Title: Treatment of IgA1
United States Patent: 7,407,653
Issued: August 5, 2008
Inventors: Plaut; Andrew G.
(Lexington, MA), Qiu; Jiazhou (Westborough, MA)
Assignee: Tufts Medical
Center, Inc. (Boston, MA)
Appl. No.: 10/921,676
Filed: August 19, 2004
Web Seminars -- Pharm/Biotech/etc.
The present invention discloses the use
of bacterial IgA1 proteases to treat IgA1 deposition in tissue and organs.
Bacterial IgA1 proteases specifically cleave IgA1 molecules and thus
provide a means to specifically cleave and remove IgA1 depositions.
Accordingly, therapeutic agents for the treatment of diseases
characterized by IgA deposition are provided. In particular, therapeutic
agents to treat IgA nephropathy, Dermatitis herpetiformis (DH), and
Henoch-Schoenlein purpura (HS) are disclosed.
Description of the
SUMMARY OF THE INVENTION
The present invention discloses the use of bacterial IgA1 proteases to treat
IgA1 deposition in tissue and organs. Bacterial IgA1 proteases specifically
cleave IgA1 molecules and thus provide a means to specifically cleave and
remove IgA1 depositions. Accordingly, therapeutic agents for the treatment
of diseases characterized by IgA deposition are provided. In particular,
therapeutic agents to treat IgA nephropathy, Dermatitis herpetiformis (DH),
and Henoch-Schoenlein purpura (HS) are disclosed.
Disclosed herein is a nucleic acid molecule encoding an IgA1 protease that
is fused to an amino acid tag located upstream of an IgA1 protease
auto-catalytic cleavage site.
In one embodiment, the tag, which is fused to the IgA1 protease, is a tag
that specifically binds to a protein ligand, such as an antibody or peptide.
The tag can be c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS.
In one aspect, a pharmaceutical composition for the treatment of IgA1
deposition is provided that comprises an IgA1 protease complexed with an
antibody, such as an anti-IgA1 protease antibody.
In another aspect, a pharmaceutical composition for the treatment of IgA1
deposition is provided that comprises a tagged IgA1 protease that is
complexed with a ligand of the tag. The tag fused to the IgA1 protease can
be c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS. Accordingly, the ligand can be
an anti-tag antibody such as anti-FLAG, anti-MYC, anti-VSV, anti-HA, or
anti-V5. Alternatively, the ligand can be a peptide or non-peptide ligand,
such as a chelating molecule.
In another aspect, a method for treatment of a disease characterized by IgA1
deposition is provided. The method involves administering to a patient a
therapeutically effective amount of an IgA1 protease.
In one embodiment, the method for treatment uses an IgA1 protease fused to a
tag complexed with a ligand of the tag, such as an anti-tag antibody. The
tag fused to the IgA1 protease can be c-Myc, Flag, HA, VSV-G, HSV, FLAG, V5,
or HIS. Accordingly, the anti-tag antibody can be anti-FLAG, anti-MYC, anti-VSV,
anti-HA, or anti-V5.
In another embodiment, the disease characterized by IgA1 deposition is IgA
nephropathy, Dermatitis herpetiformis, or Henoch-Schoenlein purpura.
The present invention relates to the use of bacterial Immunoglobulin A1
proteases (IgA1 proteases) to treat diseases that are characterized by IgA1
I. IgA1 Proteases
Herein, IgA1 proteases are used to treat diseases characterized by IgA1
deposition. IgA1 proteases are bacterial enzymes that specifically cleave
human IgA1 molecules. Human IgA2 is resistant to nearly all known IgA1
proteases because IgA2 molecules lack a hinge region that is present in all
IgA1 molecules. The hinge region of IgA1 molecules consist of a string of
amino acids, that contain cleavage sites for a variety of IgA1 proteases, as
illustrated in FIG. 1 (see Original Patent). IgA1 proteases are expressed in
gram-negative bacteria as a single-chain precursor that traverses the inner
membrane of bacterium. The precursor protein then inserts itself into the
outer bacterial membrane and undergoes auto-catalytic cleavage, releasing a
mature soluble IgA1 protease (FIG. 2a (see Original Patent)). IgA proteases
of gram-positive bacteria are also useful in this invention, although they
do not have an autocatalytic secretion mechanism. For such proteases, an
epitope tag may be added into the enzyme protein.
In one embodiment of the present invention a tag sequence is fused in frame
to an IgA1 protease, such that the tag sequence is located near the carboxyl
terminus of the secreted IgA1 protease (FIG. 2b (see Original Patent)). FIG.
3 (see Original Patent) shows a schematic of the Haemophilus influenzae Rd
IgA1 protease precursor protein illustrating that a tag sequence (e.g. His
tag) is fused in frame to an IgA1 protease upstream of the auto-catalytic
cleavage sites a, b and c.
A variety of bacteria produce IgA1 proteases and are useful in the present
invention. These include, but are not limited to Haemophilus influenzae type
1 and 2, Neisseria meningitidis type 1 and 2, Neissseria gonorrhoeae,
Streptococcus pneumoniae, Streptococcus sanguis, Clostridium ramosum,
Prevotella melaninogenica, and Ureaplasma realyticum.
