U.S. patent application number 10/417674 was filed with the patent office on 2003-09-04 for purified mammalian ctla-8 antigens and related reagents.
This patent application is currently assigned to INSERM, Institut National de la Sante et de la Recherche Medicale. Invention is credited to Banchereau, Jacques, Djossou, Odile, Fossiez, Francois, Golstein, Pierre, Lebecque, Serge J.E., Rouvier, Eric.
Application Number | 20030166862 10/417674 |
Document ID | / |
Family ID | 27373049 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166862 |
Kind Code |
A1 |
Golstein, Pierre ; et
al. |
September 4, 2003 |
Purified mammalian CTLA-8 antigens and related reagents
Abstract
CTLA-8 antigen from a mammal, reagents related thereto including
purified proteins, specific antibodies, and nucleic acids encoding
said antigen. Methods of using said reagents and diagnostic kits
are also provided.
Inventors: |
Golstein, Pierre;
(Marseille, FR) ; Rouvier, Eric; (Marseille,
FR) ; Fossiez, Francois; (Craponne, FR) ;
Lebecque, Serge J.E.; (Chazay d' Azergues, FR) ;
Djossou, Odile; (Francheville, FR) ; Banchereau,
Jacques; (Ecully, FR) |
Correspondence
Address: |
DNAX RESEARCH, INC.
LEGAL DEPARTMENT
901 CALIFORNIA AVENUE
PALO ALTO
CA
94304
US
|
Assignee: |
INSERM, Institut National de la
Sante et de la Recherche Medicale
|
Family ID: |
27373049 |
Appl. No.: |
10/417674 |
Filed: |
April 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10417674 |
Apr 16, 2003 |
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09929612 |
Aug 13, 2001 |
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09929612 |
Aug 13, 2001 |
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08432994 |
May 2, 1995 |
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6274711 |
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08432994 |
May 2, 1995 |
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08250846 |
May 27, 1994 |
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6562333 |
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08250846 |
May 27, 1994 |
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08177747 |
Jan 5, 1994 |
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08177747 |
Jan 5, 1994 |
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08077203 |
Jun 14, 1993 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/69.1; 530/388.22; 536/23.5 |
Current CPC
Class: |
C07K 16/244 20130101;
C07K 14/52 20130101; C07K 14/715 20130101; Y10S 435/975 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
530/350 ;
530/388.22; 536/23.5; 435/69.1; 435/320.1; 435/325 |
International
Class: |
C07K 014/74; C07K
016/28; C12P 021/02; C12N 005/06; C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 3, 1995 |
WO |
PCT/US95/00001 |
Claims
What is claimed is:
1. A nucleic acid at least 95% identical to one encoding a
mammalian CTLA protein or fragment thereof.
2. The nucleic acid of claim 1, wherein said encoding nucleic acid
comprises a sequence of SEQ ID NO: 1, 3, 5, 7, or 9.
3. A substantially pure primate CTLA-8 protein or peptide
thereof.
4. The protein or peptide of claim 3, wherein said protein or
peptide comprises a sequence of SEQ ID NO: 2, 4, 6, 8, or 10.
5. The protein or peptide of claim 4, wherein said protein or
peptide induces a cell to secrete an inflammatory mediator.
6. The protein or peptide of claim 5, wherein said inflammatory
mediator is IL-6; IL-8; and/or PGE2.
7. A composition comprising a protein of claim 3, and a
pharmaceutically acceptable carrier.
8. An antibody which specifically binds a mammalian CTLA-8 protein
or peptide thereof.
9. The antibody of claim 8, wherein said antibody is raised against
a peptide sequence of SEQ ID NO: 2, 4, 6, 8, or 10.
10. The antibody of claim 9, wherein said antibody is a monoclonal
antibody.
11. The antibody of claim 10, wherein said antibody blocks a CTLA-8
induced secretion of an inflammatory mediator.
12. The antibody of claim 10, wherein said inflammatory mediator is
IL-6; IL-8; and/or PGE2.
13. An antibody of claim 10, wherein said antibody is labeled.
14. A kit comprising: a) a nucleic acid at least 95% identical to
one encoding a mammalian CTLA-8 protein or peptide; b) a
substantially pure mammalian CTLA-8 protein or fragment; or c) an
antibody or receptor which specifically binds a mammalian CTLA-8
protein.
15. The kit of claim 14, wherein said encoding nucleic acid
comprises a sequence of SEQ ID NO: 1, 3, 5, 7, or 9.
16. The kit of claim 14, wherein said protein or fragment is
selected from the group consisting of: a) a protein or peptide from
a mammal, including a rat or human; b) a protein or peptide
comprising at least one polypeptide segment of SEQ ID NO: 2, 4, 6,
8, or 10; and c) a protein or peptide which exhibits a
post-translational modification pattern distinct from a natural
mammalian CTLA-8 protein.
17. The kit of claim 14 comprising an antibody or receptor,
wherein: a) said CTLA-8 protein from a mammal, including a mouse or
human; b) said antibody is raised against a peptide sequence of SEQ
ID NO: 2, 4, 6, 8, or 10; c) said antibody is a monoclonal
antibody; or d) said antibody is labeled.
18. A method of modulating physiology or development of a cell
comprising contacting said cell with an agonist or antagonist of a
mammalian CTLA-8 protein.
19. A method of claim 18, wherein said antagonist is an antibody
against a mammalian CTLA-8 protein.
20. A method of modulating CTLA-8 induced secretion of an
inflammatory mediator from a cell in a tissue comprising the step
of contacting said tissue with: a) an antibody which specifically
binds to a mammalian CTLA-8; or b) a substantially pure CTLA-8
protein or peptide thereof.
21. The method of claim 20, wherein said inflammatory mediator is
selected from the group consisting of: IL-6; IL-8; and/or PGE2.
22. The method of claim 20, wherein said cell is selected from the
group consisting of: a) a synovial cell; b) an endothelial cell; c)
an epithelial cell; d) a fibroblast cell; or e) a carcinoma cell.
Description
[0001] The present application is a continuation-in-part of
copending U.S. Ser. No. 08/250,846, filed May 27, 1994, which is a
continuation-in-part of then copending patent application U.S. Ser.
No. 08/177,747, filed Jan. 5, 1994, which is a continuation-in-part
of then copending patent application U.S. Ser. No. 08/077,203,
filed Jun. 14, 1993, each of which is incorporated herein by
reference. Also incorporated by reference is co-pending
PCT/US95/00001, filed Jan. 3, 1995.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions related to
proteins which function in controlling physiology, development, and
differentiation of mammalian cells, e.g., cells of a mammalian
immune system. In particular, it provides proteins and mimetics
which regulate cellular physiology, development, differentiation,
or function of various cell types, including hematopoietic
cells.
BACKGROUND OF THE INVENTION
[0003] The immune system of vertebrates consists of a number of
organs and several different cell types. Two major cell types
include the myeloid and lymphoid lineages. Among the lymphoid cell
lineage are B cells, which were originally characterized as
differentiating in fetal liver or adult bone marrow, and T cells,
which were originally characterized as differentiating in the
thymus. See, e.g., Paul (ed.) (1993) Fundamental Immunology (3d
ed.) Raven Press, New York.
[0004] In many aspects of the development of an immune response or
cellular differentiation, soluble proteins known as cytokines play
a critical role in regulating cellular interactions. These
cytokines apparently mediate cellular activities in many ways. They
have been shown, in many cases, to modulate proliferation, growth,
and differentiation of hematopoietic stem cells into the vast
number of progenitors composing the lineages responsible for an
immune response.
[0005] However, the cellular molecules which are expressed by
different developmental stages of cells in these maturation
pathways are still incompletely identified. Moreover, the roles and
mechanisms of action of signaling molecules which induce, sustain,
or modulate the various physiological, developmental, or
proliferative states of these cells is poorly understood. Clearly,
the immune system and its response to various stresses had
relevance to medicine, e.g., infectious diseases, cancer related
responses and treatment, allergic and transplantation rejection
responses. See, e.g., Thorn, et al. Harrison's Principles of
Internal Medicine McGraw/Hill, New York.
[0006] Medical science relies, in large degree, to appropriate
recruitment or suppression of the immune system in effecting cures
for insufficient or improper physiological responses to
environmental factors. However, the lack of understanding of how
the immune system is regulated or differentiates has blocked the
ability to advantageously modulate the normal defensive mechanisms
to biological challenges. Medical conditions characterized by
abnormal or inappropriate regulation of the development or
physiology of relevant cells thus remain unmanageable. The
discovery and characterization of specific cytokines will
contribute to the development of therapies for a broad range of
degenerative or other conditions which affect the immune system,
hematopoietic cells, as well as other cell types. The present
invention provides solutions to some of these and many other
problems.
SUMMARY OF THE INVENTION
[0007] The present invention is based, in part, upon the discovery
of a cDNA clone encoding a cytokine-like protein. This protein has
been designated CTLA-8. The invention embraces isolated genes
encoding the proteins of the invention, variants of the encoded
protein, e.g., mutations (muteins) of the natural sequence, species
and allelic variants, fusion proteins, chemical mimetics,
antibodies, and other structural or functional analogs. Various
uses of these different nucleic acid or protein compositions are
also provided.
[0008] The present invention embraces isolated genes encoding the
proteins of the invention, variants of the encoded protein, e.g.,
mutations (muteins) of the natural sequence, species and allelic
variants, fusion proteins, chemical mimetics, antibodies, and other
structural or functional analogs. Various uses of these different
nucleic acid or protein compositions are also provided.
[0009] The present invention provides a nucleic acid with at least
95% identity to one encoding a mammalian CTLA-8 protein or fragment
thereof. The encoding nucleic acid can comprise a sequence of SEQ
ID NO: 1, 3, 5, 7, or 9.
[0010] The present invention also provides a substantially pure
mammalian CTLA-8 protein or peptide thereof. The protein or peptide
can comprise at least one polypeptide segment of SEQ ID NO: 2, 4,
6, 8, or 10; exhibit a post-translational modification pattern
distinct from a natural mammalian CTLA-8 protein; or induce a cell
to secrete an inflammatory mediator, e.g., IL-6, IL-8, and/or PGE2.
A further embodiment is a composition comprising such a protein and
a pharmaceutically acceptable carrier.
[0011] The invention includes an antibody which specifically binds
to a primate CTLA-8 protein or peptide thereof; the antibody is
raised against a protein sequence of SEQ ID NO: 2, 4, 6, 8 or 10;
the antibody is a monoclonal antibody; the antibody blocks the
CTLA-8 induced secretion of an inflammatory mediator, e.g., IL-6,
IL-8, and/or PGE2; or the antibody is labeled.
[0012] The invention also embraces a kit comprising a substantially
pure nucleic acid at least 95% identical to one encoding a
mammalian CTLA-8 protein or peptide; a substantially. pure
mammalian CTLA-8 protein or fragment, e.g., as a positive control;
or an antibody or receptor which specifically binds a mammalian
CTLA-8 protein.
[0013] The availability of these reagents also provides methods of
modulating physiology or development of a cell comprising
contacting said cell with an agonist or antagonist of a CTLA-8
protein. The method of modulation encompasses regulating CTLA-8
induced secretion of an inflammatory mediator, e.g., IL-6, IL-8,
and/or PGE2, by contacting the cell or tissue with an antibody
which specifically binds mammalian CTLA-8 or a substantially pure
mammalian CTLA-8 protein. Preferably, the cell can be a synovial
cell, epithelial cell, endothelial cell, fibroblast cell, or a
carcinoma cell.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
OUTLINE
[0014] I. General
[0015] II. Nucleic Acids
[0016] A. natural isolates; methods
[0017] B. synthetic genes
[0018] C. methods to isolate
[0019] III. Purified CTLA-8 protein
[0020] A. physical properties
[0021] B. biological properties
[0022] IV. Making CTLA-8 protein; Mimetics
[0023] A. recombinant methods
[0024] B. synthetic methods
[0025] C. natural purification
[0026] V. Physical Variants
[0027] A. sequence variants, fragments
[0028] B. post-translational variants
[0029] 1. glycosylation
[0030] 2. others
[0031] VI. Functional Variants
[0032] A. analogs; fragments
[0033] 1. agonists
[0034] 2. antagonists
[0035] B. mimetics
[0036] 1. protein
[0037] 2. chemicals
[0038] C. species variants
[0039] VII. Antibodies
[0040] A. polyclonal
[0041] B. monoclonal
[0042] C. fragments, binding compositions
[0043] VIII. Uses
[0044] A. diagnostic
[0045] B. therapeutic
[0046] IX. Kits
[0047] A. nucleic acid reagents
[0048] B. protein reagents
[0049] C. antibody reagents
[0050] I. General
[0051] The present invention provides DNA sequence encoding various
mammalian proteins which exhibit properties characteristic of
functionally significant T cell expressed molecules. The cDNA
sequence exhibits various features which are characteristic of
mRNAs encoding cytokines, growth factors, and oncogenes. A murine
gene originally thought to be from a mouse, but now recognized as
rat as described herein contains an open reading frame encoding a
putative 150 amino acid protein. This protein is 57% homologous to
a putative protein encoded by a viral genome, the herpesvirus
Saimiri ORF13. The message was isolated using a subtraction
hybridization method applied to T cells.
