RU2238322C2 - Protein eliciting ability for formation of c3-convertase, dna, vector, conjugate and their using - Google Patents

Abstract

FIELD: biotechnology, molecular biology, biochemistry, medicine.

SUBSTANCE: invention describes native proteins, ways for activation of complement modified by so manner that protein is able to form C3-convertase that is resistant to down-regulation. Preferably, the modified protein represents the modified human C3 protein. Also, invention describes both DNA sequences encoding such proteins and DNA constructions. Invention describes also conjugates comprising such proteins and binding specifically fragment, for example, antibody, and application of such proteins and/or conjugates in therapy also. Invention can be used for humans in vivo for activation of complement and to obtain a preparation that has no immunogenic properties for humans in it using.

EFFECT: valuable biological and medicinal properties of protein.

35 cl, 15 dwg, 3 tbl, 17 ex

Description

The present invention relates to new modified proteins capable of forming resistant to downregulation of C3 convertase, to DNA sequences encoding such proteins, and to the use of these proteins as therapeutic agents, in particular to their use to reduce protein concentrations of the complement activation pathway or complement attack directions (C3b accumulation) to specific sites.

The complement system is involved in the immune response in humans and other vertebrates and plays a major role in effector functions, such as phagocytosis, cytolysis and cell recruitment, which induce local inflammatory reactions [15]. These properties are desirable for the elimination of pathogens that enter the body, such as bacteria, but are undesirable for triggering the mechanism of action on host tissues (for example, during post-ischemic reperfusion injury [3]) or on foreign therapeutic material (for example, ultra-sharp xenograft rejection [ 7]). Attempts have been made to eliminate these undesirable properties by using derivatives of regulatory complement proteins, the normal function of which is to suppress complement activation [10, 18].

The complement system includes proteins that are located both on the surface of cells (receptors and regulators), and in the liquid phase (blood plasma and other extracellular environment). The stage that is crucial for generating responses is the proteolytic conversion of C3 into fragments C3b and C3a. C3a is anaphylatoxin, which, like C5a, attracts mast cells to the site of infection, which leads to local histamine secretion, dilation of blood vessels and other inflammatory reactions. The resulting C3b has the ability to bind to surfaces surrounding the site of its formation. This C3b then focuses the “attack” of the cytolytic complement components (C5-C9).

Bound to the surface of C3b and its decomposition products function as ligands for C3 receptors, mediating, for example, phagocytosis [15]. There are two different ways to activate complement, both of which lead to the conversion of C3 to C3b and subsequent reactions. The classic pathway is usually triggered by antibody complexes with an antigen that activates a cascade that includes the Clq, Clr, Cls, C2, and C4 proteins. An alternative path depends on the activation loop, including C3 itself, and it requires factors B and D.

The conversion of C3 to C3b (or C3i) leads to the formation of a product that can combine with the B factor to produce C3bB (or C3iB). To obtain C3bBb, these complexes act with the participation of the D-factor, which is a C3 convertase capable of cleaving a larger amount of C3 to form C3b, which leads to an increase in the formation of C3bBb and to even greater conversion of C3. Under certain conditions, the C3bBb complex stabilizes upon binding to properdine (P), which is an up-regulator. However, this positive feedback loop is usually limited to a slow “shutdown” caused by regulatory proteins, in particular the H-factor and I-factor.

Factor H (and structurally related molecules associated with the cell) (I) displace B and Bb from C3b and (II) act as a cofactor of the I-factor, which cleaves C3b to form iC3b, thereby preventing any recombination with the B-factor, which allows you to get a larger number of C3-convertase. This pathway is launched for enhanced production of C3b in the presence of surfaces such as the walls of many bacterial cells that bind the resulting C3b and prevent its interaction with factors H and I. The resulting C3b also has the ability to bind to endogenous cells. The surfaces of endogenous cells, usually available for complement, are further protected by membrane-bound regulators, such as MCP, DAF and CR1, which act similarly to the H factor.

There are several cases that are rarely found in vivo when normal regulation in the liquid phase cannot occur, and spontaneous conversion of C3 ultimately leads to the total elimination of C3 from the bloodstream, namely, (I) genetic deficiency of factor H or I [13] , (II) the presence of antibodies (jade factors) that bind to C3bBb and inhibit dissociation [4], and (III) contact with a cobra venom protein called cobra venom factor (CVF), which combines with the B factor to form the C3 enzyme -convertase, which does not contain C3b and not exposed to factors H and I [14]. These data illustrate the importance for normal physiological reactions of down-regulation of complement in the absence of specific activation.

Cases are also known where specific activation occurs, but is undesirable, in particular when it is directed against the host tissue (for example, tissue damage due to ischemia or surgery) or against foreign material specially introduced for therapeutic purposes (such as xenograft, artificial organ or dialysis membrane). Activation of complement leads to an unwanted attack and additional damage, so in these cases it seems advisable to block or inhibit activation and response.

Existing approaches to prevent complement-mediated damage are aimed at the use of proteins with a decreasing regulatory ability (CR1, MCP, DAF and factors H and I) to inhibit complement activation. Inhibitors of complement, such as I-factor, H-factor and soluble derivatives of membrane-bound proteins CR1, DAF, MCP, are able to suppress the amplification loop of the alternative pathway characteristic of the liquid phase. Therefore, attempts were made to use these molecules, in particular CR1 (which is probably the most active), to reduce complement-mediated damage in model physiological situations [10, 18].

Factor H is an endogenous factor that is present in blood plasma in high concentrations (usually 0.3-0.5 mg / ml [15]), and therefore, although increased concentrations of inhibitors can reduce reactions in the liquid phase, their activity is insufficient. therefore, large amounts of purified proteins are required to be administered in vivo (for example, more than 5 mg of soluble CR1 / kg body weight). In addition, the alternative pathway is activated by surfaces on which the action of the H-factor is already impeded. Although this does not necessarily simultaneously lead to a decrease in the activity of other inhibitors, it can be assumed that these factors are unlikely to be fully or universally effective.

Cobra venom factor (CVF) has the ability to produce stable C3 convertase, which in experimental in vivo conditions can be used to reduce complement levels in animals in vivo and in other samples (e.g., human plasma) in vitro. CVF has a high activity (for example, 40 μg / kg may interfere with mouse complement activity [16]). However, there are drawbacks that make it unsuitable for therapeutic use in humans.

Firstly, it is obtained from cobra venom (which is an inaccessible and dangerous source), and, therefore, it must be thoroughly cleaned of poison neurotoxins. The difficulties in obtaining direct sources of poison are also obvious. This problem cannot be easily overcome by cloning and expression of the ex vivo gene, since there are post-translational modifications that occur in the body of snakes (specific proteolytic processing), which can complicate (or make impossible) in vitro reproduction. In addition, the enzymes and degradation conditions necessary for this processing are currently unknown. Secondly, the protein is foreign in origin (for humans) and, therefore, immunogenic. This excludes its repeated therapeutic use, which is necessary for the decompensation of the patient for many weeks (for example, in order to allow the xenograft to take root).

Although CVF has some structural and functional homologies with human C3 [17], it also has important differences in both respects (for example, it differs in chain structure, place of biosynthesis, lack of sensitivity to complement regulators, and formation of stable C3 convertase). At present, the equivalent of C3 is unknown, which is obtained from a source other than cobra, which can be cloned and sequenced and which in general structure and function is more similar to human C3 than CVF [8].

CVF is a poison-specific product from an animal that is extremely distant from Homo sapiens in evolutionary terms. Therefore, it does not seem advisable to use genetic manipulation to modify this protein in order to turn it into a product that can be used and be non-immunogenic for humans.

The authors of the present invention have developed an alternative strategy in which physiological regulation is not used and which, instead of inhibiting complement activation, leads to superactivation of the system. This strategy can be applied in two cases. First, it can be used in vivo to activate complement until one or more components are exhausted, which leads to a loss of the ability to produce local responses to any subsequent intervention (such as a xenograft). Secondly, unregulated superactivation can be deliberately localized to a specific target (for example, a virus or a virus infected cell) in order to increase the sensitivity of this target to destructive reactions that are mediated by complement.

The term "complement activation regulators" in the context of the present description includes all proteins whose activity inhibits the enhancement of C3 conversion, and it is understood that they are not limited to those proteins whose genes are located in the RCA genetic locus. This concept, however, does not include “up-regulators,” such as properdine. The term “C3 conversion” means the proteolytic conversion of C3 to C3b and C3a, unless otherwise specified, and the term “C3 convertase” (or simply “convertase”) refers to an enzyme (usually a complex of two or more protein components, for example, C3bBb, C3iBb , CVFBb or C4b2a), which catalyzes this reaction.

Thus, the first subject of the invention is a protein of the native complement activation pathway, modified so that the protein has the ability to form C3 convertase resistant to downregulation.

The term "native" means found in vivo, i.e. obtained in nature. Thus, the definition includes any naturally occurring complement activation pathway protein modified as described above. It is understood that the concept is not limited to species-specific proteins. In other words, the modified human protein can be used as resistant to downregulation of C3 convertase, for example, in other mammalian species. Usually, modified complement pathway proteins of the same species should be used.

Modification of the coding sequence of C3 DNA, for example, using site-directed mutagenesis, can lead to a variant C3 that is resistant to regulatory complement proteins, which still retains its positive functional properties (the ability to be cleaved by C3 convertases to produce C3b) and structural integrity (correct structure) chains and the presence of a thiol ester bond). The present invention, presented in this description, relates to genetically modified forms of native complement proteins, for example, human C3, the C3b fragment of which acquires the ability to become resistant to physiological regulation of complement. As a result of such stability, these molecules can form stabilized forms of the corresponding C3 convertase, which leads to an enhanced conversion of C3 to C3b and subsequent decomposition of the products under physiological conditions (for example, in vivo).

In a preferred embodiment, the invention relates to a modified human C3 protein that is resistant to I-factor crushing.

This can be achieved by modifying protein residues at proteolytic sites.

A particularly preferred embodiment is a modified human C3 protein, which is modified by replacing either Arg-1303 or Arg-1320, or both of them with another amino acid. Another amino acid may be tyrosine, cystine, tryptophan, glutamine, glutamic acid or glycine. Replacement of Arg-1303 with glutamic acid or glycine is preferred (less preferred is glutamine). Replacement of Arg-1320 with glutamine is preferred.

Other strategies for producing stable modified proteins of the invention include:

I) Reduced sensitivity to the inhibitory effects of factor H and related proteins (e.g., MCP, DAF, CR1). For example, in human C3, residues 767-776 and 1209-1271 are involved in the binding of the H factor [21, 24] and replacing one or more of these residues or other residues also associated with the action of these proteins can reduce the binding of one or more of these regulatory proteins.

II) Reduced dissociation rate of C3bBb. Mutations can be introduced that will enhance the interaction between C3b and Bb. The result of this can be both a decrease in spontaneous destruction of the enzyme, and a decrease in the efficiency of the H-factor (and related regulators) during the displacement of Bb from C3b.

These mutations are necessary to reduce the rate of both spontaneous and H-factor-mediated destruction of C3bBb. Even in the absence of the H factor, the half-life of the C3bBb complex in the liquid phase at 37 ° C in the presence of properdine is only about 10 min [6].

III) Residues 752-761 of human C3 are involved in B-factor binding. This region is a highly conserved region in C3 and a sequence closely related to it has been found in C4. Since C4 binds the homologue of the B-factor C2, the marked similarity of this region in C3 and C4, along with its high conservatism in C3, further confirms its role in C3 as a B-factor binding site. Thus, changes in this area can affect the affinity for b and the stability of C3bBb.

IV) Resistance to other complement activation regulators, such as CR1, DAF, and MCP, is also desirable. The mechanism of action of all these regulators is similar to that of the H-factor, and therefore, additional mutagenesis is not required. Similarly, some pathogens express their own complement activation inhibitors, which are often structurally and functionally homologous to the H factor (for example, the secretory peptide of vaccinia virus (Vaccinia)). These molecules protect the invading species from immune responses, and therefore it may be appropriate to attack them with the targeted action of C3-convertase enzymes that are resistant to this protection.

V) Mutations that enhance the stabilization of C3 convertase with properdine. Properdine activity consists in stabilizing the C3bBb complex, in slowing down spontaneous and H-factor-dependent dissociation. This stabilization is ineffective in the liquid phase, but is probably more important for enhancing the process if it has already been started on the corresponding activating surface [5]. An increase in this activity (by increasing its affinity) can upset the balance in the liquid phase and thereby enhance the spontaneous conversion of C3. This should be especially important in combination with the other modifications described above.

VI) Mutations that impede the acquisition of C3bBb C5 convertase activity. The depletion of active C3 in the bloodstream is usually accompanied by an undesirable side effect, i.e. obtaining large quantities of anaphylactic peptides. The most active of these is C5a, which is obtained by cleavage of C5 using some C3 convertase enzymes. This reaction probably depends on the affinity of the convertase to another C3b molecule [11] and therefore can be suppressed using C3 mutations that eliminate this interaction.

VII) Improved activity of C3 convertase. The active site C3bBb of the enzyme C3 convertase is located in the Bb region. The component C3b functions are likely to impart an active conformation to Bb and / or to bind and orient the substrate affected by Bb. The mechanism of this phenomenon is not yet known, but in any case, in this case, there is the possibility of enhancing the activity of convertase through mutations in C3.

VIII) Expression in a functionally active form. For wild-type C3, conversion to C3b is necessary before it can be combined into a new complex with a C3 convertase. To convert to C3b (or to C3i) in vivo, it is necessary to slow down the action of modified C3. Therefore, it is desirable either to administer the protein in a form capable of immediately forming a convertase, or to administer preformed convertase complexes. Therefore, it seems appropriate to obtain a functionally active C3b-like ex-vivo reagent. This can be achieved in vitro (for example, by proteolysis).

IX) Modifications of the native protein that serve to introduce new cleavage sites in such a way that the peptide regions necessary for B-factor binding are retained, and the regions necessary exclusively for H-factor binding can be specifically removed. For example, sites can be introduced such that the C3b-like form of modified C3 can be further cleaved to give a form that still binds the B factor but is less sensitive to inactivation by factors H and I.

X) Modifications in other areas that may affect the interaction of C3b with the B-factor and / or with the H-factor.

The invention is based on a revision of the traditional approach and is based on stimulating the conversion of C3 to reduce C3, which leads to the failure of the system. An additional field of application of the invention is the ability to stimulate the conversion of C3 at a specific location and, therefore, attract complement-dependent effector mechanisms to attack a specific target.

Thus, the final effect should be an increase in the number of cases of C3 conversion, when the modified protein is introduced into the physiological environment (for example, into the blood) containing complement activation regulators. Then this activity can be used either to reduce native C3 in this medium, or to localize the conversion of C3 on the desired target.

