Patent Grant
8889128
The present invention relates to a method for treating or preventing diseases or conditions mediated by IgG antibodies, such as autoimmune diseases, transplant rejection, post-operative treatment and acquired haemophilia.
IgG is a heterotetramer composed of two heavy chains and two light chains held together by disulfide bonds forming three protein domains separated by a flexible and protease sensitive hinge region. The two identical Fab portions bind antigens and the single Fc portion is responsible for effector functions, including binding and activation of complement factor C1q and Fc receptors on leukocytes.
In addition to the polypeptide backbone the Fc portion contains a conserved glycan on each heavy chain attached to Asn-297. This oligosaccharide is of the complex biantennary type with a core fucose linked to the innermost N-acetylglucosamine (GlcNAc). These glycans are located in the interface between the CH2 domains (second constant domain of the heavy chains).
EndoS is an endoglycosidase secreted by the human pathogen Streptococcus pyogenes. EndoS specifically hydrolyzes the asparagine-linked glycan on IgG between the two core GlcNAc residues. In contrast to many related endoglycosidases that require or are enhanced by denaturation of the glycoprotein substrate, EndoS only hydrolyzes native IgG. No other substrate for EndoS has been found to date.
The present inventors have shown that EndoS is useful in treating and preventing diseases mediated by IgG antibodies. In particular, the inventors have shown that EndoS efficiently hydrolyzes IgG in human blood and in vivo in rabbits, that deglycosylation of IgG by EndoS abrogates its arthritis-inducing capacity in mice, and that EndoS has a protective effect in a mouse model of lethal IgG-driven idiopathic thrombocytopenic purpura (ITP). EndoS pretreatment of pathogenic antibodies inhibits the development of this disease, and the enzyme also rescues mice from already established disease when severe thrombocytopenia and subcutaneous bleeding have developed.
In accordance with the present invention, there is thus provided the use of an EndoS polypeptide, or a polynucleotide encoding an EndoS polypeptide, in the manufacture of a medicament for the treatment or prevention of a disease or condition mediated by IgG antibodies.
The present invention also provides:
SEQ ID NO: 1 is an amino acid sequence of EndoS isolated from S. pyogenes AP1.
SEQ ID NO: 2 is an amino acid sequence of EndoS isolated from S. pyogenes AP1, including a signal sequence.
SEQ ID NO: 3 is a nucleic acid sequence encoding EndoS isolated from S. pyogenes AP1, including a signal sequence.
The present invention provides a method for treating or preventing diseases or conditions mediated by IgG antibodies, which method comprises administering to a subject an EndoS polypeptide or a polynucleotide encoding an EndoS polypeptide.
The present inventors have found that EndoS hydrolyzes IgG in human blood and in vivo in rabbits, that deglycosylation of IgG by EndoS abrogates its arthritis—inducing capacity in mice, and that EndoS has a protective effect in a mouse model of lethal IgG-driven idiopathic thrombocytopenic purpura (ITP). EndoS pretreatment of pathogenic antibodies inhibits the development of this disease, and the enzyme also rescues mice from already established disease when severe thrombocytopenia and subcutaneous bleeding have developed. Accordingly, EndoS can be used to treat or prevent diseases or conditions mediated by IgG antibodies.
Polypeptides
The EndoS polypeptide is preferably S. pyogenes EndoS, or a variant or fragment of S. pyogenes EndoS which retains IgG endoglycosidase activity. The variant may be an EndoS polypeptide from another organism, such as another bacterium. The bacterium is preferably a Streptococcus, such as Streptococcus equi, Streptococcus zooepidemicus or, preferably, Streptococcus pyogenes. Alternatively, the variant may be from Corynebacterium pseudotuberculosis, for example the CP40 protein; Enterococcus faecalis, for example the EndoE protein; or Elizabethkingia meningoseptica (formerly Flavobacterium meningosepticum), for example the EndoF2 protein. The sequences of EndoS variants from various S. pyogenes serotypes and from S. equi and S. zooepidemicus are shown in
The EndoS polypeptide may comprise:
(a) the amino acid sequence of SEQ ID NO: 1;
(b) a variant thereof having at least 50% identity to the amino acid sequence of SEQ ID NO: 1 and having IgG endoglycosidase activity; or
(c) a fragment of either thereof having IgG endoglycosidase activity.
Preferably, the polypeptide comprises, or consists of, the sequence of SEQ ID NO: 1. SEQ ID NO: 1 is the sequence of the mature form of EndoS, without the signal sequence, and corresponds to amino acids 37 to 995 of SEQ ID NO: 2.
The polypeptide may additionally include a signal sequence. Accordingly, the EndoS polypeptide may comprise:
(a) the amino acid sequence of SEQ ID NO: 2;
(b) a variant thereof having at least 50% identity to the amino acid sequence of SEQ ID NO: 2 and having IgG endoglycosidase activity; or
(c) a fragment of either thereof having IgG endoglycosidase activity.
The EndoS polypeptide may consist of the sequence shown in SEQ ID NO: 2.
Variant polypeptides are those for which the amino acid sequence varies from that in SEQ ID NO: 1 or SEQ ID NO: 2, but which retain the same essential character or basic functionality as EndoS. The variant polypeptides may therefore display IgG endoglycosidase activity. Typically, polypeptides with more than about 50%, 55% or 65% identity, preferably at least 70%, at least 80%, at least 90% and particularly preferably at least 95%, at least 97% or at least 99% identity, with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 are considered variants of the protein. Such variants may include allelic variants and the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains the basic functionality of EndoS. The identity of variants of SEQ ID NO: 1 or SEQ ID NO: 2 may be measured over a region of at least 100, at least 250, at least 500, at least 750, at least 800, at least 850, at least 900, at least 950, at least 955 or more contiguous amino acids of the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or more preferably over the full length of SEQ ID NO: 1 or SEQ ID NO: 2.