[0052] These proteins are designated CTLA-8 proteins. The natural
proteins should be capable of mediating various physiological
responses which would lead to biological or physiological responses
in target cells. Initial studies had localized the message encoding
this protein to various cell lines of hematopoietic cells. Genes
encoding the antigen have been mapped to mouse chromosome 1A and
human chromosome 2q31. Murine CTLA-8 was originally cloned by
Rouvier, et al. (1993) J. Immunol. 150:5445-5456. Similar sequences
for proteins in other mammalian species should also be
available.
[0053] Purified CTLA-8, when cultured with synoviocytes, is able to
induce the secretion of IL-6 from these cells. This induction is
reversed upon the addition of a neutralizing antibody raised
against human CTLA-8-8. Endothelial, epithelial, fibroblast and
carcinoma cells also exhibit responses to treatment with CTLA-8.
This data suggests that CTLA-8 may be implicated in inflammatory
fibrosis, e.g., psoriasis, sclerodermia, lung fibrosis, or
cirrhosis. CTLA-8 may also cause proliferation of carcinomas or
other cancer cells inasmuch as IL-6 often acts as a growth factor
for such cells.
[0054] The descriptions below are directed, for exemplary purposes,
to a murine or human CTLA-8 protein, but are likewise applicable to
related embodiments from other species.
[0055] II. Nucleic Acids
[0056] Table 1 discloses the nucleotide and amino acid sequences of
a murine CTLA-8 protein. The described nucleotide sequences and the
related reagents are useful in constructing a DNA clone useful for
expressing CTLA-8 protein, or, e.g., isolating a homologous gene
from another natural source. Typically, the sequences will be
useful in isolating other genes, e.g., allelic variants, from
mouse, and similar procedures will be applied to isolate genes from
other species, e.g., warm blooded animals, such as birds and
mammals. Cross hybridization will allow isolation of genes from
other species. A number of different approaches should be available
to successfully isolate a suitable nucleic acid clone from other
sources.
1TABLE 1 Nucleotide sequence encoding a murine CTLA-8 protein and
predicted amino acid sequence. Also can use complementary nucleic
acid sequences for many purposes. Submitted to GenBank/EMBL under
accession number L13839. 1 GAATTCCATC CATGTGCCTG ATGCTGTTGC
TGCTACTGAA CCTGGAGGCT ACAGTGAAGG 61 CAGCGGTACT CATCCCTCAA
AGTTCAGTGT GTCCAAACGC CGAGGCCAAT AACTTTCTCC 121 AGAACGTGAA
GGTCAACCTG AAAGTCATCA ACTCCCTTAG CTCAAAAGCG AGCTCCAGAA 181
GGCCCTCAGA CTACCTCAAC CGTTCCACTT CACCCTGGAC TCTGAGCCGC AATGACGACC
241 CTGATAGATA TCCTTCTGTG ATCTGGGAGG CACAGTGCCG CCACCAGCGC
TGTGTCAACG 301 CTGAGGGGAA GTTGGACCAC CACATGAATT CTGTTCTCAT
CCAGCAAGAG ATCCTGGTCC 361 TGAAGAGGGA GCCTGAGAAG TGCCCCTTCA
CTTTCCGGGT GGAGAAGATG CTGGTGGGCG 421 TGGGCTGCAC CTGCGTTTCC
TCTATTGTCC GCCATGCGTC CTAAACAGAG ACCTGAGGCT 481 AGCCCCTAAG
AAACCCCTGC GTTTCTCTGC AAACTTCCTT GTCTTITTAA AACAGTTCAC 541
AGTTGAATCT CAGCAAGTGA TATGGATTTA AAGGCGGGGT TAGAATTGTC TGCCTTCCAC
601 CCTGAAAAGA AGGCGCAGAG GGGATATAAA TTGCTTCTTG TTTTTCTGTG
GGCTTTAAAT 661 TATTTATGTA TTTACTCTAT CCCGAGATAA CTTTGAGGCA
TAAGTTATTT TAATGAATTA 721 TCTACATTAT TATTATGTTT CTTAATGCAG
AAGACAAAAT TCAAGACTAA GAAATTTTAT 781 TATTTAAAAG GTAAAACCTA
TATTTATATG AGCTATTTAT GGGTCTATTT ATTTTTCTTC 841 AGTGCTAAGA
TCATGATTAT CAGATCTACC TAAGGAAGTC CTAAATAATA TTAAATATTA 901
ATTGAAATTT CAGTTTTACT ATTTGCTTAT TTAAGGTTCC CTCCTCTGAA TGGTGTGAAA
961 TCAAACCTCG TTTTATGTTT TTAAATTATT GAGGCTTCGA AAAATTGGGC
AATTTAGCTT 1021 CCTACTGTGT GTTTAAAAAC CTTGTAACAA TATCACTATA
ATAAATTTTT GGAAGAAAAT predicted amino acid sequence (150 amino
acids). Mature polypeptide probably starts at about amino acid 13
(Ala). MCLML LLLLN LEATV KAAVL IPQSS VCPNA EANNF LQNVK VNLKV INSLS
SKASS RRPSD YLNRS TSPWT LSRNE DPDRY PSVIW EAQCR HQRCV NAEGK LDHHM
NSVLI QQEIL VLKRE PEKCP FTFRV EKMLV GVGCT CVSSI VRHAS
[0057] The purified protein or defined peptides are useful for
generating antibodies by standard methods, as described above.
Synthetic peptides or purified protein can be presented to an
immune system to generate a specific binding composition, e.g.,
monoclonal or polyclonal antibodies. See, e.g., Coligan (1991)
Current Protocols in Immunology Wiley/Greene; and Harlow and Lane
(1989) Antibodies: A Laboratory Manual Cold Spring Harbor
Press.
[0058] For example, the specific binding composition could be used
for screening of an expression library made from a cell line which
expresses a CTLA-8 protein. The screening can be standard staining
of surface expressed protein, or by panning. Screening of
intracellular expression can also be performed by various staining
or immunofluorescence procedures. The binding compositions could be
used to affinity purify or sort out cells expressing the
protein.
[0059] This invention contemplates use of isolated DNA or fragments
to encode a biologically active corresponding CTLA-8 protein or
polypeptide. In addition, this invention covers isolated or
recombinant DNA which encodes a biologically active protein or
polypeptide and which is capable of hybridizing under appropriate
conditions with the DNA sequences described herein. Said
biologically active protein or polypeptide can be an intact
antigen, or fragment, and have an amino acid sequence as disclosed
in Table 1. Further, this invention covers the use of isolated or
recombinant DNA, or fragments thereof, which encode proteins which
are homologous to a CTLA-8 protein or which were isolated using
cDNA encoding a CTLA-8 protein as a probe. The isolated DNA can
have the respective regulatory sequences in the 5' and 3' flanks,
e.g., promoters, enhancers, poly-A addition signals, and others. In
particular, the murine CTLA-8 gene has significant homology, about
60%, to the putative protein encoded by the open reading frame
ORF13, of herpesvirus Saimiri (Table 2); to a human CTLA-8
counterpart (Table 3), about 60%; and to a mouse CTLA-8 counterpart
(Table 4), about 80%.
2TABLE 2 Nucleotide sequence of the related herpesvirus Saimiri
open reading frame ORF13 and predicted amino acid sequence of
encoded protein, see GenBank/EMBL accession number M60286.
herpesvirus ACCTTCATGC AAATACATCT TATCTTACCA GATTCTCGCC TCATTTGCAA
50 ACATGCCTCA TCTTTTGAGA AGAAACGCAA TTCGAACTTC TTCTAATGCT 100
CCTGAACAGC AGCCTGTGCT GCAGCCTGAG CTTGATGCTA TTGAAGAGCT 150
AGAATAAGAG CTATTTTTTG ACGATGGGTG CTGCCTTTCT GTTCAAGAAA 200
TCTGCTTAAT TGTTCTTGGA TTCTTATTGT TTCTGCTAGC TGTAATTGTT 250
TTTTATAACT ATACAGACAC AGATCAATTT GTGAAGCTGA CACATCTTAT 300
GAGCCACAAA AATTCTATCA AAGGACCTTT AAGGACCTTT GGTATGTACT 350
CATAATTTTA TTTTTTTATT TCTAAAACAA TCTTAGTATA TATAATTAAT 400
ACAAATTTTA GAAAATACTA TAATAAATAT TGAAAGCTGT ATTTACATTG 450
TAAACTATAT ATAGGCAATG TAAAGTCATT CTAACTTTAG GTTTGCTTTA 500
CCTGTTACAG AAACTTCACC TGTGTGTCAA GAGCTGCAAA CATGGCTTTA 550
GACTTAAGAA ATCTTAAACA CCTGACTGCT AACTTCAGTT TTAGAATAAT 600
GATATGGATT ATGCTATGTT TGGCTCTACC TACTGATAGT AAACCTATTT 650
CAACAACTGA AGCTCCAATA CTAAACATAA CACAATCTCC AAGTTTGAAC 700
ATCTCATCAC CTTCTACTTT AGAACCTTCA GAGCCTCTTA AAAACTGTAC 750
AACATTCTTA GACTTACTTT GGCAGCGGCT GGGCGAGAAC GCTTCTATAA 800
AGGACTTGAT GTTAACATTA CAACGAGAAG AAGTCCACGG AAGAATGACT 850
ACACTTCCTT CACCTAGACC AAGCAGTAAA GTTGAAGAAC AACAGTTACA 900
AAGACCTAGA AACTTACTGC CTACTGCTGT CGGGCCACCT CATGTCAAAT 950
ATAGACTATA TAATCGCTTA TGGGAAGCTC CTAAAGGAGC TGATGTTAAT 1000
GGTAAACCTA TACAATTTGA TGACCCTCCT CTTCCTTATA CAGGGGCATA 1050
TAATGATGAT GGTGTTTTAA TGGTTAATAT TAATGGAAAA CATGTGAGGT 1100
TTGATAGCTT GTCTTATTGG GAAAGAATTA AAAGATCTGG TACCCCATGG 1150
TGTATAAAGA CACCAAGTGA AAAAGCAGCA ATATTGAAGC AGCTTTTAAA 1200
AGCTGAAAAA AAATGTAGGA CTACTTCTAA ACGTATCACT GAGTTAGAAG 1250
AGCAGATTAA AGAACTAGAA AAAACTAGTA CATCTCCATA GATTACTGTT 1300
AGAATGTGTT TATCATACTA AAATAAATGC TTTATGTATT GCAATATTAC 1350
TTGTTTGCTA TGACTTTGGT ATATGAAATG CAAATCTTAA ATAAAAAGTT 1400
TTTCTCTAGT ATTGGCGTCA CTGTATTTTA CTAGCAAAAA TATATAAATT 1450
GTTATGTAGC AACAAGTTTG TATCAATATA AAAACTCTAA AGTATATAAA 1500
CAAACATTCA ATTAGTGTAA ATCATAGCAA GCATATCTTT TCATACGTGT 1550
CTAGTTAATT TAAAGAATTA ATTATGACAT TTAGAATGAC TTCACTTGTG 1600
TTACTTCTCC TGCTGAGCAT AGATTGTATA GTAAAGTCAG AAATAACAAG 1650
CGCACAAACC CCAAGATGCT TAGCTGCTAA CAATAGCTTT CCACGGTCTG 1700
TGATGGTTAC TTTGAGCATC CGTAACTGGA ATACTAGTTC TAAAAGGGCT 1750
TCAGACTACT ACAATAGATC TACGTCTCCT TGGACTCTCC ATCGCAATGA 1800
AGATCAAGAT AGATATCCCT CTGTGATTTC GGAAGCAAAG TGTCGCTACT 1850
TACGATGTGT TAATGCTGAT GGGAATGTAG ACTACCACAT GAACTCAGTC 1900
CCTATCCAAC AAGAGATTCT AGTGGTGCGC AAAGGGCATC AACCCTGCCC 1950
TAATTCATTT AGGCTAGAGA AGATGCTAGT GACTGTAGGC TGCACATGCG 2000
TTACTCCCAT TGTTCACAAT GTAGACTAAA AGCTATCTAA ATTTTGAAAA 2050
TTAACATTTC ACTAAAAAAC AAAAACTTGA TTTTTTTCTT TTAAATAAAA 2100
AAAGTTTAAT ATAAGTTCTG GCTTGTTTGG TTTTTGACTA ATCAATGTAG 2150
ATCACACTTG TGATCTTAGC TCTCGGGAAG CAATGTAAGA AAATATATTT 2200
AACTTAAGAG TTTTAGACTT GCTTGAGTTT TATGAGTAAA AAACAAAGAA 2250
TAAGCACAGC TTCTTGTATC TTCTTTTAAA AACTTTAAGT TATTTATGTA 2300
TTTAATATAA TCTAATGTTT CTTAAACATG TTGAGTTTGA GGTCCACTAA 2350
TACAACATTA TAATTTTTTC TGTTATAACA CTTTTGCAAG AAGAACTCAT 2400
TTTATAGAAA ATGAGCAGTA TTCAAAAAAA ATGTTTGATA TGCTGTAATA 2450
TTGGAGAGGA AGAACTTTTA CAAGCATGTG ATTGTCCTAG CAGAGTCCAT 2500
CATACATGCT TACAAAGTCA 2520 Tm17 Peptide sequence MVIDG CKKYM RRTCG
DVLDN LRGDC YQVLI EDCIP VLKMY AKEGR EFDYV 50 INDLT AVPIS TSPEE
DSTWD FLRLI LDLSM KVLKQ DGKTF TQGNC VNLTE 100 ALSLY EEQLG RLYCP
VEFSK EIVCV PSYLE LNVFY TVWKK AKP 143
[0060]
3TABLE 3 Nucleotide sequence of human CTLA-8 fragment and predicted
amino acid sequence of encoded protein. AGC/CGC AAT GAG GAC CCT GAG
AGA TAT CCC TCT GTG ATC TGG GAG GCA AAG TGC CGC CAC TTG CCC TGC ATC