The IgA1 protease nucleotide sequences of the present invention can be
obtained from any bacteria where an IgA1 protease is expressed, as long as
the IgA1 protease is capable of cleaving human IgA1 molecules. Nucleotide
sequences encoding IgA1 proteases from numerous bacterial strains have
already been identified and include: Clostridium ramosum (Genbank Accession
number, AY028440); Ureaplasma urealyticum (Genbank Accession number, NC.sub.--002162);
Haemophilus influenzae (Genbank Accession number, X59800) and bacterial
strains Rd (Genbank Accession number, NC-000907), 7768 (Genbank Accession
number, AF274862), 6338 (Genbank Accession number, AF27486), 2509 (Genbank
Accession number, AF274859), aegyptius (Genbank Accession number, AF369907),
8625 (Genbank Accession number, AJ001741), HK284 (Genbank Accession number,
X82487), Da66 (Genbank Accession number, X82467), HK635 (Genbank Accession
number, X82488), and other deposited sequences from unidentified strains (Genbank
Accession numbers, X59800, X82488, X64357, M87492, M87491, M87490, and
M87489); Neisseria meningitidis (Genbank Accession number AF235032) and
bacterial strains, Z2491 (Genbank Accession number, NC-03316), B40 (Genbank
Accession number, AF012211), Z4099 (Genbank Accession number, AF012210),
Z4018 (Genbank Accession number, AF012209), Z4400 (Genbank Accession number,
AF012208), Z3524 (Genbank Accession number, AF012207), Z40204 (Genbank
Accession number, AF012206), Z3910 (Genbank Accession number, AF012205),
Z3906 (Genbank Accession number, AF012204), Z2491 (Genbank Accession number,
AF012203), IHN341 (Genbank Accession number, AJ001740), NL3327 (Genbank
Accession number, AJ001739), NL823 (Genbank Accession number, AJ001737),
NL3293 (Genbank Accession number, AJ001738), HK284 (Genbank Accession
number, X82487), ETH2 (Genbank Accession number, X82469), NG093 (Genbank
Accession number, X82482), NCG80 (Genbank Accession number, X82479), NG117 (Genbank
Accession number, X82483), HF96 (Genbank Accession number, X82475), HF54 (Genbank
Accession number, X82473), HF48 (Genbank Accession number, X82480), HF13 (Genbank
Accession number, X82474), NGC65 (Genbank Accession number, X82484), NCG16 (Genbank
Accession number, X82485), SM1894 (Genbank Accession number, X82476), EN3771
(Genbank Accession number, X82468), NG44/76 (Genbank Accession number,
X82481), SM1166 (Genbank Accession number, X82486), HF159 (Genbank Accession
number, X82471), 81139 (Genbank Accession number, X82477), HF117 (Genbank
Accession number, X82470), SM1027 (Genbank Accession number, X82472) and
Genbank Accession number, AF235032; Neissseria gonorrhoeae (Genbank
Accession number, A12416) and bacterial strain, MS11 (Genbank Accession
number, S75490); Streptococcus pneumoniae (Genbank Accession number, X94909)
and bacterial strains MGAS315 (Genbank Accession number, NC-004070), R6 (Genbank
Accession number, NC-003098); and Streptococcus sanguis (Genbank Accession
number, NC-003098) and bacterial strains SK85 (Genbank Accession number,
Y13461), SK49 (Genbank Accession number, Y13460), SK4 (Genbank Accession
number, Y13459), SK162 (Genbank Accession number, Y13458), SK161 (Genbank
Accession number, Y13457), SK115 (Genbank Accession number, Y13456, and
Sk112 (Genbank Accession number, Y13455). IgA1 proteases of the invention
may be utilized as described herein either without or with an attached tag
as described below.
In the present invention, sequences encoding IgA1 proteases are cloned into
vectors suitable for expression of the protein, such that soluble IgA1
protease can be produced and isolated. The vectors can be constructed using
standard methods (Sambrook et al., Molecular Biology: A laboratory Approach,
Cold Spring Harbor, N.Y. 1989; Ausubel, et al., Current protocols in
Molecular Biology, Greene Publishing, 1995), guided by the principles
discussed below. In brief, conventional ligation techniques are used to
insert DNA sequences encoding IgA1 protease into a bacterial cloning and/or
To prepare nucleic acids encoding IgA1 protease, a source of genes encoding
for IgA1 proteases is required. The genes can be obtained from natural or
synthetic sources. Methods for cloning novel IgA1 protease genes from
bacterial strains are described in Lomholt H., et al., Mol. Microbiol.
(1995) 15(3), 495-508; Fishman, Y. et al., (1985), p. 164-168 in G. K.
Schoolink (ed.), The Pathogenic Neisseria, Am. Soc. Microbiol., Washington
D.C.; Koomey, J. et al., Proc. Natl, Acad. Sci. USA, (1982) 79: 7881-7885;
Halter, R, et al., EMBO J., (1984) 3: 1595-1601; Bricker, J. et. al., Proc,
Natl. Acad. Sci. USA, (1983), 80:2681-2685; Koomey, J. M. and Falkow, S.,
supra; Grundy, J. F. et al., J. Bacteriol, (1987) 169:4442-4450; and
Gilbert, J. V. et al., Infect. Immun., (1988) 56:1961-1966, all of which are
herein incorporated by reference.
Alternatively, DNA encoding a known IgA1 protease can be isolated from
bacterial genomic DNA by polymerase chain reaction (PCR) amplification using
primers specific for the IgA1 protease gene of interest. Briefly, bacterial
genomic DNA is isolated using methods well known in the art, for example
using bacterial genomic DNA isolation kits provided by QIAGEN or standard
methods described in Sambrook et al., Molecular Biology: A laboratory
Approach, Cold Spring Harbor, N.Y. (1989) and Ausubel, et al., Current
protocols in Molecular Biology, Greene Publishing, (1995), herein
incorporated by reference.
PCR is well known in the art (Mullis and Faloona, Methods Enzymol., (1987),
155: 335, herein incorporated by reference). In general, oligonucleotide
primers useful according to the invention are single-stranded DNA or RNA
molecules that hybridize selectively to a nucleic acid template that encodes
IgA1 protease to prime enzymatic synthesis of a second nucleic acid strand.
The primer is complementary to a portion of a target molecule present in a
pool of nucleic acid molecules from the bacterial genome. It is contemplated
that primers are prepared by synthetic methods, either chemical or
enzymatic. Alternatively, such a molecule or a fragment thereof is naturally
occurring, and is isolated from its natural source or purchased from a
commercial supplier. Mutagenic oligonucleotide primers are 15 to 100
nucleotides in length, ideally from 20 to 40 nucleotides, although
oligonucleotides of different length are of use. Preferably, the primers
also comprise a unique restriction enzyme sequence.
