any low levels of protease contamination in the final formulation, storage conditions compatible with long-term storage, especially in the non-frozen state. Means that The presence of AAT in the starting material means that any active protease will not appear in the product because the complex is formed and removed by the process.

The mechanism by which co-expression of AAT prevents the proteolytic damage of fibrinogen has such a dramatic effect. The mechanism by which the expected effects on the proteases (plasmin and thrombin) that are most likely to be present in milk is shown. It is unknown from the lack. Presumably, it is due to the presence of a constant AAT that blocks even the production of low concentrations of proteases, and the very slight damage to fibrinogen by its presence (if this occurs, the overall effect of fibrinogen on proteolysis is May be sufficient to increase the susceptibility). Thus, there is already higher and unrestricted protease activity, and the addition of AAT to milk may not be as effective as existing fibrinogen is already heading for further proteolytic damage. unknown. In this case, about 10 transgenic animals
AAT is expressed with g / L inhibitor. Such a high AAT concentration may be particularly preferred in milk stability and co-expression with fibrinogen, as well as co-expression.

Even if the protective effect of AAT is unexpected, sufficiently high expression levels can be obtained, their specificity is wide enough to inhibit the proteases involved and the mammary gland It must be emphasized that if there is indeed no detrimental effect on ecology or host animal physiology, it does not prevent the possibility of using other protease inhibitor proteins to protect the heterologous protein. . These limits can be readily determined by one skilled in the art without undue experimentation or undue burden. Thus, the present invention is directed to protecting heterologous proteins susceptible to proteolysis (such as, for example, factor VIII and tissue plasminogen activator) expressed in non-human mammal milk. Protease inhibitors,
In particular, co-expression of serpin and Kunitz family protease inhibitors. According to the present invention, expressing only the protease inhibitor protein in milk is beneficial with respect to the storage properties of the milk for human consumption and as an aid to further processing into the product Is also recognized.

The invention will now be described by way of a number of examples. In this example, reference is made to the accompanying drawings. Example Overview Unless otherwise specified, recombinant DNA and molecular biology manipulations were performed by Mani.
atis et al. (“Molecular Cloning” Cold Spring
g Harbor (1982)) “Recombinant DNA” Met bodies in Enzymology Volume 68, (edited)
by R. by. Wu), Academic Press (1979);
mbinant DNA part B "Methods in Enzym ology Volume 100, (Wu, Grossman and Mo
ldgave, Eds), Academic Press (1983);
combinant DNA part C " Methods in Enz
ymology Volume 101, (Wu, Grossman and
Moldgave, Eds), Academic Press (1983);
and “Guide to Molecular Cloning Tech”
niques ", Methods in Enzymology Volume
e 152 (edited by SL Berger & AR Ki
mmel), Academic Press (1987). Unless otherwise noted, all chemicals are BDH Chemicals Ltd, P
cool, Dorset, England, or Sigma Chemical
Company, Poole, Dorset, England.
Unless otherwise stated, all DNA modifying enzymes and restriction endonucleases were obtained from BCL, Boehringer Mannheim House, Bell.
Purchased from Lane, Lewes, East Sussex BN71LG, UK.

[Abbreviations: bp = base pairs; kb = kilo base pairs; AAT = α1-antitrypsin; BLG = β-lactoglobulin; FIX = Factor IX; E Coli = Esh
erichia coli; dNTPs = deoxyribonucleotide triphosphate;
Restriction endonucleases, for example, abbreviated as Bam Hi; addition of -O into after site for the restriction endonuclease, for example Pvu II-O indicates that the recognition site has been destroyed] transgenic animals Construction of constructed sheep transgenic sheep is described in International Patent Application WO-A-8800239.
(Pharmaceutical Proteins Ltd) and Sim
ons, Wilmut, Clark, Archival, Bishop &
Lathe (1988) Biotechnology 6, 179-183.