AAC GCT GAT GGG AAC GTG GAC TAC CAC ATG AAC TCT GTC CCC ATC CAG CAA
GAG ATC CTG GTC CTG CGC AGG GAG CCT CCA CAC TGC CCC AAC TCC TTC CGG
CTG GAG AAG ATA CTG GTG TCC GTG GGC TCC ACC TCT GTC ACC CCC ATT GTC
CAC CAT GTG GCC TAA ser/arg asn glu asp pro glu arg tyr pro ser val
ile trp glu ala lys cys arg his leu gly cys ile asn ala asp gly asn
val asp tyr his met asn ser val pro ile gln gln glu ile leu val leu
arg arg glu pro pro his cys pro asn ser phe arg leu glu lys ile leu
val ser val gly cys thr cys val thr pro ile val his his val ala OCH
this was used to isolate a full length clone from human; it
corresponds to nucleotides 272-510: GG CACAAACTCA TCCATCCCCA
GTTGATTGGA AGAAACAACG 42 ATG ACT CCT GGG AAG ACC TCA TTG GTG TCA
CTG CTA CTG CTG CTG 87 Met thr pro gly lys thr ser leu val ser leu
leu leu leu leu 15 AGC CTG GAG GCC ATA GTC AAG GCA GGA ATC ACA ATC
CCA CGA AAT 132 ser leu glu ala ile val lys ala gly ile thr ile pro
arg asn 30 CCA GGA TCC CCA AAT TCT GAG GAC AAG AAC TTC CCC CGG ACT
GTG 177 pro gly cys pro asn ser glu asp lys asn phe pro arg thr val
45 ATG GTC AAC CTG AAC ATC CAT AAC CGG AAT ACC AAT ACC AAT CCC 222
met val asn leu asn ile his asn arg asn thr asn thr asn pro 60 aaA
AGG TCC TCA GAT TAC TAC AAC CGA TCC ACC TCA CCT TGG AAT 267 lys arg
ser ser asp tyr tyr asn arg ser thr ser pro trp asn 75 CTC CAC CGC
AAT GAG GAC CCT GAG AGA TAT CCC TCT CTG ATC TGG 312 leu his arg asn
glu asp pro glu arg tyr pro ser val ile trp 90 GAG GCA AAG TGC CGC
CAC TTG GGC TGC ATC AAC GCT GAT GGG AAC 357 glu ala lys cys arg his
leu gly cys ile asn ala asp gly asn 105 GTG GAC TAC CAC ATG AAC TCT
GTC CCC ATC CAG CAA GAG ATC CTG 402 val asp tyr his met asn ser val
pro ile gln gln glu ile leu 120 GTC CTG CGC AGG GAG CCT CCA CAC TGC
CCC AAC TCC TTC CGG CTG 447 val leu arg arg glu pro pro his cys pro
asn ser phe arg leu 135 GAG AAG ATA CTG GTG TCC GTG GGC TGC ACC TGT
GTC ACC CCG ATT 492 glu lys ile leu val ser val gly cys thr cys val
thr pro ile 150 GTC CAC CAT GTG GCC TAA 510 val his his val ala OCH
156
[0061]
4TABLE 4 Nucleotide sequence of mouse CTLA-8 fragment and predicted
amino acid sequence of encoded protein.
gaggctcaagtgcacccagcaccagctgatcaggacgcgcaaacatgagtccagggagagcttcatctg
69 tgtctctgatgctgttgctgctgctgagcctggcggctacagtgaaggcagca-
gcgatcatccctcaaa 138 gctcagcgtgtccaaacactgaggccaaggacttcct-
ccagaatgtgaaggtcaacctcaaagtcttta 207
actcccTTGGCGCAAAAGTGAGCTCCAGAAGgCCCTCAGACTACCTCAACCGTTCCACGTCACCCTGGA
276 CTCTCCACCGCAATGAAGACCCTGATAGATATCCCTCTGTGATCTGGGAAGCTCAGT-
GCCGCCACCAGC 345 GCTGTGTCAATGCGGAGggaaagctggaccaccacatgaat-
tctgttctcatccagcaagagatcctgg 414 tcctgaagagggagcctgagagctg-
ccccttcactttcagggtcgagaagatgctggtgggTGTGGGCT 483
GCACCTGCGTGGCCTCGATTGTCCGCCAGGCAGCCTAAACAGAGACCCGCGGCTGACCCCTAAGAAACC
552 CCCACGTTTCTCAGCAAACTTACTTGCATTTTTAAAACAGTTCGTGCTATTGATTTT-
CAGCAAGGAATG 621 TGGATTCAGAGGCAGATTCAGAATTGTCTGCCCTCCACAAT-
GAAAAGAAGGTGTAAAGGGGTCCCAAAC 690 TGCTTCgtgtttgtttttctgtgga-
ctttaaattatttgtgtatttacaatatcccaagataactttga 759
aggcgtaacttatttaatgaagtatctacattattattatgtttctttctgaagaagacaaaattcaag
828 actcagaaattttattatttaaaaggtaagcctatatttatatgagctatttatgaa-
tctatttatttt 897 tcttcagtatttgaagtattaagaacatgattttCAGATCT-
ACCTACGGAAGTCCTAAGTAAGATTAAA 966 TATTAATGGAAATTTCAGCTTTACT-
ATTTGGTTGATTTAAGGTTCTCTCCTCTGAATGGGGTGAAAACC 1035
AAACTTAGTTTTATGTTTAATAACTTTTTAAATTATTGAAGATTCAAAAAATTGGATAATTTAGCTCCC
1104 TACTCTGTTTTAAAAAAAAAAAAAAAAAAA 1134 Mouse CTLA-8 predicted
amino acid sequence. The mature polypeptide probably starts at a
position about amino acid 19 (Leu) to amino acid 21 (Ala).
METSerProGlyArgAlaSerSerValSerLeuMETLeuLeuLeuLeuLeu-
SerLeuAlaAlaThrValLys 24 AlaAlaAlaIleIleProGlnSerSerAlaCy-
sProAsnThrGluAlaLysAspPheLeuGlnAsnValLys 48
ValAsnLeuLysValPheAsnSerLeuGlyAlaLysValSerSerArgArgProSerAspTyrLeuAsnArg
72 SerThrSerProTrpThrLeuHisArgAsnGluAspProAspArgTyrProSer-
ValIleTrpGluAlaGln 96 CysArgHisGlnArgCysValAsnAlacluGlyLys-
LeuAspHisHisMETAsnSerValLeuIleGlnGln 120
GluIleLeuValLeuLysArgGluProGluSercysProPheThrPheArgValGlubysMETLeuValGly
144 ValGlyCysThrCysValAlaSerIleValArgGlnAlaAla 158
[0062] An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially separated from
other components which naturally accompany a native sequence, e.g.,
ribosomes, polymerases, and flanking genomic sequences from the
originating species. The term embraces a nucleic acid sequence
which has been removed from its naturally occurring environment,
and includes recombinant or cloned DNA isolates and chemically
synthesized analogs or analogs biologically synthesized by
heterologous systems. A substantially pure molecule includes
isolated forms of the molecule. Alternatively, a purified species
may be separated from host components from a recombinant expression
system. The size of homology of such a nucleic acid will typically
be less than large vectors, e.g., less than tens of kB, typically
less than several kB, and preferably in the 2-6 kB range.
[0063] An isolated nucleic acid will generally be a homogeneous
composition of molecules, but will, in some embodiments, contain
minor heterogeneity. This heterogeneity is typically found at the
polymer ends or portions not critical to a desired biological
function or activity.
[0064] A "recombinant" nucleic acid is defined either by its method
of production or its structure. In reference to its method of
production, e.g., a product made by a process, the process is use
of recombinant nucleic acid techniques, e.g., involving human
intervention in the nucleotide sequence, typically selection or
production. Alternatively, it can be a nucleic acid made by
generating a sequence comprising fusion of two fragments which are
not naturally contiguous to each other, but is meant to exclude
products of nature, e.g., naturally occurring mutants. Thus, for
example, products made by transforming cells with any unnaturally
occurring vector is encompassed, as are nucleic acids comprising
sequence derived using any synthetic oligonucleotide process. Such
is often done to replace a codon with a redundant codon encoding
the same or a conservative amino acid, while typically introducing
or removing a sequence recognition site. Alternatively, it is
performed to join together nucleic acid segments of desired
functions to generate a single genetic entity comprising a desired
combination of functions not found in the commonly available
natural forms. Restriction enzyme recognition sites are often the
target of such artificial manipulations, but other site specific
targets, e.g., promoters, DNA replication sites, regulation
sequences, control sequences, or other useful features may be
incorporated by design. A similar concept is intended for a
recombinant, e.g., fusion, polypeptide. Specifically included are
synthetic nucleic acids which, by genetic code redundancy, encode
polypeptides similar to fragments of these antigens, and fusions of
sequences from various different species variants.
[0065] A significant "fragment" in a nucleic acid context is a
contiguous segment of at least about 17 nucleotides, generally at
least 20 nucleotides, more generally at least 23 nucleotides,
ordinarily at least 26 nucleotides, more ordinarily at least 29
nucleotides, often at least 32 nucleotides, more often at least 35
nucleotides, typically at least 38 nucleotides more typically at
least 41 nucleotides, usually at least 44 nucleotides, more usually
at least 47 nucleotides, preferably at least 50 nucleotides, more
preferably at least 53 nucleotides, and in particularly preferred
embodiments will be at least 56 or more nucleotides. Said fragments
may have termini at any location, but especially at boundaries
between structural domains.
[0066] A DNA which codes for a CTLA-8 protein will be particularly
useful to identify genes, mRNA, and cDNA species which code for
related or homologous proteins, as well as DNAs which code for
homologous proteins from different species. There are likely
homologues in other species, including primates. Various CTLA-8
proteins should be homologous and ate encompassed herein. However,
even proteins that have a more distant evolutionary relationship to
the antigen can readily be isolated under appropriate conditions
using these sequences if they are sufficiently homologous. Primate
CTLA-8 protein proteins are of particular interest.
[0067] This invention further covers recombinant DNA molecules and
fragments having a DNA sequence identical to or highly homologous
to the isolated DNAs set forth herein. In particular, the sequences
will often be operably linked to DNA segments which control
transcription, translation, and DNA replication. Alternatively,
recombinant clones derived from the genomic sequences, e.g.,
containing introns, will be useful for transgenic studies,
including, e.g., transgenic cells and organisms, and for gene
therapy. See, e.g., Goodnow (1992) "Transgenic Animals" in Roitt
(ed.) Encyclopedia of Immunology Academic Press, San Diego, pp.
1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al. (1991)
Science 254:707-710; Capecchi (1989) Science 244:1288; Robertson
(1987)(ed.) Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach IRL Press, Oxford; Rosenberg (1992) J. Clinical Oncology
10:180-199; and Cournoyer and Caskey (1993) Ann. Rev. Immunol.
11:297-329.
[0068] Homologous nucleic acid sequences, when compared, exhibit
significant similarity. The standards for homology in nucleic acids
are either measures for homology generally used in the art by
sequence comparison or based upon hybridization conditions. The
hybridization conditions are described in greater detail below.