Amino acid identity may be calculated using any suitable algorithm. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
The variant sequences typically differ by at least 1, 2, 3, 5, 10, 20, 30, 50, 100 or more mutations (which may be substitutions, deletions or insertions of amino acids). For example, from 1 to 100, 2 to 50, 3 to 30 or 5 to 20 amino acid substitutions, deletions or insertions may be made. The modified polypeptide generally retains activity as an IgG-specific endoglycosidase. The substitutions are preferably conservative substitutions, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Variants of the amino acid sequence of SEQ ID NO: 1 preferably contain residues 191 to 199 of SEQ ID NO: 1, i.e., Leu-191, Asp-192, Gly-193, Leu-194, Asp-195, Val-196, Asp-197, Val-198 and Glu-199 (SEQ ID NO:24) of SEQ ID NO: 1 (which correspond to residues 227 to 235 of SEQ ID NO: 2, i.e., Leu-227, Asp-228, Gly-229, Leu-230, Asp-231, Val-232, Asp-233, Val-234 and Glu-235 (SEQ ID NO:24) of SEQ ID NO: 2). These amino acids constitute a perfect chitinase family 18 active site, ending with glutamic acid. The glutamic acid in the active site of chitinases is essential for enzymatic activity. Most preferably, therefore, the variant of SEQ ID NO: 1 contains Glu-199 of SEQ ID NO: 1 and the variant of SEQ ID NO: 2 contains Glu-235 of SEQ ID NO: 2. The variant of SEQ ID NO: 1 may contain residues 191 to 199 of SEQ ID NO: 1 having one or more conservative substitutions, provided that the variant contains Glu-199 of SEQ ID NO: 1. Alternatively, the variant of SEQ ID NO: 2 may contain residues 227 to 235 of SEQ ID NO: 2 having one or more conservative substitutions, provided that the variant contains Glu-235 of SEQ ID NO: 2.
The fragment of the EndoS polypeptide used in the invention is typically at least 10, for example at least 20, 30, 40, 50 or more amino acids in length, up to 100, 200, 250, 300, 500, 750, 800, 850, 900, 950 or 955 amino acids in length, as long as it retains the IgG endoglycosidase activity of EndoS. Preferably, the fragment of the EndoS polypeptide used in the invention encompasses residues 191 to 199 of SEQ ID NO: 1, i.e., Leu-191, Asp-192, Gly-193, Leu-194, Asp-195, Val-196, Asp-197, Val-198 and Glu-199 (SEQ ID NO:24) of SEQ ID NO: 1 (residues 227 to 235 of SEQ ID NO: 2, i.e., Leu-227, Asp-228, Gly-229, Leu-230, Asp-231, Val-232, Asp-233, Val-234 and Glu-235 (SEQ ID NO:24) of SEQ ID NO: 2). A preferred fragment of SEQ ID NO: 2 consists of amino acids 37 to 995 of SEQ ID NO: 2, i.e., SEQ ID NO: 1, which corresponds to the form of EndoS secreted from S. pyogenes after removal of the signal peptide. Another preferred fragment of the invention consists of amino acids 1 to 409 of SEQ ID NO: 1 (amino acids 37 to 445 of SEQ ID NO: 2), which corresponds to the enzymatically active α-domain of EndoS generated by cleavage by the streptococcal cysteine proteinase SpeB.
The polypeptides used in the invention may be chemically modified, e.g. post-translationally modified. For example, they may be glycosylated, phosphorylated or comprise modified amino acid residues. They may be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote insertion into the cell membrane. Such modified polypeptides fall within the scope of the term “polypeptide” used herein.
Typically, polypeptides for use in accordance with the invention display immunoglobulin endoglycosidase activity, and in particular IgG endoglycosidase activity. Preferably, the polypeptide hydrolyzes the β-1,4-di-N-acetylchitobiose core of the asparagine-linked glycan of IgG. Preferably the activity is specific for IgG. The endoglycosidase activity may be determined by means of a suitable assay. For example, a test polypeptide may be incubated with IgG at a suitable temperature, such as 37° C. The starting materials and the reaction products may then be analysed by SDS PAGE. Typically, the molecular mass of the IgG heavy chain is reduced by approximately 3 kDa if the test polypeptide has IgG endoglycosidase activity. Another assay for determining whether a test polypeptide has IgG endoglycosidase activity is by detection of glycosylated IgG using Lens culinaris agglutinin lectin (LCA), optionally using horseradish peroxidase and peroxidase substrate. Typically, the carbohydrate signal is reduced if the test polypeptide has IgG endoglycosidase activity. Another assay for determining whether a test polypeptide has IgG endoglycosidase activity is by incubation of a test polypeptide with purified IgG Fc fragments followed by reduction of the sample with 10 mM dithiotreitol and mass spectroscopy (MALDI-TOF) analysis. Typically, the mass of monomeric IgG Fc is reduced by 1417±14 Da if the test polypeptide has IgG endoglycosidase activity.
The endoglycosidase activity of the polypeptides can be further characterised by inhibition studies.
The endoglycosidase activity of the polypeptide is generally IgG-specific in that the polypeptide may not degrade the other classes of Ig, namely IgM, IgA, IgD and IgE, when incubated with these immunoglobulins under conditions that permit cleavage of IgG. The EndoS polypeptide is capable of hydrolyzing IgG molecules present in the subject to be treated. Thus, where the subject is a human, the EndoS polypeptide is capable of hydrolyzing human IgG. EndoS is capable of hydrolyzing human IgG of all four subclasses (IgG1-4). In preferred embodiments, the EndoS polypeptide has the ability to hydrolyze human, Rhesus monkey, mouse, rat, rabbit, horse, goat, dog and swine IgG.
Polypeptides for use in the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide for use in the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 50%, e.g. more than 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.