Typically, selective hybridization occurs when two nucleic acid sequences
are substantially complementary (at least about 65% complementary over a
stretch of at least 14 to 25 nucleotides, preferably at least about 75%,
more preferably at least about 90% complementary). See Kanehisa, Nucleic
Acids Res., (1984), 12: 203, incorporated herein by reference. As a result,
it is expected that a certain degree of mismatch at the priming site is
tolerated. Such mismatch may be small, such as a mono-, di- or
tri-nucleotide. Alternatively, it may comprise nucleotide loops, which we
define as regions in which mismatch encompasses an uninterrupted series of
four or more nucleotides.
Overall, five factors influence the efficiency and selectivity of
hybridization of the primer to a second nucleic acid molecule. These
factors, which are (i) primer length, (ii) the nucleotide sequence and/or
composition, (iii) hybridization temperature, (iv) buffer chemistry and (v)
the potential for steric hindrance in the region to which the primer is
required to hybridize, are important considerations when non-random priming
sequences are designed.
There is a positive correlation between primer length and both the
efficiency and accuracy with which a primer will anneal to a target
sequence: longer sequences have a higher melting temperature (TM) than do
shorter ones, and are less likely to be repeated within a given target
sequence, thereby minimizing promiscuous hybridization. Primer sequences
with a high G-C content or that comprise palindromic sequences tend to self-hybridise,
as do their intended target sites, since unimolecular, rather than
bimolecular, hybridization kinetics are generally favored in solution: at
the same time, it is important to design a primer containing sufficient
numbers of G-C nucleotide pairings to bind the target sequence tightly,
since each such pair is bound by three hydrogen bonds, rather than the two
that are found when A and T bases pair. Hybridization temperature varies
inversely with primer annealing efficiency, as does the concentration of
organic solvents, e.g. formamide, that might be included in a hybridization
mixture, while increases in salt concentration facilitate binding. Under
stringent hybridization conditions, longer probes hybridize more efficiently
than do shorter ones, which are sufficient under more permissive conditions.
Stringent hybridization conditions typically include salt concentrations of
less than about 1M, more usually less than about 500 mM and preferably less
than about 200 mM. Hybridization temperatures range from as low as 0.degree.
C. to greater than 22.degree. C., greater than about 30.degree. C., and
(most often) in excess of about 37.degree. C. Longer fragments may require
higher hybridization temperatures for specific hybridization. As several
factors affect the stringency of hybridization, the combination of
parameters is more important than the absolute measure of any one alone.
Primers preferably are designed using computer programs that assist in the
generation and optimization of primer sequences. Examples of such programs
are "PrimerSelect" of the DNAStar.TM. software package (DNAStar. Inc.;
Madison, Wis.) and OLIGO 4.0 (National Biosciences. Inc.). Once designed,
suitable oligonucleotides are prepared by a suitable method, e.g. the
phosphoramidite method described by Beaucage and Carruthers (1981)
Tetrahedron Lett., 22: 1859) or the triester method according to Matteucci
and Caruthers (1981) J. Am. Chem. Soc., 103: 3185, both incorporated herein
by reference, or by other chemical methods using either a commercial
automated oligonucleotide synthesizer or VLSIPS.TM. technology.
PCR is performed using template bacterial DNA (at least 1 fg: more usefully,
1-1000 ng) and at least 25 pmol of oligonucleotide primers; it may be
advantageous to use a larger amount of primer when the primer pool is
heavily heterogeneous, as each sequence is represented by only a small
fraction of the molecules of the pool, and amounts become limiting in the
later amplification cycles. A typical reaction mixture includes: 2 .mu.l of
DNA, 25 pmol of oligonucleotide primer, 2.5 .mu.l of 10.times. PCR buffer 1
(Perkin-Elmer, Foster City, Calif.), 0.4.mu. of 1.25 mM dNTP, 0.15 .mu.l (or
2.5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, Calif.) and
deionized water to a total volume of 25 .mu.l. Mineral oil is overlaid and
the PCR is performed using a programmable thermal cycler.
The length and temperature of each step of a PCR cycle, as well as the
number of cycles, is adjusted in accordance to the stringency requirements
in effect. Annealing temperature and timing are determined both by the
efficiency with which a primer is expected to anneal to a template and the
degree of mismatch that is to be tolerated; obviously, when nucleic acid
molecules are simultaneously amplified and mutagenised, mismatch is
required, at least in the first round of synthesis. An annealing temperature
of between 30.degree. C. and 72.degree. C. is used. Initial denaturation of
the template molecules normally occurs at between 92.degree. C. and
99.degree. C. for 4 minutes, followed by 20-40 cycles consisting of
denaturation (94-99.degree. C. for 15 seconds to 1 minute), annealing
(temperature determined as discussed above: 1-2 minutes), and extension
(72.degree. C. for 1-5 minutes, depending on the length of the amplified
product). Final extension is generally for 4 minutes at 72.degree. C., and
may be followed by an indefinite (0-24 hour) step at 4.degree. C.
Subsequent to PCR amplification, the DNA can be isolated by standard means,
such as gel electrophoresis, or column purification. The DNA encoding the
bacterial IgA1 protease can then be digested with appropriate restriction
enzymes and ligated into a suitable cloning and/or expression vector.
Vectors and Host Cells
Any vector can be used in the present invention. As used herein, vector
refers to a discrete element that is used to introduce heterologous DNA into
bacterial cells for the expression and/or replication thereof. Numerous
vectors suitable for the present invention are publicly available, including
bacterial plasmids and bacteriophage. Each vector contains various
functional components, which generally include a cloning (or "polylinker")
site, an origin of replication and at least one selectable marker gene. If
given vector is an expression vector, it additionally possesses one or more
of the following: enhancer element, promoter, transcription termination and
signal sequences, each positioned in the vicinity of the cloning site, such
that they are operatively linked to the gene encoding an IgA1 protease
according to the invention.
Both cloning and expression vectors generally contain nucleic acid sequences
that enable the vector to replicate in one or more selected host cells.
Typically in cloning vectors, this sequence is one that enables the vector
to replicate independently of the host chromosomal DNA and includes origins
of replication or autonomously replicating sequences. Such sequences are
well known for a variety of bacteria. For example, the origin of replication
from the plasmid pBR322 is suitable for most Gram-negative bacteria.