[0069] Substantial homology in the nucleic acid sequence comparison
context means either that the segments, or their complementary
strands, when compared, are identical when optimally aligned, with
appropriate nucleotide insertions or deletions, in at least about
50% of the nucleotides, generally at least 56%, more generally at
least 59%, ordinarily at least 62%, more ordinarily at least 65%,
often at least 68%, more often at least 71%, typically at least
74%, more typically at least 77%, usually at least 80%, more
usually at least about 85%, preferably at least about 90%, more
preferably at least about 95 to 98% or more, and in particular
embodiments, as high at about 99% or more of the nucleotides.
Alternatively, substantial homology exists when the segments will
hybridize under selective hybridization conditions, to a strand, or
its complement, typically using a sequence derived from Table 1, 2,
or 3. Typically, selective hybridization will occur when there is
at least about 55% homology over a stretch of at least about 14
nucleotides, preferably at least about 65%, more preferably at
least about 75%, and most preferably at least about 90%. See,
Kanehisa (1984) Nuc. Acids Res. 12:203-213. The length of homology
comparison, as described, may be over longer stretches, and in
certain embodiments will be over a stretch of at least about 17
nucleotides, usually at least about 20 nucleotides, more usually at
least about 24 nucleotides, typically at least about 28
nucleotides, more typically at least about 40 nucleotides,
preferably at least about 50 nucleotides, and more preferably at
least about 75 to 100 or more nucleotides.
[0070] Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters,
typically those controlled in hybridization reactions. Stringent
temperature conditions will usually include temperatures in excess
of about 30.degree. C., more usually in excess of about 37.degree.
C., typically in excess of about 45.degree. C., more typically in
excess of about 55.degree. C., preferably in excess of about
65.degree. C., and more preferably in excess of about 70.degree. C.
Stringent salt conditions will ordinarily be less than about 1000
mM, usually less than about 500 mM, more usually less than about
400 mM, typically less than about 300 mM, preferably less than
about 200 mM, and more preferably less than about 150 mM. However,
the combination of parameters is much more important than the
measure of any single parameter. See, e.g., Wetmur and Davidson
(1968) J. Mol. Biol. 31:349-370.
[0071] CTLA-8 protein from other mammalian species can be cloned
and isolated by cross-species hybridization of closely related
species, e.g., human, as disclosed in Table 3. Homology may be
relatively low between distantly related species, and thus
hybridization of relatively closely related species is advisable.
Alternatively, preparation of an antibody preparation which
exhibits less species specificity may be useful in expression
cloning approaches.
[0072] III. Purified CTLA-8 Protein
[0073] The predicted sequence of murine CTLA-8 protein amino acid
sequence is shown in Table 1. The homologous herpesvirus predicted
ORF13 protein sequence is shown in Table 2, and is assigned SEQ ID
NO: 4. A human counterpart is described in Table 3. The peptide
sequences allow preparation of peptides to generate antibodies to
recognize such segments.
[0074] As used herein, the terms "murine CTLA-8 protein" and "human
CTLA-8 protein shall encompass, when used in a protein context, a
protein having amino acid sequences shown in Table 1 or Table 3, or
a significant fragment of such a protein. It also refers to a mouse
derived polypeptide which exhibits similar biological function or
interacts with CTLA-8 protein specific binding components. These
binding components, e.g., antibodies, typically bind to a CTLA-8
protein with high affinity, e.g., at least about 100 nM, usually
better than about 30 nM, preferably better than about 10 nM, and
more preferably at better than about 3 nM. Homologous proteins
would be found in mammalian species other than rat or humans, e.g.,
mouse, primates, and in the herpesvirus genome, e.g., ORF13.
Non-mammalian species should also possess structurally or
functionally related genes and proteins.
[0075] The term "polypeptide" as used herein includes a significant
fragment or segment, and encompasses a stretch of amino acid
residues of at least about 8 amino acids, generally at least 10
amino acids, more generally at least 12 amino acids, often at least
14 amino acids, more often at least 16 amino acids, typically at
least 18 amino acids, more typically at least 20 amino acids,
usually at least 22 amino acids, more usually at least 24 amino
acids, preferably at least 26 amino acids, more preferably at least
28 amino acids, and, in particularly preferred embodiments, at
least about 30 or more amino acids. The specific ends of such a
segment will be at any combinations within the protein, preferably
encompassing structural domains.
[0076] The term "binding composition" refers to molecules that bind
with specificity to CTLA-8 protein, e.g., in a ligand-receptor type
fashion, an antibody-antigen interaction, or compounds, e.g.,
proteins which specifically associate with CTLA-8 protein, e.g., in
a natural physiologically relevant protein-protein interaction,
either covalent or non-covalent. The molecule may be a polymer, or
chemical reagent. No implication as to whether CTLA-8 protein is
either the ligand or the receptor of a ligand-receptor interaction
is represented, other than the interaction exhibit similar
specificity, e.g., specific affinity. A functional analog may be a
protein with structural modifications, or may be a wholly unrelated
molecule, e.g., which has a molecular shape which interacts with
the appropriate binding determinants. The proteins may serve as
agonists or antagonists of a receptor, see, e.g., Goodman, et al.
(eds.) (1990) Goodman & Gilman's: The Pharmacological Bases of
Therapeutics (8th ed.), Pergamon Press.
[0077] Solubility of a polypeptide or fragment depends upon the
environment and the polypeptide. Many parameters affect polypeptide
solubility, including temperature, electrolyte environment, size
and molecular characteristics of the polypeptide, and nature of the
solvent. Typically, the temperature at which the polypeptide is
used ranges from about 4.degree. C. to about 65.degree. C. Usually
the temperature at use is greater than about 18.degree. C. and more
usually greater than about 22.degree. C. For diagnostic purposes,
the temperature will usually be about room temperature or warmer,
but less than the denaturation temperature of components in the
assay. For therapeutic purposes, the temperature will usually be
body temperature, typically about 37.degree. C. for humans, though
under certain situations the temperature may be raised or lowered
in situ or in vitro.
[0078] The electrolytes will usually approximate in situ
physiological conditions, but may be modified to higher or lower
ionic strength where advantageous. The actual ions may be modified,
e.g., to conform to standard buffers used in physiological or
analytical contexts.
[0079] The size and structure of the polypeptide should generally
be in a substantially stable state, and usually not in a denatured
state. The polypeptide may be associated with other polypeptides in
a quaternary structure, e.g., to confer solubility, or associated
with lipids or detergents in a manner which approximates natural
lipid bilayer interactions.
[0080] The solvent will usually be a biologically compatible
buffer, of a type used for preservation of biological activities,
and will usually approximate a physiological solvent. Usually the
solvent will have a neutral pH, typically between about 5 and 10,
and preferably about 7.5. On some occasions, a detergent will be
added, typically a mild non-denaturing one, e.g., CHS or CHAPS, or
a low enough concentration as to avoid significant disruption of
structural or physiological properties of the antigen.
[0081] Solubility is reflected by sedimentation measured in
Svedberg units, which are a measure of the sedimentation velocity
of a molecule under particular conditions. The determination of the
sedimentation velocity was classically performed in an analytical
ultracentrifuge, but is typically now performed in a standard
ultracentrifuge. See, Freifelder (1982) Physical Biochemistry (2d
ed.), W. H. Freeman; and Cantor and Schimmel (1980) Biophysical
Chemistry, parts 1-3, W. H. Freeman & Co., San Francisco. As a
crude determination, a sample containing a putatively soluble
polypeptide is spun in a standard full sized ultracentrifuge at
about 50K rpm for about 10 minutes, and soluble molecules will
remain in the supernatant. A soluble particle or polypeptide will
typically be less than about 30 S, more typically less than about
15 S, usually less than about 10 S, more usually less than about 6
S, and, in particular embodiments, preferably less than about 4 S,
and more preferably less than about 3 S.
[0082] IV. Making CTLA-8 Protein; Mimetics
[0083] DNA which encodes the CTLA-8 protein or fragments thereof
can be obtained by chemical synthesis, screening cDNA libraries, or
by screening genomic libraries prepared from a wide variety of cell
lines or tissue samples.
[0084] This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length protein or fragments which can
in turn, for example, be used to generate polyclonal or monoclonal
antibodies; for binding studies; for construction and expression of
modified molecules; and for structure/function studies. Each
antigen or its fragments can be expressed in host cells that are
transformed or transfected with appropriate expression vectors.
These molecules can be substantially purified to be free of protein
or cellular contaminants, other than those derived from the
recombinant host, and therefore are particularly useful in
pharmaceutical compositions when combined with a pharmaceutically
acceptable carrier and/or diluent. The antigen, or portions
thereof, may be expressed as fusions with other proteins.
[0085] Expression vectors are typically self-replicating DNA or RNA
constructs containing the desired antigen gene or its fragments,
usually operably linked to suitable genetic control elements that
are recognized in a suitable host cell. These control elements are
capable of effecting expression within a suitable host. The
specific type of control elements necessary to effect expression
will depend upon the eventual host cell used. Generally, the
genetic control elements can include a prokaryotic promoter system
or a eukaryotic promoter expression control system, and typically
include a transcriptional promoter, an optional operator to control
the onset of transcription, transcription enhancers to elevate the
level of mRNA expression, a sequence that encodes a suitable
ribosome binding site, and sequences that terminate transcription
and translation. Expression vectors also usually contain an origin
of replication that allows the vector to replicate independently of
the host cell. Methods for amplifying vector copy number are also
known, see, e.g., Kaufman, et al. (1985) Molec. and Cell. Biol.
5:1750-1759.
[0086] The vectors of this invention contain DNA which encodes a
CTLA-8 protein, or a fragment thereof, typically encoding a
biologically active polypeptide. The DNA can be under the control
of a viral promoter and can encode a selection marker. This
invention further contemplates use of such expression vectors which
are capable of expressing eukaryotic cDNA coding for a CTLA-8
protein in a prokaryotic or eukaryotic host, where the vector is
compatible with the host and where the eukaryotic cDNA coding for
the antigen is inserted into the vector such that growth of the
host containing the vector expresses the cDNA in question. Usually,
expression vectors are designed for stable replication in their
host cells or for amplification to greatly increase the total
number of copies of the desirable gene per cell. It is not always
necessary to require that an expression vector replicate in a host
cell, e.g., it is possible to effect transient expression of the
antigen or its fragments in various hosts using vectors that do not
contain a replication origin that is recognized by the host cell.
It is also possible to use vectors that cause integration of a
CTLA-8 protein gene or its fragments into the host DNA by
recombination, or to integrate a promoter which controls expression
of an endogenous gene.
[0087] Vectors, as used herein, comprise plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles which
enable the integration of DNA fragments into the genome of the
host. Expression vectors are specialized vectors which contain
genetic control elements that effect expression of operably linked
genes. Plasmids are the most commonly used form of vector but all
other forms of vectors which serve an equivalent function and which
are, or become, known in the art are suitable for use herein. See,
e.g., Pouwels, et al.. (1985 and Supplements) Cloning Vectors: A
Laboratory Manual, Elsevier, N.Y., and Rodriquez, et al.
(1988)(eds.) Vectors: A Survey of Molecular Cloning Vectors and
Their Uses, Buttersworth, Boston, Mass.
[0088] Transformed cells include cells, preferably mammalian, that
have been transformed or transfected with vectors containing a
CTLA-8 gene, typically constructed using recombinant DNA
techniques. Transformed host cells usually express the antigen or
its fragments, but for purposes of cloning, amplifying, and
manipulating its DNA, do not need to express the protein. This
invention further contemplates culturing transformed cells in a
nutrient medium, thus permitting the protein to accumulate in the
culture. The protein can be recovered, either from the culture or
from the culture medium.
[0089] For purposes of this invention, DNA sequences are operably
linked when they are functionally related to each other. For
example, DNA for a presequence or secretory leader is operably
linked to a polypeptide if it is expressed as a preprotein or
participates in directing the polypeptide to the cell membrane or
in secretion of the polypeptide. A promoter is operably linked to a
coding sequence if it controls the transcription of the
polypeptide; a ribosome binding site is operably linked to a coding
sequence if it is positioned to permit translation. Usually,
operably linked means contiguous and in reading frame, however,
certain genetic elements such as repressor genes are not
contiguously linked but still bind to operator sequences that in
turn control expression.
[0090] Suitable host cells include prokaryotes, lower eukaryotes,
and higher eukaryotes. Prokaryotes include both gram negative and
gram positive organisms, e.g., E. coli and B. subtilis. Lower
eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and
species of the genus Dictyostelium. Higher eukaryotes include
established tissue culture cell lines from animal cells, both of
non-mammalian origin, e.g., insect cells, and birds, and of
mammalian origin, e.g., human, primates, and rodents.