The C3 analogue, the C3b fragment of which is resistant to the effects of the I-factor (for example, the derivative described in Example 1), must bind the B-factor, which then must be cleaved by the D-factor and finally dissociated to an inactive form. In the absence of I-factor inactivation, modified C3b must be able to re-bind new B-factor molecules and thereby enhance its inactivation. Therefore, another potential use of the modifications described in this invention may be the inactivation of an alternative pathway by exhausting B-factor activity. A similar approach can also be applied to the modification of C4 to enhance the consumption of C2 and thereby disable the classic complement activation pathway.

The invention includes any other protease used similarly to the C3bBb enzyme, which leads to the cleavage of C3 to C3b, despite the presence of complement activation regulators.

The invention also includes DNA sequences that encode the protein of the invention, as well as DNA constructs containing such DNA sequences.

The term "DNA sequence" includes all other nucleotide sequences which, due to the degeneracy of the genetic code, also encode a given amino acid sequence, or which are practically homologous to this sequence. These sequences, therefore, also fall within the scope of the invention.

Nucleotide sequences that are “substantially homologous” also fall within the scope of the present invention. “Practical homology” can be considered either at the nucleotide level or at the amino acid level. At the nucleotide level, sequences having significant homology can be considered those that hybridize with the nucleotide sequences of the invention under stringent conditions (for example, at a temperature of 35-65 ° C. in a salt solution of a concentration of about 0.9 M). At the amino acid level, a protein sequence can be considered substantially homologous to another protein sequence if a significant number of constitutive amino acids have homology. Can be homologous (in increasing order of preference) at least 55%, 60%, 70%, 80%, 90%, 95% or even 99% amino acids.

As indicated above, the proteins of the invention can be used to achieve the effects of localized complement activation. One way to achieve this is by conjugating the protein to a fragment that must bind to the desired target. Thus, a second subject of the invention is a conjugate comprising a protein of the invention crosslinked with a specific binding fragment, for example a specific binding protein. An example of such a protein may be an antibody or antigen binding fragment thereof.

The proteins of the invention are intended to be administered to a subject to achieve the desired therapeutic effect. Therefore, the invention also includes:

a) a protein of the invention intended for use in therapy;

b) the use of a protein or conjugate according to the invention for the manufacture of a medicament intended to reduce protein levels of the complement activation pathway, and in particular, to prevent the rejection of foreign material;

c) a pharmaceutical composition comprising one or more proteins or conjugates of the invention together with one or more pharmaceutically acceptable carriers and / or excipients; and

d) a method of reducing complement activation pathway proteins in a mammal, comprising administering to the mammal a protein of the invention, preferably in the form of a pharmaceutical composition.

The pharmaceutical compositions may be in the form of unit dosage forms containing a predetermined amount of the active substance in a dose. Such a unit dose may contain at least, for example, 1 mg of active ingredient and preferably 2-3 mg. The upper limit that such a standard dose can reach will depend on many factors, such as the condition to be treated, route of administration and age, weight and condition of the patient, as well as economic factors. For example, a unit dose may contain 10 mg or even 100 mg of active ingredient.

The proteins of the invention can be used in vivo to disable the complement system. This may be appropriate in the following circumstances:

(a) In order to prevent complement-mediated destruction or damage to the graft, in particular the xenograft (material transplanted from other animal species), and especially the discordant xenograft (when the types of donor and recipient are discordant (mismatched)). The recipient must first have the complement removed before surgery, and he must be in such a state until either the graft is accommodated or replaced with a more compatible organ.

Initial treatment should be carried out within a few days before transplantation. Additional complement removal may be required during a rejection crisis. Treatment may be with antihistamines to combat general inflammatory reactions (e.g., vasodilation), likely resulting from the production of C3a and / or C5a.

Removing complement may also be appropriate when using artificial organs or tissues (e.g., dialysis membranes of an artificial kidney), which activates the complement system. As described previously, the protein can be used either in an inactive form, functionally similar to the C3b form, or in the form of a preformed active C3 convertase (type C3bBb). These forms can be entered using any route, and active convertase must be found in the bloodstream C3 (for example, administered intravenously, subcutaneously, etc.).

Another alternative method may be ex vivo treatment, for example, by transfusion of blood through a matrix carrying active convertase. This may have the advantage that anaphylactic peptides (C3a and C5a) and other low molecular weight inflammatory mediators (such as histamine and nitric oxide) can be removed (e.g. by dialysis) before blood (or plasma) is removed complement will be returned to the patient.

(b) In order to prevent complement-mediated damage resulting from major surgery. The complement should be removed from the patient, as described above, preferably before the operation (but, if necessary, after it), and he should be in such a state until the risk of additional internal damage due to the complement-dependent immune attack is eliminated.

(c) In order to minimize complement-mediated damage resulting from a non-surgical lesion. In these cases, the removal of complement should be carried out after the initial lesion, but the compositions and methods of administration are likely to generally correspond to those described above. This may be especially appropriate when the restoration involves the replication of ischemic tissue by circulation (for example, myocardial ischemia, frostbite, burns, etc.).

(d) In order to minimize complement-mediated damage resulting from antibody-antigen interactions. Complement-mediated defense reactions are especially undesirable in autoimmune diseases, which include glomerulonephritis, hemolytic anemia, intermittent claudication, type I diabetes, rheumatoid arthritis, and multiple sclerosis. Disabling the complement system during serious stages of the disease can alleviate the condition, which can be done, for example, by local injection into the joint with rheumatoid arthritis.

(e) In order to increase the sensitivity of a specific pathogenic target to complement-mediated immune mechanisms. With this approach, the goal is not to use a superactive C3 convertase to achieve an overall decrease in C3, but instead to ensure that C3 is converted to the desired target, convertase is applied locally. The target may be a pathogenic organism, such as a bacterium, virus or other parasite, or a harmful host cell or tissue, such as a tumor cell or a virus infected cell. C3 convertase can be localized in the target either by local application (for example, direct injection into the medium, which delays its distribution in the general blood flow, is possible), or by combining with a guiding fragment, for example, an antibody. For example, a modified protein can be crosslinked with a specific immunoglobulin either by chemical cross-linking of the proteins, or by combining the DNA coding sequences and the expression (and purification) of the fusion protein (for example, in the case of IgG, either the heavy or light chain must be linked with C3 and co-expressed with C3, or both chains can be combined in one complete fusion polypeptide), or by including specific coding sequences (for example, for domains like leucine howl "zipper") in the DNA of both partners involved in the fusion (e.g., modified

C3 and specific antibodies) in such a way that the expressed products, when mixed together, bind together to form stable conjugates. The fusion protein may then be administered locally or into the general bloodstream.

Liposomes (carrying an antibody on the surface with a modified protein located either on the surface or inside the liposome) and / or virions (for example, designed to express proteins on their surface) can also be used to co-deliver the antibody and the modified protein. This strategy can be used directly, alone or in combination with other treatment systems, at any stage of the disease. It may be especially advisable to use it to eliminate any cancer cells that have survived in the bloodstream after removal of the tumor by surgery. Modified protein antibody conjugates can also be used ex vivo to eliminate pathogenic tissue. For example, to destroy leukemia cells in bone marrow extracts and then return the remaining healthy cells to the patient.

Alternatively, lymphocytes that are not suitable for the types of recipient's major histocompatibility complex (MHC) can be removed from the bone marrow before transplantation. In addition, the modified protein can be crosslinked with the antigen, and this combination can be used either in vivo or ex vivo to attack lymphocytes with unnecessary reactivity (for example, against transplanted or native tissue).

The same technique can be used to treat other species using either a derivative of a human modified protein or a similar analogue designed specifically for these species.

Preferred characteristics of each subject of the invention are also preferred for each other subject of the invention mutatis mutandis (as amended).

Below the invention is illustrated by examples, which are not aimed at limiting the scope of the invention, with reference to the accompanying drawings.

Figure 1 shows the theoretically predicted sequence of the human C3 protein encoded in PC3 (the standard single-letter code for amino acids was used).

Figure 2 shows the cDNA sequence in PC3 (the standard single-letter code for sense strand deoxynucleotides is used, written in the 5’-3 ’direction).

Figure 3 shows the visualization of the modified proteins of the invention.

Figure 4 shows the effect of various mutations in human C3 leading to the replacement of Arg ¹³⁰³ or Arg ¹³²⁰ by I-factor mediated cleavage at these sites.

Note:

1) samples labeled biosynthetically using [35S],

2) the reactions were carried out at normal ionic strength,

3) immunoprecipitation using anti-C3,

4) SDS-PAGE in reducing conditions,

5) autoradiogarphia.

Figure 5 shows the increased resistance of human C3 having a mutation Arg ¹³⁰³ → Gln ¹³⁰³ , in relation to inactivation by factors I and N.

Figure 6 shows an analysis of cleavage with a C3 convertase having mutations at residues 752-754 and 758-760.

A photograph of western blotting performed in 7.5% polyacrylamide SDS-PAGE gel (reducing conditions) after electrophoretic transfer to nitrocellulose, probing with sheep’s anti-human anti-C3 antibody and using horseradish peroxidase cross-linked with anti-human immunoglobulin, and enhancing chemiluminescence "Enhanced ChemiLuminescence" (method and reagents for determination supplied by Amersham, UK), imprinted on an x-ray film. The cleavage reaction and determination was carried out according to example 4 when compared with the results shown in figure 3.

Explanation:

Lanes 1-4: Wild-type C3 (expressed in COS cells).

Lanes 5-8: Mutant C3 (residues 752-754 replaced with Gly-Ser-Gly and residues 758-760 also replaced with Gly-Ser-Gly) (expressed in COS cells).

Lanes 1.5: no add.

Lanes 2, 6: + CVFBb.

Lanes 3, 7: + H + I factors.

Lanes 4, 8 + CVFBb + H + I factors.

The bands indicated by arrows correspond to the following:

A: alpha chain C3,

B: C3 alpha’s chain,

C: beta chain C3,

D: product of cleavage of the alpha’-chain C3 with a molecular weight of 68 kDa E: heavy IgG chain.

Figure 7 shows an analysis of the cleavage of the radiolabeled B-factor by the D-factor in the presence of wild-type C3 and mutant C3 (C3i).

A photograph of an autoradiographic study of SDS-PAGE gel is provided. All samples contained D-factor and ²²⁵ I-labeled B-factor and were incubated for 3 hours at 37 ° C.

Samples in numbered tracks also included:

1) only the buffer,

2) dilution 1/125 C3 wild-type,

3) dilution of 1/25 C3 wild-type,

4) dilution 1/5 C3 wild-type,

5) dilution of 1/25 mutant C3 (residues 1427 Gln, 1431 Asp and 1433 Gln),

6) dilution 1/5 of mutant C3,

7) undiluted mutant C3,

The bands indicated by arrows correspond to the following:

A. Unsplit labeled with ¹²⁵ I B-factor (93 kDa).

B. Cleaved product 60 kDa ("Bb").

C: Cleaved product 33 kDa ("Ba").

On Fig shows a study using SDS-PAGE, illustrating the formation of the conjugate between C3i and IgG.

The result was obtained by staining with Coomassie 4% acrylamide SDS-PAGE gel during distillation under non-reducing conditions.

Numbered tracks contain the following patterns:

1. PDP-IgG

2. C3i

3. PDP-IgG + reaction mixture with C3i

The arrows show:

A. Possible conjugate C3i-IgG (350 kDa)

B. C3i (200 kDa)

C. IgG (150 kDa)

Figure 9 shows that the conjugate directs the activity of C3 convertase against sheep erythrocytes.

This graph shows the% lysed sheep erythrocytes after sensitization with dilutions of the conjugate C3i-IgG, PDP-IgG or C3i followed by washing, obtaining C3-convertases using properdine and factors B and D and the final lysis using NGPS in CFD / EDTA, as described in the methods. Lysis is caused only by conjugate, and this lysis is dose dependent.

Figure 10 and 11 shows the features of the cleavage of the mutant C3 DV-1AM (see examples 12-14).

In relation to figure 10, COS cell supernatants containing expressed wild-type C3 (A) and C3 mutant DV-1AM (B) were treated with 1) were left without treatment; 2) CVFBb; 3) 10 μg / ml I-factor and 50 μg / ml H-factor; or 4) CVFBb plus 10 μg / ml of I-factor and 50 μg / ml of H-factor, were immunoprecipitated, analyzed by SDS-PAGE (in these tracks) and transferred by electroblotting to nitrocellulose as described in Example 4. B in this case, blotting was performed using a combination of rat monoclonal antibodies Clone-3 and Clone-9, which react with the C3dg region of C3 and its fragmented products (Lachmann PJ. et al., J. Immunol. 41: 503 (1980)), and then horseradish peroxidase crosslinked with anti-rat immunoglobulin (from Sigma) and detected using ECL reagents and instructions uu supplied by Amersham.

As applied to FIG. 11, COS cell supernatants containing the expressed C3 DV-1B mutant (A), wild-type C3 mutant (B) and C3 DV-6 mutant (C) were treated with 1) were left without treatment; 2) 10 μg / ml I-factor and 50 μg / ml H-factor; 3) CVFBb; 4) CVFBb plus 10 μg / ml I-factor and 2 μg / ml H-factor; 5) CVFBb plus 10 μg / ml I-factor and 10 μg / ml H-factor; or 6) CVFBb plus 10 μg / ml of I-factor and 50 μg / ml of H-factor, were immunoprecipitated, analyzed using SDS-PAGE (in these tracks), transferred by electroblotting to nitrocellulose and detected using polyclonal sheep antibodies to C3, as described in example 4.

12 shows an analysis of a product related to C3 and resulting from a “reading frame shift” mutation. This product is designated as HDV-3X. The results obtained for HDV-3X are compared with the results obtained for DV-3, which like HDV-3X includes mutations T1031G, E1032N, Q1033H, E1035N and K1036I, but unlike HDV-3X, it is not modified at the C-terminus.

COS cell supernatants containing the expressed C3 mutant DV-3 (A) and C3 mutant HDV-3X (B) were treated with 1) were left untreated; 2) CVFBb + 10 μg / ml I-factor; 3) CVFBb + 10 μg / ml I-factor + 1 μg / ml H-factor; 4) CVFBb + 10 μg / ml I-factor + 5 μg / ml H-factor; 5) CVFBb + 10 μg / ml I-factor + 25 μg / ml H-factor, was subjected to immunoprecipitation, analyzed using SDS-PAGE (in these tracks) and transferred by electroblotting to nitrocellulose, as described in example 4. In this blotting was performed using a combination of rat monoclonal antibodies Clone-3 and Clone-9, which react with the C3dg region of C3 and its fragmented products (Lachmann PJ et al., 1980, J. Immunol. 41: 503), and then peroxidase from horseradish crosslinked with anti-rat immunoglobulin (from Sigma), and detected using ECL reagents and instructions, post sold by Amersham.

