WO1993010138A1

WO1993010138A1 - Anti-sense oligonucleotides for isotype-specific suppression of immunoglobulin production

Info

Publication number: WO1993010138A1
Application number: PCT/US1992/010024
Authority: WO
Inventors: Tse Wen Chang
Original assignee: Tanox Biosystems, Inc.
Priority date: 1991-11-18
Filing date: 1992-11-18
Publication date: 1993-05-27
Also published as: CA2122132A1; EP0641353A1; EP0641353A4; JPH07501694A; AU3143993A

Abstract

The invention includes oligonucleotides which are complementary to the 3'-end untranslated regions or to at least some of the splicing recognition region of an mRNA transcript precursor for a particular immunoglobulin heavy chain. More particularly, the oligonucleotides are complementary to at least a continuous 12 nucleotide segment of the sequence upstream to the 3' splice junction up to and including the branch point A residue. Sequences of some of the oligonucleotides of the invention are the underlined portions of SEQ ID NOS: 3-33. These oligonucleotides will hybridize with the splicing recognition region and prevent or inhibit maturation of the mRNA. This will prevent production of mRNA encoding a complete immunoglobulin, so that it will not be translated into a functional immunoglobulin. The invention also pertains to the preparation of these anti-sense oligonucleotides for specifically suppressing IgG and/or IgM production in patients suffering from autoimmune diseases and for specifically suppressing IgE in patients suffering from allergic diseases manifested by immediate-type hypersensitivities.

Description

ANTI-SENSE OLIGONUCLEOTIDES FOR ISOTYPE-SPECIFIC

SUPPRESSION OF IMMUNOGLOBULIN PRODUCTION

Background of the Invention In man and many mammals, five classes of immunoglobulins, namely IgD,

IgM, IgG, IgA, and IgE, are synthesized. An antibody monomer contains two

identical light chains of one of the two isotypes, K and λ, and two identical heavy chains of one of the five isotypes, δ, μ, y, a, and e. Some of these isotypes have

subclasses. For example, in man, γ has γl, γ2, γ3, and γ4 subclasses. Antibodies have enormously diverse binding specificity characterized by

their individually unique binding sites. Each heavy chain or light chain has its own unique variable region segment, as well as constant region segments which

define the isotype and subclass. A mature, immunocompetent, resting B cell has a rearranged genes encoding for the heavy and light chain variable regions and, therefore, a defined antigen specificity. However, while undergoing activation and clonal expansion process, a B cell can further mature and differentiate into a cell expressing one or more immunoglobulins of the five classes, all of which

bear the same variable regions and antigen specificity.

The five classes of antibodies, which could probably bear the same

variable regions and the same antigen specificities, have different immune

functions and are differently involved in the pathogenesis of immune system- related disease. IgM exists as a pentamer and is multivalent, and fixes

complement effectively; IgG generally is most effective in mediating antibody- dependent cellular cytotoxicity and crosses the placenta to the fetus; IgA is most important in mucosal surface; IgE is dominant in sensitizing mast cells and basophils in pharmacological mediator release; IgD is on the B cell surface and

is probably crucial in the B cell activation process. Among the different classes of antibodies, those of IgG isotype are most conspicuously involved as autoantibodies in autoimmune diseases, such as in rheumatoid arthritis, system lupus erythematosus, Graves' disease, Hashimoto's

_* thyroiditis, myasthenia gravis, pemphigus vulgaris, and idiopathic thrombocytopenia purpura. IgE is mainly responsible for immediate type

hypersensitivities manifesting allergic rhinitis, extrinsic asthma, drug and food allergies, and atopic dermatitis.

For treating certain diseases, such as one of the autoimmune or allergic diseases, it is desirable to suppress the antibodies causing the disease. Since IgG is responsible for many autoimmune diseases, and IgE is mainly responsible for immediate-hypersensitivities, it is desirable that the antibody suppression be isotype-specific. By doing so, the adverse effects to the immune system will be minimized. A number of experimental approaches for studying isotype-specific

suppression have been proposed. One approach involves inhibiting the

lymphokines involved in isotype-specific switching, e.g. experiments have been

done with an anti-interleukin 4 monoclonal antibody in an attempt to inhibit IgE

synthesis. Another approach is to use monoclonal antibodies specific for

membrane-bound immunoglobulin of specific isotypes to target B cells producing the specific isotypes. The present invention employs anti-sense oligonucleotides for targeting the mRNA precursors of the specific isotypes and even of particular subclasses of isotypes.

Anti-sense technology is based on the principle that a properly designed

oligonucleotide or analogue capable of binding to a key segment of an RNA or

a DNA can enter a cell, binding to the crucial element of the mRNA, or its precursor RNA, or DNA, and effectively inhibit the function of the RNA or DNA

species. In mammals, DNA generally exist as double helices of two complementary strands, complexing with nuclear proteins such as histones, forming extremely complex but orderly structural units that compact together to form a chromosome. The DNA strand is a genetically inherited sequence of millions of four deoxyribonucleotides distinguished by their base side chains, adenine ("A"), thymine ("T"), cytosine ("C"), and guanine ("G"). The DNA strand is functionally a linear stretch of thousand of genes, some of which encode structural polypeptides and some of which carry regulatory information or

functions.