Polypeptides for use in the present invention may be isolated from any suitable organism that expresses an EndoS polypeptide or a variant of an EndoS polypeptide. Typically, the EndoS polypeptide is isolated from suitable EndoS expressing strains of Streptococcus, preferably strains of S. pyogenes. Suitable organisms and strains may be identified by a number of techniques. For example, S. pyogenes strains may initially be tested for the presence an ndoS gene. Polynucleotide primers or probes may be designed based on, for example, SEQ ID NOs: 1, 2 or 3. The presence of the ndoS gene can then be verified by PCR using such primers or by hybridisation of probes to genomic DNA of the S. pyogenes strain.
Streptococcal strains expressing active EndoS or a variant thereof can be identified by assaying for IgG endoglycosidase activity in the culture supernatant or by immunodetection using antibodies directed towards EndoS. The Streptococcal strains that have been verified as expressing active EndoS are the S. pyogenes M1 serotype strains AP1 and SF370, the S. equi strain 4047 and the S. zooepidermicus strain H70. In addition, the ndoS gene is found in the following S. pyogenes strains: M1 serotype strains SSI-1 and MGAS5005, M2 serotype strain MGAS10270, M3 serotype strain MGAS315, M4 serotype strain MGAS10750, M5 serotype strain Manfredo, M6 serotype strain MGAS10394, M12 serotype strain MGAS9429, M18 serotype strain MGAS8232, M28 serotype strain MGAS6180 and M49 serotype strain 591.
Isolation and purification of EndoS from an expressing S. pyogenes culture, or from cultures of other cells expressing EndoS is typically on the basis of IgG endoglycosidase activity. Preferably the purification method involves an ammonium sulphate precipitation step and an ion exchange chromatography step. According to one method, the culture medium is fractionated by adding increasing amounts of ammonium sulphate. The amounts of ammonium sulphate may be 10 to 80%. Preferably the culture medium is fractionated with 50% ammonium sulphate, and the resulting supernatant is further precipitated with 70% ammonium sulphate. Pelleted polypeptides may then be subjected to ion exchange chromatography, for example by FPLC on a Mono Q column. Eluted fractions may be assayed for IgG endoglycosidase activity and peak activity fractions may be pooled. Fractions may be analysed by SDS PAGE. Fractions may be stored at −80° C. In an alternative method to purify EndoS, EndoS without the signal sequence (i.e., having the sequence of SEQ ID NO: 1) is expressed in Escherichia coli using GST Gene Fusion System (Amersham-Pharmacia Biotech, Uppsala, Sweden). A 2929 base pair PCR product covering bases 304 to 3232 of the ndoS sequence is amplified from S. pyogenes genomic DNA using primers 5′-ACT-GGG-ATC-CCG-GAG-GAG-AAG-ACT-3′ (SEQ ID NO:26) with a BamHI site (underlined) and 5′-TTA-ATC-TCG-AGG-TTG-CTA-TCT-AAG-3′ (SEQ ID NO:27) with an XhoI site (underlined). This fragment is digested with BamHI and XhoI and ligated into the pGEX-5X-3 generating plasmid pGEXndoS that is used to transform E. coli BL21(DE3)pLys. pGEXndoS/BL21(DE3)pLys is induced with 0.1 mM isopropyl β-D-thiogalactopyranoside. After induction, bacteria are lysed using BugBuster™ (Novagen) and the GST-EndoS fusion protein is purified on Glutathione-Sepharose®. The GST tag is removed using factor Xa according to protocols (Amersham-Pharmacia Biotech), and residual factor Xa is removed using Xarrest™-agarose (Novagen). This results in a preparation of recombinant EndoS (rEndoS) that is homogenous as assessed by SDS-PAGE and Western blot using EndoS-specific antibodies. Prior to in vivo experiments protein samples are sterile-filtered through a 0.2 μm filter (Millipore). Purified EndoS protein is stored at −80° C. in phosphate buffered saline.
Polypeptides for use in the invention may also be prepared as fragments of such isolated polypeptides. Further, the EndoS polypeptides may also be made synthetically or by recombinant means. For example, a recombinant EndoS polypeptide may be produced by transfecting mammalian cells in culture with an expression vector comprising a nucleotide sequence encoding the polypeptide operably linked to suitable control sequences, culturing the cells, extracting and purifying the EndoS polypeptide produced by the cells.
The amino acid sequence of polypeptides for use in the invention may be modified to include non-naturally occurring amino acids or to increase the stability of the compound. When the polypeptides are produced by synthetic means, such amino acids may be introduced during production. The polypeptides may also be modified following either synthetic or recombinant production.
Polypeptides for use in the invention may also be produced using D-amino acids. In such cases the amino acids will be linked in reverse sequence in the C to N orientation. This is conventional in the art for producing such polypeptides.
A number of side chain modifications are known in the art and may be made to the side chains of the EndoS polypeptides, provided that the polypeptides retain IgG endoglycosidase activity.
Polynucleotides
A polynucleotide encoding an EndoS polypeptide or variant may be used to treat or prevent a disease or condition mediated by pathogenic IgG antibodies. In particular the polynucleotide may comprise or consist of: (a) the coding sequence of SEQ ID NO: 3; (b) a sequence which is degenerate as a result of the genetic code to the sequence as defined in (a); (c) a sequence having at least 60% identity to a sequence as defined in (a) or (b) and which encodes a polypeptide having IgG endoglycosidase activity; or (d) a fragment of any one of the sequences as defined in (a), (b) or (c) which encodes a polypeptide having IgG endoglycosidase activity.
Typically the polynucleotide is DNA. However, the polynucleotide may be a RNA polynucleotide. The polynucleotide may be single or double stranded, and may include within it synthetic or modified nucleotides.
A polynucleotide of the invention can typically hybridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 3 at a level significantly above background. Background hybridization may occur, for example, because of other DNAs present in a DNA library. The signal level generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: 3 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 3. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32P. Selective hybridisation may typically be achieved using conditions of medium to high stringency. However, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, Molecular Cloning: A Laboratory Manual, 1989). For example, if high stringency is required suitable conditions include from 0.1 to 0.2×SSC at 60° C. up to 65° C. If lower stringency is required suitable conditions include 2×SSC at 60° C.