Advantageously, a cloning or expression vector may contain a selection gene
also referred to as a selectable marker. This gene encodes a protein
necessary for the survival or growth of transformed host cells grown in a
selective culture medium. Host cells not transformed with the vector
containing the selection gene will therefore not survive in the culture
medium. Typical selection genes encode proteins that confer resistance to
antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or
tetracycline, complement auxotrophic deficiencies, or supply critical
nutrients not available in the growth media.
Since the replication of vectors according to the present invention is most
conveniently performed in E. coli, an E. coli-selectable marker, for
example, the .beta.-lactamase gene that confers resistance to the antibiotic
ampicillin, is of use. These can be obtained from E. coli plasmids, such as
pBR322 or a pUC plasmid such as pUC18 or pUC19.
Expression vectors usually contain a promoter that is recognized by the host
organism and is operably linked to the coding sequence of interest. Such a
promoter may be inducible or constitutive. 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 control sequence "operably
linked" to a coding sequence is ligated in such a way that expression of the
coding sequence is achieved under conditions compatible with the control
Promoters suitable for use with prokaryotic hosts include, for example, the
.beta.-lactamase and lactose promoter systems, alkaline phosphatase, the
tryptophan (trp) promoter system and hybrid promoters such as the tac
promoter. Promoters for use in bacterial systems will also generally contain
a Shine-Delgarno sequence operably linked to the coding sequence. A
preferred promoters of the present invention are the
isopropylthiogalactoside (IPTG)-regulatable promoters.
Any bacterial strain is considered a suitable host cell for expression of
and cloning of the IgA1 proteases of the present invention. An exemplary
host is E. coli.
Introduction of Vectors to Host Cells.
Vectors can be introduced to selected host cells by any of a number of
suitable methods known to those skilled in the art. For example, vector
constructs may be introduced to appropriate bacterial cells by infection
using bacteriophage vector particles such as lambda or M13, or by any of a
number of transformation methods for plasmid vectors or for bacteriophage
DNA. For example, standard calcium-chloride-mediated bacterial
transformation is still commonly used to introduce naked DNA to bacteria (Sambrook
et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.), but electroporation may also be
used (Ausubel et al., Current Protocols in Molecular Biology, (1988), (John
Wiley & Sons, Inc., NY, N.Y.)).
Purification of Soluble IgA1 Protease
After introduction of an expression vector encoding IgA1 protease into a
suitable bacterial host cell, the bacteria are propagated for the
overproduction of soluble IgA1 protease by standard means (Sambrook et al.,
Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y. (1989)
and Ausubel, et al., Current protocols in Molecular Biology, Greene
Publishing, (1995), herein incorporated by reference). Briefly, bacteria,
such as E. coli, which harbor an expression vector that encodes IgA1
protease, or bacteria that have DNA encoding IgA1 protease integrated into
the bacterial genome, are grown in bacterial growth media at 37.degree. C.
When the bacterial cultures reach log phase, soluble IgA1 protease is
purified from the growth media by means well known in the art.
For example, H. influenzae Rd bacteria that express 6.times.His-IgA1
protease are cultured as 20 L (two 10 L) in a fermentor charged with
brain-heart infusion broth supplemented with NAD and hemin. The cells are
grown at 37.degree. C. until they reach stationary phase, 16-20 h. The
bacterial mass is then removed with a Pellicon system, and each 10 L of
culture supernatant containing the active enzyme is concentrated to 400 ml.
The buffers are adjusted to have the protein in 25 mM Tris/HCl buffer, pH
7.5, with 0.05% NaN3. To remove unwanted protein, 80 ml batches of this
concentrate is applied to a 40 ml bed-volume DE-52 anion-exchange column
equilibrated in 25 mM Tris buffer. IgA protease does not bind to this
column, and is collected as flow through using 500 ml Tris buffer. Yield of
recovery is typically 85-90% based on assay using human IgA substrate.
Ammonium sulfate is then used to precipitate the enzyme (60% saturation
ammonium sulfate; 390 gm per L). The precipitate is dissolved with the
following buffer: 50 mM sodium phosphate, 12.5 mM Tris/HCl, 0.3 M NaCl and
0.025% sodium azide, adjusted to pH 7.5, and the enzyme is then dialyzed
against this buffer for several days. The final volume of enzyme solution is
approximately 200 ml for each 10 L of starting culture.
For affinity purification, 40 ml aliquots of the enzyme solution is applied
to Ni-NTA-agarose in a column with bed volume of 40 ml. The bound enzyme is
washed three times with volumes of 500 ml of buffers containing 50 mM sodium
phosphate, 12.5 mM Tris/HCl, 0.3 M NaCl and 0.025% sodium azide. pH of these
buffer washes is reduced in stepwise fashion, beginning with pH 7.5, then
6.6, then 6.0, intended to remove weakly adherent, non-enzyme proteins from
the nickel ligand. The final wash again uses buffer at pH 7.5, now 200 ml.
The 6.times.His-IgA protease is then eluted from Ni-NTA agarose using 50 ml
0.1 M imidazole in 50 mM Tris/HCl, pH 7.5. The recovered enzyme is
concentrated by positive pressure filtration using a 100 kDa cut-off
Centricon membrane, washed three times with 25 mM Hepes, pH 7.15, and then
stored in Hepes buffer.
Assay for IgA1 Protease Activity
The IgA1 protease is tested for enzyme activity by standard means as
described in Plaut, AG and Bachovchin WW, IgA-specific prolyl endopeptidases:
serine type. Methods Enzymol. 1994;244:137-51, herein incorporated by
reference. The assay can be performed with purified protease or IgA1
protease present in bacterial growth media. An IgA1 protease has sufficient
activity to be useful according to the invention if it has one Unit
activity, with Unit equal to one microg human IgA1 cleaved per minute at
II. Tagged IgA1 Protease
In one embodiment, the IgA1 protease is fused to a tag, although the
invention may be practiced in the absence of a tag and/or ligand complexed
thereto. Fusing a tag to the IgA1 proteases of the present invention aids in
purification and detection of the protease, as well as provides a means in
which the IgA1 protease can form a complex with a ligand, such as an
anti-tag antibody, for therapeutic purposes.