Messenger RNA is a transcribed product of a segment, generally a gene, in DNA. Messenger RNA is a sequence of ribonucleotides with A, C, G, and

uracil ("U") bases. In mammals, RNA is generally single stranded and hence is

complementary to a segment of only one of the two double-stranded DNA. A naturally occurring mRNA and its complementary DNA strand is a "sense" strand. An artificially designed segment, either synthesized in vitro or in vivo, is an "anti-sense" strand.

The chemical basis of the complementarity between the two strands in a double helix or between an "anti-sense" segment and a sense segment is that A

or T (or U in the case of RNA) and G and C, of two neighboring nucleotides in the two strands form stable hydrogen bonds. Both DNA and RNA strands have

directions, with their ends being referred as 5' and 3' ends, pertaining to the

hydroxy linkages on the ribosyl monosaccharide backbone of the nucleotides.

During the transcription process (the production of RNA), a set of regulatory proteins and enzymes separate the sense strand of DNA from its

complementary strand at the control region of a gene and allow RNA polymerase to assemble an RNA molecule according to the DNA sequence in that gene. The newly synthesized RNA precursor is processed by another set of proteins and enzymes to form a mature mRNA species, which is then transported from the

nucleus to the cytosol. Li the translation process, a set of regulatory proteins and enzymes bind to the control segment near the 5' end of the mRNA and read the information along the mRNA sequence (from the 5' end to the 3' end) to assemble amino acids to form a unique polypeptide chain.

In applying the "anti-sense" concept, oligonucleotides for inhibiting the production of crucial viral proteins or products or oncogenic proteins (potentially

for use in infectious diseases and cancer) can be placed into one of two categories in terms of target specificity. Cohen, J.S. Trends Pharm. Sci. 10:435 (1989);

LeDoan, T. et al. Bull. Cancer 76:849 (1989); Takayama, K.M. and Inouye, M. Crit. Rev. Biochem. Mol. Biol. 25:155 (1990); Rothenberg, M. et al. J. Natl.

Cancer Inst. 81:1539 (1989); Eguchi, Y. et al. Ann. Rev. Biochem. 60:631

(1991). The first group target the control region of mRNA or its precursor RNA and interfere the proper expression of the messenger species. The anti-sense

compounds can interfere with proper maturation of the precursor RNA transcripts

and prevent them from becoming functional mRNA. They can also affect the translation process by interfering with the regulatory components of the gene.

They can also affect the stability of the mRNA, making it more susceptible to

RNase. The other group of anti-sense compounds target the control region of a

gene and hence inhibit the transcription process. In the latter cases, the oligonucleotide binds to one strand of the DNA as the two strands of DNA open up, and forms an apparent "triplex" structure.

In the past few years, the development of anti-sense technology for

pharmaceutical application has made substantial progress because of the successful development of methods of synthesizing analogues of oligonucleotides. The

oligonucleotides made from the native nucleotides are highly charged and penetrate the cellular plasma membrane very poorly. They are also very sensitive to nuclease digestion in the blood. The modified oligonucleotides include phosphorothioate oligomers, methylphosphonate oligomers, α-anomeric

oligomers, and others. These oligonucleotides hybridize with the complementary RNA or DNA segments equally well. However, since they are less hydrophilic and more lipophilic, they can get into cells more effectively. They are also much more resistant to nuclease cleavage than the native oligonucleotides.

As for the identification of segments in a precursor RNA or in a mature mRNA species for designing anti-sense compounds, there are a number of

potential regions that are more likely to contain "effective" target segments.

Based on experience with many mRNA and precursor RNA targets, the most

probable regions are the 5' regulatory regions, the splicing junctions, and the 3'- end untranslated regions that control mRNA stability. Even segments of the

coding regions can provide targets for anti-sense compounds. One widely used process would be to synthesize several oligonucleotides of 10 to 20 nucleotides in length covering portions of the regions mentioned above, and to test the effects on inhibiting the expression of the encoded protein. When a oligonucleotide

shows significant inhibition, additional oligonucleotides with slight variations in length and in the exact positions in the region would also be synthesized and

tested. The ones with optimal effect would be selected. Generally speaking, in mammals, a DNA gene consists of not only

segments encoding peptide sequences (exons), but also dispersing segments of non-coding DNA (introns). During transcription, the RNA which is initially

transcribed corresponds to the entire sequence, including the regulatory regions at the 5' end, the exons, and the introns. This RNA precursor is processed in a

sequence of steps, in which the introns are excised and the exons are spliced together to form a mature mRNA, which contains a contiguous coding sequence of a polypeptide, along with regulatory elements flanking the 5' and 3' ends. The mechanism of mRNA splicing is complex. It may involve Ul- and U2- containing small RNA-protein complexes (Ul, U2 snRNPs). The Ul RNA has a sequence at its 5' end that is precisely complementary to the nine-nucleotide consensus for the 5' splice site of an exon. Binding of Ul-snRNP to the 5' splice site results in a cut at the 5' splice junction. The cut occurs through ATP

hydrolysis accompanied by formation of an unusual 2' -5' phosphodiester bond between the 5 '-terminal G residue of the intron and a site about 30 nucleotides

upstream from the 3' end of the intron. This creates an intermediate RNA lariat structure in which an A residue invariably forms the branch point. During the splicing reaction, U2 snRNPs associate with the intron branch point, while Ul snRNPs interact with the 5' splice site and some other component binds to the 3' splice junction in the pre-mRNAs. All these bindings seem to

assemble along an ordered pathway. Signals crucial for the splicing of mammalian pre-mRNAs reside directly at the splice junction. Analysis of the intron sequences of mammalian genes showed that exon-intron boundaries in pre- mRNAs are highly conserved. The conserved sequences are generally regarded as mRNA splice sites, and are shown in SEQ ID NO:l and SEQ ID NO:2.