The identification of transgenic sheep is described in International Patent Application WO-A-8800239.
(Pharmaceutical Proteins Ltd). Example 1 In vitro experiments were performed to determine the effectiveness of AAT in inhibiting plasmin. To a series of tubes containing 4 mg mL of fibrinogen, add 0.5 m
AAT was added at concentrations of g / mL and 1 mg / mL. Subsequently, these tubes were charged with 83: 1 or 166: 1 on a weight basis and 55: 1 or 110: on a molar basis.
Plasmin (3 μg) was added to give any AAT: plasmin ratio of 1. Subsequently, the tubes were incubated at 37 ° C. and the results were obtained from SDS-PAGE (see FIG. 1).

The results of this experiment show that fibrinogen is considerably degraded after 4 hours at 37 ° C., even though the maximum amount of AAT is equivalent to 110 AATs for each plasmin molecule. . Example 2 Increased Stability of AAT Milk to Non-AAT Milk Method To mimic accelerated aging, AAT skim milk (from transgenic sheep expressing AAT at a level of about 13 g / L) and (ewe) Aliquots (1 mL) of non-AAT skim milk (obtained from) were incubated at 37 ° C. for 18 days. At the end of this period, the milk was visually inspected, where the non-AAT milk had separated into two phases, while the AAT milk had the appearance of fresh milk. Mix both tubes, pH 4.6, 5 mL 25 mM Tris
-The contents were added to the acetate buffer. After mixing this solution, it was centrifuged at 2000 rpm for 10 minutes to promote precipitation of the casein fraction. continue,
The precipitated casein sediment was washed with water before resuspending casein in 2.5 mL of 8M urea in TBE buffer. Subsequently, dithiothreitol (DTT) was added to each tube to a final concentration of 2 mM.

Subsequently, each sample was diluted at a ratio of 1: 5 with 6 M urea in 20 mM Tris-Acetate buffer, pH 5.0, and 1 mL of Resource S (Ame
rsham) Pharmacia Biotech) 500 μL was injected onto a cation exchange column. Chromatography was performed using a gradient to 0.3 M NaCl over 40 column volumes, eluting the bound protein at 1 mL / min. Samples of fresh AAT milk and fresh non-AAT milk were processed exactly as described above and also applied to a cation exchange column. Results As shown in the chromatogram shown as FIG. 2, after their precipitation from the milk, complete separation of the casein protein in the milk is achieved. The elution order from the column is β followed by κ, then αS ₁ , then αS ₂ . When comparing this chromatogram with the chromatogram shown in FIG. 3 which represents the non-AAT milk after 18 days of incubation at 37 ° C., it is very easy for this chromatographic separation to have no separation. , Indicating that significant structural damage has occurred to casein, probably as a result of extensive proteolysis. In contrast, the chromatograms shown in FIGS. 4 and 5, which represent AAT milk before and after incubation at 37 ° C., respectively, show that the pre-incubation chromatograms are unheated non-AAT.
It turns out to be very similar to that obtained with milk. After incubation at 37 ° C. for 18 days, the chromatogram retains its overall pattern, indicating that the structure of casein is relatively intact. Such indicated from the reduction in peak height and area, [alpha] S ₁ casein, probably it is interesting to note that the most damaged. This is consistent with reports in the literature suggesting that αS ₁ casein is most prone to degradation by proteolysis. Conclusion The conclusions of this experiment are as follows. 1) Visual inspection of accelerated and aged samples of AAT and non-AAT milk indicated that non-A
It can be seen that AT milk is significantly less stable than AAT milk. 2) Cation exchange chromatography of casein confirmed that in the aged sample of non-AAT milk, significant structural changes occurred in the casein fraction, and separation was no longer present at the chromatographic stage. Is done. These structural changes are probably due to proteolysis. 3) Cation exchange chromatography of casein in aged AAT milk has some degree of casein degradation, but the overall separation obtained on this column was obtained on unaged AAT milk. It is clear that they are similar. 4) The degradation of αS ₁ and αS ₂ casein appears to occur before β casein, as expected from the literature. Example 3-Preparation and screening of AAT and fibrinogen transgenic sheep Sheep (a and b) were screened in two ways to confirm that both AAT and fibrinogen construct were transfected.
Screening for fibrinogen Sheep DNA was digested with the restriction enzyme AvrII. This enzyme produces fragments from three fibrinogen chains with specific molecular weights. These fragments are separated by size using a gel. DNA on gel
Transfer to binding membrane (referred to as blot). Subsequently, B common to all three chains
The blot is probed with radioactive DNA made from the LG (β-lactoglobulin) gene regulatory region. The regulatory region binds to DNA having the same sequence;
Any region of similar sequence is made radioactive. Radioactivity can be detected by placing a photographic film over the area. The radioactive band from the sheep can be compared to the known control, 1, and 10 copies of FIG. Lane A is a negative control; Lane B is a 1 copy control; Lane C is a 10 copy control; Lane D is sheep a and lane E is sheep b.