The coding sequence of SEQ ID NO: 3 may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50, 100, 200, 500 or 750 substitutions. The polynucleotide of SEQ ID NO: 3 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends. Additional sequences such as signal sequences may also be included. The modified polynucleotide generally encodes a polypeptide which has IgG specific endoglycosidase activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table above.
A nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 3 will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the coding sequence of SEQ ID NO: 3 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, at least 100, at least 200, at least 500, more preferably at least 750 contiguous nucleotides or most preferably over the full length of SEQ ID NO: 3 or the length of SEQ ID NO: 3 encoding a polypeptide having the sequence shown in SEQ ID NO: 1 or 2. Sequence identity may be determined by any suitable method, for example as described above.
Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher sequence identity over longer lengths) being preferred. Thus, for example a polynucleotide which has at least 90% sequence identity over 60, preferably over 100 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 500 nucleotides.
Polynucleotide fragments will preferably be at least 20, for example at least 25, at least 30 or at least 50 nucleotides in length. They will typically be up to 100, 150, 250 or 500 nucleotides in length. Fragments can be longer than 500 nucleotides in length, for example up to 600, 700, 800, 900, 1000, 1500, 2000, 2500 or 3000 nucleotides in length, or even up to a few nucleotides, such as five, ten or fifteen nucleotides, short of the coding sequence of SEQ ID NO: 3.
Polynucleotides for use in the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. The polynucleotides are typically provided in isolated and/or purified form.
In general, short polynucleotides will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
Longer polynucleotides will generally be produced using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15-30 nucleotides) to a region of the ndoS gene which it is desired to clone, bringing the primers into contact with DNA obtained from a bacterial cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
Such techniques may be used to obtain all or part of the ndoS gene sequence described herein. Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al. (1989).
EndoS polynucleotides as described herein have utility in production of the polypeptides for use in the present invention, which may take place in vitro, in vivo or ex vivo. The polynucleotides may be used as therapeutic agents in their own right or may be involved in recombinant protein synthesis.
The polynucleotides for use in the invention are typically incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Therefore, polynucleotides for use in the invention may be made by introducing an EndoS polynucleotide into a replicable vector, introducing the vector into a compatible host cell and growing the host cell under conditions which bring about replication of the vector. The host cell may, for example, be an E. coli cell.
Preferably the vector is an expression vector comprising a nucleic acid sequence that encodes an EndoS polypeptide. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals, which may be necessary and which are positioned in the correct orientation in order to allow for protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. (1989).
Preferably, a polynucleotide for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence, such as a promoter, “operably linked” to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.
The vectors may be for example, plasmid, virus or phage vectors provided with a origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vector is typically adapted to be used in vivo.
Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. Mammalian promoters, such as β-actin promoters, may be used. Tissue-specific promoters are especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). Viral promoters are readily available in the art.
The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell. Other examples of suitable viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.
Diseases and Conditions
The EndoS polypeptide, or polynucleotide, may be used to treat or prevent diseases or conditions mediated by pathogenic IgG antibodies. It is well known in the art that IgG antibodies are involved in the pathogenesis of a number of different diseases and conditions. The present inventors have found that the role of pathogenic IgG antibodies in such diseases can be inhibited using an EndoS polypeptide or polynucleotide.
The disease or condition can be an autoimmune disease. Such diseases include Addison's disease, alopecia greata, ankylosing spondilitis, antiphospholipid syndrome, aplastic anaemia, autoimmune gastritis, autoimmune hearing loss, autoimmune haemolytic anaemias, autoimmune hepatitis, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune polyendocrinopathy, Beçhet's disease, bullous pemphigoid, cardiomyopathy, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, coeliac disease, Crohn's disease, CREST syndrome, Degas disease, epidermolysis bullosa acquisita, essential mixed cryoglobulinaemia, giant cells arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillan-Barre syndrome, Hashimoto's thyroiditis, idiopathic thrombocytopenic purpura, inflammatory bowel disease, Kawasaki's disease, Meniere's syndrome, mixed connective tissue disease, Mooren's ulcer, multiple sclerosis, myasthenia gravis, pemphigus foliaceous, pemphigus vulgaris, pernicious anaemia, polyarteritis nodosa, polyglandular autoimmune syndrome type 1 (PAS-1), polyglandular autoimmune syndrome type 2 (PAS-2), polyglandular autoimmune syndrome type 3 (PAS-3), polymyositis/dermatomyositis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's syndrome, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, subacute thyroiditis, sympathetic opthalmia, systemic lupus erythematosus, Takayasu's arteritis, type 1 diabetes mellitus, vitiligo, Vogt-Koyanagi-Harada disease or Wegener's granulomatosis. Preferably the autoimmune disease is rheumatoid arthritis (RA), systemic lupus erythematosus or idiopathic thrombocytopenic purpura.
The disease or condition can be asthma. The asthma can be acute or chronic asthma.
IgG activates the classical pathway of the complement system. EndoS polypeptides and polynucleotides can therefore be used to treat diseases and conditions where complement activation is detrimental to the patient. For example, the EndoS polypeptides and polynucleotides can be used to treat transplantation-derived disorders, for example transplant rejection (such as acute or chronic allograft or xenograft rejection) and graft-versus-host disease. The transplantation-derived disorder may occur due to the transplantation of a tissue or an organ in a patient.
EndoS polypeptides and polynucleotides are also of use in post-operative treatment, for example in the treatment of patients who have undergone heart by-pass operations.
Further, EndoS polypeptides and polynucleotides can be used for the treatment of acquired haemophilia, i.e to remove IgG in haemophilia patients who have developed autoantibodies against coagulation factors.
The subject is typically a mammalian subject, such as a mouse, rat or primate (e.g. a marmoset or monkey). The subject may be human or a non-human animal. Where the subject is a laboratory animal such as a mouse, rat or primate, the animal may be treated to induce a disease or condition mediated by pathogenic IgG antibodies. For example, the mouse anti-CII antibody induced arthritis (CAIA) model described by Nandakumar et al. (Am. J. Pathol. 163(5): 1827-1837, 2003), or a modified version of that model, may be used.