13 shows the results of an experiment in which supernatants of COS cells containing expressed C3 mutants E1Q2 (A), E1Q2QG3 (B) and E1Q2E3 (C) were treated (at 37 ° C, 2.5 hours) with 1) CVFBb + 10 μg / ml I-factor; 2) CVFBb + 10 μg / ml I-factor + 50 μg / ml H-factor; 3) CVFBb + 10 μg / ml I-factor + 25 μg / ml sCRl, subjected to immunoprecipitation, analyzed using SDS-PAGE (in these tracks) and transferred by electroblotting to nitrocellulose, as described in example 4. In this case, blotting was performed using a combination of rat monoclonal antibodies Clone-3 and Clone-9 as described in Example 12, followed by horseradish peroxidase crosslinked with anti-rat immunoglobulin (from Sigma) and detected using ECL reagents and instructions supplied by Amersham .

The band corresponding to 86 kDa (indicated by an arrow) represents the cleavage product mediated by the I-factor at the third site, unless cleavage at sites 1 or 2 previously occurred.

On Fig shows the results of an experiment in which the supernatants of COS cells containing the expressed mutants C3 NC3 (A), FT-1 (B), FT-2 (C), FT-3 (D), FT-4 (E) and FT-5 (F), were treated (at 37 ° C, 2.75 h) with 1) were left without treatment; 2) CVFBb + 10 μg / ml I-factor + 50 μg / ml H-factor, was immunoprecipitated, analyzed by SDS-PAGE (in these tracks) and transferred by electroblotting to nitrocellulose, as described in example 4. In this case, blotting was performed using a combination of rat monoclonal antibodies Clone-3 and Clone-9, as described in example 12, and then horseradish peroxidase crosslinked with anti-rat immunoglobulin (from Sigma), and detected using ECL reagents and instructions supplied by Amersham.

On Fig shows the results of an experiment in which the supernatants of COS cells containing expressed mutants C3 NC3 (A), FR-1 (B), FR-2 (C), FR-3 (D), FR-4 (E) and FT-2 (F), were treated (at 37 ° C, 2.5 hours) with 1) were left without treatment; 2) CVFBb + 10 μg / ml I-factor + 50 μg / ml H-factor, was immunoprecipitated, analyzed by SDS-PAGE (in these tracks) and transferred by electroblotting to nitrocellulose, as described in example 4. In this case, blotting was performed using a combination of rat monoclonal antibodies Clone-3 and Clone-9, as described in example 12, and then horseradish peroxidase crosslinked with anti-rat immunoglobulin (from Sigma), and detected using ECL reagents and instructions supplied by Amersham.

For ease of reference, the following is the relationship between the groups of claims that relate to certain proteins that can act as resistant to downregulation of C3 convertases, and the examples and tables of the present description:

The standard methods and definitions below apply to all examples.

All complement components are labeled according to those of a person, unless otherwise indicated, using standard terminology for all proteins and fragments that are their derivatives (for example, in accordance with the link [15]). In addition, the term “C3i” refers to any molecular form of C3 without an intact thiol ester bond, but which retains the C3a polypeptide on the alpha chain.

human C3 cDNA and coding sequence are numbered in accordance with figure 2 using the numbering adopted in the EMBL nucleotide database (according to the link [2]). The sequence presented is the authors construct (PC3), in which there are no first 11 nucleotides of the 5'-untranslated region shown in reference [2], and therefore the first base is numbered 12. The putative initiation codon is nucleotides numbered 61-63 the codon for the N-terminal residue of the serine beta chain is represented by nucleotides 127-129 and the codon for the N-terminal residue of the serine beta chain is represented by nucleotides 2074-2076.

The protein sequence is numbered according to the numbering of the precursor sequence, as shown in FIG. 1, in accordance with the translation of the theoretically predicted DNA sequence given in the appendix (it is assumed that amino acids 1-22 contain a signal sequence that is removed during biosynthesis, and it is assumed that amino acids 668-671 can be removed by cleaving the precursor into alpha and beta chains).

The following abbreviations have the following meanings: CVF means cobra venom factor; ELISA means enzyme-linked immunosorbent assay; E. coli means Escherichia coli; tbp means the length in thousands of pairs of nucleotides; HSV-1 means herpes simplex virus type I; PBS means phosphate buffered saline; COS-1 means a cell line originating from monkey kidney cells. The following restriction endonucleases are: AflII, DraI, DraIII, EcoRI, EcoRV, HindIII, NaeI, NheI, XbaI.

Standard methods

Standard molecular biology techniques, such as plasmid isolation, agarose gel electrophoresis and DNA ligation, can be found in reference [21]. Double-stranded DNA was sequenced using the Sequenase version 2.0 kit, available from United States Biochemicals. C3 expression was determined by ELISA using plastic plates precoated (sensitized) with polyclonal sheep anti-human C3 antibody purified by affinity chromatography to which culture supernatant samples were added. C3 binding was determined using rat monoclonal antibody to C3 conjugated with alkaline phosphatase and a chromogenic substrate, para-nitrophenol phosphate. Samples were calibrated using purified C3 human plasma.

Methods for purification of complement and CVF proteins and preparation of preparations purified by affinity chromatography of antibodies to C3 used in the analysis can be found in reference [28]. Equivalent reagents can also be obtained from Sigma chemical company LTD.

The coding sequence of c3 cDNA

The c3 cDNA coding sequence of the invention was constructed from two segments isolated from a randomly primed human liver cDNA library in the pGEM4 vector (Promega). Five oligodeoxynucleotides corresponding to known segments in the human C3 coding sequence were radioactively labeled with T4 polynucleotide kinase and [γ - ³² P] ATP and used to probe the filter library from agarose plates. Two clones containing inserts (inserts) of approximately 4 kb were isolated. Digestion with restriction enzymes, hybridization with specific oligonucleotide probes and partial sequence analysis showed that one of these clones ('A13') included the 5'-end of the transcript 5.1 kb in length and the other ('B44') to the 3'-end.

Thus, these inserts overlap in a region of approximately 3 kbp including a unique EcoRI restriction site. The incomplete 5′-region of A13 was cleaved with EcoRI and NheI and replaced with the complete segment isolated from B44 by digestion with EcoRI and XbaI. Both fragments were purified by low melting point agarose gel electrophoresis before ligation with T4 DNA ligase to give a vector ('PGC3') containing a 5.1 kb full length C3 protein precursor encoding a DNA fragment. .

The linker sequences of the 5 ′ end of the C3 coding region contained two ATGs, which are false translation start codons. Therefore, they were removed using gap plasmid mutagenesis, as described in the method of example 1, using oligodeoxynucleotide PL-ATC-3 (tagggagacc ggaagcttgc cctctccctc tgtccctctg t), which led to the deletion of approximately 50 base pairs of the DNA of the linker / adapter, without changing the coding sequence C3. This mutant vector is 7.7 kb in length, containing a 5.1 kb c3 cDNA sequence fragment plus a sequence of PGEM4 vector of 2.6 kbp (Promega), is referred to herein as PC3.

The coding region C3 of the PGC3 plasmid was completely sequenced and only 4 differences were found from the previously published human C3 cDNA sequence (allele "S") [2]:

(I) substitutions C2481 → G and C2805 → T do not change the encoding;

(II) the replacement T1001 → C encodes the previously described polymorphic form of HAV 4-1 (replacement: leucine 314 → proline) [20]; and

(III) the substitution G2716 → A encodes the substitution: valine 886 → isoleucine, which was not previously described for human C3, although Il was found in this position in mouse and rat C3.

The sequence of the invention includes start and stop codons with a complete signal sequence and, therefore, must encode functionally active C3.

Using ELISA analysis in supernatants of COS-1 cell cultures (infected using lipofectamine and an Invitrogen pcDNA3 expression vector), wild-type C3 was detected at a concentration of up to 1.7 μg / ml. Cells infected with one pcDNA3 vector did not produce C3 in an amount that could be detected. In addition, analysis of the expressed product using cleavage reactions followed by immunoprecipitation, SDS page-SDS and immunoblotting showed that:

(I) the primary translation product was correctly processed into a mature double-stranded form;

(Ii) this product, like native C3, could be cleaved with C3 convertase (CVFBb) to give C3b; and

(III) the expressed protein, like native C3, was not cleaved by factor H plus I, but became cleaved after conversion to C3b using the enzyme C3 convertase. These data confirm that the original plasmid according to the invention can be translated into functionally active C3.

For data on an alternative way of constructing and expressing the C3 coding sequence, see link [25].

Example 1: Preparation of C3, in which the arginine residues at both cleavage sites recognized by the I-factor (positions on amino acid sequences 1303 and 1320) are replaced by glutamine residues in order to prevent cleavage of the C3b fragment by the I-factor.

a) Mutagenesis

As mutagens oligodeoxynucleotides used QRI1 (caactgcccagccaaagctccaagatcacc), QRI2 (gccagcctcctgcaatcagaagagaccaag) and AFL4149 (taataaattcgaccttaaggtcaccataaaac), as well as the corresponding antisense oligodeoxynucleotides QRI1n (ggtgatcttggagctttggctgggcagttg), QRI2n (cttggtctcttctgattgcaggaggctggc) and AFL4149n (gttttatggtgaccttaaggtcgaatttatta).

QRI1 and QRI1n determine the replacement of arginine with glutamine at the cleavage site recognized by the I-factor at position 1303 of the amino acid sequence of the C3 precursor (by replacing G3968C3969 with AA in the cDNA sequence), and QRI2 and QRI2n lead to the same replacement of the amino acid residue at position 1320 ( by replacing nucleotide G4019 with A) at the cleavage site recognized by the I-factor.

AFL4149 and AFL4149n introduce the AflII restriction site, at position 4149 of the cDNA sequence (by replacing C4149 with T) without changing the encoded amino acid sequence. These two primers were used as markers to determine, on the basis of cleavage of the DNA product with AflII, that successful mutagenesis occurred.

Mutagenesis was performed using the "gap plasmid" method. A series of PGC3 plasmids ('UPGC3') containing uridine instead of thymidine was obtained by growing E. coli strain CJ236 in the presence of 0.25 μg / ml uridine. This plasmid was digested with SmaI and a 7.2 kbp product. ('USD1') was purified on an agarose gel to remove a 0.5 kbp fragment. from the sequence C3 (residues 1463-1947). The second component of the “breach” plasmid (gap plasmid) ('DN2') was obtained by cleavage of PGC3 with DraIII plus NaeI and double purification of the 5.1 kb fragment. agarose gel electrophoresis. 200 ng DN2 was mixed with approximately 500 ng US1 in 50 μl of water heated to 100 ° C and slowly cooled to below 50 ° C before adding 20 μl-25 μl of 2XT7 buffer (100 mM Tris / HCl / pH 7.4 / 14 mM MgCl ₂ , 100 mM NaCl, 2 mM dithiothreitol and 1 mM each of ATP, dATP, dTTP, dTTP and dGTF) plus 10 nmoles of each of the 5'-phosphorylated mutagenic primers (QRI1, QRI2 plus AFL4149 were used in one reaction, in another reaction, QRIln, QRI2n plus AFL4149n) were used. The mixture was reheated to 70 ° C for 5 min and slowly cooled (within 30-60 min) to 20 ° C. 10 units of T7 DNA polymerase plus 80 units of T4 DNA ligase were added at 0 ° C. The mixture (total volume 50 μl) was incubated first at 0 ° C for 5 minutes, then at room temperature for 5 minutes and finally at 37 ° C for 3 hours. 1 μl of each mixture was used to transform 100 μl of supercompetent strain E. coli XL1 (Stratagene) according to the manufacturer's instructions.

Ampicillin-resistant colonies were screened for AflII cleavage and positive (successful) mutants were grown in 100 ml of cultures from which plasmids were isolated and sequenced (using C3ra-3876 sequencing primer, cttcatggtgttccaagcct, corresponding to nucleotides 3876-3895 of the c3NA cDNA study) in cleavage sites recognized by the I-factor.

For information on other mutagenesis protocols using the "gap plasmid" method, see references [26, 27].

b) Transfer of mutant DNA into a eukaryotic expressing vector

Fragments of the C3 coding sequence from mutant plasmids were excised by double digestion with HindIII and NaeI. DraI was also included in the remaining plasmid to disable it. The C3 coding sequence was purified on an agarose gel and ligated with the pcDNA3 vector (Invitrogen), which was linearized with HindIII and EcoRI enzymes and dephosphorylated with calf intestinal phosphorylase. Ligated mixtures were used to transform the supercompetent strain of E. coli XL1 and then plated on culture plates containing ampicillin.

Randomly selected (three or four) ampicillin-resistant colonies were grown in 2-3 ml of cultures and small amounts of plasmid DNA were isolated from them. Plasmids carrying the correct insert were detected by digestion of plasmid DNA with restriction enzymes EcoRI, HindIII and AflII. The corresponding colonies were grown in 100 ml of cultures and plasmids were purified by standard methods. These mutants were originally constructed from PGC3, and therefore they retained two ATG codons at the 5 ′ end of the coding region. Therefore, this region (plus the 5 ′ end of 3 kb of C3 coding sequence) was cleaved with HindIII and EcoRI and replaced by dating with the same fragment excised from PC3. Such reconstructed vectors were obtained by a standard method and used to transfect COS cells.

c) Expression of wild-type C3 and mutant C3

Mutant wild-type C3 and C3 were expressed for a short period of time in plasmid-transfected CO S-1 cells using lipofectamine ^® (GIBCO) according to the manufacturer's instructions. Typically, 1-1.5 × 10 ⁵ cells per well of a standard 6-well culture plate were transfected with 2-4 μg of plasmids using 9 μl of lipofectamine reagent. C3 secretion was evaluated in supernatants, and normal yields 3–6 days after transfection were 0.3–1.7 μg per ml of supernatant.

results

a) Obtaining mutants

The following mutants were isolated, which are designated in accordance with the included mutagenic oligodeoxynucleotide sequences:

(I) 3 mutants having both QRI1 and QRI2 mutations plus AFL4149: C3M-26, C3M-58 and C3M-61;

(II) 1 mutant with both mutations QRI1 and QRI2, but without AFL4149: C3M-8; and

(III) 1 mutant with QRI2 and AFL4149 mutations, but without QRI1: C3M-51 (which was used in example 3).

b) Evidence that functional influences were the result of mutations specifically introduced into cleavage sites recognized by the I-factor

Sequencing confirmed the absence of other modifications at a distance of 178-350 bases around the mutant region of each mutant. The sequence of one of the mutants obtained using this technique, C3M-51 (see Example 3), was analyzed throughout the “gap” (bases 2463-5067) used for mutagenesis, and no other deviations from the wild sequence were found type.