The bands are of the correct size, confirming that all three fibrinogen genes have been introduced into this sheep. The probe uses the sheep's own B
AAT in LG and sheep is also detected, but they are not separated on the gel. AAT Screening The AAT screening step is performed by using the restriction enzyme BamHI,
Same as fibrinogen screening. This cuts the DNA into bands of different sizes, producing different bands. See FIG. Lane A is sheep 1; lane B is sheep b; lane C is a negative control; lane D is a 10 copy control; lane E is a 20 copy control. The bands observed are the BLG regulatory region of the AAT transgene, the sheep's own BLG, and the BLG regulatory region of the fibrinogen transgene.
This enzyme does not separate fibrinogen chains separately. Knowing the restriction pattern predicted by BamHI confirms the presence of a fragment consistent with the AAT transgene. Example 4-Analysis of milk of transgenic sheep Each AA named sheep 1 and 2 by the process of Southern blotting
DNA from two sheep crossed from the T and fibrinogen lines was analyzed,
Both human proteins α1 antitrypsin (AAT) and fibrinogen were found to be positive. Lactation was induced in both sheep by hormonal treatment and milked twice daily. When lactation was continued for about 6 weeks, the average daily milk yield was measured. a) RID Analysis of Sheep's Milk To determine the concentration of each human protein expressed in sheep's milk, the radial immunodiffusion (RID) method was used. A commercially available RID kit manufactured by Binding Site was used. Accurate concentrations were achieved by comparing the diameter of the ring made by the milk sample from the sheep to both the human AAT and fibrinogen standards provided with the RID kit. The results are detailed below. AAT RID Results Human AAT Standard Ring Diameter 0.28 mg / mL 4.1 mm 0.84 mg / mL 4.9 mm 1.40 mg / mL 7.1 mm 1.68 mg / mL 7.6 mm 2.80 mg / mL 9.1 mm Sheep milk ring diameter 1:10 dilution Sheep 980082 6.0 mm Sheep 980417 5.8 mm This data shows that for sheep 1, AAT concentration 1 in sheep milk before dilution was used.
For 0.5 mg / mL sheep 2, this corresponds to an AAT concentration in sheep milk of 9.6 mg / mL before dilution. Results of fibrinogen RID Human fibrinogen standard Ring diameter 0.45 mg / mL 4.1 mm 1.35 mg / mL 5.1 mm 2.25 mg / mL 5.9 mm 2.70 mg / mL 6.4 mm 4.50 mg / mL 7.9 mm Sheep milk ring diameter 988000 5.0 mm 980417 5.4 mm for a 1 to 5 dilution This is a 6.3 mg / mL undiluted fibrinogen concentration in sheep milk for sheep 1 and an undiluted sheep for sheep 2 This corresponds to a concentration of 8.1 mg / mL. b) SDS-PAGE analysis of sheep's milk The protein composition of sheep's milk could be analyzed by SDS-PAGE electrophoresis analysis under non-reducing conditions. More precisely, it was possible to identify all fibrinogen degradation products, such as fragments X and Y. Milk obtained from double transgenic sheep and F expressing only fibrinogen
Gel comparing pre-milked sheep from the line (ie without AAT)
This is shown in FIG.