Therapy and Prophylaxis
The present invention provides the use of EndoS polypeptides and polynucleotides to treat or prevent a disease or condition mediated by pathogenic IgG antibodies. Treatment may be therapeutic or prophylactic.
The EndoS polypeptide or polynucleotide may be administered to an individual in order to prevent the onset of one or more symptoms of the disease or condition. In this embodiment, the subject may be asymptomatic. The subject may have a genetic predisposition to the disease. A prophylactically effective amount of the polypeptide or polynucleotide is administered to such an individual. A prophylactically effective amount is an amount which prevents the onset of one or more symptoms of a disease or condition.
A therapeutically effective amount of the EndoS polypeptide or polynucleotide is an amount effective to ameliorate one or more symptoms of a disease or condition. Preferably, the individual to be treated is human.
The EndoS polypeptide or polynucleotide may be administered to the subject by any suitable means. The polypeptide or polynucleotide may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical or other appropriate administration routes.
The EndoS polypeptide or polynucleotide may be administered to the subject in such a way as to target therapy to a particular site. For example, an EndoS polypeptide may be administered directly to the site of a transplanted organ. The EndoS polypeptide may be injected locally, for example intraarticularly or in one or more joints. Local administration of EndoS to the joints is particularly preferable for the prophylaxis or treatment of rheumatoid arthritis (RA). The EndoS polypeptide may be conjugated with reagents that bind cartilage specifically. For EndoS polynucleotides, expression vectors encoding the EndoS polypeptide may be used to direct expression of EndoS to a particular tissue, for example by using tissue-specific promoters or RNAi.
The formulation of any of the polypeptides and polynucleotides mentioned herein will depend upon factors such as the nature of the polypeptide or polynucleotide and the condition to be treated. The polypeptide or polynucleotide may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The polypeptide or polynucleotide may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.
Typically the polypeptide or polynucleotide is formulated for use with a pharmaceutically acceptable carrier or diluent and this may be carried out using routine methods in the pharmaceutical art. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.
Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.
Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit “S”, Eudragit “L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
Pharmaceutical compositions suitable for delivery by needleless injection, for example, transdermally, may also be used.
A therapeutically effective amount of polypeptide or polynucleotide is administered. The dose may be determined according to various parameters, especially according to the polypeptide or polynucleotide used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg, preferably from about 0.1 mg/kg to 10 mg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.
The EndoS nucleotide sequences described above and expression vectors containing such sequences can also be used as pharmaceutical formulations as outlined above. Preferably, the nucleic acid, such as RNA or DNA, in particular DNA, is provided in the form of an expression vector, which may be expressed in the cells of the individual to be treated. The vaccines may comprise naked nucleotide sequences or be in combination with cationic lipids, polymers or targeting systems. The vaccines may be delivered by any available technique. For example, the nucleic acid may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery. The nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.
Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the nucleic acid to be administered can be altered. Typically the nucleic acid is administered in the range of 1 pg to 1 mg, preferably to 1 pg to 10 μg nucleic acid for particle mediated gene delivery and 10 μg to 1 mg for other routes.
The present invention also provides a method of treating, ex vivo, blood taken from a patient suffering from a disease or condition mediated by pathogenic IgG antibodies comprising contacting the blood with an EndoS polypeptide. EndoS may thus be used for extracorporeal treatment of blood. The EndoS may be used to treat one or more components of blood, such as plasma or serum. The ex vivo method described herein may be practised on blood that has already been removed from the body of a patient. The blood or blood product may optionally be returned to the patient after being contacted with an EndoS polypeptide.
The following Examples illustrate the invention:
In order to be efficient as a therapeutic agent against pathological IgG, EndoS needs to be active at low concentrations in a human whole blood environment. To investigate this, recombinant EndoS (rEndoS) without the signal sequence (i.e. having the sequence of SEQ ID NO: 1) was produced and purified as previously described (Collin & Olsén, 2001, Infect. Immun. 69: 7187-7189). Increasing final concentrations (0, 0.31, 0.63, 1.25, 2.5, 5, 10, and 20 μg/ml) of rEndoS were incubated in 500 μl of heparinized human blood from healthy volunteers with rotation end over end for 1 hour at 37° C. Samples were centrifuged at 720×g for 10 min at 4° C. followed by purification of IgG in plasma using protein G Sepharose according to manufacturer's instructions (GE Healthcare Biosciences, Uppsala, Sweden). There was no difference in binding efficiency to protein G between fully glycosylated IgG and EndoS treated IgG, which is in concordance with previous findings for the IgG binding proteins protein H (from S. pyogenes) and protein A (from Staphylococcus aureus) (Collin & Olsén 2001, EMBO J. 20: 3046-3055). Purified IgG was separated on 10% SDS-PAGE, stained with Coomassie or electroblotted onto PVDF (Immobilon-P, Millipore, Bedford, Mass.). Glycosylated IgG was detected using 5 μg/ml of biotinylated Lens culinaris agglutinin lectin (LCA) and 1 μg/ml of Streptavidin-Horseradish peroxidase (Vector Laboratories, Burlingame, Calif.) and SuperSignal West Pico peroxidase substrate (Pierce, Rockford, Ill.). Membranes were analyzed using a Chemidoc XRS imaging system and Quantity One image analysis software (Bio-Rad, Hercules, Calif.).
These experiments showed that increasing concentrations of EndoS gradually shift the IgG heavy chain to an approximately 3 kDa smaller apparent molecular mass and that almost no full size heavy chain could be seen above a concentration of 2.5-5 μg/ml of rEndoS (
These results indicate that 5 μg/ml of rEndoS in 1 hour completely hydrolyzes the IgG pool in human blood. Assuming an IgG plasma concentration of 10 mg/ml this would mean complete hydrolysis of IgG in 1 hour at an rEndoS to IgG ratio of 1:2000. Thus, rEndoS shows a remarkably efficient hydrolysis of the functionally important IgG glycan in such a complex environment as human blood.