Moreover, representative sequencing of a complete fragment with a length of 2922 bases from all mutants did not reveal any single point mutation that could be obtained as a result of errors made by polymerase. All expressed mutants had a double-stranded structure and were cleaved by C3-convertases characteristic of native C3. In General, it is unlikely that the applied mutants contained any undesirable substitutions, although they were not completely re-sequenced.

Example 2: Preparation of C3, in which the arginine residue at one cleavage site recognized by the I-factor (position on amino acid sequence 1303) is replaced by a glutamine residue

They worked similarly to the method described in example 1, except that only mutagenic oligodeoxynucleotides AFL4149 plus QRI1 or AFL4149n plus QRI1n (i.e., without QRI2 and QRI2n) were used for mutagenesis.

results

a) Received mutants

2 mutants were identified with the QRI1 and AFL4149 mutations, but without the QRI2 mutation: C3M-I23, -27. The C3M-I23 mutant was expressed as described in Example 1.

This protein was cleaved by CVFBb. The C3b-like product was relatively (compared to the wild type) resistant to cleavage at position 1303 by factors I and H, but could still be cleaved at position 1320. Therefore, this C3b derivative is partially resistant to I-factor.

Example 3: Preparation of C3, in which the arginine residue at one cleavage site recognized by the I-factor (position on amino acid sequence 1320) is replaced by a glutamine residue

They worked similarly to the method described in example 1, except that only mutagenic oligodeoxynucleotides AFL4149 plus QRI2 or AFL4149n plus QRI2n (i.e., without QRI1 and QRI1n) were used for mutagenesis. In addition, by the method used in example 1, one mutant was also obtained having the QRI2 and AFL4149 mutations, but not without QRI1.

results

a) Received mutants

3 mutants with QRI2 and APL4149 mutations, but without QRI1 mutations were isolated: C3M-51, C3M-Q2, C3M-Q13. The C3M-51 mutant was expressed as described in Example 1. This protein was digested with CVFBb. The C3b-like product could not be easily cleaved at position 1320 by factors I and H, but could still be cleaved at position 1303. Therefore, this C3b derivative is partially resistant to the I-factor.

Example 4: Analysis of the functional effects of mutations

The supernatants (100-400 μl) of transfected COS cells were incubated at 37 ° C for 2 hours, and the COS cells were transfected with pcDNA3 carrying such inserts as:

1) non-mutant sequence C3;

2) mutant C3M-I23 (encoding Arg ¹³⁰³ → Gln);

3) mutant C3M-26 (encoding Arg ¹³⁰³ → Gln, Arg ¹³²⁰ → Gln,); and

4) mutant C3M-51 (encoding Arg ¹³²⁰ → Gln).

200 μl of culture supernatants taken 3 days after transfection were pretreated with 2 mM phenylmethanesulfonyl fluoride (0 ° C, 15 min) and then incubated at 37 ° C for 2 hours under the following conditions:

A) without addition;

B) with pre-prepared C3 convertase, CVFBb (10 μl out of 200 μl of a mixture containing 6.6 μg CVF, 100 μg B-factor and 1.4 μg D-factor in a phosphate-buffered physiological solution (PBS) containing 10 mM MgCl ₂ , which was pre-incubated at 37 ° C for 15 min);

B) with factors H (5 μg) and I (1 μg); and

D) with CVFBb plus factors H and I.

The resulting product was then immunoprecipitated by adding 0.6 μg of sheep immunoglobulin purified by affinity chromatography to human C3 at room temperature and after 1 h adding 20 μl of a 5% suspension of washed formalin-fixed Streptococcus sp. Group C cells. (protein G) (from Sigma). After incubation for 45 min at room temperature, the particles were washed once in PBS, 5 mM NaN ₃ and once in a buffer containing 20 mM Tris-Hcl, 137 mM NaCl, 0.1% (v / v) Tween 20, pH 7.6, before elution with 1% SDS / 2% 2-mercaptoethanol (90-100 ° C, 5 min). These eluates were separated using SDS-PAGE, subjected to electroblotting on nitrocellulose and the bands corresponding to C3 were detected by probing sheep immunoglobulin purified with affinity chromatography to human C3, and then horseradish peroxidase crosslinked with a donkey anti-immunoglobulin and determination using chemiluminescence enhancing substrates "Enhanced Chemiluminescence", supplied by Amersham. A photograph taken at 2 min exposure of the film in x-rays is shown. Visible bands corresponding to C3 are indicated by the highlighted arrows, and individual samples (1-4, AD) are those that have just been described. (The protruding band corresponding to 50 kDa (between the bands corresponding to 46 and 68 kDa) present in all samples is the IgG heavy chain, which was used for immunoprecipitation, and it is detected by horseradish peroxidase crosslinked with a donkey anti-mutant immunoglobulin).

Results (see FIG. 3)

1. All untreated samples (1-A, 2-A, 3-A, 4-A) contain bands of proper migration for C3 alpha and beta chains, which indicates that all mutants are expressed, and correctly post-translationally are being processed. The presence of bands corresponding to 43 or 46 kDa in these samples indicates the presence of some H-factor + I-factor-like activity in the culture medium. The spontaneous hydrolysis of C3 over a 3-day biosynthesis period leads to the production of C3i, which is cleaved by this activity. In non-mutant C3 this leads to the formation of bands corresponding to 43 and 75 kDa (the 75 kDa band is not visible because: (I) it is hidden by a beta chain having a mass of 75 kDa, and (II) the antibody used for Western blotting has a very insignificant activity in relation to this region of the C3 alpha chain: - its presence is further confirmed by re-probing with rat monoclonal antibody, "Clone-3" specific to this region). The addition of factors H and I without CVFBb (1-C, 2-C, 3-C, 4-C) did not split the remaining part of C3, which indicates that it is an active C3 (with an intact thiol ester bond).

2. Non-mutant C3 (1) is cleaved by CVFBb and the C3b product is further cleaved by endogenous enzymes (1-B) or by the addition of factors H and I (1-D). The band corresponding to 43 kDa indicates Arg ¹³²⁰ cleavage, and the band corresponding to 68 kDa (which appears with a longer exposure) indicates Arg ¹³⁰³ cleavage.

3. The mutant C3M-I23 (Arg ¹³⁰³ → Gln) can be cleaved using CVFBb, and this product turned out to be relatively resistant to endogenous activity similar to the activity of factors H and I (2-B), and a certain part of the alpha'-chain (C3b) exhibits stability, but can still be split by adding an excess of factors H and I (2-D). The presence of a product with a molecular weight of 43 kDa indicates Arg ¹³²⁰ cleavage (the weak band corresponding to 71 kDa represents another fragment of the alpha chain that can be seen with a longer exposure), but there was no 68 kDa band, which indicates that this mutant is resistant to cleavage of the mutant Gln ¹³⁰³ .

4. The C3M-26 mutant (Arg ¹³⁰³ → Gln, Arg ¹³²⁰ → Gln) can be cleaved using CVFBb, and the C3b-like product (alpha ') was resistant to endogenous activity similar to the activity of factors H and I (3-B). It was also very resistant to additional factors H and I (3-D) compared to non-mutant C3 (1) and other mutants (2 and 4). A small amount of 46 kDa product was detected, which indicates some cleavage of the mutant Gln ¹³⁰³ (a concomitant fragment of 68 kDa also became visible after a longer exposure). Very little or no product was found with a molecular weight of 43 kDa corresponding to any Gln ¹³²⁰ cleavage.

Thus, the Arg - Gln mutation at position 1303 is less effective than the mutation at position 1320 in preventing I-factor cleavage. (This slow residual cleavage may also occur in the C3M-I23 mutant (Arg ¹³⁰³ → Gln), but the 46 kDa intermediate is likely to rapidly convert to 43 kDa as a result of further cleavage of the non-mutant Arg ¹³²⁰ ).

5. The C3M-51 mutant (Arg ¹³²⁰ → Gln) can be cleaved using CVFBb, and this product was cleaved with endogenous activity similar to the activity of factors H and I (4-B) and additional factors H and I (4-D). The presence of a product of 46 kDa (and a weak band corresponding to 68 kDa) indicate the cleavage of Arg ¹³⁰³ . However, the absence of a 46 kDa band indicates that no splitting of mutant Gln ¹³²⁰ occurred.

Example 5: Comparison of various amino acid substitutions at position 1303

1. Introduction

In the previous examples, mutations Arg ¹³⁰³ and Arg ^{1320 are} described, resulting in the production of a glutamine residue. Both mutations conferred resistance to I-factor cleavage at these positions. However, a small but detectable degree of Gln ¹³⁰³ cleavage was detected. Therefore, a number of other amino acid substitutions at this position were created and evaluated. Cleavage occurs in decreasing order of efficiency when the residue at position 1303 is: Arg>Tyr> [Cys or Trp]>Gln> [Glu or Gly]. These results are unexpected because: (I) all known naturally occurring cleavages mediated by the human I-factor occur at the carboxyl end of arginine residues, so it has been suggested that the enzyme needs arginine; and (II) if there is a splitting into other residues, then we can assume that they should be similar to Arg in electrostatic characteristics, i.e. represent an alkaline residue (Lys or His) (for example, trypsin selectively cleaves the carboxyl end of Arg, Lys or His), so the possibility of cleavage by tyrosine substitution cannot be predicted.

Therefore, the replacement of Arg ¹³⁰³ with glycine or glutamic acid is preferable to create a C3 derivative resistant to I-factor inactivation.

2. Methods

2.1 Mutagenesis: the mutagenic degenerate primer used was: caactgcccagc (gt) (ag) (cg) agctccaagatcacc (letters in parentheses indicate a mixture of bases in this position). Mutants were constructed either using the gap-plasmid method (as described previously in the examples) or using the “megaprimer method” (V. Picard et al., Nucl. Asid Res. 22: 2587-91 (1994)), in which the synthesis versus transcription primer was caccaggaactgaatctagatgtgtccctc, and the synthesis primer was gttttatggtgaccttaaggtcgaatttatta. All mutations were performed on matrices in which C3-encoding DNA already contained mutations in which the amino acid residue at position 1320 was glutamine, the AflII restriction site was inserted at position 4149 (as described previously in the examples), and that was confirmed by DNA sequencing.

2.2 Expression: mutants were expressed in COS cells using the pcDNA3 vector, as previously described in the examples, was biosynthetically labeled with [ ³⁵ S] methionine in serum-free medium.

2.3 Analysis: supernatants were treated with CVFBb (obtained by reacting CVF with factors B and D in a buffer containing magnesium) and factors H and I, followed by immunoprecipitation with an anti-C3 antibody and separation by SDS polyacrylamide gel, which was carried out under reducing conditions (as described earlier in the examples). The gel was fixed, treated with Amersham Amplify reagent, dried and exposed to an autoradiographic film, obtaining the result shown in the drawing.

3. Results

The cleavage mediated by the I-factor at position 1303 (site 1), in the absence of cleavage at position 1320 (site 2) (where glutamine was replaced), results in bands corresponding to 46 and 68 kDa. It can be seen that the splitting occurs in the following order: Arg (R)> Tyt (Y)> Cys (C) and Trp (W)> Gln (Q)> Gly (G) and Glu (E). The wild-type protein (arginine in both positions) is cleaved in both positions to obtain fragments with a molecular weight of 43 (which are too small to appear on this gel) and 68 kDa.

4. Drawing

The results are shown in figure 4. Residues on site 1 (position 1303) and site 2 (position 1320) are indicated above the respective tracks.

Example 6: Demonstration of Increased Resistance to Inactivation by Factors I and H as a result of Arg ¹³⁰³ → Gln Mutation

1. Introduction

The previous examples show that replacing Arg ¹³⁰³ or Arg ^{1320 with} glutamine makes this site resistant to I-factor cleavage. Mutation of both sites results in a molecule resistant to cleavage at both sites. In the present example, it was further demonstrated that a single mutation Arg ¹³⁰³ → Gln (without modification of Arg ¹³²⁰ ) leads to significant resistance to functional inactivation by factors I and H compared with the wild type.

2. Method

2.1 Expression: Obtaining the mutation Arg ¹³⁰³ → Gln described in the previous example. CHO (a standard laboratory cell line derived from Chinese hamster ovary cells) was transfected with this mutant using a calcium phosphate method and stable transfectants were selected for G418 resistance ("Geneticin" supplied by Sigma). Cell culture supernatants were collected and expressed C3 was partially purified by precipitation with sodium sulfate (10-20% (w / v) fraction) and ion exchange chromatography on Q-sepharose and mono-Q-sepharose (AW Dodds Methods Enzymol. 223: 46 (1993)).

2.2 Analysis: Sheep erythrocytes were coated (sensitized) with SO16 monoclonal antibody (RA Harrison and PJ Lachmann Handbook of Experimental Immunology, 4th ed., Chapter 39 (1986)) and then 4.4 ml of 5% (v / v) suspension was incubated with approximately 10 μg C2, 24 μg C4 and 1 μg C1 (purified human complement components) for 10 min at 37 ° C in CFD (RAHarrison and PJLachmann, supra). Then, 0.8 ml of this mixture was incubated for 105 min with 0.25 ml of the mixture containing semi-purified C3 mutant or wild type and EDTA to a final concentration of 12.5 mM. The cells were then washed with CFD and used to incorporate into gelatin containing CFD (0.1% w / v) (CFD gel). The radioligand binding to [ ¹²⁵ I] labeled clone 4 of the anti-C3 monoclonal antibody was used to confirm that the corresponding amounts of wild or mutant type C3b were precipitated.

For analysis, 40 μl of a 5% cell suspension was diluted in 250 μl of CFD gel and 50 μl aliquots were incubated with 50 μl of CFD gel containing dilutions of each factor I and H to final concentrations of 100, 10, 1 and 0 μg / ml at 37 ° C for 30 minutes Then 0.9 ml CFD was added, the cells were pelleted by centrifugation and washed two more times, using 1 ml CFD each time. Cells were then resuspended in 100 μl CFD gel containing 100 μg / ml B-factor, 100 μg / ml properdine, 1 μg / ml D-factor and 0.3 mM NiCl ₂ . After incubation for 10 min at 37 ° C, 0.9 ml of CFD containing 10 mM EDTA and 2% (v / v) normal guinea pig serum was added. After an additional incubation for 30 min at 37 ° C, the cells not destroyed by lysis were pelletized by centrifugation and the degree of lysis was determined by measuring the absorbance of the supernatant at 412 nm. Absorption equivalent to 100% lysis was determined from aliquots of cells lysed in water, and the percentage of lysis was calculated from this.

This analysis allows one to evaluate the ability of precipitated C3b to form a functionally active convertase C3bBbP. Conversion to iC3b prevents convertase formation and subsequent serum / EDTA lysis.