[0091] Prokaryotic host-vector systems include a wide variety of
vectors for many different species. As used herein, E. coli and its
vectors will be used generically to include equivalent vectors used
in other prokaryotes. A representative vector for amplifying DNA is
pBR322 or many of its derivatives. Vectors that can be used to
express the CTLA-8 proteins or its fragments include, but are not
limited to, such vectors as those containing the lac promoter
(pUC-series); trp promoter (pBR322-trp); Ipp promoter (the
pIN-series); lambda-pP or pR promoters (pOTS); or hybrid promoters
such as ptac (pDR540). See Brosius, et al. (1988) "Expression
Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters",
in Rodriguez and Denhardt (eds.) Vectors: A Survey of Molecular
Cloning Vectors and Their Uses, Buttersworth, Boston, Chapter 10,
pp. 205-236.
[0092] Lower eukaryotes, e.g., yeasts and Dictyostelium, may be
transformed with vectors encoding CTLA-8 proteins. For purposes of
this invention, the most common lower eukaryotic host is the
baker's yeast, Saccharomyces cerevisiae. It will be used to
generically represent lower eukaryotes although a number of other
strains and species are also available. Yeast vectors typically
consist of a replication origin (unless of the integrating type), a
selection gene, a promoter, DNA encoding the desired protein or its
fragments, and sequences for translation termination,
polyadenylation, and transcription termination. Suitable expression
vectors for yeast include such constitutive promoters as
3-phosphoglycerate kinase and various other glycolytic enzyme gene
promoters or such inducible promoters as the alcohol dehydrogenase
2 promoter or metallothionine promoter. Suitable vectors include
derivatives of the following types: self-replicating low copy
number (such as the YRp-series), self-replicating high copy number
(such as the YEp-series); integrating types (such as the
YIp-series), or mini-chromosomes (such as the YCp-series).
[0093] Higher eukaryotic tissue culture cells are the preferred
host cells for expression of the functionally active CTLA-8
protein. In principle, many higher eukaryotic tissue culture cell
lines are workable, e.g., insect baculovirus expression systems,
whether from an invertebrate or vertebrate source. However,
mammalian cells are preferred, in that the processing, both
cotranslationally and posttranslationally. Transformation or
transfection and propagation of such cells has become a routine
procedure. Examples of useful cell lines include HeLa cells,
Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell
lines, insect cell lines, bird cell lines, and monkey (COS) cell
lines. Expression vectors for such cell lines usually include an
origin of replication, a promoter, a translation initiation site,
RNA splice sites (if genomic DNA is used), a polyadenylation site,
and a transcription termination site. These vectors also usually
contain a selection gene or amplification gene. Suitable expression
vectors may be plasmids, viruses, or retroviruses carrying
promoters derived, e.g., from such sources as from adenovirus,
SV40, parvoviruses, vaccinia virus, or cytomegalovirus.
Representative examples of suitable expression vectors include
pCDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol.
5:1136-1142; pMC1neo Poly-A, see Thomas, et al. (1987) Cell
51:503-512; and a baculovirus vector such as pAC 373 or pAC 610,
see O'Reilly, et al. (1992) Baculovirus Expression Vectors: A
Laboratory Manual Freeman and Co., CRC Press, Boca Raton, Fla.
[0094] It will often be desired to express a CTLA-8 protein
polypeptide in a system which provides a specific or defined
glycosylation pattern. In this case, the usual pattern will be that
provided naturally by the expression system. However, the pattern
will be modifiable by exposing the polypeptide, e.g., an
unglycosylated form, to appropriate glycosylating proteins
introduced into a heterologous expression system. For example, the
CTLA-8 protein gene may be co-transformed with one or more genes
encoding mammalian or other glycosylating enzymes. Using this
approach, certain mammalian glycosylation patterns will be
achievable or approximated in prokaryote or other cells.
[0095] The CTLA-8 protein, or a fragment thereof, may be engineered
to be phosphatidyl inositol (PI) linked to a cell membrane, but can
be removed from membranes by treatment with a phosphatidyl inositol
cleaving enzyme, e.g., phosphatidyl inositol phospholipase-C. This
releases the antigen in a biologically active form, and allows
purification by standard procedures of protein chemistry. See,
e.g., Low (1989) Biochim. Biophys. Acta 988:427-454; Tse, et al.
(1985) Science 230:1003-1008; and Brunner, et al. (1991) J. Cell
Biol. 114:1275-1283.
[0096] Now that the CTLA-8 protein has been characterized,
fragments or derivatives thereof can be prepared by conventional
processes for synthesizing peptides. These include processes such
as are described in Stewart and Young (1984) Solid Phase Peptide
Synthesis, Pierce Chemical Co., Rockford, Ill.; Bodanszky and
Bodanszky (1984) The Practice of Peptide Synthesis,
Springer-Verlag, New York; and Bodanszky (1984) The Principles of
Peptide Synthesis, Springer-Verlag, New York. For example, an azide
process, an acid chloride process, an acid anhydride process, a
mixed anhydride process, an active ester process (for example,
p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl
ester), a carbodiimidazole process, an oxidative-reductive process,
or a dicyclohexylcarbodiimide (DCCD)/additive process can be used.
Solid phase and solution phase syntheses are both applicable to the
foregoing processes.
[0097] The CTLA-8 protein, fragments, or derivatives are suitably
prepared in accordance with the above processes as typically
employed in peptide synthesis, generally either by a so-called
stepwise process which comprises condensing an amino acid to the
terminal amino acid, one by one in sequence, or by coupling peptide
fragments to the terminal amino acid. Amino groups that are not
being used in the coupling reaction are typically protected to
prevent coupling at an incorrect location.
[0098] If a solid phase synthesis is adopted, the C-terminal amino
acid is bound to an insoluble carrier or support through its
carboxyl group. The insoluble carrier is not particularly limited
as long as it has a binding capability to a reactive carboxyl
group. Examples of such insoluble carriers include halomethyl
resins, such as chloromethyl resin or bromomethyl resin,
hydroxymethyl resins, phenol resins,
tert-alkyloxycarbonyl-hydrazidated resins, and the like.
[0099] An amino group-protected amino acid is bound in sequence
through condensation of its activated carboxyl group and the
reactive amino group of the previously formed peptide or chain, to
synthesize the peptide step by step. After synthesizing the
complete sequence, the peptide is split off from the insoluble
carrier to produce the peptide. This solid-phase approach is
generally described by Merrifield, et al. (1963) in J. Am. Chem.
Soc. 85:2149-2156.
[0100] The prepared protein and fragments thereof can be isolated
and purified from the reaction mixture by means of peptide
separation, for example, by extraction, precipitation,
electrophoresis and various forms of chromatography, and the like.
The CTLA-8 proteins of this invention can be obtained in varying
degrees of purity depending upon its desired use. Purification can
be accomplished by use of the protein purification techniques
disclosed herein or by the use of the antibodies herein described
in immunoabsorbant affinity chromatography. This immunoabsorbant
affinity chromatography is carried out by first linking the
antibodies to a solid support and then contacting the linked
antibodies with solubilized lysates of appropriate source cells,
lysates of other cells expressing the protein, or lysates or
supernatants of cells producing the CTLA-8 protein as a result of
DNA techniques, see below.
[0101] V. Physical Variants
[0102] This invention also encompasses proteins or peptides having
substantial amino acid sequence homology with the amino acid
sequence of the CTLA-8 protein. The variants include species or
allelic variants.
[0103] Amino acid sequence homology, or sequence identity, is
determined by optimizing residue matches, if necessary, by
introducing gaps as required. This changes when considering
conservative substitutions as matches. Conservative substitutions
typically include substitutions within the following groups:
glycine, alanine; valine, isoleucine, leucine; aspartic acid,
glutamic acid; asparagine, glutamine; serine, threonine; lysine,
arginine; and phenylalanine, tyrosine. Homologous amino acid
sequences are typically intended to include natural allelic and
interspecies variations in each respective protein sequence.
Typical homologous proteins or peptides will have from 25-100%
homology (if gaps can be introduced), to 50-100% homology (if
conservative substitutions are included) with the amino acid
sequence of the CTLA-8 protein. Homology measures will be at least
about 35%, generally at least 40%, more generally at least 45%,
often at least 50%, more often at least 55%, typically at least
60%, more typically at least 65%, usually at least 70%, more
usually at least 75%, preferably at least 80%, and more preferably
at least 80%, and in particularly preferred embodiments, at least
85% or more. See also Needleham, et al. (1970) J. Mol. Biol.
48:443-453; Sankoff, et al. (1983) Chapter One in Time Warps,
String Edits, and Macromolecules: The Theory and Practice of
Sequence Comparison Addison-Wesley, Reading, Mass.; and software
packages from IntelliGenetics, Mountain View, Calif.; and the
University of Wisconsin Genetics Computer Group, Madison, Wis.
[0104] The isolated DNA encoding a CTLA-8 protein can be readily
modified by nucleotide substitutions, nucleotide deletions,
nucleotide insertions, and inversions of nucleotide stretches.
These modifications result in novel DNA sequences which encode
these antigens, their derivatives, or proteins having similar
physiological, immunogenic, or antigenic activity. These modified
sequences can be used to produce mutant antigens or to enhance
expression. Enhanced expression may involve gene amplification,
increased transcription, increased translation, and other
mechanisms. Such mutant CTLA-8 protein derivatives include
predetermined or site-specific mutations of the respective protein
or its fragments. "Mutant CTLA-8 protein" encompasses a polypeptide
otherwise falling within the homology definition of the murine
CTLA-8 or human CTLA-8 protein as set forth above, but having an
amino acid sequence which differs from that of CTLA-8 protein as
found in nature, whether by way of deletion, substitution, or
insertion. In particular, "site specific mutant CTLA-8 protein"
generally includes proteins having significant homology with a
protein having sequences of Table 1, 2, or 3, and as sharing
various biological activities, e.g., antigenic or immunogenic, with
those sequences, and in preferred embodiments contain most of the
disclosed sequences. Similar concepts apply to different CTLA-8
proteins, particularly those found in various warm blooded animals,
e.g., mammals and birds. As stated before, it is emphasized that
descriptions are generally meant to encompass all CTLA-8 proteins,
not limited to the mouse embodiment specifically discussed.
[0105] Although site specific mutation sites are predetermined,
mutants need not be site specific. CTLA-8 protein mutagenesis can
be conducted by making amino acid insertions or deletions.
Substitutions, deletions, insertions, or any combinations may be
generated to arrive at a final construct. Insertions include amino-
or carboxy-terminal fusions. Random mutagenesis can be conducted at
a target codon and the expressed mutants can then be screened for
the desired activity. Methods for making substitution mutations at
predetermined sites in DNA having a known sequence are well known
in the art, e.g., by M13 primer mutagenesis or polymerase chain
reaction (PCR) techniques. See also Sambrook, et al. (1989) and
Ausubel, et al. (1987 and Supplements).
[0106] The mutations in the DNA normally should not place coding
sequences out of reading frames and preferably will not create
complementary regions that could hybridize to produce secondary
mRNA structure such as loops or hairpins.
[0107] The present invention also provides recombinant proteins,
e.g., heterologous fusion proteins using segments from these
proteins. A heterologous fusion protein is a fusion of proteins or
segments which are naturally not normally fused in the same manner.
Thus, the fusion product of an immunoglobulin with a CTLA-8
polypeptide is a continuous protein molecule having sequences fused
in a typical peptide linkage, typically made as a single
translation product and exhibiting properties derived from each
source peptide. A similar concept applies to heterologous nucleic
acid sequences.
[0108] In addition, new constructs may be made from combining
similar functional domains from other proteins. For example,
antigen-binding or other segments may be "swapped" between
different new fusion polypeptides or fragments. See, e.g.,
Cunningham, et al. (1989) Science, 243:1330-1336; and O'Dowd, et
al. (1988) J. Biol. Chem. 263:15985-15992. Thus, new chimeric
polypeptides exhibiting new combinations of specificities will
result from the functional linkage of biologically relevant domains
and other functional domains.
[0109] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable
synthetic DNA fragments. A double stranded fragment will often be
obtained either by synthesizing the complementary strand and
annealing the strand together under appropriate conditions or by
adding the complementary strand using DNA polymerase with an
appropriate primer sequence, e.g., PCR techniques.
[0110] VI. Functional Variants
[0111] The blocking of physiological response to CTLA-8 proteins
may result from the inhibition of binding of the antigen to its
natural binding partner, e.g., through competitive inhibition.
Thus, in vitro assays of the present invention will often use
isolated protein, membranes from cells expressing a recombinant
membrane associated CTLA-8 protein, soluble fragments comprising
binding segments, or fragments attached to solid phase substrates.
These assays will also allow for the diagnostic determination of
the effects of either binding segment mutations and modifications,
or protein mutations and modifications, e.g., analogs.