In order to further substantiate the use of EndoS as a therapeutic agent, the IgG glycan hydrolyzing activity of EndoS in the circulation of live animals was investigated. Swedish loop rabbits with a body weight of approximately 3 kg were injected intravenously with 1 mg of rEndoS, corresponding to an approximate rEndoS to IgG ratio of 1:2000 given that rEndoS distributes in blood only. Animals showed no signs of disease. Serum samples were withdrawn at 0, 1, 2, 4, 6, 8 and 12 hours, and 1, 2, 3, 4, 5, 6, 8, and 10 days. Serum IgG was analyzed for glycosylation status using SDS-PAGE and lectin blot analysis as described above for human blood.
These experiments showed that before injection, the apparent molecular mass of the heavy chains of IgG was comparable to fully glycosylated intact rabbit IgG (
In order to see if rEndoS was active within an animal that had already been exposed to the enzyme, a second injection with 1 mg of rEndoS was performed 35 days after the first injection. Again, animals seemed unaffected by the injection and serum samples were withdrawn and analyzed as above. SDS-PAGE revealed that before the second injection, IgG heavy chain migrated as fully glycosylated control rabbit IgG heavy chain (
In order to investigate if rEndoS still had activity within an animal that had been exposed intravenously twice to rEndoS, a third injection with 1 mg of rEndoS was performed 130 days after the first injection. Again, animals were unaffected by the injection and serum samples were withdrawn and analyzed as above. SDS-PAGE revealed that prior to the third injection, IgG heavy chain migrated as fully glycosylated control rabbit IgG heavy chain (
Taken together, these results indicate that low concentrations of EndoS efficiently hydrolyze the heavy chain glycan on the whole rabbit IgG pool in vivo. Furthermore, previous intravenous exposure to EndoS does not significantly affect the in vivo enzymatic activity of EndoS.
Since EndoS had full activity when injected a second and a third time, it was of interest to determine whether this was due to no or low immune response against the enzyme, or if there were specific antibodies against EndoS that did not interfere with enzymatic activity. This was of particular interest since it is known that both healthy individuals and those infected with S. pyogenes have antibodies against EndoS (Åkesson et al, 2004, J. Infect. Dis. 189: 797-804).
In order to investigate this, purified rEndoS was separated on 10% SDS-PAGE and electroblotted onto PVDF that was cut into 1.5 mm strips. Strips were incubated with 1:500 dilutions of all the serum samples from the first, second and third injections, followed by incubation with peroxidase-labeled goat anti-rabbit antibodies (Pierce). Strips were developed using chemiluminescence as described above for lectin blots.
This experiment revealed that before the first injection there were already antibodies reacting with rEndoS (
Given the very high signal levels in the Western blots from samples obtained after the second and third injections, samples prior to and after all three injections were also analyzed by ELISA. For ELISA experiments, 2 μg of EndoS was used to coat microtiter plates (Nunc, Roskilde, Denmark), followed by blocking with 20 mg/ml of bovine serum albumin in PBS. Sera from animals before EndoS injections and 0.5, 1, 5, and 10 days after injections were used as primary antiserum in serial dilutions of 1:100 to 1:200,000. Peroxidase-labeled goat anti-rabbit antibodies (Pierce) were used as secondary antibodies and ABTS (Roche, Ind.) as peroxidase substrate. A standard curve for rabbit IgG was generated by coating microtiter plates as above with serial dilutions of polyclonal rabbit IgG (Sigma) and peroxidase-labeled goat anti-rabbit antibodies as secondary antibodies. Plates were analyzed at 405 nm in a Victor3 multi label reader (Perkin-Elmer, Waltham, Miss.).
The ELISA experiments confirmed that just prior to the second injection of EndoS, the reactivity against EndoS was comparable or slightly higher than before the first injection, and the reactivity still had not increased at 5 days after injection (
These results indicate that there are antibodies directed towards EndoS in unexposed animals and that rEndoS elicits an immune response in rabbits upon repated intravenous exposure. However, these antibodies do not interfere with the activity of rEndoS in the circulation during three consecutive administrations. Furthermore, repeated administration does not affect the approximately 12 hours circulation time (defined as the ability to detect EndoS) of the enzyme as analyzed by immunoprecipitation and Western blot analysis of EndoS from rabbit serum samples.
SDS-PAGE and lectin blot analysis of IgG monoclonal antibodies (CIIC1 and M2139) incubated with and without EndoS and separated by 10% SDS-PAGE was carried out and the results are shown in
EndoS specifically hydrolyzes the β-1,4-di-N-acetylchitobiose core of the asparagine-linked glycan of immunoglobulin (IgG). After the removal of the carbohydrate side chain using EndoS, IgG molecular weight is reduced. The difference in size of the γ-chains can be clearly seen in the stained gel picture between the IgG sample treated with EndoS and non-treated IgG.
To confirm that the size alteration of IgG was caused by EndoS activity and resulted in the removal of the glycan moiety on γ-chains rather than proteolytic degradation, a lectin blot analysis was performed. The lectin from Galanthus nivalis (GNL) preferentially recognizes α-1,3 mannose residues found in the biantennary glycan on γ-chains. Lectin blot analysis of the same samples with the GNL lectin revealed a significantly reduced signal when incubated with EndoS. In contrast, the γ-chains were still glycosylated when incubated in the absence of EndoS. These data indicate that EndoS has the ability to remove structures containing α-1,3 mannose from the γ-chains of mouse IgG.
This experiment was performed to understand whether the removal of carbohydrate moieties from collagen type II (CII) specific IgG monoclonal antibody (mAb) affected its binding capacity to collagen type II in vivo.