[0083] Fibrinogen produced by double transgenic animals has only F1-type proteins. This milk does not contain F2 fibrinogen and any products associated with degradation, such as fragments X and Y. Milk from animals transfected with fibrinogen alone clearly shows the degradation product fragment X and F2 fibrinogen.

[0084] SDS-PAGE electrophoresis analysis performed under reducing conditions gives data for each chain subunit of fibrinogen protein. When milk obtained from sheep transfected with both AAT and fibrinogen is compared with sheep expressing only fibrinogen (sheep from the F2 line), a difference can be clearly observed. FIG. 9 shows two types of fibrinogen.

The fibrinogen expressed by the two types of sheep constructs clearly contains α, β, and γ chains characteristic of the fibrinogen protein. However, a comparison of the α-chain subunits reveals interesting differences. In single transgenic fibrinogen milk, two α-chain subunits are observed, appearing as broad bands on the scanned gel image. This is in sharp contrast to fibrinogen expressed by AAT / fibrinogen dual construct animals, which clearly shows only a single α-chain. This is also evidence that virtually no fibrinogen degradation occurs in sheep milk obtained from animals positive for both AAT and fibrinogen. c) Western blot analysis of sheep milk Further evidence that proteolytic damage to fibrinogen produced in sheep milk transfected with AAT and fibrinogen is unlikely is obtained by western blot analysis. Can be First, milk obtained from single and double transgenic lines is separated into protein components by gel analysis based on molecular weight. Subsequently, a stable “print” of the gel is created by electrophoretic transfer to a plastic membrane. Subsequently, the membrane is exposed to a polyclonal antibody against fibrinogen and washed extensively to remove unbound antibody. The bound antibody is detected by a secondary antibody conjugated to the reporter enzyme horseradish peroxidase. Subsequently, after thorough washing to remove unbound secondary antibody, all fibrinogen subunits and their subunits were used using light-emitting peroxidase substrate and permanent records taken on photographic film. Visualize the fragments from proteolytic digestion.

[0086] Fibrinogen expressed in the absence of AAT has undergone different degrees of proteolysis, depending on the sheep individual and the stage of lactation (later, more proteolysis). Although much of the degraded material is removed by purification, the α-chain excised by proteolysis is still present at a constant rate and its products are F1 and F2, as seen in fibrinogen from F plasma. Contains both seeds. However, the range of processing enzymes present in milk is more limited than in plasma, and much of the F2 in milk is due to a truncation near amino acid 220. This is based on SDS Page analysis of the purified product,
Found in both Western blots of milk (in the case discussed)
This produces a clear truncated α-chain species (FIG. 10). The truncated chains are
Indicated on the left by an arrow.

FIG. 10 clearly shows that undetectable levels of truncated α-chain are present in the analyzed AAT and fibrinogen double transgenic milk (tracks 3 and 8). . This contrasts with the various, and in some cases a considerable degree, of degradation (all other trucks) found in single transgenic milk. d) Hydrophobic interaction chromatography (HIC) analysis The method of HIC is used to separate forms F1, F2 and X of fibrinogen. Purify fibrinogen from milk by three rounds of precipitation. Subsequently, the preparation is applied to a butyl sepharose column. After removing contaminants by pre-washing the column, F1, F2, and fragment X are eluted in order with a gradient in which ammonium sulfate gradually decreases.