To generate an IgA protease comprising a tag, a sequence encoding a tag can
be ligated in frame to a sequence encoding an IgA1 protease using
conventional molecular biology techniques. The tag sequence is ligated
upstream of the DNA sequence encoding the IgA1 protease auto-catalytic
cleavage site such that, upon cleavage of the IgA1 protease precursor
protein, a soluble IgA1 protease comprising a tag is secreted into bacterial
Alternatively, an IgA1 protease comprising a tag is generated by PCR-based
site directed mutagenesis. There are a number of site-directed mutagenesis
methods known in the art which allow one to mutate specific regions within a
protein. These methods are embodied in a number of kits available
commercially for the performance of site-directed mutagenesis, including
both conventional and PCR-based methods. Examples include the EXSITE.TM. PCR-based
site-directed mutagenesis kit available from Stratagene (Catalog No. 200502;
PCR based) and the QUIKCHANGE.TM. site-directed mutagenesis kit from
Stratagene (Catalog No. 200518; PCR based), and the CHAMELEON.RTM.
double-stranded site-directed mutagenesis kit, also from Stratagene (Catalog
No. 200509). Briefly, a tag sequence is introduced into a PCR fragment by
inclusion of a sequence encoding the tag near the 5' or 3' end of one of the
PCR primers. The PCR fragment is generated in a manner to provide
appropriate restriction sites such that the fragment can be digested then
ligated into parental vector for replacement of specific amino acid codons.
In one embodiment, the tag of the present invention has a specific binding
affinity for an antibody, so that the protease forms an immuno-complex upon
binding ligand. For example, the tag may comprise a unique epitope for which
antibodies are readily available. Alternatively, the tag can comprise
metal-chelating amino acids (e.g. His) so that the IgA proteases can complex
with a metal-chelating resin or bead, for example nickle-NTA beads.
In another embodiment, the tag comprises a detectable marker, such as an
enzyme, or comprises an amino acid that can be labeled with a detectable
marker. Detectable markers include, for example, radioisotopes, fluorescent
molecules, chromogenic molecules, luminescent molecules, and enzymes. Useful
detectable markers in the present invention include biotin for staining with
labeled streptavidin conjugate, fluorescent dyes (e.g., fluorescein, texas
red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g.,
.sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P), enzymes (e.g., horse
radish peroxidase, alkaline phosphatase and others commonly used in an
ELISA), and calorimetric labels such as colloidal gold. Patents teaching the
use of such detectable markers include U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, the entireties of
which are incorporated by reference herein.
Non-limiting examples of suitable tags according to the invention include c-Myc,
HA, and VSV-G, HSV, FLAG, V5, and HIS. The amino acid and nucleic acid
sequence for each tag is shown below
-- see Original Patent.
Placing a tag on an IgA1 protease has the benefit of enabling easy detection
of the IgA1 protease both in vivo and in vitro. A tag that comprises an
epitope for an antibody can be detected either using anti-tag antibodies or
antibodies that are conjugated to a detectable marker. The detectable marker
can be a naturally occurring or non-naturally occurring amino acid that
bears, for example, radioisotopes (e.g., .sup.125I, .sup.35S), fluorescent
or luminescent groups, biotin, haptens, antigens and enzymes. There are many
commercially available Abs to tags, such as c-myc, HA, VSV-G, HSV, V5, His,
and FLAG. In addition, antibodies to tags used in the invention can be
produced using standard methods to produce antibodies, for example, by
monoclonal antibody production (Campbell, A. M., Monoclonal Antibodies
Technology: Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, the Netherlands (1984); St. Groth et
al., J. Immunology, (1990) 35: 1-21; and Kozbor et al., Immunology Today
(1983) 4:72). The anti-tag antibodies can then be detectably labeled through
the use of radioisotopes, affinity labels (such as biotin, avidin, etc.),
enzymatic labels (such as horseraddish peroxidase, alkaline phosphatase,
etc) using methods well known in the art, such as described in international
application WO 00/70023 and (Harlour and Lane (1989) Antibodies, Cold Spring
Harbor Laboratory, pp. 1-726), herein incorporated by reference.
Assays for detecting tags include, but are not limited to, Western Blot
analysis, Immunohistochemistry, Elisa, FACS analysis, enzymatic assays, and
autoradiography. Means for performing these assays are well known to those
of skill in the art. For example, radiolabels may be detected using
photographic film or scintillation counters and fluorescent markers may be
detected using a photodetector to detect emitted light. Enzymatic labels are
typically detected by providing the enzyme with a substrate and detecting
the reaction product produced by the action of the enzyme on the substrate,
and calorimetric labels are detected by simply visualizing the colored
The tag can be further used to isolate the IgA1 protease away from other
cellular material. For example, by immunoprecipitation, or by using anti-tag
antibody affinity columns or anti-tag antibody conjugated beads. When a HIS
tag is used, isolation can be performed using a metal-chelate column (See
Hochuli in Genetic Engineering: Principles and Methods ed. J K Setlow,
Plenum Press, NY, chp 18, pp 87-96). Means for performing these types of
purification are well known in the art.
In a preferred embodiment, an anti-tag antibody is used to generate an IgA1
protease immuno-complex such that the IgA1 protease retains enzymatic
activity once complexed. Such an immuno-complex can be used in
pharmaceutical preparations for the treatment of IgA1 deposition diseases.
For example, an IgA1 immuno-complex, when administered to a patient, is
believed to become trapped in the glomerulous of the kidney, a site of IgA1
deposition in IgA nephropathy and Henoch-Schoenlein purpura disease.
III. Treatment of IgA1 Deposition Diseases
Herein, IgA1 proteases are used as therapeutic agents to treat IgA1
deposition diseases. The abnormal deposition of IgA1 molecules is known to
cause renal failure, skin blistering, rash, arthritis, gastrointestinal
bleeding and abdominal pain.