Referring to SEQ ID NO: 1 , the nine nucleotide consensus sequence for

the 5' junctions extends three residues upstream and six residues downstream

from the splice point, which is between the 3rd and 4th G-base nucleotides. The C at the 5' end of SEQ ID NO: 1 can also be an A, and the 6th A from the 5' end

can be a G. Referring to SEQ ID NO:2, the 3' splice junction includes a pyrimidine-

rich region of variable length (but^" always greater than ten nucleotides). In SEQ

ID NO:2, the C can be a T. This pyrimidine-rich region is followed by a short consensus sequence extending only three residues upstream and one residue downstream from the splice point, which is between the two Gs at the 3' end.

It is noted that at both the 3' and 5' splice junctions, the conserved sequences extend further into the intron than into the exon, thereby placing only minimal constraints on the sequence of the mature mRNA.

The sequence of the introns upstream of the 3' splice junction in the CHI

region and the 5' end of the exons for the constant regions of the human immunoglobulins γl, γ2, y_\, yA, δ, al, al, e and μ are shown respectively in

SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10 and 11.

An examination of SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10 and 11 shows

that the AG dinucleotide occurs invariantly at the 3' intron-exon junction, and the

branch point A residue occurs either 32 residues upstream (in the case of the

IgGs, IgD, and IgE) or 35 residues upstream (in the case of the IgAs) or 34 residues upstream (for IgM) from the 3' splice junction. Between these invariant residues lie the nucleotide sequences which are considerably non-homologous among immunoglobulins of different isotypes. Since the intron sequences between the 5' splice junction and the branch point are not needed for splicing reactions, oligonucleotide sequences which would successfully interfere with

splicing would be complementary to the region about 30 nucleotides upstream of the 3' splice junction.

In the germ-line organization, the variable (V) gene family of 50 or more genes and the cluster of heavy chain constant region genes are in separate chromosomes. During the somatic maturation process of B cells, a V gene is

rearranged with the constant region gene cluster at a certain pre-B cell stage.

Hinds, K.R. and Litman, G.W. Nature 320:546 (1986); Blackwell, T.K. and Alt, F.W. Ann. Rev. Genet. 23:605 (1989). The heavy chain cluster in man is

organized in the sequence of μ, δ, γ3, γl, αl , yl, γ4, e and al. Flanagon, J.G. and Rabbitts, T.H. Nature 300:709 (1982). In a further maturation process, the fused VDJ segment may be switched to one of the γ's or a, or e, this switching being regulated by certain T cell factors and some other yet-to-be characterized factors. Thus, for a B cell expressing a particular isotype and subclass, the VDJ

segment is adjacent to the particular heavy chain constant region gene segment. For example, for a B cell expressing IgG,, the VDJ segment is thought to be adjacent to the constant region gene of γl.

In the genomic organization, the peptide sequence coding segments are

encoded by exons which are separated by introns. Generally speaking, for a heavy chain immunoglobulin gene, the leader sequence serves as secretion signal

at the endoplasmic reticulum. The fused VDJ segment, each of the constant

region domains (including CHI, CH2, and CH3, and for μ and e, CH4), and the membrane anchor peptide segments, all are encoded for by discrete exons separated by introns. For δ chain, the hinge region of the immunoglobulin is encoded by a separate exon. The control regions for transcription and translation are at the 5' end flanking the exon of the leader sequence. The transcription

termination sequence is at the 3' end flanking the membrane exons.

During the transcription process of an immunoglobulin heavy chain, an RNA transcript complementary to the entire length of the genomic structure from the 5' end control regions to the 3' end transcription termination signal sequence is made. This RNA precursor is then processed through a sequence of splicing steps, in which the introns are excised and the peptide-coding exons are spliced together to form mature mRNA. In this RNA splicing process, the 3' end of an exon is spliced to the 5' end of the next exon in sequence. For example, the 3' end of the fused VDJ segment is spliced to the 5' end of the CHI exon.

As discussed above, the same VDJ may be rearranged to the constant region genes of various heavy chain isotypes during B cell maturation. In one

emrjodiment of the present invention, the splicing between the VDJ exon and an isotype specific CHI exon is inhibited by anti-sense oligonucleotides that bind to the splicing recognition sequence 5' to the CHI exon or that bind to the splicing junctional segment of CHI. The invention is explained further below.

Summary of the Invention

The invention includes oligonucleotides which are complementary to the

splicing recognition region of an mRNA transcript precursor for a particular

immunoglobulin heavy chain, in one or more of the CH regions of the

immunoglobulin gene. More particularly, the oligonucleotides are complementary to a portion of the sequence upstream of the 3' splice junction and towards the branch point A residue, or to a portion of the sequence upstream and downstream of the 3' splice junction. The oligonucleotides generally should be

complementary to at least a continuous 12 nucleotide segment of these sequences in order to bind stably. Longer segments may be used, if desired.