The elution profile in FIG. 11 obtained by analysis of purified fibrinogen obtained from ATT / fibrinogen double transgenic sheep shows that only F1 fibrinogen is present and no F2 or X can be detected. However, in purified transgenic sheep fibrinogen (results shown in FIG. 12), F1, F2, and X-type proteins are all present, and F1 is not a major molecular species. This is further evidence that co-expression of AAT and fibrinogen inhibits fibrinogen degradation, and the absence of AAT results in fibrinogen protein degradation. e) Reverse phase chromatography (RPC) of fibrinogen To accurately analyze the stoichiometric composition of the chains of fibrinogen produced by transgenic sheep, the method of RPC was used. 10 mM
A sample of fibrinogen after HIC purification was reduced in the presence of dithiotothreitol and 6M guanidine (this is the step of releasing three different fibrinogen chains from the undamaged protein). Vydac 4.6 × 150
Samples were analyzed using a mm C4 column and 0.1% TFA and CH ₃ CN buffer. The elution profile in FIG. 13 shows that fibrinogen obtained from single transgenic fibrinogen sheep is an indicator of α-chain degradation typical of the presence of F2 and X fibrinogen. As a result, the alpha chain is no longer the major species of proteins. This result indicates that AA showed no sign of α-chain degradation.
In sharp contrast to that obtained with fibrinogen from T / fibrinogen double transgenic sheep. Therefore, the alpha chain is the major species and F2
Or it is clear that there is no fibrinogen of type X at all. f) Stability analysis of fibrinogen Milk obtained from AAT / fibrinogen double transgenic animals was incubated at 30 ° C for 18 hours. After this time, the whey was partially purified by three rounds of precipitation before being subjected to HIC analysis using a butyl sepharose column. This will detect the degradation of any F1 fibrinogen into F2 and fragment X due to protein instability in milk.

The HIC elution profile of fibrinogen obtained from AAT / fibrinogen hybrid sheep shown in FIG. 14 shows only F1 type fibrinogen protein. Thus, no decomposition due to instability occurs. g) Fibrinogen co-expressed with AAT is functionally equivalent to fibrinogen expressed alone. Purified fibrinogen from double transgenic milk is functionally equivalent to that made from single transgenic milk. It was shown that there is. Subsequently, it was also shown that it was functionally equivalent to plasma-derived fibrinogen. In this example, changes in optical density track clot formation at low thrombin and fibrinogen concentrations.

To quantify the time-dependent change in opacity of fibrinogen in the gel,
An optical density (OD) measurement is used. This opacity is measured as the increase in OD over time. This change in OD is due to light scattering from fibrin fibers, rather than an increase in absorbance. The coarser the gel structure, the thicker the fibrin fiber and the higher the OD. The thinner the gel structure, the thinner the fibrin fiber and the lower the OD. Interesting parameters in this assay are the initial increase in the rate of OD, which is an indicator of the rate of gelation, and the final OD, which is an indicator of fiber roughness (the coarser the fiber, the more light is scattered).

To a plastic microcuvette was added 500 μL of either 0.15 mg / mL single or double transgenic fibrinogen in tris buffered saline supplemented with 5 mM CaCl ₂ . Each cuvette contains 0.6 U / mL of purified plasma-derived thrombin (Enz
yme Research Laboratories) After adding 50 μL,
Mix quickly. The absorbance at 350 nm was recorded for 20 minutes. The results are shown in FIG.

A comparison of the OD profiles in which only fibrinogen was expressed or in the presence of AAT shows that both the coagulation rate and the final fiber roughness are very similar (FIG. 15), and therefore the two materials function. It is shown that they are equivalent to each other. Single transgenic ones appear to make coarser fibers, but from a single assay it cannot confer any significance and the results should be considered indistinguishable.

[Brief description of the drawings]

FIG. 1. SDS-PAGE showing that AAT has little effect (in vitro) as an inhibitor on plasmin. Lane 1 shows fibrinogen (4 m
g / mL) with AAT (1 mg / mL) without plasmin; lane 2 with fibrinogen (4 mg / mL) and AAT (1 mg / mL) with 6 μg / μL plasmin; lane 3 with fibrinogen ( 4mg / mL) and AAT
(0.5 mg / mL), with 6 μg / mL plasmin.