[0112] This invention also contemplates the use of competitive drug
screening assays, e.g., where neutralizing antibodies to antigen or
binding partner fragments compete with a test compound for binding
to the protein. In this manner, the antibodies can be used to
detect the presence of any polypeptide which shares one or more
antigenic binding sites of the protein and can also be used to
occupy binding sites on the protein that might otherwise interact
with a binding partner.
[0113] Additionally, neutralizing antibodies against the CTLA-8
protein and soluble fragments of the antigen which contain a high
affinity receptor binding site, can be used to inhibit antigen
function in tissues, e.g., tissues experiencing abnormal
physiology.
[0114] "Derivatives" of the CTLA-8 antigens include amino acid
sequence mutants, glycosylation variants, and covalent or aggregate
conjugates with other chemical moieties. Covalent derivatives can
be prepared by linkage of functionalities to groups which are found
in the CTLA-8 amino acid side chains or at the N- or C-termini, by
means which are well known in the art. These derivatives can
include, without limitation, aliphatic esters or amides of the
carboxyl terminus, or of residues containing carboxyl side chains,
O-acyl derivatives of hydroxyl group-containing residues, and
N-acyl derivatives of the amino terminal amino acid or amino-group
containing residues, e.g., lysine or arginine. Acyl groups are
selected from the group of alkyl-moieties including C3 to C18
normal alkyl, thereby forming alkanoyl aroyl species. Covalent
attachment to carrier proteins may be important when immunogenic
moieties are haptens.
[0115] In particular, glycosylation alterations are included, e.g.,
made by modifying the glycosylation patterns of a polypeptide
during its synthesis and processing, or in further processing
steps. Particularly preferred means for accomplishing this are by
exposing the polypeptide to glycosylating enzymes derived from
cells which normally provide such processing, e.g., mammalian
glycosylation enzymes. Deglycosylation enzymes are also
contemplated. Also embraced are versions of the same primary amino
acid sequence which have other minor modifications, including
phosphorylated amino acid residues, e.g., phosphotyrosine,
phosphoserine, or phosphothreonine.
[0116] A major group of derivatives are covalent conjugates of the
CTLA-8 protein or fragments thereof with other proteins or
polypeptides. These derivatives can be synthesized in recombinant
culture such as N- or C-terminal fusions or by the use of agents
known in the art for their usefulness in cross-linking proteins
through reactive side groups. Preferred antigen derivatization
sites with cross-linking agents are at free amino groups,
carbohydrate moieties, and cysteine residues.
[0117] Fusion polypeptides between the CTLA-8 proteins and other
homologous or heterologous proteins are also provided. Homologous
polypeptides may be fusions between different surface markers,
resulting in, e.g., a hybrid protein exhibiting receptor binding
specificity. Likewise, heterologous fusions may be constructed
which would exhibit a combination of properties or activities of
the derivative proteins. Typical examples are fusions of a reporter
polypeptide, e.g., luciferase, with a segment or domain of an
antigen, e.g., a receptor-binding segment, so that the presence or
location of the fused antigen may be easily determined. See, e.g.,
Dull, et al., U.S. Pat. No. 4,859,609. Other gene fusion partners
include bacterial .beta.-galactosidase, trpE, Protein A,
.beta.-lactamase, alpha amylase, alcohol dehydrogenase, and yeast
alpha mating factor. See, e.g., Godowski, et al. (1988) Science
241:812-816.
[0118] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable
synthetic DNA fragments. A double stranded fragment will often be
obtained either by synthesizing the complementary strand and
annealing the strand together under appropriate conditions or by
adding the complementary strand using DNA polymerase with an
appropriate primer sequence.
[0119] Such polypeptides may also have amino acid residues which
have been chemically modified by phosphorylation, sulfonation,
biotinylation, or the addition or removal of other moieties,
particularly those which have molecular shapes similar to phosphate
groups. In some embodiments, the modifications will be useful
labeling reagents, or serve as purification targets, e.g., affinity
ligands.
[0120] Fusion proteins will typically be made by either recombinant
nucleic acid methods or by synthetic polypeptide methods.
Techniques for nucleic acid manipulation and expression are
described generally, for example, in Sambrook, et al. (1989)
Molecular Cloning: A Laboratory Manual (2d ed.), Vols. 1-3, Cold
Spring Harbor Laboratory. Techniques for synthesis of polypeptides
are described, for example, in Merrifield (1963) J. Amer. Chem.
Soc. 85:2149-2156; Merrifield (1986) Science 232: 341-347; and
Atherton, et al. (1989) Solid Phase Peptide Synthesis: A Practical
Approach, IRL Press, Oxford.
[0121] This invention also contemplates the use of derivatives of
the CTLA-8 proteins other than variations in amino acid sequence or
glycosylation. Such derivatives may involve covalent or aggregative
association with chemical moieties. These derivatives generally
fall into the three classes: (1) salts, (2) side chain and terminal
residue covalent modifications, and (3) adsorption complexes, for
example with cell membranes. Such covalent or aggregative
derivatives are useful as immunogens, as reagents in immunoassays,
or in purification methods such as for affinity purification of
antigens or other binding proteins. For example, a CTLA-8 antigen
can be immobilized by covalent bonding to a solid support such as
cyanogen bromide-activated Sepharose, by methods which are well
known in the art, or adsorbed onto polyolefin surfaces, with or
without glutaraldehyde cross-linking, for use in the assay or
purification of anti-CTLA-8 protein antibodies or its receptor or
other binding partner. The CTLA-8 antigens can also be labeled with
a detectable group, for example radioiodinated by the chloramine T
procedure, covalently bound to rare earth chelates, or conjugated
to another fluorescent moiety for use in diagnostic assays.
Purification of CTLA-8 protein may be effected by immobilized
antibodies or binding partners.
[0122] A solubilized CTLA-8 antigen or fragment of this invention
can be used as an immunogen for the production of antisera or
antibodies specific for the protein or fragments thereof. The
purified antigen can be used to screen monoclonal antibodies or
binding fragments prepared by immunization with various forms of
impure preparations containing the protein. In particular, the term
"antibodies" also encompasses antigen binding fragments of natural
antibodies. The purified CTLA-8 proteins can also be used as a
reagent to detect any antibodies generated in response to the
presence of elevated levels of the protein or cell fragments
containing the antigen, both of which may be diagnostic of an
abnormal or specific physiological or disease condition.
Additionally, antigen fragments may also serve as immunogens to
produce the antibodies of the present invention, as described
immediately below. For example, this invention contemplates
antibodies raised against amino acid sequences encoded by
nucleotide sequences shown in Table 1, 2, or 3, or fragments of
proteins containing them. In particular, this invention
contemplates antibodies having binding affinity to or being raised
against specific fragments which are predicted to lie outside of
the lipid bilayer.
[0123] The present invention contemplates the isolation of
additional closely related species variants. Southern blot analysis
established that similar genetic entities exist in other mammals,
e.g., rat and human. It is likely that the CTLA-8 proteins are
widespread in species variants, e.g., rodents, lagomorphs,
carnivores, artiodactyla, perissodactyla, and primates.
[0124] The invention also provides means to isolate a group of
related antigens displaying both distinctness and similarities in
structure, expression, and function. Elucidation of many of the
physiological effects of the antigens will be greatly accelerated
by the isolation and characterization of distinct species variants.
In particular, the present invention provides useful probes for
identifying additional homologous genetic entities in different
species.
[0125] The isolated genes will allow transformation of cells
lacking expression of a corresponding CTLA-8 protein, e.g., either
species types or cells which lack corresponding antigens and should
exhibit negative background activity. Expression of transformed
genes will allow isolation of antigenically pure cell lines, with
defined or single specie variants. This approach will allow for
more sensitive detection and discrimination of the physiological
effects of CTLA-8 proteins. Subcellular fragments, e.g., cytoplasts
or membrane fragments, can be isolated and used.
[0126] Dissection of the critical structural elements which effect
the various physiological or differentiation functions provided by
the proteins is possible using standard techniques of modern
molecular biology, particularly in comparing members of the related
class. See, e.g., the homolog-scanning mutagenesis technique
described in Cunningham, et al. (1989) Science 243:1339-1336; and
approaches used in O'Dowd, et al. (1988) J. Biol. Chem.
263:15985-15992; and Lechleiter, et al. (1990) EMBO J.
9:4381-4390.
[0127] In particular, functional domains or segments can be
substituted between species variants to determine what structural
features are important in both binding partner affinity and
specificity, as well as signal transduction. An array of different
variants will be used to screen for molecules exhibiting combined
properties of interaction with different species variants of
binding partners.
[0128] Antigen internalization may occur under certain
circumstances, and interaction between intracellular components and
"extracellular" segments of proteins involved in interactions may
occur. The specific segments of interaction of CTLA-8 protein with
other intracellular components may be identified by mutagenesis or
direct biochemical means, e.g., cross-linking or affinity methods.
Structural analysis by crystallographic or other physical methods
will also be applicable. Further investigation of the mechanism of
biological function will include study of associated components
which may be isolatable by affinity methods or by genetic means,
e.g., complementation analysis of mutants.
[0129] Further study of the expression and control of CTLA-8
protein will be pursued. The controlling elements associated with
the antigens may exhibit differential developmental, tissue
specific, or other expression patterns. Upstream or downstream
genetic regions, e.g., control elements, are of interest.
[0130] Structural studies of the antigen will lead to design of new
variants, particularly analogs exhibiting agonist or antagonist
properties on binding partners. This can be combined with
previously described screening methods to isolate variants
exhibiting desired spectra of activities.
[0131] Expression in other cell types will often result in
glycosylation differences in a particular antigen. Various species
variants may exhibit distinct functions based upon structural
differences other than amino acid sequence. Differential
modifications may be responsible for differential function, and
elucidation of the effects are now made possible.
[0132] Thus, the present invention provides important reagents
related to antigen-binding partner interaction. Although the
foregoing description has focused primarily upon the murine CTLA-8
and human CTLA-8 protein, those of skill in the art will
immediately recognize that the invention encompasses other
antigens, e.g., mouse and other mammalian species or allelic
variants, as well as variants thereof.
[0133] VII. Antibodies
[0134] Antibodies can be raised to the various CTLA-8 proteins,
including species or allelic variants, and fragments thereof, both
in their naturally occurring forms and in their recombinant forms.
Additionally, antibodies can be raised to CTLA-8 proteins in either
their active forms or in their inactive forms. Anti-idiotypic
antibodies are also contemplated.
[0135] Antibodies, including binding fragments and single chain
versions, against predetermined fragments of the antigens can be
raised by immunization of animals with conjugates of the fragments
with immunogenic proteins. Monoclonal antibodies are prepared from
cells secreting the desired antibody. These antibodies can be
screened for binding to normal or defective CTLA-8 proteins, or
screened for agonistic or antagonistic activity, e.g., mediated
through a binding partner. These monoclonal antibodies will usually
bind with at least a K.sub.D of about 1 mM, more usually at least
about 300 .mu.M, typically at least about 10 .mu.m, more typically
at least about 30 .mu.M, preferably at least about 10 .mu.m, and
more preferably at least about 3 .mu.m or better.
[0136] The antibodies, including antigen binding fragments, of this
invention can have significant diagnostic or therapeutic value.
They can be potent antagonists that bind to a binding partner and
inhibit antigen binding or inhibit the ability of an antigen to
elicit a biological response. They also can be useful as
non-neutralizing antibodies and can be coupled to toxins or
radionuclides so that when the antibody binds to the antigen, a
cell expressing it, e.g., on its surface, is killed. Further, these
antibodies can be conjugated to drugs or other therapeutic agents,
either directly or indirectly by means of a linker, and may effect
drug targeting.
[0137] The antibodies of this invention can also be useful in
diagnostic applications. As capture or non-neutralizing antibodies,
they can be screened for ability to bind to the antigens without
inhibiting binding by a partner. As neutralizing antibodies, they
can be useful in competitive binding assays. They will also be
useful in detecting or quantifying CTLA-8 protein or its binding
partners. See, e.g., Chan (ed.)(1987) Immunoassay: A Practical
Guide Academic Press, Orlando, Fla.; Ngo (ed.)(1988) Nonisotopic
Immunoassay Plenum Press, NY; and Price and Newman (eds.)(1991)
Principles and Practice of Immunoassay Stockton Press, NY.
[0138] Antigen fragments may be joined to other materials,
particularly polypeptides, as fused or covalently joined
polypeptides to be used as immunogens. An antigen and its fragments
may be fused or covalently linked to a variety of immunogens, such
as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid,
etc. See Microbiology, Hoeber Medical Division, Harper and Row,
1969; Landsteiner (1962) Specificity of Serological Reactions,
Dover Publications, New York, and Williams, et. al. (1967) Methods
in Immunology and Immunochemistry, Vol. 1, Academic Press, New
York, for descriptions of methods of preparing polyclonal antisera.
A typical method involves hyperimmunization of an animal with an
antigen. The blood of the animal is then collected shortly after
the repeated immunizations and the gamma globulin is isolated.