The oligonucleotides of the invention would be complementary to some or all of the underlined portions of SEQ ID NOS: 3-33, and/or to some of these

portions as well as some of the portions downstream of the 3' splice junction and in the exon region (shown in bold face type). The underlined portions represent the conserved portions of the ougonucleotides. In these sequences the exons are shown in bold face type, and the introns in normal type. The 3' splice point is the point of transition between these regions. The branch point A residue is also in bold face type.

These oligonucleotides will hybridize with the splicing recognition region

and prevent or inhibit maturation of the mRNA. This will prevent production of mRNA encoding a complete immunoglobulin, so that it will not be translated into a functional immunoglobulin.

The oligonucleotides can be tested for activity in vitro on B cell lines or peripheral blood mononuclear cells (PBMCs), to determine their ability to inhibit

the production of antibodies of a particular class of interest. When an

oligonucleotide demonstrates significant activity, additional oligonucleotides of varying lengths covering the target segment can be synthesized and tested, with those of optimal length and sequence being selected as product candidates.

The present disclosure focuses on the splice junctions of the various immunoglobulin domains as the target segments for designing anti-sense compounds. The 3'-end untranslated regions of each antibody isotype, which are

known to regulate the stability of the mRNA species, are also attractive targets

for regulation with anti-sense compounds. A systematic approach for identifying

effective target segments for anti-sense compounds is to synthesize over-lapping

oligonucleotides of about 15 nucleotide long, covering the entire length of the 3'- end untranslated region. These oligonucleotides may overlap with adjacent

oligonucleotides by up to 5 nucleotides on each end. Since oligonucleotide synthesis with synthesizers is a very established procedure, to make overlapping oligonucleotides covering a gene region of 1000 to 2000 nucleotide long is feasible. These oligonucleotides can then be tested in vitro against B cell lines or PBMCs, to determine their ability to inhibit the production of a particular class of antibodies. Those which inhibit production to the greatest extent would be

selected.

In applying the oligonucleotides of the invention for in vivo therapy, use

of one of the oligonucleotides at a time achieves isotype-specific suppression of

immunoglobulins, in that only one immunoglobulin isotype is suppressed. This

is advantageous because one can effect treatment while selectively deleting only one isotype of humoral immunity. Alternatively, several oligonucleotides of

different specificities could be used to cause a generalized humoral immunosuppression .

This selective suppression of immunoglobulin isotypes is suitable for treating autoimmune diseases and allergies. The immunoglobulins IgG and IgM have been associated with autoimmune diseases, including especially rheumatoid arthritis. Suppressing IgG and/or IgM production would be useful in treating

rheumatoid arthritis, and probably also in treating other autoimmune diseases including systemic lupus erythematosus, scleroderma, and myasthenia gravis.

Allergies of the immediate-type hypersensitivity class are all mediated by IgE. Suppression of IgE by using the appropriate oligonucleotide of the invention

would inhibit or prevent allergic reactions.

Another use for the oligonucleotides of the invention is in production of monoclonal antibodies. The oligonucleotides can be used to selectively inhibit production of various immunoglobulin isotypes, either by B cells extracted from the immunized animal prior to fusion with an immortalized cell line, or by the hybridomas after the fusion. This allows production of immunoglobulins of a particular desired isotype, or the prevention of production of immunoglobulins of a particular isotype. As a result, no monoclonal antibodies of these isotypes

would be produced. Detailed Description of the Preferred Embodiments

The invention pertains to use of anti-sense compounds to achieve isotype-

specific suppression for treating a number of clinical conditions. Because the 5' regulatory regions and the variable domains of the immunoglobulins are highly variable and are not unique to particular isotypes, these regions are not suitable

targets for anti-sense compounds.

The most suitable targets in the immunoglobulin mRNA or the precursor

RNA are at the splice junctions and in the 3'-end untranslated regions. The

coding regions or other areas of the untranslated regions may also provide targets

for anti-sense intervention. These other regions are much longer and may require larger numbers of oligonucleotides to determine the precise target areas.

As an example of how to identify a target segment and how to design anti- sense compounds to that segment, one can consider the 3' -end untranslated region of human e chain. The region SEQ ID NO:34 between the stop codon TGA at the end of the 4th constant domain and the poly A signal is 126 nucleotides long.

Seno M. et al. NucL Acids Res. 11:719-727 (1983). A set of 23 complementary oligonucleotides of 15 nucleotides in length, overlapping by 5 nucleotides, are

synthesized. These oligonucleotides, at concentrations ranging from 0J to 200 μg/ml are tested for their effect on the synthesis of IgE by treating SKO-007 cells (ATCC, Rockville, Maryland). These IgE-expressing cells are incubated with the potential inhibitors for 1 to 3 days and the IgE secreted into the medium is assayed by a standard IgE ELISA, such as that described in Sun L. et al. /.

Immunol. 146:199-205 (1991). The tests may also be done with PBMCs which

are isolated from donors with high serum IgE (atopic individuals).

When an oligonucleotide, or an adjacent oligonucleotide, causes inhibition

of IgE synthesis by the SKO-007 cells, the susceptible region is then identified. An additional set of oligonucleotides, ranging from 10 to 12 nucleotides in length which overlap by only one or two nucleotides and covering the identified region, are then synthesized and tested for effect on IgE production. By doing so, the segment with the optimal sequence and length can be identified. Analogues of oligonucleotides with enhanced lipophilicity and resistance to RNase can also be

synthesized and tested. In this way, the optimal oligonucleotides can be identified.