FIG. 2 is a chromatogram showing the separation of casein protein in milk after precipitation from milk. The wavelength of UV was 280 nm.

FIG. 3 is a chromatogram showing non-AAT milk after 18 days of incubation at 37 ° C. The wavelength of UV was 280 nm.

FIG. 4 is a chromatogram showing AAT milk before incubation. The wavelength of UV was 280 nm.

FIG. 5 is a chromatogram showing AAT milk before and after incubation at 37 ° C. for 18 days. The wavelength of UV was 280 nm.

FIG. 6 is a Southern blot showing screening of DNA of sheep a and b into which AAT and fibrinogen have been introduced. Sheep were screened for fibrinogen. Lane A is a negative control; Lane B is a 1 copy control; Lane C is a 10 copy control; Lane D is sheep a and lane E is sheep b.

FIG. 7 is a Southern blot showing screening of DNA of sheep a and b into which AAT and fibrinogen have been introduced. Sheep were screened for AAT. Lane A is sheep 1; lane B is sheep b; lane C is a negative control; lane D is a 10 copy control; lane E is a 20 copy control.

FIG. 8 shows SDS-PAGE electrophoretic analysis under non-reducing conditions of the protein composition of milk of sheep transfected with both AAT and fibrinogen and milk of sheep transfected with only fibrinogen.

FIG. 9 shows SD under reducing conditions showing sheep milk transfected with both AAT and fibrinogen and sheep milk transfected with only fibrinogen.
Fig. 7 shows S-PAGE electrophoresis analysis. Lane 1 is AAT fibrinogen sheep 1; lane 2 is AAT-fibrinogen sheep 2; lane 3
And 4 are F2 strain fibrinogen sheep: Lane 5 is AAT standard protein.

FIG. 10 shows Western blot analysis of milk from each sheep of AAT-fibrinogen transgenic sheep and fibrinogen only transgenic sheep. Track 1 shows low quality milk showing significant degradation; Tracks 2, 4-8 and 10 show single transgenic milk showing inconsistent degradation; Tracks 3 and 9 show Shown is milk from sheep transfected with AAT and fibrinogen.

FIG. 11 shows the elution profile of milk from AAT-fibrinogen transgenic sheep separated by HIC.

FIG. 12 shows the elution profile of fibrinogen-only transgenic sheep milk separated by HIC.

FIG. 13. Transgenic sheep with only strain F3 fibrinogen (FIG. 13A) and A
14 shows the eluted profile obtained from reverse phase chromatography performed on milk of AT-fibrinogen transgenic sheep (FIG. 13B). Each fibrinogen chain is annotated. Both wavelengths are 214 nm.

FIG. 14 shows the HIC elution profile of AAT-fibrinogen transgenic sheep.

FIG. 15 is an optical density curve showing coagulation of fibrinogen purified from a transgenic sheep containing only fibrinogen (line 1) and an AAT-fibrinogen transgenic sheep (line 2).

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] January 17, 2001 (2001.1.17)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72 ) Inventor McLeese, Graham Edward UK, EH 25.9 PP, Edinburgh, Roslyn (no address), PP Therapeutics (Scotland) Limited F-term (reference) 4B024 AA01 AA05 BA80 DA02 GA11 GA30 4B065 AA90X AA90Y AB01 AC14 BA01 CA24 CA41 CA43 CA44 4H045 AA10 AA20 AA30 BA10 CA40 DA56 EA20 EA34 FA71 FA74

Claims (27)