[0139] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies may be found in, e.g., Stites, et al. (eds.)
Basic and Clinical Immunology (4th ed.), Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.) Academic Press, New York; and particularly in Kohler and
Milstein (1975) in Nature 256: 495-497, which discusses one method
of generating monoclonal antibodies. Summarized briefly, this
method involves injecting an animal with an immunogen. The animal
is then sacrificed and cells taken from its spleen, which are then
fused with myeloma cells. The result is a hybrid cell or
"hybridoma" that is capable of reproducing in vitro. The population
of hybridomas is then screened to isolate individual clones, each
of which secrete a single antibody species to the immunogen. In
this manner, the individual antibody species obtained are the
products of immortalized and cloned single B cells from the immune
animal generated in response to a specific site recognized on the
immunogenic substance.
[0140] Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to
selection of libraries of antibodies in phage or similar vectors.
See, Huse, et al. (1989) "Generation of a Large Combinatorial
Library of the Immunoglobulin Repertoire in Phage Lambda," Science
246:1275-1281; and Ward, et al. (1989) Nature 341:544-546. The
polypeptides and antibodies of the present invention may be used
with or without modification, including chimeric or humanized
antibodies. Frequently, the polypeptides and antibodies will be
labeled by joining, either covalently or non-covalently, a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and are reported
extensively in both the scientific and patent literature. Suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent moieties, chemiluminescent moieties,
magnetic particles, and the like. Patents, teaching the use of such
labels 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. Also, recombinant
immunoglobulins may be produced, see Cabilly, U.S. Pat. No.
4,816,567.
[0141] The antibodies of this invention can also be used for
affinity chromatography in isolating the protein. Columns can be
prepared where the antibodies are linked to a solid support, e.g.,
particles, such as agarose, Sephadex, or the like, where a cell
lysate may be passed through the column, the column washed,
followed by increasing concentrations of a mild denaturant, whereby
the purified CTLA-8 protein will be released.
[0142] The antibodies-may also be used to screen expression
libraries for particular expression products. Usually the
antibodies used in such a procedure will be labeled with a moiety
allowing easy detection of presence of antigen by antibody
binding.
[0143] Antibodies raised against each CTLA-8 protein will also be
useful to raise anti-idiotypic antibodies. These will be useful in
detecting or diagnosing various immunological conditions related to
expression of the respective antigens.
[0144] VIII. Uses
[0145] The present invention provides reagents which will find use
in diagnostic applications as described elsewhere herein, e.g., in
the general description for physiological or developmental
abnormalities, or below in the description of kits for
diagnosis.
[0146] This invention also provides reagents with significant
therapeutic value. The CTLA-8 protein (naturally occurring or
recombinant), fragments thereof, and antibodies thereto, along with
compounds identified as having binding affinity to CTLA-8 protein,
should be useful in the treatment of conditions associated with
abnormal physiology or development, including abnormal
proliferation, e.g., cancerous conditions, or degenerative
conditions. Abnormal proliferation, regeneration, degeneration, and
atrophy may be modulated by appropriate therapeutic treatment using
the compositions provided herein. For example, a disease or
disorder associated with abnormal expression or abnormal signaling
by a CTLA-8 antigen should be a likely target for an agonist or
antagonist of the protein.
[0147] Other abnormal developmental conditions are known in the
cell types shown to possess CTLA-8 antigen mRNA by Northern blot
analysis. See Berkow (ed.) The Merck Manual of Diagnosis and
Therapy, Merck & Co., Rahway, N.J.; and Thorn, et al.
Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y.
These. problems may be susceptible to prevention or treatment using
compositions provided herein.
[0148] Recombinant antibodies which bind to CTLA-8 can be purified
and then administered to a patient. These reagents can be combined
for therapeutic use with additional active or inert ingredients,
e.g., in conventional pharmaceutically acceptable carriers or
diluents, e.g., immunogenic adjuvants, along with physiologically
innocuous stabilizers and excipients. These combinations can be
sterile filtered and placed into dosage forms as by lyophilization
in dosage vials or storage in stabilized aqueous preparations. This
invention also contemplates use of antibodies or binding fragments
thereof, including forms which are not complement binding.
[0149] Screening using CTLA-8 for binding partners or compounds
having binding affinity to CTLA-8 antigen can be performed,
including isolation of associated components. Subsequent biological
assays can then be utilized to determine if the compound has
intrinsic biological activity and is therefore an agonist or
antagonist in that it blocks an activity of the antigen. This
invention further contemplates the therapeutic use of antibodies to
CTLA-8 protein as antagonists. This approach should be particularly
useful with other CTLA-8 protein species variants.
[0150] The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, physiological state of the patient,
and other medicants administered. Thus, treatment dosages should be
titrated to optimize safety and efficacy. Typically, dosages used
in vitro may provide useful guidance in the amounts useful for in
situ administration of these reagents. Animal testing of effective
doses for treatment of particular disorders will provide further
predictive indication of human dosage. Various considerations are
described, e.g., in Gilman, et al. (eds.) (1990) Goodman and
Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed.,
Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.
(1990), Mack Publishing Co., Easton, Pa. Methods for administration
are discussed therein and below, e.g., for oral, intravenous,
intraperitoneal, or intramuscular administration, transdermal
diffusion, and others. See also Langer (1990) Science
249:1527-1533. Pharmaceutically acceptable carriers will include
water, saline, buffers, and other compounds described, e.g., in the
Merck Index, Merck & Co., Rahway, N.J. Dosage ranges would
ordinarily be expected to be in amounts lower than 1 mM
concentrations, typically less than about 10 .mu.m concentrations,
usually less than about 100 nM, preferably less than about 10 pM
(picomolar), and most preferably less than about 1 fM (femtomolar),
with an appropriate carrier. Slow release formulations, or a slow
release apparatus will often be utilized for continuous
administration.
[0151] CTLA-8 protein, fragments thereof, and antibodies to it or
its fragments, antagonists, and agonists, may be administered
directly to the host to be treated or, depending on the size of the
compounds, it may be desirable to conjugate them to carrier
proteins such as ovalbumin or serum albumin prior to their
administration. Therapeutic formulations may be administered in any
conventional dosage formulation. While it is possible for the
active ingredient to be administered alone, it is preferable to
present it as a pharmaceutical formulation. Formulations typically
comprise at least one active ingredient, as defined above, together
with one or more acceptable carriers thereof. Each carrier should
be both pharmaceutically and physiologically acceptable in the
sense of being compatible with the other ingredients and not
injurious to the patient. Formulations include those suitable for
oral, rectal, nasal, or parenteral (including subcutaneous,
intramuscular, intravenous and intradermal) administration. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any methods well known in the art of pharmacy.
See, e.g., Gilman, et al. (eds.) (1990) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press,
Parrytown, N.Y.; Remington's Pharmaceutical Sciences, 17th ed.
(1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds.)(1993)
Pharmaceutical Dosage Forms: Parenteral Medications 2d ed., Dekker,
N.Y.; Lieberman, et al. (eds.)(1990) Pharmaceutical Dosage Forms:
Tablets 2d ed., Dekker, N.Y.; and Lieberman, et al. (eds.)(1990)
Pharmaceutical Dosage Forms: Disperse Systems Dekker, N.Y. The
therapy of this invention may be combined with or used in
association with other chemotherapeutic or chemopreventive
agents.
[0152] Both the naturally occurring and the recombinant forms of
the CTLA-8 proteins of this invention are particularly useful in
kits and assay methods which are capable of screening compounds for
binding activity to the proteins. Several methods of automating
assays have been developed in recent years so as to permit
screening of tens of thousands of compounds in a short period. See,
e.g., Fodor, et al. (1991) Science 251:767-773, which describes
means for testing of binding affinity by a plurality of defined
polymers synthesized on a solid substrate. The development of
suitable assays can be greatly facilitated by the availability of
large amounts of purified, soluble CTLA-8 protein as provided by
this invention.
[0153] This invention is particularly useful for screening
compounds by using recombinant antigen in any of a variety of drug
screening techniques. The advantages of using a recombinant protein
in screening for specific ligands include: (a) improved renewable
source of the antigen from a specific source; (b) potentially
greater number of antigen molecules per cell giving better signal
to noise ratio in assays; and (c) species variant specificity
(theoretically giving greater biological and disease specificity).
The purified protein may be tested in numerous assays, typically in
vitro assays, which evaluate biologically relevant responses. See,
e.g., Coligan Current Protocols in Immunology; Hood, et al.
Immunology Benjamin/Cummings; Paul (ed.) Fundamental Immunology;
and Methods in Enzymology Academic Press.
[0154] One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing the CTLA-8 antigens. Cells may
be isolated which express an antigen in isolation from other
functionally equivalent antigens. Such cells, either in viable or
fixed form, can be used for standard protein-protein binding
assays. See also, Parce, et al. (1989) Science 246:243-247; and
Owicki, et al. (1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011,
which describe sensitive methods to detect cellular responses.
Competitive assays are particularly useful, where the cells (source
of CTLA-8 protein) are contacted and incubated with a labeled
binding partner or antibody having known binding affinity to the
ligand, such as .sup.125I-antibody, and a test sample whose binding
affinity to the binding composition is being measured. The bound
and free labeled binding compositions are then separated to assess
the degree of antigen binding. The amount of test compound bound is
inversely proportional to the amount of labeled receptor binding to
the known source. Any one of numerous techniques can be used to
separate bound from free antigen to assess the degree of binding.
This separation step could typically involve a procedure such as
adhesion to filters followed by washing, adhesion to plastic
followed by washing, or centrifugation of the cell membranes.
Viable cells could also be used to screen for the effects of drugs
on CTLA-8 protein mediated functions, e.g., second messenger
levels, i.e., Ca.sup.++; cell proliferation; inositol phosphate
pool changes; and others. Some detection methods allow for
elimination of a separation step, e.g., a proximity sensitive
detection system. Calcium sensitive dyes will be useful for
detecting Ca.sup.++ levels, with a fluorimeter or a fluorescence
cell sorting apparatus.
[0155] Another method utilizes membranes from transformed
eukaryotic or prokaryotic host cells as the source of the CTLA-8
protein. These cells are stably transformed with DNA vectors
directing the expression of a membrane associated CTLA-8 protein,
e.g., an engineered membrane bound form. Essentially, the membranes
would be prepared from the cells and used in any receptor/ligand
type binding assay such as the competitive assay set forth
above.
[0156] Still another approach is to use solubilized, unpurified or
solubilized, purified CTLA-8 protein from transformed eukaryotic or
prokaryotic host cells. This allows for a "molecular" binding assay
with the advantages of increased specificity, the ability to
automate, and high drug test throughput.
[0157] Another technique for drug screening involves an approach
which provides high throughput screening for compounds having
suitable binding affinity to CTLA-8 and is described in detail in
Geysen, European Patent Application 84/03564, published on Sep. 13,
1984. First, large numbers of different small peptide test
compounds are synthesized on a solid substrate, e.g., plastic pins
or some other appropriate surface, see Fodor, et al. (1991). Then
all the pins are reacted with solubilized, unpurified or
solubilized, purified CTLA-8 binding composition, and washed. The
next step involves detecting bound binding composition.
[0158] Rational drug design may also be based upon structural
studies of the molecular shapes of the CTLA-8 protein and other
effectors or analogs. Effectors may be other proteins which mediate
other functions in response to antigen binding, or other proteins
which normally interact with the antigen. One means for determining
which sites interact with specific other proteins is a physical
structure determination, e.g., x-ray crystallography or 2
dimensional NMR techniques. These will provide guidance as to which
amino acid residues form molecular contact regions. For a detailed
description of protein structural determination, see, e.g.,
Blundell and Johnson (1976) Protein Crystallography, Academic
Press, New York.
[0159] Purified CTLA-8 protein can be coated directly onto plates
for use in the aforementioned drug screening techniques. However,
non-neutralizing antibodies to these ligands can be used as capture
antibodies to immobilize the respective ligand on the solid
phase.
[0160] IX. Kits
[0161] This invention also contemplates use of CTLA-8 proteins,
fragments thereof, peptides, and their fusion products in a variety
of diagnostic kits and methods for detecting the presence of a
binding composition. Typically the kit will have a compartment
containing either a defined CTLA-8 peptide or gene segment or a
reagent which recognizes one or the other, e.g., antigen fragments
or antibodies.
[0162] A kit for determining the binding affinity of a test
compound to a CTLA-8 protein would typically comprise a test
compound; a labeled compound, for example an antibody having known
binding affinity for the antigen; a source of CTLA-8 protein
(naturally occurring or recombinant); and a means for separating
bound from free labeled compound, such as a solid phase for
immobilizing the antigen. Once compounds are screened, those having
suitable binding affinity to the antigen can be evaluated in
suitable biological assays, as are well known in the art, to
determine whether they exhibit similar biological activities to the
natural antigen. The availability of recombinant CTLA-8 protein
polypeptides also provide well defined standards for calibrating
such assays.