Similar systematic approaches can be undertaken to find anti-sense compounds for suppression of other regions of the _ gene and other immunoglobulin isotypes. A similar approach may also be used for designing

anti-sense compounds to target the 3 '-end untranslated regions of the four subclasses of γ chains. The region between the ATG stop codon and AATAA signal is 100 nucleotides long for γl (SEQ ID NO:35), and is 101 nucleotides long for γ2, γ3, and γ4, (SEQ ID NOS: 36, 37 and 38, respectively). See Huck S. et al. Nucl. Acids Res. 14:1779-1789 (1986) . The regions have only a few

nucleotide differences among the four subclasses. A set of 18 oligonucleotides can be synthesized for each subclass, with most of these oligonucleotides shared among the subclasses. These oligonucleotides can then be tested for abUity to

inhibit cell lines secreting the specific subclass of IgG.

The anti-sense compounds of the invention also target the segments at the

splicing junctions, or at the segments known to be involved in splicing. These oligonucleotides are complementary to at least about a continuous 12 nucleotide segment of the underlined portions of the sequences of SEQ ID NOS:3-33. The sequences represented by SEQ ID NOS:3-ll are explained above. SEQ ID NOS: 12, 13, 14 and 15 represent, respectively, the sequences of the intron and

exon near the 3' splice sites of the CH2 regions of γl, γ2, γ3, γ4. SEQ ID NOS:16, 17, 18 and 19 represent, respectively, the sequences of the intron and exon near the 3' splice sites of the CH3 regions of γl, γ2, γ3, γ4. SEQ ID NOS:20 and 21 represent, respectively, the sequences of the intron and exon near the 3' splice sites of the CH2 and CH3 regions of δ. SEQ ID NOS:22 and 23 represent, respectively, the sequences of the intron and exon near the 3' splice sites of the hinge 1 and hinge 2 regions of δ. SEQ ID NOS: 24 and 25 represent, respectively, the sequences of the intron and exon near the 3' splice sites of CH2 and CH3 of αl. SEQ ID NOS:26 and 27 represent, respectively, the sequences of the intron and exon near the 3' splice sites of CH2 and CH3 of α2. SEQ ID

NOS:28, 29 and 30 represent, respectively, the sequences of the intron and the

exon near the 3' splice sites of CH2, CH3 and CH4 of e. SEQ ID NO: 31 , 32,

33 represent the sequence of the intron and exon near the 3' splice sites of CH2,

CH3, and CH4 for μ. The exons are shown in bold face type, and the introns in normal type. The 3' splice point is the point of transition between these regions. The branch point A residue is also in bold face type.

In general, the preferred length for the oligomers of the invention is 15

nucleotides, although they can be as short as about 12 nucleotides. As noted

above, the oligomers or the invention hybridize with the segment of the nucleotide upstream of the 3' splice junction (underlined in the sequence listing portion), and/or with a portion of this segment as well as a continuous portion of the segment downstream of the 3' splice junction (shown in bold letters in the sequence listing). The ougonucleotides of the invention can therefore either be

RNA (having a U instead of a T base nucleotide as in DNA) or DNA.

The oligonucleotides of the invention can include the entire 32 or 35 nucleotide portion upstream from the 3' splice point which they hybridize with,

and/or they can even include a portion downstream of the 3' splice point.

However, they can also be shorter, to a length of 12 nucleotides or even less.

The shorter oligonucleotides, again, can include just the portion upstream of the 3' splice point, or some of the upstream portion and some of the downstream (exon) portion. The disadvantage of segments much shorter than 12 nucleotides is that they may not hybridize in a stable fashion. The disadvantage of segments much longer than 15 nucleotides is the added cost of synthesizing longer oligonucleotides, and the fact that they may not readily permeate the cellular

membranes so as to get into the cytoplasm.

All of the oligonucleotides of the invention can be synthesized with

techniques which are well known in the art, such as in an Apphed Biosystems 380A synthesizer.

As noted above, the oligonucleotides of the invention are useful in treating autoimmune diseases, allergies, and in causing humoral immunσsuppression. For treatment of autoimmune diseases, oligonucleotides complementary to the appropriate portions of SEQ ID NOS.3, 4, 5, 6, 11, 12, 13, 14, 15, 16, 17, 18,

19, 30, 31, 32, and 33 are preferred, as they will cause suppression of IgG and IgM production. For IgG, it is noted that there is also a hinge region gene

between the sequence encoding the CHI region (SEQ ID NOS:3, 4, 5, 6) and the

sequence encoding the CH2 region (SEQ ID NOS:12, 13, 14, 15). An oligonucleotide complementary to the portion of the hinge region sequence upstream of the hinge region 3' splice junction, or a portion upstream and a continuous downstream portion, could also be used as an oligonucleotide of the invention. For allergy treatment, one would use oligonucleotides complementary to the appropriate portion of SEQ ID NOS: 10, 27, 28 and 29, as they would cause suppression of IgE production. For inducing humoral immunosuppression, one would use oligonucleotides complementary to the appropriate portion of SEQ ID NOS:3-33, as they would suppress production of all immunoglobulins. As noted above, the oligonucleotides admmistered can be DNA or RNA, as long as they are complementary to the target segments, and hybridize with them.