3. Results

The results shown in the drawing indicate that to eliminate the hemolytic activity of the Arg ¹³⁰³ → Gln mutant, more than 10-fold amounts of I-factor and H-factor are required, compared with the wild type. This mutation, therefore, gives an advantage when creating a derivative of C3, the C3b product of which is resistant to inactivation by factors H and I. This effect can be a result of either greater resistance to cleavage at position 1303 (when Arg is replaced by Gln), or greater stability to cleavage at position 1320, when cleavage first occurs at position 1303.

4. Drawing

The results are shown in figure 5. The concentration of factors H and I is plotted on the X axis. Q1 denotes the Arg ¹³⁰³ → Gln mutation. Lysis (%) is measured as described in the methods.

Conclusion

Important features of human C3 inherent in the modified variants presented in the present description are the following:

(I) The molecule contains a derivative that is functionally similar to C3b, which can be combined with a functionally active human B-factor, and can also be cleaved by the human D-factor to produce an enzyme capable of cleaving human C3.

(II) The amino acid sequences of the derivatives have greater homology with C3 of humans than with C3 of any other species for which sequence data are currently available, or with any other protein sequence currently known. Structural features of C3 characteristic of a wild-type protein, but optional for modified derivatives, include the following features:

(a) The coding DNA sequence and the translated protein sequence for the human C3 variant used in the examples of the invention described in the present description are shown in FIGS. 2 and 1, respectively. This protein sequence differs from the published sequence [2] by at least two amino acids (details are given in the examples). It can be assumed that significantly more variations are compatible with C3 functions, even if most of them are not present in the population.

(b) The primary translation product is processed by proteolytic cleavage into two chains connected by disulfide bridges, the alpha chain (residues 672-1663) and the beta chain (residues 23-667), with the removal of the signal sequence (residues 1-22).

(c) The mature protein contains a thiol ester linkage between Cys1010 and Gln1013 residues.

(d) C3 convertase cleaves C3 to remove C3a (residues 672-748). After this reaction, a rupture of the thiol ester bond occurs.

(e) In the presence of the H factor, the I factor cleaves C3b between Argl303 and Ser 1304 residues and between Argl320 and Ser 1321.

Modifications created on the native C3 molecule

Replacing Arg ¹³⁰³ with Gln

This modification affects one C3b I-factor cleavage site. Its action is aimed at reducing the level of I-factor cleavage in this position. The substitution for glutamine was chosen in order to eliminate the positive charge of arginine, which is probably important for the serine-protease activity of the I-factor, but the hydrophilicity characteristic and close chain size are preserved, which should minimize any disturbances in the tertiary structure of the protein. This assumption is supported by evidence that the mutation did not interfere with the processing of the double-stranded structure, the formation of a thiol ester bond, or the cleavage of C3 by C3 convertase. Replacing Arg with another amino acid can give a similar or even more pronounced effect, as shown in example 5.

It also seems possible to reduce this cleavage by substituting Ser ¹³⁰⁴ (the other side of the cleavage site) or other residues involved in the enzyme-substrate interaction.

Replacing Arg ¹³²⁰ with Gln

This modification affects another C3b I-factor cleavage site. Its action is aimed at a significant reduction in the level (actually elimination) of I-factor cleavage in this position. The substitution for glutamine was made in accordance with the same criteria as described above, and this mutation also did not interfere with the processing of the double-stranded structure, the formation of a thiol ester bond, or the cleavage of C3 by C3 convertase. And in this case, substitution with another amino acid, as well as substitution of Ser ¹³²¹ or other residues involved in the enzyme-substrate interaction, can give a similar effect.

The combination of two mutations, Arg ¹³⁰³ → Gln and Arg ¹³²⁰ → Gln, protects C3b from inactivation and, therefore, supports its ability to form part of the active C3bBb convertase. Other mutations (including combinations of mutations) can be used that eliminate both cleavage reactions (for example, Arg ¹³⁰³ → Glu or Arg ¹³⁰³ → Gly can be used in combination with Arg ¹³²⁰ → Gln).

Example 7: Various mutations that reduce the interaction of C3b / C3i with the H-factor

7.1 Introduction

In other laboratories, data were obtained based either on the actions of synthetic peptides (Ganu VS and Muller-Eberhard HJ, 1985, Complement 2:27; Becherer JD et al., 1992, Biochemistry 31: 1787-1794), or on limited mutagenesis (Taniguchi Sidle A. and Isenman DE, 1994 J. Immunol. 153: 5285-5302), suggesting that residues 752-761 in the primary sequence of the C3 transcript (see FIG. 1) may be involved in interaction with the H-factor. However, other published data suggest that only residues 767-776 are involved in interaction with the H-factor, and residues 752-761 are important for interaction with the B-factor (Fishelson, 1991, Mol. Immunol. 28: 545-552). The inventors have suggested that a more intense mutagenesis of this region can reduce the affinity for the H-factor and is therefore desirable for the purpose of creating a derivative of C3 resistant to the H-factor. In addition, it was hypothesized that prominent acid residues (aspartic and glutamic acids) may be important for mutation residues and that it may be appropriate to replace them with neutral residues that are able to mediate strong interactions with less probability. This example describes the replacement of residues 752-754 of Asp-Glu-Asp with Gly-Ser-Gly in combination with the replacement of residues 758-760 of Glu-Glu-Asn with Gly-Ser-Gly. The product has reduced cleavage characteristics, which are consistent with a decrease in sensitivity to the H factor. This confirms that C3 can be modified in order to reduce the binding of the H-factor and, therefore, reduce the sensitivity to factors H and I. These modifications are desirable to obtain a C-convertase that is stable under physiological conditions.

7.2 Method

Methods of mutagenesis, expression and analysis are described previously in the examples. The synthesized mutagenic oligonucleotide had the sequence:

agtaacctgggttcgggcatcattgcaggatcgggcatcgtttcc.

7.3. results

The results of the cleavage reactions are shown in Fig.6. They show that:

1. Adding CVFBb to wild-type C3 leads to the elimination of the alpha chain (lane 2), since the resulting C3b is sensitive to low concentrations of factors I and H in the culture supernatant. C3i formed during expression or subsequent incubation was similarly decomposed to give iC3i. The addition of exogenous factors I and H (lanes 3 and 4) does not change relative to lanes 1 and 2, respectively, since the medium itself contains sufficient activity of factor H and I to effect complete cleavage.

2. In contrast, treatment of the C3 mutant with CVFBb (lane 6) does not lead to the disappearance of the alpha chain. The formation of a certain amount of alpha ’is observed, which corresponds to C3b, but some or all of them are preserved, which indicates that the stability of the alpha chain is not only the result of the lack of CVFBb cleavage. The remaining undigested alpha chain in lane 2, therefore, may be C3i not cleaved by the endogenous activities of factors H and I, although it is also likely that some of the product is native C3, preserved if the mutant acquires partial resistance to CVFBb. The addition of high concentrations of exogenous factors H and I (lanes 7 and 8) leads to a decrease in the alpha and alpha'-chains, which indicates that (I) the mutant is not completely resistant to these factors and (II) the alpha chain, unsplit CVFBb (lane 2) is more likely to come from C3i (which is cleaved by factors H and I, but not cleaved by CVFBb), and not from native C3 (which is cleaved by CVFBb, not by factors H and I). The fact that not all of the alpha chain is cleaved, even on lane 8, is probably the result of resistance to factors H and I.

Thus, a change in residues 752-754 and residues 758-760 can lead to the formation of a C3 molecule, which can still be cleaved by C3 convertases, but is partially resistant to the effects of factors H and I. Given other published data, this seems most likely, therefore that mutations modified the region involved in the interaction with the H-factor, which led to a decrease in affinity for the H-factor.

Example 8: C3 site in which a mutation can be induced to modify the interaction of C3i with the B factor

8.1 Introduction

In the previous examples, it was shown that C3 mutations can change interactions with factors H and I. To identify other sites in C3 that can interact with the B factor, a comparison was made with known sequences of C3 molecules from various species, as well as with available C4 and other homologous proteins. The region corresponding to residues 1427-1433 of human C3 has been identified, which can participate in the specific functions of C3 and C4. These may include interaction with a B-factor (or its homologue C2 in case of C4), but not necessarily, since other possible functions include the formation of a thiol ester bond, conversion to C3b (or into C4b form), interaction with a substrate C3 and / or C5 with convertase activity and interaction with the I-factor and with its cofactors. Thus, the selected residues were replaced with the corresponding residues (based on the analysis of the primary structure of the sequence) found in another homologous protein, in this case, human C5. So, at position 1427, the Arg residue was replaced with Gln, at position 1431, the Lys residue was replaced with Asp, and at position 1433, the Glu residue was replaced with Gln. It was found that the resulting mutant is sensitive to cleavage by C3 convertase (CVFBb) and the C3b product can be cleaved by factors H and I. However, this mutant does not support the conversion of the B factor into Bb plus Ba, due to the binding of the B factor to C3i (or to C3b ) Thus, confirmation has been obtained that a change in this region reduces the interaction with the B factor. Although this is undesirable for the preparation of a superactive C3 convertase, this fact indicates that other modifications of the indicated C3 region can also change the interaction with the B factor and some of them are likely to increase affinity. As a consequence, such mutations can also increase the stability and activity of the bimolecular C3bBb (or C3iBb) convertase enzyme.

8.2 Methods

The primary sequence structures shown in Table I below (at the end of the text) illustrate the reason why the inventors believe that this area is promising for mutagenesis. It is assumed that the nature of certain residues in this region was strongly conserved in C3 and C4, but was very different in other proteins. Residues at positions 1427, 1431, and 1433 were selected, since the nature of their charge is characteristic of groups that can participate in protein-protein interactions. Replacements were made for the corresponding residues of human C5, because they showed very pronounced differences in electrostatic properties, but within the context of some other conservative residues, which indicate the similarity of their local structure.

Methods of mutagenesis, expression and analysis of C3 cleavage reactions correspond to those described in the examples presented earlier (examples 1-4). The synthesized mutagenic oligonucleotide had the sequence: tggtgttgaccaatacatctccgactatcagctggacaa.

B Factor Conversion Analysis

The expressed product was purified from the medium of COS cells by affinity chromatography on a Clone-3-Sepharose type column as described in Example 9. This method results in a significant conversion of the C3i form with a cleaved thiol ester bond. Wild-type C3 was isolated using the same technique. Wild type C3 dilutions (1/5, 1/25, and 1/125) were dispersed by SDS page stripping (reducing conditions) in parallel with the C3 mutant, and silver staining showed that the mutant was present at a concentration equivalent to that corresponding to the dilution wild-type is slightly lower than 1/25, but significantly higher than 1/125. The same dilutions were used in the analysis of the transformation of the B-factor. 5 μl of these C3 were incubated with 25 μl of CFD-G containing 5 μg / ml D-factor and approximately 1.6 μg / ml labeled with ¹²⁵ I B-factor (approximately 1000-2000 decays per min / μl) for 3 h at 37 ° C. Then the samples were analyzed using SDS-PAGE (reducing conditions) followed by autoradiography of the dried gel. The results are shown in Fig.7.

8.3 Results

As can be seen in Fig.7, a pronounced cleavage of the B-factor occurs even when diluting wild-type C3 (C3i) 1/125. In contrast, no noticeable cleavage was observed in the presence of mutant, even undiluted C3, which should correspond to a higher concentration than a dilution of 1/125 wild-type sample.

Thus, this mutant probably has a reduced ability to cleave the B-factor, most likely due to a decrease in its affinity for B-factor binding. Therefore, in C3 there is a region in which the mutation is induced, in which the interaction between the C3i (or C3b) and the B-factor changes, and possibly also the stability of the convertase (C3iBb or C3bBb).

Example 9: Purification of Expressed C3 Mutant Molecules

9.1 Introduction

This example demonstrates how mutant C3 molecules can be isolated from expression media, such as culture medium, of transfected eukaryotic cells. Using simple affinity purification, C3 molecules of sufficient purity are obtained for use in function tests and for conjugation with the antibody using the method described in Example 10. Although elution from the antibody is accompanied by hydrolysis of a significant portion of the internal thioether, the C3i product still remains suitable as a precursor to obtain active C3 convertase, as well as to obtain conjugates of C3i antibody. This approach can probably be considered as part of the method of obtaining the drug necessary for in vivo use.

9.2 Method

Purification by affinity chromatography on a Clone-3-Sepharose-type column.

Clone-3 is a rat monoclonal antibody that is specific for C3 and its derivatives, including C3b and C3i (Lachmann PJ et al., 1980, J. Immunol. 41: 503-515). Other monoclonal antibodies to C3 are also available, and in some cases they have been successfully used to isolate C3 from small amounts of human plasma (Dodds AW, 1993, Methods Enzymol. 223: 46-61) and, therefore, can also be used to isolate molecules expressed ex vivo. The IgG fraction was crosslinked with CL-4B sepharose using dicyanbromide (procedure described by Harrison and Lachmann, 1986, Handbook of Experimental Immunology, 4th ed., Edited by Weir, Herzenberg, Blackwell and Herzenberg; Blackwell, Oxford). Culture supernatants were either directly passed through a column of this resin (re-circulation) or first concentrated by precipitation with 25% (w / v) Na ₂ SO ₄ and re-dissolved and dialyzed in countercurrent PBS, 5 mM NaN ³ . Then, the column was washed successively with (I) PBS, 5 mM NaN ₃ and (II) PBS containing 1 M NaCl. Bound C3 was eluted with 50 mM sodium borate buffer, pH 10.5, and it was immediately neutralized by collecting 0.9 ml fractions in 0.1 ml of 1 M Tris / Hcl buffer, pH 7. The product was then dialyzed in countercurrent PBS, 5 mM NaN ₃ .

Obtaining C3 Bearing His-Tag

"His-Tag" (tagged histidine) is a filament of histitide residues exhibiting affinity for columns carrying nickel ions. This method was used to isolate the expressed product. It is assumed that this method may be suitable for the isolation of expressed mutant C3 molecules, therefore, the inventors used insertion mutagenesis to obtain a plasmid encoding C3 with a tail consisting of 6 histidine residues at the carboxyl end (C-terminus immediately adjacent to residue 1663) . Such localization for His-Tag was chosen in order to minimize interference with the synthesis, folding, processing, and disulfide bond formation of the resulting C3. Residue 1661 is a cysteine residue involved in the formation of a disulfide bond with the previous residue of the sequence (probably Cys 1537; K. Dolmer and Sottrup-Jensen L., 1993, FEBS-Lett. 315: 85-90) and therefore seems appropriate make an insert behind this structural element. The mutation was introduced using the "gap-plasmid" method, which was used in example 1, using the synthesized mutagenic oligonucleotide with the following sequence: tgggtgccccaaccatcatcatcatcatcattgaccacaccccc.