In one embodiment, the invention provides a method for treating IgA
nephropathy by administering to a patient in need of such treatment an IgA1
protease. IgA nephropathy is a disease of the kidney. The disease is
considered to be an immune-complex-mediated glomerulonephritis, which is
characterized by granular deposition of IgA1 in the glomerular mesangial
areas. Nephropathy results and is defined by proliferative changes in the
glomerular mesangial cells.
IgA nephropathy is one of the most common types of chronic
glomerulonephritis and a frequent cause of end-stage renal disease.
The invention further provides a method for treating Dermatitis
herpetiformis (DH) by administering to a patient in need of such treatment
an IgA1 protease. Dermatitis herpetiformis is a chronic blistering skin
disease associated with deposits of IgA1 at the dermal-epidermal junction
(Hall, R P & T. J. Lawley, J. Immunol. (1985) 135(3): 1760-5). DH patients
have granular IgA1 deposits and have an associated gluten-sensitive
In another embodiment, the invention provides a method for treating
Henoch-Schoenlein purpura (HS) by administering to a patient in need of such
treatment an IgA1 protease. Henoch-Schoenlein purpura is a skin and kidney
disease. HSP is characterized by deposition of IgA1-containing immune
complexes in tissue. The disease is diagnosed by observing evidence of IgA1
deposition in the skin tissue or kidney via immunofluorescence microscopy.
The clinical manifestations typically include rash; arthralgias; abdominal
pain; and renal disease.
The therapeutic effect of IgA proteases of the present invention can be
tested in any suitable animal model known to those skilled in the art. Some
exemplary animal models are described below.
1. IgA Nephropathy
A number of rat and mice animal models of IgA nephropathy are available and
are useful in the present invention. These models are described in
Emancipator, S. N. et al., (1987) Animal models of IgA nephropathy In IgA
nephropathy. A. R. Clarkson, editor. Martinus Nijhoff publishing, Boston.
188-203, herein incorporated by reference in its entirety. An exemplary
model is described in Gesualdo L. et al, (1990) J. Clin. Invest. 86:
715-722, also herein incorporated in its entirety. Briefly, an IgA antibody/dextran
sulfate complex is injected into mice. The immuno-complex lodges in the
kidney and the mice present with glomerulonephritis that resembles typical
cases of human IgA nephropathy. It is preferred that in the above models,
human IgA1 is introduced and expressed in the model as described further in
the Examples. How the model is made and used for testing therapeutic agents
is described in more detail below.
Soluble immune complexes of dextran sulfate (500 kD, Sigma Chemical Co., St.
Louis, Mo.) and monoclonal IgA anti-.beta.1-6 glycoside (J558: Litton
Bionetics, Kensington, Md.) are prepared at threefold excess (26.5 .mu.g
dextran/mg J558 (Nephropathy model); 22.0 .mu.g dextran/mg MOPC 104 E
(normal control)). Complexes containing 3 mg antibody are injected into
Swiss-Webster mice via tail vein injection. After 1 hour, the point of
maximal deposition of IgA complexes in the kidney, mice are injected
intraperitoneally with multiple doses of either saline or therapeutic agent
at given intervals, such as 10 minute intervals. The mice are killed 1 hour
after the last injection.
Kidneys are then isolated from each mouse to look at IgA1 deposition and
morphology by light, immunofluoresence, and electron microscopy.
Briefly, to monitor IgA1 deposition, snap-frozen samples of renal cortex,
cryostat sectioned at 4 um, are stained with fluoresceinated IgG fractions
of goat antisera specific for mouse IgA (US Biochemical Corp) by direct
immunofluoresence to semiquantitatively score for IgA1 deposits (Nakazawa,
M. et al., (1986) Lab. Invest. 55:551-556, and Nakazawa, M. et al., (1986)
J. Exp. Med. 164:1973-1987). A therapeutic agent is regarded as an effective
agent when the number of IgA1 deposits scored is reduced towards the number
of IgA1 deposits observed in a normal kidney.
Morphological changes, such as expansion of mesangial matrix and mesangial
hypercellularity, is scored by staining sections of renal cortex with PAS
reagent (Gesualdo, L. et al, (1990) J. Clin. Invest. 86: 715-722). Briefly,
renal cortex is fixed in 10% formalin, embedded in paraffin and stained.
Expansion of mesangial matrix and mesangial hypercellularity is scored
semiquantitatively according to the methods described in Nakazawa, M. et al.
(1986) Lab. Invest. 55:551-556, and Nakazawa, M. et al. (1986) J. Exp. Med.
164:1973-1987, herein incorporated by reference in their entirety.
Normal mesangial matrix is scored as 0. Expansion of mesangial matrix is
scored as +1 when widened mesangial stalks are observed, +2 when matrix
encroachment on capillary lumens is observed, and +3 when conspicuous
widening of mesingial stalk is observed along with a decrease in capillary
lumen. A therapeutic agent is regarded as effective agent when the expansion
of mesangial matrix is reduced towards the morphology of the matrix observed
in a normal kidney, for example to a score of +2, or +1, or 0.
Normal mesangial cellularity is scored as 0 and is defined as 3 or fewer
cell nuclei per mesengial area. Hypercellularity is scored as +1 when 4 to 6
cell nuclei per mesengial area are observed, as +2 when 4 to 6 cell nuclei
per mesengial area are observed in most areas but some areas have 7 or more
nuclei, and as +3 when 7 or more cell nuclei per mesengial area are observed
in most areas. A therapeutic agent is regarded as effective agent when the
mesangial hypercellularity is reduced towards that observed in a normal
kidney, for example to a score of +2, or +1, or 0.
Total glomerular area, matrix area, and glomerular cellularity are also
quantified in randomly selected glomeruli from each mouse by computer
morphometry (Cue image analysis system, Olympus Corp., Columbia, Md.) (Gesualdo
L. et al, (1990) J. Clin. Invest. 86: 715-722). Briefly, cubes of cortex are
fixed in 2.5% gluteraldehyde in 0.1 M sodium cacodylate, post fixed in 1%
OsO.sub.4, and embedded in Spurr's epoxy (Polysciences, Inc. Warrington,
Pa.). 50-70 nm sections are stained with uranyl acetate and lead hydroxide.