The oligonucleotides of the invention can be used in vitro and in vivo in

non-modified form. Li the alternative, in order to increase their hydrophobicity

and make them more Iipophilic, they can be chemically modified, for example,

by changing them to phosphorothioate, methylphosponate, or α-anomeric

oligonucleotides. The manner of making these modifications is well-known in the

art. To use the oligonucleotides of the invention in vivo, one would administer

a large excess of the oligonucleotides to the patient. They could be administered intravenously, or possibly even orally. The dosage could be readily calculated.

Depending on which isotype one wishes to suppress, one would calculate the total number of B cells producing that isotype, and then administer enough of the

oligonucleotide to attach to the appropriate gene segment in each B cell.

To use the oligonucleotides of the invention in vitro, i.e. , in production

of monoclonal antibodies, one would administer enough of the oligonucleotides to suppress the B cells which produce the isotype(s) which one desires to suppress. It would be preferable to do this before fusion with the immortalized cell line, which is done to produce hybridomas. However, it could also be done after the fusion.

It should be understood that the foregoing terms and expressions are descriptive only and not limiting, and that the invention is defined only in the claims which follow, and includes all equivalents of the subject matter of those claims.

SEQUENCE LISTING (1) General Information: (i) Applicant: Chang, Tse Wen

(ii) Title of Invention: ANTI-SENSE OUGONUCLEOTIDES FOR ISOTYPE- SPECIFIC SUPPRESSION OF IMMUNOGLOBULIN PRODUCTION (in) Number of Sequences: 38 (iv) Correspondence Address:

(A) Addressee: Tanox Biosystems, Inc.

(B) Street: 10301 Stella Link Rd. (C) City: Houston

(D) State: Texas

(E) Country: USA

(F) Zip: 77025

(v) Computer Readable Form: (A) Medium Type: Diskette, 3.5 inch

(B) Computer: IBM PS/2

(C) Operating System: DOS 3.30

(D) Software: Wordperfect 5J (vi) Current application data: (A) Application Number:

(B) Filing Date:

(C) Classification:

(vii) Prior Application Data: (A) Application Number: 07/794,395 (B) Filing Date: 11/18/91

(viii) Attorney/Agent Information:

(A) Name: Mirabel, Eric P.

(B) Registration Number: 31,211

(C) Reference/Docket Number: TNX91-6-PCT (ix) Telecommunication Information:

(A) Telephone: (713) 664-2288 <B) Telefax: (713) 664-8914

(2) Information for SEQ ID NO:l: (i) Sequence Characteristics: (A) Length: 9 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO:l CAGGTAAGT 9

(2) Information for SEQ ID NO:2: (i) Sequence Characteristics:

(A) Length: 4 nucleotides

(B) Type: nucleic acid (C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 2 CAGG 4 (2) Information for SEQ ID NO: 3: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded (D) Topology: linear

(xi) Sequence Description: SEQ ID NO:3

CACACCGCGG TCACATGGCA CCACCTCTCT TGCAGCCTCC 40 ACCAAGGGCC CATCG 55 (2) Information for SEQ ID NO:4: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear (xi) Sequence Description: SEQ ID NO:4

CACACCGCGG TCACATGGCA CCACCTCTCT TGCAGCCTCC 40

ACCAAGGGCCCATCG55

(2) Information for SEQ ID NO:5:

(i) Sequence Characteristics: (A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO:5 CACACCGCAG TCACATGGCG CCATCTCTCT TGCAGCTTCC 40

ACCAAGGGCC CATCG 55

(2) Information for SEQ ID NO: 6:

(i) Sequence Characteristics:

(A) Length: 55 nucleotides (B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 6

CACACCGGGG TCACATGGCA CCACCTCTCT TGCAGCTTCC 40 ACCAAGGGCC CATCC 55 (2) Information for SEQ ID NO:7: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid (C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO:7

TCCAGCCTCA GACTCCCACT GTGTCTGTCT TCCAGCACCC 40 ACCAAGGCTC CGGAT 55 (2) Information for SEQ JD NO:8: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid (C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 8

AGGGCCGCGT CCTCACAGTG CATTCTGTGT TCCAGCATCC 40

CCGACCAGCC CCAAG 55 (2) Information for SEQ ID NO:9:

(i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded (D) Topology: linear

(xi) Sequence Description: SEQ ID NO:9

AGGGCCGCGT CCTCACAGCG CATTCTGTGT TCCAGCATCC 40 CCGACCAGCC CCAAG 55 (2) Information for SEQ ID NO:10: (i) Sequence Characteristics:

(A) Length: 54 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear (xi) Sequence Description: SEQ ID NO: 10

CTGAGGCTGG CACTGACTAG GTTCTGTCCT CACAGCCTCC 40

ACACAGAGCC CATC 54

(2) Information for SEQ ID NO:ll:

(i) Sequence Characteristics: (A) Length: 56 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 11 TCACCCTCCC TTTCTCTTTT GTCTGCGGGT CCTCAGGGAG 40 TGCATCCGCC CCAACC 56 (2) Information for SEQ ID NO: 12: (i) Sequence Characteristics: (A) Length: 55 nucleotides (B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 12 GGGTGCTGAC ACGTCCACCT CCATCTCTTC CTCAGCACCT 40 GAACTCCTGG GGGAC 55 (2) Information for SEQ ID NO: 13: (i) Sequence Characteristics: (A) Length: 52 nucleotides (B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 13