The insertion of the correct sequence was confirmed by DNA sequencing. The DNA sequence could then be transferred to an expression vector. After transfection of eukaryotic cells, it is possible to isolate expressed C3 as a result of affinity for a column carrying nickel ions, or using any other matrix having a specific affinity for His-Tag.

9.3 Results

Numerous C3 mutants were purified on a Clone-3-Sepharose column, including the mutants described in Examples 1 and 2 and expressed in CHO cells. Products retained the ability to maintain B factor cleavage by the D factor. The same method was used to isolate the mutant described in Example B2 expressed in COS cells. Silver stained SDS-PAGE gels revealed that the purity of the isolated products was not 100%, but often was higher or equal to 50%. This is due to starting materials typically containing less than 10 μg / ml C3 in 10% (v / v) fetal calf serum plus other cellular proteins. In addition, C3 did not decompose during the isolation process and the activity of endogenous factors H and I was probably eliminated.

Purification by His-Tag includes milder elution conditions from a column containing nickel ions. For example, EDTA was used. Thus, the application of this method for C3 should ensure the isolation without breaking the internal thiol ester bond.

Example 10: Conjugation of C3i with an Antibody and Use to Ensure C3 Convertase Activity Against a Specific Cell

10.1 Introduction

One of the objects of the invention is the fact that stable C3 convertases derived from mutant C3 molecules should induce enhanced conversion of C3, which, if localized at a particular target site, will enhance the complement-dependent attack on that target. A preferred method for directing the response is to bind a mutant C3 molecule, either as a C3i derivative or as a C3b derivative, with an antibody specific for the desired target. This example demonstrates a working procedure for the preparation of such conjugates, which is applicable for C3i or C3b mutant molecules and can be used for material purified from an affinity based expression system, even if the C3 thiol ester was destroyed during the cleaning process. By binding C3i to an antibody that specifically binds to sheep erythrocytes, it is further shown that a conjugate can be formed that binds C3i to the surface of the red blood cell, such as C3iBbP convertase, which initiates lysis of these cells when other complement components are delivered in the form of normal marine serum mumps (in EDTA to prevent the formation of de-novo C3 convertases). Thus, conjugation with an antibody can be used to direct the C3i molecule for the purpose of initiation, depending on the complement of the attack on a particular type of cell. In this example, wild-type C3i is used from human plasma, which forms C3 convertase in vitro. In vivo, C3i and C3b are destroyed by factors H and I. Thus, a C3 mutant constructed in accordance with the present invention so that it is resistant to factors H and I and, therefore, is able to form stable C3 convertase, is preferred with physiological point of view.

10.2 Methods

(I) Preparation and purification of a C3i antibody conjugate

The antibody used was an IgG fraction isolated from polyclonal rabbit anti-mutant erythrocyte serum. 1.1 mg was incubated with 75 nmoles of SPDP in conjugation buffer, pH 7.5 (20 mM KH ₂ PO ₄ , 60 mM Na ₂ H ₄ O, 0.12 M NaCl) for 2 hours at room temperature. PDP-IgG was purified by gel filtration on a Superose B column (Pharmacia) (in phosphate buffer, pH 7.4, containing 0.5 M NaCI). The reduction of the sample with dithiothreitol was used to establish that 4 PDP groups are crosslinked with the IgG molecule. C3i was obtained by treating purified C3 with 0.1 M methylamine, pH 7.2 (2 hours at 37 ° C). Excess methylamine was removed by gel filtration, followed by dialysis in countercurrent conjugation buffer. 18 nmoles of C3i were mixed with 1.7 mmol of PDP-IgG in 1.26 ml of conjugation buffer and incubated for 1 day at room temperature and then 1.5 days at 4 ° C. Fig. 8 shows Coomassie blue stained (Coomassie Blue) SDS-PAGE gel corresponding to the conjugated reaction mixture, which shows the appearance of fractions with a molecular weight of approximately 350 kDa, which were not present either in PDP-IgG or C3i. These species were partially purified by gel filtration on a superose 6 column in phosphate buffer, pH 7.4, containing 0.5 M NaCl, and then dialyzed in countercurrent PBS. They eluted to C3 in a volume in which fractions with a molecular weight of 300-400 kDa can be determined using calibration based on molecular weight standards of globular molecules. Concentrations of conjugate, free antibody, and uncrosslinked C3 were determined from SDS-PAGE gel stained with Coomassie (non-reducing conditions). Two-dimensional SDS-PAGE (the first direction is non-reducing, the second direction is reducing) showed that the ratio between IgG and C3i in the conjugate is 1: 1.

(II) Demonstrating that a C3 Antibody Conjugate Can Be Used to Ensure Convertase Activity Against a Specific Cell

20 μl of various dilutions of the conjugate (0 (no conjugate), 1/100, 1/50, 1/10) were incubated with 100 μl of approximately 1% (v / v) sheep erythrocytes (pre-washed in CFD) for 1 h at 37 ° C. In parallel, incubations were carried out with equivalent amounts of PDP-IgG (without C3) and one C3. The cells were then washed four times in CFD and resuspended in 100 μl CFD-G. 50 μl of this mixture was lysed with 150 μl of H ₂ O, and then by adding 800 μl of CFD containing 10 mM EDTA and 2% (v / v) NGPS. Another 50 μl of conjugate-sensitized cells were incubated for 15 min at 37 ° C with 50 μl of CFD-G containing 190 μg / ml B-factor, 2 μg / ml D-factor, 20 μg / ml properdine and 0.6 mM NiCl ₂ , and then lysed with 900 μl CFD containing 10 mM EDTA and 2% (v / v) NGPS. After incubation for 30 min at 37 ° C, the cells were pelleted by centrifugation (2000 × g for approximately 3 min) and the optical absorbance of the supernatant was measured at 412 nm. Assuming that 100% lysis occurs in the water-treated samples and the clear buffer is free of cells,% lysis is calculated as shown in Fig. 9. The conjugate induces dose-dependent lysis, while neither PDP-IgG nor any C3i result in any lysis significantly exceeding that which occurs in the absence of any such treatment.

10.3 Summary of results

The method used allowed the successful crosslinking of C3i with IgG, as can be seen from the following:

1. The formation of a band of an appropriate size (approximately 350 kDa) for a conjugate with a C3: IgG ratio of 1: 1 is shown by SDS-PAGE in Fig. 8.

2. Two-dimensional SDS-PAGE (the first direction is non-reducing, the second direction is reducing) showed that these species contained both IgG and C3i.

3. The characteristic of elution of these species using gel filtration, in this case, corresponds to a molecular weight of approximately 350 kDa.

4. The conjugate exhibits hemolytic activity, which is not characteristic of either PDP-IgG or C3i (Fig. 9).

Analysis of hemolytic activity (Fig.9) additionally shows that:

1) a specific antibody to sheep erythrocytes localized C3i on the membrane of the target cell (sheep erythrocyte), protecting it from removal during washing (as opposed to free C3i),

2) the conjugate retains the activity of C3i in the sense that it is still able to form C3 convertase by reaction with properdine and factors B and D,

3) this convertase can initiate a complement-dependent attack on the target, in this case, by activating the lysis pathway (C5-9) to erythrocyte lysis.

Additional data from other laboratories have shown that cobra venom factor can be associated with an antibody, and that these conjugates can direct complement activation against a specific cell type (Vogel, 1988, Targeted. Diagn. Ther. 1: 191-224; Muller B. and Muller-Ruchholtz W., 1987, Leuk. Res. 11: 461-468; Parker CJ, White VF and Falk RJ, 1986, Complement 3: 223-235; Petrella EC et al., 1987, J. Immunol Methds 104: 159-172). These data confirm the claim that C3, modified in such a way that it is capable of forming a stable C3 convertase, such as cobra venom factor, can be used to direct complement-mediated responses as described in this invention.

Example 11: Demonstration that mutant C3 molecules induce B-factor conversion in normal human serum

11.1 Introduction

The main objective of the present description of the invention is the destructive depletion of complement activity in biological fluids. The invention describes methods for producing C3 molecules that are resistant to downregulation by factors H and I. In this state, they will bind to the B factor and generate active C3 convertases. The activity of these convertases is demonstrated in the hemolytic analysis shown in Example 6. Such a convertase can therefore destroy C3. If the convertase is unstable, then it can dissociate without leading to a significant conversion of C3. However, this may allow the binding of a new B-factor and its conversion into Bb and Ba. Thus, mutant C3 will enhance the consumption of the B-factor, ultimately leading to the failure of the alternative pathway for complement activation and its inability to enhance the stimulation of the classical pathway. If stable C3 convertase is obtained, B-factor conversion should decrease, but C3 consumption should increase. Thus, both of these situations are desirable. This example shows that mutant C3 molecules, which are modified to give them resistance to the I-factor, but do not have any modification that changes the stability of convertase, provide accelerated conversion of B-factor in human serum. In contrast, wild-type C3 does not cause a noticeable conversion, probably due to the fact that wild-type C3i is rapidly degraded by factors H and I.

11.2 Method

The resulting mutants are as follows:

Q1R2 Arg ¹³⁰³ replaced by Gln (example 2),

Q1Q2 Arg ¹³⁰³ replaced by Gln plus Arg ¹³²⁰ replaced by Gln (example 1),

E1Q2 Arg ¹³⁰³ replaced by Glu plus Arg ¹³²⁰ replaced by Gln (Example 5).

All of these mutants were obtained in CHO cells and then purified by precipitation with Na ₂ SO ₄ , followed by affinity purification on Clone-3-Sepharose, as described in Example BZ. Wild-type C3 (R1R2) was similarly isolated. According to SDS-PAGE data for silver staining, the concentration of Q1R2 was from 1/5 to 1/25 compared to the wild type, the concentration of Q1Q2 was about 1/5 of the wild type, and the concentration of E1Q2 was from 1/25 to 1/125 of the wild type. All preparations probably contained mostly molecules with a broken thiol ester bond (C3i).

10 μl of these C3 preparations were incubated for 1 h at 37 ° C with 10 μl of a solution of 20% (v / v) normal human serum in PBS containing 1 mM MgCl ₂ and approximately 300 ng labeled with ¹²⁵ I B-factor ; approximately 2-300000 decays per minute). Then 5 μl was analyzed using SDS-PAGE (reducing conditions). The dried gel was exposed to an autoradiographic film in order to identify the positions of the bands corresponding to the intact B factor and its cleavage products. Then the bands were cut out and radioactivity was calculated in order to accurately determine the degree of cleavage. The value obtained using one buffer was subtracted as the background value (which includes not only background cleavage, but also the decomposition of products and other contaminants present in the radioligand preparation).

11.3 Results

The obtained values of the splitting of the B-factor,%:

1/25 wild-type 1.49

1/5 wild-type 2.74

Q1R2 6.19

Q1Q2 7.41

E1Q2 6.42

Therefore, all I-factor resistant mutants cause higher levels of B-factor cleavage than equivalent amounts of wild-type C3 (C3i). Elevated doses or longer incubations can lead to complete failure of the alternative complement activation pathway.

The abbreviations used in the above examples have the following meanings:

CFD stands for complement fixative diluent (as determined by Harrison and Lachmann Handbook of Experimental Immunology, 4th ed., Edited by Weir, Herzenberg, Blackwell and Herzenberg; Blackwell, Oxford);

CFD-G — CFD containing 0.1% (w / v) gelatin; PBS means phosphate buffered saline; NGPS stands for normal guinea pig serum; SDS-PAGE refers to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; SPDP is N-succinimidyl-3- [2-pyridyldithio] propionate.

Example 12: Mutations in residues 992-1000

1. Introduction

In other laboratories, data based on the actions of synthetic peptides have been obtained (Ganu VS and Muller-Eberhard HJ, 1985, Complement 2:27; Becherer JD et al., 1992, Biochemistry 31: 1787-1794; Fishelson Z., 1991, Molecular Immunology 28: 545-552; Lambris JD, Ganu VS, Hirani S. and Muller-Eberhard HJ. 1988, J. Biol. Chem. 263: 12147-12150) suggesting that various residues in human C3b may be involved in interaction with H factor. Applicants have used a different sequence comparison approach to theoretically predict residues involved in C3-specific functions. A site-directed mutagenesis was performed, which showed that most of these candidates have very little or no effect on functional sensitivity to the H factor. However, several mutations did decrease the sensitivity to the H factor. These mutations were created in parts of the molecule for which participation in the relationship with the H-factor or in the change in its binding was not previously detected. Therefore, a change in these specific residues could be used to obtain mutant C3 derivatives that are partially or fully resistant to inhibition by the H-factor in the physiological environment and which can form complex C3-convertase enzymes (C3bBb, etc.), which are also resistant to H inactivation -factor.

Factor H is structurally homologous to other complement inhibitory proteins, including CR1, MCP, and DAF. From the point of view of this obvious evolutionary relationship and mutual competition for binding, it is likely that the structure of the interaction with C3b is similar to the structure of the interaction with the H factor (Fairies et al., 1990, Complement Inflamm. 7: 30-41). Therefore, the described mutations that change the sensitivity to the H-factor will also probably be suitable for changing interactions with these other proteins, especially so that the activity of their complements is not subjected to down regulation. They can also be used to modify interactions with the SCR domains of the B-factor (and with homologous domains in C2 involved in binding to C4b). Mutations in the corresponding regions of C4 and C5 may also be suitable for modifying their interactions with the SCR domains of Clr and Cls (C4), the C4b binding protein (C4b) and C6 and C7 (C5b).

2. Method

2.1 Search for residues involved in C3-specific functions.

These theoretical predictions were made based on a comparative analysis of the primary structure of human C3 and all homologous proteins, the sequences of which were available from published databases. These include functionally equivalent molecules of mouse, rat, guinea pig, rabbit, cobra, spur frog, chicken and rainbow trout, human and mouse C4, human and mouse C5, C3-like river lamprey and myxin, cobra venom factor (CVF) and human alpha-2-macroglobulin and their homologues. Then a search was made for residues that were conserved for different C3 but distinctly different in homology (especially in C5 and CVF), which lack C3-specific functions of interest. Some of these residues were modified to encode residues corresponding to residues in C5 or CVF, expressed in COS cells, and secreted products were tested for cleavage in the presence of CVFBb and factors H and I. All methods correspond to standard methods and methods described in the example 1. The generalized results are shown in table II.