Coded grids are examined in a JEOL JEM 100 EX microscope and matrix,
cellularity, and immune deposits are semiquantified as described in Nakazawa,
M. et al., (1986) J. Exp. Med. 164:1973-1987, herein incorporated by
reference in its entirety.
Hematuria (the presence of red blood cells in urine) and proteinura (the
presence of protein in urine) are also a suitable measure of IGA
Nephropathy. Briefly, mice are placed in metabolic cages and urine is
collected for 24 hours. The urine is then centrifuged and assayed for
protein by turbidimetry in 3% sulfalicylic acid and hematuria by microscopy,
as described in Nakazawa, M. et al., (1986) J. Exp. Med. 164:1973-1987,
herein incorporated by reference in its entirety. Typically, a normal mouse
without IgA nephropathy will have less then three red blood cells per high
power field (40.times.), while mice with IgA nephropathy will have greater
than 10 red blood cells per high power field. A reduction in the number of
red blood cells per high power field is indicative that the therapeutic
agent is effective for IgA nephropathy. Mice are tested for hematuria and
proteinura before treatment to determine the reference value indicative of
disease. A reduction in the reference value, as compared to the value for
hematuria and proteinura obtained before treatment, of 5%, 10%, 30%, 40%
preferably 50%, and more preferably greater than 50% after treatment with
the therapeutic agent is indicative that the agent is effective for
treatment of IgA1 Nephropathy.
IV Dosage, Formulation and Administration
Herein, bacterial IgA proteases are used to treat IgA deposition diseases.
The IgA1 protease of the present invention can be used in a composition that
is combined with a pharmaceutically acceptable carrier. Such a composition
may also contain diluents, fillers, salts, buffers, stabilizers,
solubilizers, and other materials well known in the art. In one aspect, the
IgA1 protease is complexed with an antibody to form a therapeutic immuno-complex.
Such a therapeutic immuno-complex is particularly useful for treatment of
diseases characterized by IgA1 deposition in the kidney since the large
immuno-complex is believed to lodge in the renal glomerulus upon
In an alternate embodiment, the pharmaceutical formulation may include two
or more different IgA proteases, administered together or sequentially,
providing a synergistic effect. For example, an IgA protease of H.
influenzae, a serine-type protease, may be admininstered with an IgA
protease of Streptococcus sanguis, an entirely different metal-dependent
protease. Such combined or sequential administration of different proteases
may be useful because the enzymes may interact with (e.g., bind to) the IgA1
substrate in different ways, thus providing an advantage over single
The term "pharmaceutically acceptable" means a non-toxic material that does
not interfere with the effectiveness of the biological activity of the
active ingredient(s). The characteristics of the carrier will depend on the
route of administration. Such carriers include, but are not limited to,
saline, buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. For drugs administered orally, pharmaceutically
acceptable carriers include, but are not limited to pharmaceutically
acceptable excipients such as inert diluents, disintegrating agents, binding
agents, lubricating agents, sweetening agents, flavoring agents, coloring
agents and preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn starch and
alginic acid are suitable disintegrating agents. Binding agents may include
starch and gelatin, while the lubricating agent, if present, will generally
be magnesium stearate, stearic acid or talc. If desired, tablets may be
coated with a material such as glyceryl monostearate or glyceryl distearate,
to delay absorption in the gastrointestinal tract.
Pharmaceutically acceptable salts can be formed with inorganic acids such as
acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate
butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,
glycerophosphate, hemisulfate heptanoate, hexanoate, hydrochloride
hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, thiocyanate,
tosylate and undecanoate. Base salts include ammonium salts, alkali metal
salts such as sodium and potassium salts, alkaline earth metal salts such as
calcium and magnesium salts, salt with organic bases such as
dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids
such as arginine, lysine, and so forth. Also, the basic nitrogen-containing
groups can be quarternized with such agents as lower alkyl halides, such as
methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl
sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates, long chain
halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and
iodides, aralkyl halides like benzyl and phenethyl bromides and others.
Water or oil-soluble or dispersible products are thereby obtained.
The composition may also contain other agents, which either enhance the
activity of the composition, or compliment its activity or use in treatment,
or maintain the activity of the therapeutic agent in storage. Such
additional factors and/or agents may be included in the composition to
produce a synergistic effect or to minimize side effects. Additionally,
administration of the composition of the present invention may be
administered concurrently with other therapies.
Administration of the therapeutic agent of the present invention can be
carried out in a variety of conventional ways, such as oral ingestion,
inhalation, topical application or cutaneous, subcutaneous, intraperitoneal,
parenteral or intravenous injection.
The compositions containing the therapeutic agent of the present invention
can be administered intravenously, as by injection of a unit dose, for
example. The term "unit dose" when used in reference to a therapeutic
composition of the present invention refers to physically discrete units
suitable as unitary dosage for the subject, each unit containing a
predetermined quantity of active material calculated to produce the desired
therapeutic effect in association with the required diluent, i.e., carrier
Modes of administration of the therapeutic agent of the present invention
include intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous and intra-arterial injection and infusion; preferably
intravenous injection. Pharmaceutical compositions for parenteral injection
comprise pharmaceutically acceptable sterile aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions as well as sterile powders
for reconstitution into sterile injectable solutions or dispersions just
prior to use. Examples of suitable aqueous and nonaqueous carriers,
diluents, solvents or vehicles include water, ethanol, polyols (e.g.,
glycerol, propylene glycol, polyethylene glycol and the like),
carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g.,
olive oil) and injectable organic esters such as ethyl oleate. Proper
fluidity may be maintained, for example, by the use of coating materials
such as lecithin, by the maintenance of the required particle size in the
case of dispersions and by the use of surfactants. These compositions may
also contain adjuvants such as preservatives, wetting agents, emulsifying
agents and dispersing agents, and/or compounds to shield the immunogenic
determinant of the therapeutic agent. Prevention of the action of
microorganisms may be improved by the inclusion of various antibacterial and
antifungal agents such as paraben, chlorobutanol, phenol sorbic acid and the
like. It may also be desirable to include isotonic agents such as sugars,
sodium chloride and the like. Prolonged absorption of an injectable
pharmaceutical form may be brought about by the inclusion of agents, such as
aluminum monostearate and gelatin, which delay absorption. Injectable depot
forms are made by forming microencapsule matrices of the therapeutic agent
in biodegradable polymers such as polylactide-polyglycolide,
poly(orthoesters) and poly(anhydrides). Depending upon the ratio of
therapeutic agent to polymer and the nature of the particular polymer
employed, the rate of therapeutic agent release can be controlled. Depot
injectable formulations are also prepared by entrapping the therapeutic
agent in liposomes or microemulsions which are compatible with body tissues.