GGGTGCTGAC ACATCTGCCT CCATCTCTTC CTCAGCACCA 40 CCTGTGGCGG AC 52

(2) Information for SEQ ID NO: 14: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid (C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 14

GGGTGCTGAC ACATCTGCCT CCATCTCTTC CTCAGCACCT 40

GAACTCCTGG GAGAC 55 (2) Information for SEQ ID NO: 15 :

(i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded (D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 15

GGGTGCTGAC ACATCTGCCT CCATCTCTTC CTCAGCACCT 40 GAGTTCCTGG GGGAC 55 (2) Information for SEQ ID NO: 16: (i) Sequence Characteristics:

(A) Length: 54 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear (xi) Sequence Description: SEQ JD NO.J6

GAGAGTGACC GCTGTACCAA CCTCTGTCCT ACAGGGCAGC 40

CCCGAGAACC ACAG 54

(2) Information for SEQ _D NO: 17:

(i) Sequence Characteristics: (A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear (xi) Sequence Description: SEQ ID NO: 17

GGGAGTGACC GCTGTGCCAA CCTCTGTCCC TACAGGGCAG 40 CCCCGAGAAC CACAG 55 (2) Information for SEQ ID NO: 18: (i) Sequence Characteristics: (A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 18 GGGAGTGACC GCTGTGCCAA CCTCTGTCCC TACAGGACAG 40

CCCCGAGAAC CACAG 55

(2) Information for SEQ JD NO:19:

(i) Sequence Characteristics:

(A) Length: 55 nucleotides (B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 19

GGGAGTGACC GCTGTGCCAA CCTCTGTCCC TACAGGGCAG 40 CCCCGAGAAC CACAG 55

(2) Information for SEQ ID NO:20: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid (C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO:20

CGGCCACAGC TGCTCTCGTT TGCTCTCCCC TGCAGAGTGT 40

CCGAGCCACA CCCAG 55 (2) Information for SEQ ID NO:21 :

(i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded (D) Topology: linear

(xi) Sequence Description: SEQ ID NO:21

GGCCAGAGCC TGACAGCCCC CCCACCTCCC CGCAGCTGCG 40

CAGGCACCCG TCAAG 55

(2) Information for SEQ ID NO:22: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded (D) Topology: linear

(xi) Sequence Description: SEQ ID NO:22

CCTAAGCCTC ACCTGCACTT TTCCTTGGAT TTCAGAGTCT 40 CCAAAGGCAC AGGCC 55 (2) Information for SEQ ID NO:23: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear (xi) Sequence Description: SEQ ID NO: 23

AACATGTATA ATTTTTGTCA ATTAAAAATT TTTAGGAAGA 40

GGAGGAGAAG AGAAG 55

(2) Information for SEQ ID NO:24:

(i) Sequence Characteristics: (A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO:24 TGTGCTGACC AGCTCAGGCC ATCTCTCCAC TCCAGTTCCC 40

TCAACTCCAC CTACC 55

(2) Information for SEQ ID NO:25:

(i) Sequence Characteristics:

(A) Length: 55 nucleotides (B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO:25

TGCCCAGCCC CCTGACCTGG CTCTCTACCC TCCAGGAAAC 40 ACATTCCGGC CCGAG 52

(2) Information for SEQ ID NO:26: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid (C) Strandedness: double-stranded (D) Topology: linear

(xi) Sequence Description: SEQ ID NO:26

CGTTCTGACC AGCTCAGGCC ATCTCTCCAC TCCAGTCCCC 40 CACCTCCCCA TGCTG 55 (2) Information for SEQ TD NO:27: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid (C) Strandedness: double-stranded (D) Topology: linear

(xi) Sequence Description: SEQ ID NO:27

TGCCCAGCCC CCTGACCTGG CTCTCTACCC TCCAGGAAAC 40 ACATTCCGGC CCGAG 55 (2) Information for SEQ ID NO:28: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded (D) Topology: linear

(xi) Sequence Description: SEQ ID NO:28

GCCAACAACC CTCATGACCA CCAGCTCACC CCCAGTCTGC 40 TCCAGGGACT TCACC 55 (2) Information for SEQ ID NO:29: (i) Sequence Characteristics:

(A) Length: 55 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear (xi) Sequence Description: SEQ ID NO:29

GGGTGGGCCT CACACAGCCC TCCGGTGTAC CACAGATTCC 40

AACCCGAGAG GGGTG 55

(2) Liformation for SEQ ID NO:30:

(i) Sequence Characteristics: (A) Length: 55 nucleotides

(B) Type: nucleic acid

( Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO:30 AGCGGGGCCC GGGCTGACCC CACGTCTGGC CACAGGCCCG 40

CGTGCTGCCC CGGAA 55

(2) Information for SEQ ID NO:31:

(i) Sequence Characteristics:

(A) Length: 58 nucleotides (B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO:31

AGTGGCGGGCACCGGGCTGACACGTGTCCTCACTGCAGTG40 ATTGCCGAGC TGCCTCCC 58

(2) Information for SEQ ID NO:32: (i) Sequence Characteristics: (A) Length: 57 nucleotides (B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 32