[Claims] 1. Use of a serine proteinase inhibitor expressed in milk of a transgenic non-human animal for stabilizing milk. 2. The use according to claim 1, wherein the serine protease inhibitor is α-1-antitrypsin or α-1-antichymotrypsin. 3. The use according to claim 1, wherein the animal is a sheep, cow, goat, rabbit, mouse, camel, buffalo, pig, or horse. 4. Use according to any one of claims 1 to 3 for stabilizing a heterologous protein further expressed in milk of a non-human animal. 5. Use of a non-human transgenic animal capable of expressing a serine proteinase inhibitor in its mammary gland in the manufacture of stabilized milk. 6. Use of a non-human transgenic animal in the manufacture of stabilized milk, wherein the exogenous DNA sequence encoding a serine proteinase inhibitor is stably integrated into its genome. 7. The use according to claim 5, wherein the non-human transgenic animal secretes and produces milk. 8. The use according to claim 4, wherein the serine protease inhibitor is α-1-antitrypsin or α-1-antichymotrypsin. 9. The use according to any one of claims 5 to 8, wherein the animal is a sheep, cow, goat, rabbit, mouse, camel, buffalo, pig, or horse. 10. A non-human transgenic animal capable of expressing a serine proteinase inhibitor and fibrinogen in the milk. 11. A non-human transgenic animal in which an exogenous DNA sequence encoding a serine proteinase inhibitor and an exogenous DNA sequence encoding fibrinogen have been stably introduced into the genome thereof. 12. The non-human transgenic animal according to claim 10, wherein the animal is capable of co-expressing a serine proteinase inhibitor and fibrinogen in its milk. 13. The method according to claim 13, wherein the serine protease inhibitor and fibrinogen are 1
The non-human transgenic animal according to any one of claims 10 to 12, which is under the control of the above milk gene promoter. 14. The non-human transgenic animal according to claim 10, wherein the fibrinogen is derived from a bovine or human. 15. The non-human transgenic animal according to claim 10, wherein the serine protease inhibitor is α-1-antitrypsin or α-1-antichymotrypsin. 16. The non-human transgenic animal according to claim 10, wherein the animal is a sheep, cow, goat, rabbit, mouse, camel, buffalo, pig, or horse. 17. Use of a non-human transgenic animal according to any one of claims 10 to 16 for the production of fibrinogen. 18. A method for producing a non-human transgenic animal according to any one of claims 10 to 16, wherein (a) a first DNA sequence or a sequence family encoding a serine proteinase inhibitor is provided. (B) introducing the DNA sequence into cells, zygotes or embryos of an animal capable of producing a transgenic animal having the first DNA sequence stably integrated into its genome; and (c) (D) obtaining a second DNA sequence encoding fibrinogen; (d) in a cell, zygote or embryo of an animal capable of producing a transgenic animal having said second DNA sequence stably integrated into its genome; Introducing said second DNA sequence or sequence family; and (e) growing said cell, zygote or embryo to an animal. 19. The method according to claim 19, wherein the first and second sequences are located in the cell, zygote or embryo.
19. The method of claim 18, wherein the method is introduced separately, simultaneously, or sequentially. 20. The method according to claim 18 or 19, wherein said first and / or second DNA sequence is operably linked to a milk gene promoter. 21. The method according to any one of claims 18 to 20, wherein said animal is induced to secrete milk. 22. The method according to claim 18, wherein the serine protease inhibitor is α-1-antitrypsin or α-1-antichymotrypsin. 23. The method according to claim 18, wherein the animal is a sheep, cow, goat, rabbit, mouse, camel, buffalo, pig, or horse. 24. A transgenic animal produced by the method according to any one of claims 18 to 23. 25. A method for producing a recombinant fibrinogen, comprising: (f) inducing the transgenic animal according to any one of claims 10 to 16 or 24 to induce lactation; A) milking the animal; (h) collecting the milk; and (i) isolating the fibrinogen (optionally including a purification step). 26. A recombinant fibrinogen produced in the milk of an animal according to any one of claims 10 to 16 or 24 or 25. 27. A fibrin sealant comprising the recombinant fibrinogen according to claim 26.