2.2 Design and analysis of the mutant DV-1AM

This mutant was created in the same way as other mutants using the "megaprimer method" described by V. Picard et al., 1994, Nuc. Acid Res. 22: 2587-2591. The mutagenic primer had the sequence ccagatgacaagtgctgccgtcagccagtcagggctgaagcacc coding for the mutations E992S, D993A, D996S, A997Q, E998S and R999G. The primer, which provides synthesis against the course of transcription, had the sequence tgtcatcgtgccgctaaaga (corresponding to deoxynucleotides at positions 2754-2773), and the primer, which provides synthesis along the transcription, had the sequence gttttatggtgaccttaaggtcgaatttatta (complementary to position 4149). The mutated DNA fragment was ligated with a vector that contained the C3 coding sequence also with the inserted AflII site at position 4149, by cleaving both fragments using AflII and EcoRI (sections at position 2997), purification of the desired products and their ligation using T4 DNA ligase. Plasmid DNA was isolated from transformed bacterial colonies and true mutants were detected by DNA sequencing. It was found that the DNA sequence contained an additional mutation encoding L1000M. COS cells were transfected with the resulting expression vector and the secreted expressed product was analyzed for cleavage as previously described.

3. Properties of the mutant DV-1AM

Analysis of the expressed DV-1AM mutated product is shown in FIG. 10. It should be noted that:

(I) Western blot analysis was performed using monoclonal antibodies to the C3dg region of C3, with its help, the precursor, alpha, alpha'-chain, fragments with a molecular weight of 77 and 68 kDa were detected, but no beta chain, fragments were detected with a molecular weight of 43 or 46 kDa,

(Ii) all bands corresponding to the DV-1AM product (bands B1-4) are slightly below equivalent wild-type bands C3 (bands A1-4); a shift in mobility is the result of mutations obtained,

(III) cleavage of wild-type C3 with CVFBb results in a small amount of alpha'-chain from C3b, and a much larger fragment with a molecular weight of 68 kDa resulting from cleavage of C3b to obtain iC3b due to the endogenous activity of factors H and I ( lane A3). The addition of exogenous factors H and I completes the conversion of C3b to iC3b (band A4). In contrast, cleavage of the DV-1AM product with CVFBb results in a larger alpha chain and only a small fragment with a molecular weight of 68 kDa (band B3). Subsequent addition of exogenous factors H and I allows C3b to be converted to iC3b (B4 band). Thus, mutant C3b is significantly more resistant compared to the wild type to endogenous factors H and I, although resistance is not complete, as evidenced by the sensitivity to cleavage by high concentrations of exogenously added factors H and I.

4. Conclusion

The DV-1AM mutation creates resistance to H-factor-dependent I-factor cleavage. The mechanism of this phenomenon remains unclear, although since the modified residues are located far (from the point of view of the primary structure) from the I-factor cleavage sites, the interaction with the H-factor is probably deteriorating. In this case, the mutation will also confer resistance to other inhibitory activities of the H-factor, such as: (I) competition with the B-factor for binding to C3b (or C3i and (II) accelerated dissociation of C3bBb- and C3bBb-convertases. -1AM is a modification of residues directly or indirectly involved in maintaining affinity for the H factor, and the numerous different mutations of these residues are likely to have the same effects as mutations of only some residues resulting from modification in accordance with this zobreteniem. Mutations in other balances in this segment of the primary structure can probably also lead to a similar effect.

Example 13: Mutation of residues at positions 1001-1005

1. Introduction

As described in the previous example, residues 1001, 1002, and 1005 are also identified as candidates that may be essential for C3-specific functions. Induction of mutations has confirmed that modification of these residues can be used to confer resistance to the H factor.

2. Method

2.1 Design and analysis of the mutant DV-1B

Used the same method as described in the previous example, except that the mutagenic primer had the sequence aacggctgaacatattaattcataccccctcgggc encoding the mutations K1001N, H1002I and V1005H. Sequence analysis of the isolated plasmid DNA confirmed that the mutation was introduced correctly. No other mutations were found.

3. Properties of the mutant DV-1B

The analysis of the expressed product with mutations DV-1B is shown in Fig.11. The following points should be noted.

(I) Western blot analysis was performed using a polyclonal antibody to C3, with its help the precursor, alpha, alpha'-chain, beta-chain, fragments with a molecular weight of 43 and 46 kDa were clearly identified, but fragments with molecular weight 77 or 68 kDa. It should be noted that alpha and alpha’s chains did not move (remained at the start) and were not detected with 100% efficiency, therefore the intensity of these bands is lower than expected and incorrectly reflects the actual quantities present.

(II) Cleavage of wild-type C3 with CVFBb results in a small amount of the alpha chain from C3b, but most of it is lost due to cleavage of C3b to produce iC3b due to the endogenous activity of factors H and I (lane B3). It appears mainly in the form of a fragment with a molecular weight of 43 kDa, although small amounts of an intermediate product with a molecular weight of 46 kDa are also visible. The addition of 2 μg / ml of exogenous H-factor (band B4) with the I-factor leads to significant additional cleavage, and the addition of 10-50 μg / ml of exogenous H-factor (bands B5, B6) completes the conversion of C3b to fully digested iC3b (absent bands corresponding to the alpha chain or a molecular weight of 46 kDa). The cleavage of the DV-1B product with CVFBb also results in a small amount of the alpha chain (lane A3; the total amount of C3 present is substantially less and the bands corresponding to the alpha and alpha chains are very weak). It is important that the amount of chain with a molecular weight of 43 kDa obtained in the absence of exogenous factors H and I is lower than that of the wild type, and the amount of the intermediate fragment with a molecular weight of 46 kDa is relatively higher, which indicates a less efficient cleavage. The addition of exogenous factors H and I (bands A4-6) completes the conversion of the indicated C3b to iC3b, but it can be seen that the amount of product with a molecular weight of 43 kDa increases with an increase in the concentration of the H factor to 50 μg / ml (lane A6), when corresponding to the intermediate product with a molecular weight of 46 kDa, is still noticeable. Thus, mutant C3b is more resistant than wild type to endogenous and exogenous factors H and I, although resistance is not complete, as evidenced by the sensitivity to cleavage by high concentrations of exogenously added factors H and I.

4. Conclusion

The DV-1B mutation creates resistance to H-factor-dependent I-factor cleavage. The mechanism of this phenomenon remains unclear, although since the modified residues are located far (from the point of view of the primary structure) from the I-factor cleavage sites, and the effect depended on the dose of the added H-factor, it is possible that the interaction with the H-factor worsens. In this case, the mutation will also confer resistance to other inhibitory activities of the H-factor, such as: (I) competition with the B-factor for binding to C3b (or C3i) and (II) accelerated dissociation of C3bBb and C3bBb convertases. The DV-1B mutation is a modification of residues directly or indirectly involved in maintaining affinity for the H factor. Numerous different mutations of the residues are likely to have the same effects as the mutation of only certain residues modified in accordance with the present invention. Mutations in other residues in this segment of the primary structure are likely to lead to a similar effect.

Example 14: Mutation of residues at positions 1152-1155

1. Introduction

As described in the previous examples, residues 1152, 1153, and 1555 are also identified as candidates that may be essential for C3-specific functions. Induction of mutations has confirmed that modification of these residues can be used to confer resistance to the H factor.

2. Method

2.1 Design and analysis of the mutant DV-6

The same method was used as described in the previous example, except that the mutagenic primer had the sequence atctcgctgcgcaaggctttcgatatttgcgag encoding mutations Q1152R, E1153K and K1155F. Sequence analysis of the isolated plasmid DNA confirmed that the correct mutation was introduced. No other mutations were found.

3. Properties of the mutant DV-6

The analysis of the expressed product with mutations of DV-6 is shown in Fig.11. The following points should be noted.

(I) As described in the previous example, Western blot analysis was carried out using a polyclonal antibody to C3, it clearly identified the precursor, alpha, alpha'-chain, beta chain, fragments with a molecular weight of 43 and 46 kDa, but only fragments with a molecular weight of 77 and 68 kDa are barely visible. It should be noted that alpha and alpha chains were not moved and were not detected with 100% efficiency, i.e. the intensity of these bands is lower than expected and does not accurately reflect the actual amounts present.

(II) Cleavage of wild-type C3 with CVFBb results in a small amount of C3b alpha chain, but most of it is lost due to C3b cleavage to produce iC3b due to the endogenous activity of factors H and I (lane B3). It appears mainly in the form of a fragment with a molecular weight of 43 kDa, although small amounts of an intermediate product with a molecular weight of 46 kDa are also visible. The addition of 2 μg / ml of exogenous H-factor (band B4) with the I-factor leads to significant additional cleavage, and the addition of 10-50 μg / ml of exogenous H-factor (bands B5, B6) completes the conversion of C3b to fully digested iC3b (not bands corresponding to the alpha'-chain or fragment with a molecular weight of 46 kDa appear). The cleavage of the DV-6 product with CVFBb also results in a small amount of alpha chain (C3 band). It is important that the amount of chain with a molecular weight of 43 kDa obtained in the absence of exogenous factors H and I is lower than that of the wild type, and the manifest amount of an intermediate fragment with a molecular weight of 46 kDa is relatively higher, indicating a lower cleavage efficiency. The addition of exogenous factors H and I (bands C4-6) completes the conversion of the indicated C3b to iC3b. However, while using wild-type C3, an intermediate product with a molecular weight of 46 kDa was eliminated at a H-factor concentration of 10 μg / ml (B5 band), which indicates complete cleavage, the species studied with a mutation that changes the sensitivity to the H-factor are still maintained at this concentration (C5 band) and complete cleavage was found only when using an H-factor concentration of 50 μg / ml. Thus, mutant C3b is more resistant than wild-type to endogenous factors H and I, although resistance is not complete, as evidenced by the sensitivity to high levels of cleavage of exogenously added factors H and I.

4. Conclusion

The DV-6 mutation creates resistance to H-factor-dependent I-factor cleavage. The mechanism of this phenomenon remains unclear, although since the modified residues are located far (from the point of view of the primary structure) from the I-factor cleavage sites, and the effect depended on the dose of the added H-factor, it is possible that the interaction with the H-factor worsens. In this case, the mutation will also confer resistance to other inhibitory activities of the H-factor, such as: (I) competition with the B-factor for binding to C3b (or C3i) and (II) accelerated dissociation of C3bBb and C3bBb convertases. The DV-6 mutation is a modification of residues directly or indirectly involved in maintaining affinity for the H factor. Numerous different mutations of these residues are likely to have the same effects as the mutation of only certain residues modified in accordance with the present invention. Mutations in other residues in this segment of the primary structure are likely to also lead to a similar effect.

Example 15: Modification of residues 1546-1663

1. Introduction

In contrast to the previous examples (12-14), the modification of residues 1546-1663 was not based on a comparative consideration of C3 sequences and related proteins. Instead, the described modification was created accidentally as a result of an unintentional deletion of nucleotides, which led to a mutation in the "reading frame shift" during translation of the C-terminal residues. The resulting product showed pronounced resistance to H-factor-dependent I-factor cleavage. Thus, similar, specially designed modifications are likely to be suitable for imparting resistance to the regulatory actions of the H-factor and / or I-factor.

2. Method

A vector equivalent to NC3 but carrying additional mutations 3151g, 3152g, 3154a, 3156c, 3159s, 3163a, 3165t, 3167t, 3168t, which translate into amino acid substitutions T1031G, E1032N, Q1033H, E1035N and K1036I, was digested with restriction enzymes Pvul (sections sequences of the vector) and BsrGI (sections on nucleotide 4692) and a band of 6.1 kb was isolated. by agarose gel electrophoresis. Another NC3 equivalent vector, but carrying the catcatcatcatcatcat insert after nucleotide 5049, encoding the amino acid insert of IUUUHH at the C-terminus, was similarly digested with Pvul and BsrGI and a 4.4 kb fragment was isolated. These two DNA fragments were ligated together and a complete plasmid was isolated. DNA sequencing showed that one nucleotide (a4696 or a4697) was lost. It has been suggested that amino acids 1546-1663 cannot be translated in the reading frame. Instead, 48 residues of the normal frame will be read until the stop codon is reached.

Product "HDV-3X" was expressed in COS cells and tested according to the previous examples.

3. Properties of the mutant HDV-3X

An analysis of the expressed product with modifications of HDV-3X is shown in FIG. The following points should be noted.

(I) Western blot analysis was performed using monoclonal antibodies to the C3dg region of C3, with its help, the precursor, alpha, alpha'-chain, fragments with a molecular weight of 77 and 66 kDa were detected, but no beta chain, fragments were found with a molecular weight of 43 or 46 kDa.

(II) HDV-3X was compared with the mutant DV-3, as with an equivalent product without additional C-terminal modification. HDV-3X has a smaller alpha chain but normal sized products with a molecular weight of 68 and 77 kDa, which is consistent with a shortened C-terminus.

(III) Cleavage of the C3 DV-3 mutant with CVFBb in the presence of I-factor results in a small amount of alpha'-chain from C3b, but a larger fragment with a molecular weight of 68 kDa resulting from cleavage of C3b to obtain iC3b due to endogenous H-factor (lane A2). The addition of exogenous H-factor completes the conversion of C3b to C3b (bands A3-5). The conversion is virtually complete when using only 1 μg / ml H-factor (lane A3). In contrast, cleavage of the HDV-3X product with CVFBb results in a larger alpha chain and only a small fragment with a molecular weight of 68 kDa (band B2). The addition of exogenous H-factor does not affect the conversion of the indicated C3b to iC3b (bands B3-5). Only a slight formation of products with a molecular weight of 68 and 77 kDa can be detected at a concentration of H-factor of 25 μg / ml (lane A5). Thus, the C3b HDV-3X mutant is more resistant than the wild type to endogenous factors H and I. The fact that resistance is partially overcome by higher concentrations of the H-factor suggests that the affinity for the H-factor may be greatly reduced.

4. Conclusion

The HDV-3X modification creates resistance to H-factor-dependent I-factor cleavage. The mechanism of this phenomenon remains unclear, although since the modified residues are located far (from the point of view of the primary structure) from the I-factor cleavage sites, and the effect depended on the dose of the added H-factor, it is possible that the interaction with the H-factor worsens. In this case, the mutation will also confer resistance to other inhibitory activities of the H-factor, such as: (I) competition with the B-factor for binding to C3b (or C3i) and (II) accelerated dissociation of C3bBb and C3bBb convertases. Modification of HDV-3X directly or indirectly affects the residues involved in maintaining affinity for the H-factor. Numerous different deletions or mutations of these residues are likely to have the same effects as deletion or mutation of only certain residues resulting from modification in accordance with the present invention. Probably they can also lead to a similar effect.

Modification HDV-3X is not created purposefully. But the methods chosen to create it and related modifications probably should be specific methods of site-directed mutagenesis, including the methods described in the previous examples.