ACCTGCCCCA CCTCTGACTC CCTTCTCTTG ACTCCAGATC 40 AAGACACAGC CATCCGG 57 (2) Information for SEQ ID NO:33: (i) Sequence Characteristics:

(A) Length: 56 nucleotides

(B) Type: nucleic acid (C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ JD NO: 33

CCACTGCCCC GCCCTCACCA CCATCTCTGT TCACAGGGGT 40

GGCCCTGCAC AGGCCC 56 (2) Information for SEQ ID NO:34:

(i) Sequence Characteristics:

(A) Length: 126 nucleotides

(B) Type: nucleic acid

(C) Strandedness: double-stranded (D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 34

CGTACTCCTG CCTCCCTCCC TCCCAGGGCT CCATCCAGCT 40 GTGCAGTGGG GAGGACTGGC CAGACCTTCT GTCCACTGTT 80 GCAATGACCC AGGAAGCTAC CCCCAATAAA CTGTGCCTGC 120 TCAGAA 126

(2) Information for SEQ ID NO:35: (i) Sequence Characteristics:

(A) Length: 100 nucleotides

(B) Type: nucleic acid (C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 35

GTGCGACGGC CGGCAAGCCC CGCTCCCCGG GCTCTCGCGG 40 TCGCACGAGG ATGCTTGGCA CGTACCCCCT GTACATACTT 80 CCCGGGCGCC CAGCATGGAA 100 (2) Information for SEQ ID NO:36: (i) Sequence Characteristics:

(A) Length: 101 nucleotides

(B) Type: nucleic acid (C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO: 36

GTGCCACGGC CGGCAAGCCC CCGCTCCCCA GGCTCTCGGG 40 GTCGCGTGAG GATGCTTGGC ACGTACCCCG TGTACATACT 80 TCCCAGGCAC CCAGCATGGA A 101 (2) Information for SEQ ID NO:37: (i) Sequence Characteristics: (A) Length: 101 nucleotides (B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO:37

GTGCGACAGC CGGCAAGCCC CCGCTCCCCG GGCTCTCGGG 40 GTCGCGCGAG GATGCTTGGC ACGTACCCCG TGTACATACT 80

TCCCGGGCACCCAGCATGGAA101

(2) Information for SEQ ID NO:38:

(i) Sequence Characteristics:

(A) Length: 101 nucleotides (B) Type: nucleic acid

(C) Strandedness: double-stranded

(D) Topology: linear

(xi) Sequence Description: SEQ ID NO:38

GTGCCAGGGC CGGCAAGCCC CCGCTCCCCG GGCTCTCGGG 40 GTCGCGCGAG GATGCTTGGC ACGTACCCCG TCTACATACT 80 TCCCAGGCACCCAGCATGGAA101

Claims

What Is Claimed Is:

1. An oligonucleotide complementary to the splicing recognition region of an mRNA transcript precursor for a particular immunoglobulin heavy chain.

2. The oligonucleotide of claim 1 which is either RNA or DNA.

3. The oligonucleotide of claim 1 complementary to at least a continuous 12

nucleotide sequence of the region extending about 30 nucleotides upstream to a CH exon or about 30 nucleotides upstream to the intron-exon junctional segment

of a CH or to some of the sequence upstream of the CH exon or the intron-exon junctional segment and some of the continuous sequence downstream thereof.

4. The oligonucleotide of claim 1 in which the CH exon is CHI .

5. The oligonucleotide of claim 1 in which the immunoglobulin heavy chain is e, α or μ.

6. The oligonucleotide of claim 1 wherein the ohgonucleotide is a phosphorothioate, methylphosponate, an α-anomeric oligomer, or another type of hydrophobized oligomer.

7. An oligonucleotide of claim 4, which is complementary to at least a continuous 12 nucleotide sequence of the underlined portions of one of SEQ ID

NOS: 3-11 , or to some of the underlined portions and a continuous segment of the bold face portions.

8. An oligonucleotide of claim 5, which is complementary to at least a continuous 12 nucleotide sequence of the underlined portions of one of SEQ ID NOS:8, 9, 10, 11, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33, or to some of the underlined portions and to a continuous segment of the bold face portions.

9. A composition for causing suppression of IgG production, comprising oligonucleotides complementary to at least a continuous 12 nucleotide sequence of the underlined portion of SEQ ID NOS: 3, 4, 5, 6, 12, 13, 14, 15, 16, 17, 18 and 19, or to some of the underlined portions and to a continuous segment of the

bold face portions.

10. A composition for causing suppression of IgM production, comprising oligonucleotides complementary to at least a continuous 12 nucleotide sequence

of the underlined portion of SEQ ID NOS:ll, 30, 31, 32, 33, or to some of the underlined portions and to a continuous segment of the bold face portions.

11. A composition for causing suppression of IgE production, comprising oligonucleotides complementary to at least a continuous 12 nucleotide sequence of the underlined portion of SEQ JX> NOS:10, 27, 28 and 29, or to some of the

underlined portions and to a continuous segment of the bold face portions.

12. A method of suppressing production of a particular immunoglobulin isotype comprising administering an oligonucleotide of claim 1.

13. The method of claim 12 comprising administering one or more of the oligonucleotides of claims 8, 9, 10, or 11.