The injectable formulations may be sterilized, for example, by filtration
through a bacterial-retaining filter or by incorporating sterilizing agents
in the form of sterile solid compositions which can be dissolved or
dispersed in sterile water or other sterile injectable media just prior to
The formulations include those suitable for oral, rectal, ophthalmic
(including intravitreal or intracameral), nasal, topical (including buccal
and sublingual), intrauterine, vaginal or parenteral (including
subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal,
intracranial, intratracheal, and epidural) administration. The formulations
may conveniently be presented in unit dosage form and may be prepared by
conventional pharmaceutical techniques. Such techniques include the step of
bringing into association the active ingredient and the pharmaceutical
carrier(s) or excipient(s). In general, the formulations are prepared by
uniformly and intimately bringing into association the active ingredient
with liquid carriers or finely divided solid carriers or both, and then, if
necessary, shaping the product.
Formulations suitable for parenteral administration include aqueous and
non-aqueous sterile injection solutions which may contain anti-oxidants,
buffers, bacteriostats and solutes which render the formulation isotonic
with the blood of the intended recipient; and aqueous and non-aqueous
sterile suspensions which may include suspending agents and thickening
agents. The formulations may be presented in unit-dose dose or multi-dose
containers. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders, granules and tablets of the kind previously
As used herein, a "therapeutically effective amount" means the total amount
of each active component of the pharmaceutical composition or method that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or amelioration of the relevant medical condition, or an increase
in rate of treatment, healing, prevention or amelioration of such
conditions. When applied to an individual active ingredient, administered
alone, the term refers to that ingredient alone. When applied to a
combination, the term refers to combined amounts of the active ingredients
that result in the therapeutic effect, whether administered in combination,
serially or simultaneously. Generally, a composition will be administered in
a single dose in the range of 100 .mu.g-10 mg/kg body weight, preferably in
the range of 1 .mu.g-100 .mu.g/kg body weight. This dosage may be repeated
daily, weekly, monthly, yearly, or as considered appropriate by the treating
When a therapeutically effective amount of the therapeutic agent of the
present invention is administered orally, the composition of the present
invention can be in the form of a liquid, the composition contains from
about 0.5 to 90% by weight of protein of the present invention, and
preferably from about 1 to 50% protein of the present invention.
When a therapeutically effective amount of the therapeutic agent of the
present invention is administered by intravenous, cutaneous or subcutaneous
injection, the protein will be in the form of a pyrogen-free, parenterally
acceptable aqueous solution. The preparation of such parenterally acceptable
protein solutions, having due regard to pH, isotonicity, stability, and the
like, is within the skill in the art. A preferred composition for
intravenous, cutaneous, or subcutaneous injection should contain, in
addition to protein of the present invention, an isotonic vehicle such as
Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose
and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle
as known in the art. The composition of the present invention may also
contain stabilizers, preservatives, buffers, antioxidants, or other
additives known to those of skill in the art.
Topical administration, in which the composition is brought in contact with
tissue(s), may be suitable for Dermatitis herpetiformis. By "contacting" is
meant not only topical application, but also those modes of delivery that
introduce the composition into the tissues, or into the cells of the
Use of timed release or sustained release delivery systems are also included
in the invention. Such systems are highly desirable in situations where
surgery is difficult or impossible, e.g., patients debilitated by age or the
disease course itself, or where the risk-benefit analysis dictates control
A sustained-release matrix, as used herein, is a matrix made of materials,
usually polymers, which are degradable by enzymatic or acid/base hydrolysis
or by dissolution. Once inserted into the body, the matrix is acted upon by
enzymes and body fluids. The sustained-release matrix desirably is chosen
from biocompatible materials such as liposomes, polylactides (polylactic
acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide
(co-polymers of lactic acid and glycolic acid) polyanhydrides,
poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitin
sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,
nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine,
isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and
silicone. A preferred biodegradable matrix is a matrix of one of either
polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of
lactic acid and glycolic acid).
The amount of the therapeutic agent of the present invention in the
pharmaceutical composition of the present invention will depend upon the
nature and severity of the condition being treated, and on the nature of
prior treatments, which the patient has undergone. Ultimately, the attending
physician will decide the amount of the therapeutic agent of the present
invention with which to treat each individual patient. Initially, the
attending physician will administer low doses of the therapeutic agent of
the present invention and observe the patient's response. Larger doses of
may be administered until the optimal therapeutic effect is obtained for the
patient, and at that point the dosage is not increased further.
The duration of intravenous therapy using the pharmaceutical composition of
the present invention will vary, depending on the severity of the disease
being treated and the condition and potential idiosyncratic response of each
individual patient. It is contemplated that the duration of each application
of the therapeutic agent of the present invention will be in the range of 12
to 72 hours of continuous intravenous administration, at a rate of
approximately 30 mg/hour. Ultimately the attending physician will decide on
the appropriate duration of intravenous therapy using the pharmaceutical
composition of the present invention.
Claim 1 of 9 Claims
1. A method comprising a step of
administering to an individual having deposits of human IgA1 an IgA1
protease selected from the group consisting of Streptococcus pneumoniae
IgA1 protease, Streptococcus sanguis IgA1 protease, Clostridium ramosum
IgA1 protease, Haemophilus influenzae IgA1 protease, Haemophilus aegyptius
IgA1 protease, Neisseria meningitidis IgA1 protease, and Neisseria
gonorrhoeae IgA1 protease, such that the deposits of human IgA1 are
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