1-2 day old neonatal rats were injected with 1 mg of CII-binding antibody (both normal and EndoS treated) i.p. Twenty-four hours after the antibody transfer, paws were dissected and snap frozen in OCT compound using isopentane and dry ice. Immuno-histochemical analysis was performed using biotinylated anti-mouse kappa (187.1) antibody and HRP conjugated secondary antibody as the detecting system using standard protocol. The results are shown in
CII-specific monoclonal antibodies induce an acute form of arthritis in mice, the so-called collagen antibody induced arthritis (CAIA) described in Nandakumar et at (2003). CAIA resembles the effector phase of arthritis without involving the priming phase of the immune response. This antibody-mediated arthritis is dependent on complement components, FcγRs, effector cytokines TNF-α and IL-1β and on neutrophils and macrophages. CAIA was used in the present study to understand the importance of deglycosylation of IgG by EndoS treatment. A monoclonal antibody cocktail containing two antibodies: M2139 mAb (IgG2b), which binds to J1 epitope (551-564; GERGAAGIAGPK; SEQ ID NO:28), and CIIC1 (IgG2a), which binds to C11 (359-363; ARGLT; SEQ ID NO:29) of collagen type II, was used to induce an acute form of arthritis, CAIA.
In order to determine whether the removal of carbohydrate side chains affects the arthritis-inducing capacity of pathogenic monoclonal antibodies to collagen type II, this cocktail of monoclonal antibodies, treated with EndoS or untreated, was injected into mice. Groups of male (BALB/c×B10.Q) F1 mice were injected with 9 mg of either untreated (n=7) or EndoS treated (n=5) anti-CII monoclonal antibodies (M2139 and CIIC1) on day 0. All of the mice were injected with 50 μg of E. coli LPS i.p. on day 5. Arthritis incidence (a) and mean arthritis score (b) are shown in
As can be seen from
To confirm the loss of arthritogenicity of monoclonal antibodies by removal of carbohydrate side chains of IgG, CAIA was induced in mice having another genetic background, B10.RIII.
Male B10.RIII mice were injected with 9 mg of either untreated (n=11) or EndoS treated (n=12) anti-CII monoclonal antibodies (M2139 and CIIC1) on day 0. All of the mice were injected with 50 μg of E. coli LPS i.p. on day 5. Arthritis incidence (a) and mean arthritis score (b) are shown in
Complement activation by CII-reactive monoclonal antibodies in vitro
In order to understand why the removal of carbohydrate from γ-chains of IgG reduced or abolished the clinical arthritis-inducing capacity of mAbs, in vitro experiments were performed with the EndoS treated and untreated antibodies to assess their capacity to induce complement activation.
In order to determine whether there is a functional difference in the capacity of glycosylated and deglycosylated antibodies in inducing the oxidative burst by polymorphonuclear leukocytes, PMNL (neutrophils), polystyrene microparticles were coated with EndoS treated and untreated antibodies and incubated with whole blood from mice having three different genotypes (FcgR+/+, FcgR−/− and FcgR+/−). Oxidative burst assays were then performed using FACS (Fluorescence Activated Cell Sorting) analysis. PMNLs were identified using RB6 antibodies.
The results are shown in
To check the histological status of joints from mouse paws that received glycosylated or EndoS treated mAbs, standard hematoxylin-eosin staining was used to stain 6 μm sections of formalin fixed decalcified joints from (BALB/c×B10.Q) F1 mice (n=3-4) injected with 9 mg of untreated, deglycosylated or an equal mixture of untreated and EndoS treated antibody cocktail.
The results showed that there was a massive infiltration of cells and cartilage and bone erosion in the joints from mice injected with glycosylated antibodies. In contrast, mouse paws injected with EndoS treated antibodies showed only minor bone erosion and no massive cell infiltration. The cartilage looked normal in these mice.
In order to determine whether the reduced arthritogenicity of deglycosylated antibodies was due to early and enhanced clearance of these antibodies from the mouse compared to glysosylated antibodies, analysis of collagen type II binding antibodies by ELISA (enzyme linked immunosorbent assay) was performed using the sera collected from B10.RIII mice on day 1 and 5. The results are shown in
We wished to determine whether any obvious differences exist between glycosylated and EndoS treated antibodies, apart from binding differences to FcγR molecules. It is most likely that the first step in the initial triggering event in the antibody transfer arthritis model is the formation of collagen—IgG immune complexes on the cartilage surface or in the synovium. Collagen epitopes are located in a repetitive structure formed on the cartilage surface, and hence it is possible that the two different antibodies can form multimeric complexes on the joint surfaces favouring arthritogenicity either by optimal complement activation or by binding to FcγR bearing cells.
In order to investigate the issue of stable immune complex formation, single immunodiffusion of antibodies was performed on agarose. Rat CII was impregnated in 1% agarose (low gelling temperature agarose 26-30° C.) gel at 1 mg/ml in PBS containing 0.05% sodium azide. 25 ul of antibodies at 1 mg/ml concentration were loaded per well. Gel was stained with Coomassie Blue. The results showed that deglycosylated antibodies did not form stable immune complexes compared to glysosylated mAbs. This inability to form stable immune complexes could be another explanation for the loss of arthritogenicity of deglycosylated antibodies.
Having established that EndoS efficiently hydrolyses the IgG glycan in vivo and that animals tolerated administration of the enzyme, we investigated the use of EndoS to treat a serious IgG-mediated disease. The disease model chosen was a mouse model of immune thrombocytopenic purpura (ITP). In this model polyclonal rabbit IgG directed against mouse platelets (αPLT-IgG) is injected intraperitoneally, leading to severe thrombocytopenia, bleedings, and ultimately death at higher doses of IgG.