Example 16: Modification of residues at positions 954 and 955 to prevent I-mediated cleavage at this site

1. Introduction

The above-described change in P ₁ residues (1303 and 1320) provided resistance to I-factor cleavage at the first two sites (Examples 1-6). It was not known whether the prevention of cleavage at these two sites also prevented cleavage at the third site responsible for the isolation of C3c from C3dg. This is the third cleavage, which normally depends on CR1 (a membrane bound receptor created in soluble form, sCR1) as a cofactor, is relatively slow and was previously detected only on iC3b (or iC3i) (i.e., after cleavage at sites 1 and 2), and not detected on C3i or C3b. To study this issue, we used the E1Q2 mutant (described in Example 11), which is highly resistant to cleavage at sites 1 and 2. If this mutant still retained sensitivity to cleavage at site 3, this would indicate that it is advisable to induce mutations on this site in order to prevent decomposition of the molecule in physiological fluids. However, there is conflicting evidence in the literature about whether cleavage occurs exclusively on the link 954-955 (Davis AE, Harrison RA and Lachmann PJ, 1984, J. Immunol. 132: 1960-1966), or cleavage can also occur in other positions. such as 959-960 (Harrison RA et al., 1996, Molecular Immunology 33, Ext. 1, 59, abstract 235; Ekdahl KN, Nilsson UR and Nilsson B., 1990, J. Immunol. 144: 4269-4274) . Initially, the arginine residue was replaced with the glutamic acid residue at position 954 (creating the mutation E1Q2E3) because: (I) it is clear from the above publications that there is a residue P ₁ from one of the cleavage sites and (II) from Example 5 it is clear that the replacement with glutamic acid in Site 1 confers greater resistance to cleavage than other substitutions. In addition, C3 of other mammalian species (mouse, rat, guinea pig, rabbit) has glutamine and glycine instead of arginine and glutamic acid at positions 954 and 955 equivalent to human C3 (for example, see Mavroidis M., Sunyer JO and Lambbris JD 1995 J. Immunol. 154: 2164-2174). These data suggest that this site (954–955) will not be sufficiently cleaved in other species and that there is another site, such as 959–960, which is probably more important (Harrison RA et al., 1996 , Molecular Immunology 33, Ext. 1, 59, abstract 235). Thus, for human C3, the equivalent mutations Arg ⁹⁵⁴ → Gln and Glu ⁹⁵⁵ → Gly were made in order to create the mutant E1Q2QG3.

2. Method

To create the mutant, the same method was used as described in the previous examples, except that the mutagenic primer for E1Q2E3 had the sequence gaacgcctgggcgaagaaggagtgcag encoding the mutation R954E, and the mutagenic primer for E1Q2QG3 had the sequence aacgcgaggggagaccccggccggccggccggccggccggccggccggccgggccgg. The product was ligated into a construct that contained mutations encoding E1Q2 (E1303, Q1320, as described in examples 5 and 11). Sequence analysis of the isolated plasmid DNA confirmed that the correct mutation was introduced. No other mutations were found. COS cells were transfected with the resulting expression vectors and the secreted expressed product was analyzed for cleavage reactions as previously described.

3. I-factor-mediated cleavage of mutants E1Q2, E1Q2E3 and E1Q2QG3

Analysis of expressed products is shown in Fig. 13. The following points should be noted.

(I) Western blot analysis was performed using monoclonal antibodies to the C3dg region of C3, with its help, the precursor, alpha, alpha'-chain, fragments with a molecular weight of 77 and 68 kDa were detected, but the beta chain, fragments were not detected with a molecular weight of 43 or 46 kDa. In addition, a cleavage product of site 3 with a molecular weight of 86 kDa without cleavage at sites 1 or 2 could be detected.

(II) The drawing shows that the product with a molecular weight of 86 kDa is indeed formed by the I-factor-mediated cleavage of E1Q2 in the presence of sCR1 (lane A3), but not in the case where the H-factor is a cofactor (lane A2).

(III) A product with a molecular weight of 86 kDa is not formed in either the E1Q2E3 (C) mutant or the E1Q2QG3 (B) mutant even in the presence of sCRl (C3 and B3).

4. Conclusion

(I) I-factor mediated cleavage at site 3 may still occur when cleavages at sites 1 and 2 are blocked. Therefore, additional blocking of cleavage at site 3 is advisable to prevent decomposition in the physiological environment of any mutant product, which otherwise is resistant to cleavage only at sites 1 and 2.

(II) Cleavage at site 3 can be blocked by replacing the residue at position 954 with Glu and by replacing the residues at positions 954 and 955 with Gln and Gly. Thus, other substitutions of residues at position 954 and / or 955 are also likely to confer resistance to cleavage at site 3.

(Ill) These mutations did not allow cleavage at other putative positions on the third cleavage site (such as 959-960), even though these sites did not carry mutations. This may indicate that either only the nucleotides at positions 954-955 represent an important cleavage site, or that other cleavages require preliminary cleavage at positions 954-955, or that the induction of other mutations in these residues prevents cleavage at other positions by another mechanism (such as conformational distortion). In any case, mutations at position 954 and / or 955 are an effective means of preventing decomposition of C3b or C3-like products.

Example 17: Modifications of the C-terminal region of C3, inhibiting I-factor cleavage

1. Introduction

Example 15 provides evidence that a mutation or deletion of residues at positions 1546-1663 confers resistance to I-factor cleavage. This example presents additional smaller mutants that also contribute to this resistance, as well as various mutations that do not have this property.

2. Method

To create the mutant used the same method as described in the previous examples, except that the mutagenic primers encoded by these mutations had the sequences shown in table III. Sequence analysis of the isolated plasmid DNA confirmed that the correct mutation was introduced. No other mutations were detected. COS cells were transfected with the resulting expression vectors and the secreted expressed product was analyzed for cleavage reactions as previously described.

3. Resistance of various mutants to I-factor cleavage

The results of experiments on the cleavage of these mutants, carried out using factors I and H, are shown in Figs. 14 and 15. It should be noted that Western blot analysis was performed using monoclonal antibodies to the C3dg region of C3, and its predecessor, alpha, was identified -, alpha'-chain, fragments with a molecular weight of 77 and 68 kDa, but no beta chain, fragments with a molecular weight of 43 or 46 kDa were detected.

On Fig shows that wild-type C3 (Nc3, A), products FT-3 (D), FT-4 (E) and FT-5 (F) were split by the I-factor, as evidenced by the appearance of bands corresponding to molecular mass of 77 kDa and / or 68 kDa and the disappearance of the alpha chain. In contrast, FT-1 and FT-2 products did not break down.

On Fig it is seen that wild-type C3 (NC3, A), products FR-1 (B), FR-2 (C), FR-3 (D) and FR-4 (E) were split, despite the fact that that in that case, the product FT-2 (F) was cleaved.

4. Conclusion

(I) Even a small truncation of FT-2 is sufficient to confer resistance to I-factor cleavage. Such stability, therefore, can be achieved by deletion of some or all of the residues at positions 1636-1663. This conclusion is confirmed by the stability shown by FT-1 carrying a deletion of residues at positions 1636-1663 in addition to deletion / modification of residues at positions 1591-1635.

(II) Since the residues at positions 1636-1663 are necessary for I-factor cleavage, many other modifications of these residues are likely to cause stability.

(III) Not all modifications of these residues confer stability. Ineffective modifications include those designated as FT-5, FR-1, FR-2, FR-3, and FR-4, as well as modifications designated as FT-3 and FT-4, which modify residues other than residues in the provisions of 1636-1663.

(IV) These data suggest that residues within region 1636-1663 that are necessary for cleavage are those that have not been modified by FT-5, FR-1, FR-2, FR-3, or FR-4. Thus, some of the residues in provisions 1649-1660 may be critical.

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Claims (35)

1. A human protein modified in such a way that it is capable of forming a C3 convertase resistant to downregulation, where the modified protein is selected from the group consisting of (a) C3 protein, which includes one or more mutations of amino acid residues at positions 992-1005 ( EDA VDAERLKHLIV) of native human C3, whereby C3b and C3i products or C3 convertases derived therefrom become resistant to complement inhibitory H-factor activity; (b) C3 protein, which includes one or more mutations of amino acid residues at positions 1152-1155 (QEAK) of human C3, whereby C3b and C3i products or C3 convertases derived therefrom become resistant to complement inhibitory H-factor activity; (c) a C3 protein that has one or more deletions, substitutions or insertions of amino acids at positions 1546-1663 of native human C3, which protein has reduced sensitivity to the H-factor and / or I-factor compared to native human C3; and (d) C3 protein, which has one or more deletions, substitutions or insertions of amino acid residues at position 954 and / or 955 of native human C3 and which has reduced sensitivity to cofactor-dependent (e.g., CR1 or H-factor) cleavage in this position, mediated by the I-factor. 2. The protein according to claim 1, characterized in that it comprises one or more mutations of amino acid residues at positions 992-1005 (EDA VDAERLKHLIV) of human C3, as a result of which the products C3b and C3i or C3 convertases derived therefrom become resistant to inhibitory complement H-factor activity. 3. The protein according to claim 2, characterized in that it comprises one or more mutations of amino acid residues at positions 992 (E), 993 (D), 996 (D), 997 (A), 998 (E), 999 (R ), 1000 (L), 1001 (K), 1002 (H), 1005 (V) of human C3. 4. The protein according to claim 3, characterized in that it comprises one or more of the following mutations E992S, D993A, D996S, A997Q, E998S, R999G, L1000M, K1001N, H1002I and V1005H. 5. The protein according to claim 1, characterized in that it comprises one or more mutations of amino acid residues at positions 1152-1155 (QEAK) of human C3, as a result of which the products C3b and C3i or C3 convertases derived from them become resistant to complement inhibitory activity H factor. 6. The protein according to claim 5, characterized in that it comprises one or more mutations of amino acid residues at positions 1152 (Q), 1153 (E) and 1155 (K) C3. 7. The protein according to claim 6, characterized in that it comprises one or more of the following mutations Q1152R, E1153K and K1155F. 8. The protein according to any one of claims 1 to 7, characterized in that it is resistant to complement inhibitory activity of CR1, MCP and / or DAF. 9. The protein according to claim 1, characterized in that it has one or more deletions, substitutions or insertions of amino acids at positions 1546-1663 of native human C3, and this protein has a reduced sensitivity to the H-factor and / or I-factor compared with native human C3. 10. The protein according to claim 9, characterized in that it includes one or more deletions of amino acids 1546-1663 of native human C3. 11. The protein according to claim 9 or 10, characterized in that it includes a deletion of all amino acids corresponding to amino acids at positions 1546-1663 of native human C3. 12. The protein according to claim 9, characterized in that it comprises one or more amino acids different from native human C3 in the region corresponding to amino acid residues at positions 1546-1663 of native human C3. 13. The protein of claim 12, wherein the amino acids in the region corresponding to the amino acid residues at positions 1546-1663 of native human C3 can be the result of a mutation such as a reading frame shift in a DNA sequence encoding native human C3. 14. The protein according to any one of claims 9 to 13, characterized in that it has one or more deletions, substitutions or insertions of amino acids at positions 1636-1663 of native human C3, and this protein has a reduced sensitivity to the H-factor and / or I factor in comparison with native human C3. 15. The protein of claim 14, characterized in that it includes one or more deletions of amino acids at positions 1636-1663 of native human C3. 16. The protein according to 14 or 15, characterized in that it includes a deletion of all amino acids corresponding to amino acids at positions 1636-1663 of native C3. 17. The protein of claim 14, wherein it comprises one or more amino acids different from native human C3 in the region corresponding to amino acid residues at positions 1636-1663 of native human C3. 18. The protein of claim 14, characterized in that it has one or more deletions, substitutions or insertions of amino acids at positions 1649-1660 of native human C3, and this protein has a reduced sensitivity to the H-factor and / or I-factor compared with native human C3. 19. The protein according to claim 1, characterized in that it has one or more deletions, substitutions or insertions of amino acid residues at position 954 and / or 955 of native human C3 and which has a reduced sensitivity to a cofactor dependent (for example, CR1 or H- factor) splitting in this position, mediated by the I-factor. 20. The protein according to claim 19, wherein the amino acid residue at the position corresponding to the residue at position 954 of human C3 is different from residue 954 of human C3, and the protein has a reduced sensitivity to a cofactor dependent (for example, CR1 or H-factor ) splitting in this position, mediated by the I-factor. 21. The protein of claim 20, wherein said amino acid residue is glutamic acid and which has reduced sensitivity to cofactor-dependent (eg, CR1 or H-factor) cleavage at this position mediated by I-factor. 22. The protein according to claim 19, characterized in that the amino acid residues at the positions corresponding to the residue at position 954 and the residue at position 955 of human C3 are glutamine and glycine, respectively, wherein the protein has a reduced sensitivity to a cofactor dependent (for example, CR1 or H-factor) cleavage in this position, mediated by I-factor. 23. The protein of claim 19, wherein the amino acid residue at the position corresponding to the residue at position 955 of human C3 is different from the residue at position 955 of human C3, and the protein has a reduced sensitivity to a cofactor dependent (for example, CR1 or H factor) cleavage in this position, mediated by the I-factor. 24. The protein according to any one of claims 1 to 23, designed to minimize or prevent complement-mediated damage. 25. A fragment or variant of a protein characterized by any one of claims 1 to 24, wherein the fragment or variant has C3 convertase activity and resistance to complement inhibitory activity of H-factor, I-factor, CR1, MCP and / or DAF. 26. A fragment or variant according to claim 25, characterized in that it is intended to minimize or prevent complement-mediated damage. 27. The DNA sequence encoding the protein according to any one of claims 1 to 23, or a fragment or variant according to claim 25. 28. Plasmid expression vector comprising the DNA sequence according to item 27. 29. The conjugate to achieve the effects of localized complement activation, comprising the protein according to any one of claims 1 to 23 or a fragment or variant according to claim 25, associated with a specific binding fragment. 30. The conjugate according to clause 29, where the specific binding fragment is a specific binding protein. 31. The conjugate of claim 30, wherein the specific binding protein is an antibody or antigen binding fragment thereof. 32. A pharmaceutical composition for minimizing or preventing complement-mediated damage, containing one or more proteins according to any one of claims 1 to 23, or a fragment or variant according to claim 25, or a conjugate according to any one of claims 29 to 31 together with one or more pharmaceutically acceptable carriers or excipients. 33. The pharmaceutical composition according to p, characterized in that the minimization or prevention of complement-mediated damage is to reduce the levels of the protein of the pathway of activation of complement or to prevent rejection of foreign material or localization and / or enhance the conversion of endogenous protein of complement and deposition in a particular place. 34. A method of reducing the protein of the complement activation pathway in a mammal, comprising administering to the mammal a protein according to any one of claims 1 to 23, or a fragment or variant of claim 25, or a conjugate according to any one of claims 29 to 31. 35. The method according to clause 34, where the introduction is carried out using the pharmaceutical composition according to clause 33.

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