Rabbit antiserum against mouse platelets was purchased from Inter-Cell Technologies (Jupiter, Fla.). The IgG fraction was isolated from this serum using protein G Sepharose. Protein purity was confirmed by SDS-PAGE analysis and protein concentration was determined using Advanced Protein Assay Reagent (Cytoskeleton, Denver, Colo.). For experiments using pre-treated IgG, purified rabbit anti-mouse platelet IgG (αPLT-IgG) was incubated with purified GST-EndoS or GST, at an enzyme to substrate ratio of 1:500 at 37° C. for 24 hours followed by removal of GST-EndoS and GST on a Glutathione-Sepharose (GE Healthcare). IgG glycan hydrolysis was confirmed by SDS-PAGE and lectin blotting using LCA as described above. Female BALB/c mice (approx. weight 20 g) were housed under standard conditions of light and temperature and were fed standard laboratory chow and water ad libitum. 1.2 mg of anti-mouse platelet IgG (untreated, EndoS treated, or GST treated) in 0.25 ml PBS was administered to the animals by intraperitoneal (i.p.) injection. Animals were monitored for mucocutaneous bleeds, physical activity, isolation from the group, and the survival time was recorded.
Immediately prior to the injection of rabbit anti-mouse platelet IgG and at regular intervals during the course of experiments, blood samples were collected from mice. From the pre-warmed tail vein, 5 μl of whole blood was collected into tubes containing 45 μl of 0.1 M sodium citrate/citric acid in PBS (pH 6.5). The platelet population in these blood samples was identified by flow cytometry. Samples were labeled with hamster anti-mouse CD-61 PE (BD Biosciences, San Jose, Calif.) for 10 min at room temperature. Ten μl of SPHEROa Rainbow Calibration Particles (BD Biosciences) were added to each tube, to enable counting. The red cell populations was lyzed using Utilysea (Dako Cytomation, Glostrup, Denmark) and the samples were analyzed on a FacsCalibur flow cytometer (BD Biosciences) in the logarithmic mode. The platelet number in the blood samples after lysis of red blood cells, was continued by manual counting in a Neubauer chamber.
In a pilot experiment, three female BALB/c mice were injected with 1.2 mg of αPLT-IgG and platelet counts were followed over time using flow cytometry and microscopy as described above. This revealed that all three mice rapidly developed thrombocytopenia and death occurred within 24 hours after αPLT-Ig administration (
Next, we tested if pre-treatment of αPLT-IgG with GST-EndoS, or GST as a control, prior to administration to mice had any effects on the development of disease and survival rate. All animals (n=4) injected with GST-EndoS-treated αPLT-IgG survived without developing any signs of disease, while all animals (n=4) injected with GST-treated αPLT-Ig developed severe subcutaneous bleeding and died within 24 hours (
In order to challenge our hypothesis further, we attempted to mimic the clinical situation of ITP patients. When these patients seek medical attention, the platelet count is often very low and subcutaneous and other bleeding complications are already manifest. We therefore induced disease in mice (n=14) with αPLT-IgG, but did not initiate treatment with GST-EndoS or GST until animals exhibited clearly visible cutaneous hematomas 5-7 hours after αPLT-IgG injection. In these experiments 5/7 of mice treated with GST-EndoS survived and recovered, while all (7/7) mice treated with GST died within 2 days, again representing a statistically significant difference in the survival rate between the two groups (p=0.0015) (
The mechanisms underlying the positive effects of EndoS are from a theoretical viewpoint quite clear, since the inventors have previously found that EndoS hydrolysis of IgG inhibits IgG of all subclasses from binding to FcRs and also reduces complement activation. What is of particular interest is that not only does EndoS inhibit IgG from binding FcRs, but it can also release already FcR-bound IgG by hydrolysis of the heavy chain glycan. It should also be noted that there seems to be one IgG-FcR interaction that is not affected like the others; EndoS hydrolyzed IgG does under certain circumstances bind better to human FcR11b than non-hydrolyzed IgG (data not shown). In the context of anti-inflammatory activity this might be of relevance, since IgG interactions with FcR11b have been shown to be important for the anti-inflammatory activity of Intravenous immunoglobulin (WIG) that is used to treat autoimmune conditions. Without being bound by any hypothesis, the inventors suggest that EndoS under certain circumstances may have a dual anti-inflammatory activity by directly inhibiting the binding of pathogenic IgG to activating FcRs, and shifting towards the inhibitory action mediated through FcR11b.
The properties of EndoS make it an attractive alternative to current therapies of conditions involving pathogenic antibodies, especially in the light of several recent studies establishing the IgG glycan as a key to IgG effector modulation. This includes the inventors own findings that EndoS hydrolysis of this glycan nearly abolishes complement activation through the classical pathway and reduces binding to Fc-receptors on leukocytes. Based on the inventors observations, it is shown that EndoS can be used to treat conditions where IgG antibodies play a pathogenic role, including autoimmune diseases as exemplified here by ITP, and acute antibody-mediated organ allograft rejections.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0624874.4 | Dec 2006 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/010904 | 12/12/2007 | WO | 00 | 1/20/2010 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/071418 | 6/19/2008 | WO | A |
| Number | Date | Country |
|---|---|---|
| 2006-520202 | Sep 2006 | JP |
| WO 03051914 | Jun 2003 | WO |
| WO 2006042027 | Apr 2006 | WO |
| Entry |
|---|
| Collin et al. Embo Journal 20:3046-3055, 2001. |
| Schmaldienst et al. Rheumatology, 40:513-521, 2001. |
| Naparstek and Plotz. Annual Reviews of Immunology, 11:79-104, 1993. |
| Collin, Mattias et al., “EndoS and SpeB from Streptococcus pyogenes Inhibit Immunoglobulin-Mediated Opsonophagocytosis,” Infection and Immunity, 2002, vol. 70, No. 12, pp. 6646-6651. |
| Collin, Mattias et al., “Effect of EndoS and SpeB from Streptococcus pyogenes on Human Immunoglobulins,” Infection and Immunity, 2001, vol. 69, No. 11, pp. 7187-7189. |
| Office Action in corresponding Japanese Patent Application No. 2009-540659, mailed Nov. 2, 2012, 17 pages. |
| Number | Date | Country | |
|---|---|---|---|
| 20100135981 A1 | Jun 2010 | US |