[0163] A preferred kit for determining the concentration of, for
example, a CTLA-8 protein in a sample would typically comprise a
labeled compound, e.g., antibody, having known binding affinity for
the antigen, a source of antigen (naturally occurring or
recombinant) and a means for separating the bound from free labeled
compound, for example, a solid phase for immobilizing the CTLA-8
protein. Compartments containing reagents, and instructions, will
normally be provided.
[0164] One method for determining the concentration of CTLA-8
protein in a sample would typically comprise the steps of: (1)
preparing membranes from a sample comprised of a membrane bound
CTLA-8 protein source; (2) washing the membranes and suspending
them in a buffer; (3) solubilizing the antigen by incubating the
membranes in a culture medium to which a suitable detergent has
been added; (4) adjusting the detergent concentration of the
solubilized antigen; (5) contacting and incubating said dilution
with radiolabeled antibody to form complexes; (6) recovering the
complexes such as by filtration through polyethyleneimine treated
filters; and (7) measuring the radioactivity of the recovered
complexes.
[0165] Antibodies, including antigen binding fragments, specific
for the CTLA-8 protein or fragments are useful in diagnostic
applications to detect the presence of elevated levels of CTLA-8
protein and/or its fragments. Such diagnostic assays can employ
lysates, live cells, fixed cells, immunofluorescence, cell
cultures, body fluids, and further can involve the detection of
antigens related to the protein in serum, or the like. Diagnostic
assays may be homogeneous (without a separation step between free
reagent and protein-protein complex) or heterogeneous (with a
separation step). Various commercial assays exist, such as
radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),
enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique
(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the
like. For example, unlabeled antibodies can be employed by using a
second antibody which is labeled and which recognizes the antibody
to a CTLA-8 protein or to a particular fragment thereof. Similar
assays have also been extensively discussed in the literature. See,
e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual,
CSH.
[0166] Anti-idiotypic antibodies may have similar use to diagnose
presence of antibodies against a CTLA-8 protein, as such may be
diagnostic of various abnormal states. For example, overproduction
of CTLA-8 protein may result in production of various immunological
reactions which may be diagnostic of abnormal physiological states,
particularly in proliferative cell conditions such as cancer or
abnormal differentiation.
[0167] Frequently, the reagents for diagnostic assays are supplied
in kits, so as to optimize the sensitivity of the assay. For the
subject invention, depending upon the nature of the assay, the
protocol, and the label, either labeled or unlabeled antibody, or
labeled CTLA-8 protein is provided. This is usually in conjunction
with other additives, such as buffers, stabilizers, materials
necessary for signal production such as substrates for enzymes, and
the like. Preferably, the kit will also contain instructions for
proper use and disposal of the contents after use. Typically the
kit has compartments for each useful reagent. Desirably, the
reagents are provided as a dry lyophilized powder, where the
reagents may be reconstituted in an aqueous medium providing
appropriate concentrations of reagents for performing the
assay.
[0168] Any of the aforementioned constituents of the drug screening
and the diagnostic assays may be used without modification or may
be modified in a variety of ways. For example, labeling may be
achieved by covalently or non-covalently joining a moiety which
directly or indirectly provides a detectable signal. In any of
these assays, the antigen, test compound, CTLA-8 protein, or
antibodies thereto can be labeled either directly or indirectly.
Possibilities for direct labeling include label groups: radiolabels
such as .sup.125I, enzymes (U.S. Pat. No. 3,645,090) such as
peroxidase and alkaline phosphatase, and fluorescent labels (U.S.
Pat. No. 3,940,475) capable of monitoring the change in
fluorescence intensity, wavelength shift, or fluorescence
polarization. Possibilities for indirect labeling include
biotinylation of one constituent followed by binding to avidin
coupled to one of the above label groups.
[0169] There are also numerous methods of separating the bound from
the free antigen, or alternatively the bound from the free test
compound. The CTLA-8 protein can be immobilized on various matrixes
followed by washing. Suitable matrixes include plastic such as an
ELISA plate, filters, and beads. Methods of immobilizing the CTLA-8
protein to a matrix include, without limitation, direct adhesion to
plastic, use of a capture antibody, chemical coupling, and
biotin-avidin. The last step in this approach involves the
precipitation of protein-protein complex by any of several methods
including those utilizing, e.g., an organic solvent such as
polyethylene glycol or a salt such as ammonium sulfate. Other
suitable separation techniques include, without limitation, the
fluorescein antibody magnetizable particle method described in
Rattle, et al. (1984) Clin. Chem. 30:1457-1461, and the double
antibody magnetic particle separation as described in U.S. Pat. No.
4,659,678.
[0170] The methods for linking proteins or their fragments to the
various labels have been extensively reported in the literature and
do not require detailed discussion here. Many of the techniques
involve the use of activated carboxyl groups either through the use
of carbodiimide or active esters to form peptide bonds, the
formation of thioethers by reaction of a mercapto group with an
activated halogen such as chloroacetyl, or an activated olefin such
as maleimide, for linkage, or the like. Fusion proteins will also
find use in these applications.
[0171] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of a CTLA-8 protein. These sequences can be used as probes for
detecting levels of antigen message in samples from patients
suspected of having an abnormal condition, e.g., cancer or
developmental problem. The preparation of both RNA and DNA
nucleotide sequences, the labeling of the sequences, and the
preferred size of the sequences has received ample description and
discussion in the literature. Normally an oligonucleotide probe
should have at least about 14 nucleotides, usually at least about
18 nucleotides, and the polynucleotide probes may be up to several
kilobases. Various labels may be employed, most commonly
radionuclides, particularly .sup.32P. However, other techniques may
also be employed, such as using biotin modified nucleotides for
introduction into a polynucleotide. The biotin then serves as the
site for binding to avidin or antibodies, which may be labeled with
a wide variety of labels, such as radionuclides, fluorescers,
enzymes, or the like. Alternatively, antibodies may be employed
which can recognize specific duplexes, including DNA duplexes, RNA
duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes. The
antibodies in turn may be labeled and the assay carried out where
the duplex is bound to a surface, so that upon the formation of
duplex on the surface, the presence of antibody bound to the duplex
can be detected. The use of probes to the novel anti-sense RNA may
be carried out in any conventional techniques such as nucleic acid
hybridization, plus and minus screening, recombinational probing,
hybrid released translation (HRT), and hybrid arrested translation
(HART). This also includes amplification techniques such as
polymerase chain reaction (PCR).
[0172] Diagnostic kits which also test for the qualitative or
quantitative presence of other markers are also contemplated.
Diagnosis or prognosis may depend on the combination of multiple
indications used as markers. Thus, kits may test for combinations
of markers. See, e.g., Viallet, et al. (1989) Progress in Growth
Factor Res. 1:89-97.
[0173] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the invention to specific embodiments.
EXAMPLES
[0174] I. General Methods
[0175] Some of the standard methods are described or referenced,
e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press;
Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, (2d
ed.), vols. 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene
Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and
Supplements) Current Protocols in Molecular Biology, Greene/Wiley,
New York; Innis, et al. (eds.)(1990) PCR Protocols: A Guide to
Methods and Applications Academic Press, N.Y. Methods for protein
purification include such methods as ammonium sulfate
precipitation, column chromatography, electrophoresis,
centrifugation, crystallization, and others. See, e.g., Ausubel, et
al. (1987 and periodic supplements); Deutscher (1990) "Guide to
Protein Purification" in Methods in Enzymology, vol. 182, and other
volumes in this series; and manufacturer's literature on use of
protein purification products, e.g., Pharmacia, Piscataway, N.J.,
or Bio-Rad, Richmond, Calif. Combination with recombinant
techniques allow fusion to appropriate segments, e.g., to a FLAG
sequence or an equivalent which can be fused via a
protease-removable sequence. See, e.g., Hochuli (1989) Chemische
Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant
Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic
Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;
and Crowe, et al. (1992) QIAexpress: The High Level Expression
& Protein Purification System QUIAGEN, Inc., Chatsworth,
Calif.
[0176] FACS analyses are described in Melamed, et al. (1990) Flow
Cytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro
(1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson,
et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New
York, N.Y.
[0177] II. Isolation of a DNA Clone Encoding CTLA-8 Protein.
[0178] Isolation of murine CTLA-8 is described in Rouvier, et al.
(1993) J. Immunol. 150:5445-5456.
[0179] Source of the CTLA-8 Message
[0180] Various cell lines are screened using an appropriate probe
for high level message expression. Appropriate cell lines are
selected based upon expression levels of the CTLA-8 message.
Applicants used subtractive hybridization methods on activated
cytotoxic T cells.
[0181] Isolation of a CTLA-8 Encoding Clone
[0182] Standard PCR techniques are used to amplify a CTLA-8 gene
sequence from a genomic or cDNA library, or from mRNA. Appropriate
primers are selected from the sequences provided, and a full length
clone is isolated. Various combinations of primers, of various
lengths and possibly with differences in sequence, may be prepared.
The full length clone can be used as a hybridization probe to
screen for other homologous genes using stringent or less stringent
hybridization conditions.
[0183] In another method, oligonucleotides are used to screen a
library. In combination with polymerase chain reaction (PCR)
techniques, synthetic oligonucleotides in appropriate orientations
are used as primers to select correct clones from a library.
[0184] III. Isolation of a Human CTLA-8.
[0185] A human genomic library was obtained from Clontech (Cat.
HL1006d) and screened with a cDNA probe composed of a 453 base pair
entire coding sequence of a murine CTLA-8. A number of independent
lambda clones were found to hybridize strongly with the murine
CTLA-8 probe. One clone contained a hybridizing XbaI fragment of
approximately 2000 base pairs which corresponded to a fragment
previously detected using a similar probe on a human genomic DNA
Southern blot. This 2000 base pair fragment was subcloned into
Bluescript (Stratagene) and sequenced. This revealed a 240 base
pair region (see Table 3) 83.8% homologous to the murine CTLA-8 of
Table 1. Translation of this region yielded an amino acid sequence
70.8% homologous to the 79 carboxy-terminal amino acids of the
murine CTLA-8 putative protein. The exon was used as a probe to
screen a library of cDNA made with a primer corresponding to the
last 21 nucleotides of the coding region. Three independent cDNA
clones were obtained containing the complete coding region of the
human CTLA-8. The 468 base pair open reading frame encodes a 155
amino acid polypeptide with a theoretical molecular weight of
17,100 daltons. See Table 3. This human CTLA-8 is 66.4% homologous
to the ORF-13 of the virus, and 58.3% homologous to murine CTLA-8
encoded protein. Moreover, the 6 cysteines are conserved between
the three genes, as well as the putative glycosylation and
phosphorylation sites.
[0186] Analysis of the human CTLA-8 amino acid sequence exhibits a
hydrophobic stretch of 19 residues, from 7 to about 25, at the
amino terminus, similar to a signal peptide. It is highly likely
that the human CTLA-8 is a secreted protein of a molecular weight
resembling a cytokine.
[0187] IV. Biochemical Characterization of CTLA-8 Proteins.
[0188] Two forms of human CTLA-8 were expressed in heterologous
cells; the native form, and a recombinant form displaying the FLAG
peptide at the carboxy terminus. See, e.g., Crowe et al. (1992)
QIAexpress: The High Level Expression and Protein Purification
System QIAGEN, Inc. Chatsworth, Calif.; and Hopp et al. (1988)
Bio/Technology 6:1204-1210. These two forms of the human CTLA-8
protein were introduced into the expression vectors pME18S or
pEE12, and subsequently transfected into COS-7 or NSO cells,
respectively, by electroporation. Electroporated cells were then
cultivated for 48 hours in RPMI medium supplemented with 10% Fetal
Calf Serum. Cells were then incubated with .sup.35S-Met and
.sup.35S-Cys in order to label cellular proteins. Comparison of the
proteins under reducing conditions on SDS-PAGE showed that cells
transfected with human CTLA-8 secreted a polypeptide of 15,000
daltons. Non-reducing SDS-PAGE revealed 2 specific bands around
28,000 daltons and 33,000 daltons. Treatment with endoglycosidase F
(Boehringer Mannheim) demonstrated that the higher molecular weight
species represents an N-glycosylated form of human CTLA-8.
[0189] In order to determine if the natural form of human CTLA-8
produced by activated CD4+ T cells was also secreted as a dimer
similar to transfected COS-7 and NSO cells, peripheral blood
mononuclear cells (PBMC) were purified from 500 ml of human blood
on a Ficoll gradient. B cells, CD8+ T cells, monocytes, and NK
cells were depleted using 100 .mu.l of ascitic fluid containing
anti-CD19, anti-CD8, anti-CD14, and 25 gg of NKH1 monoclonal
antibody (Coulter, Hialeah, Fla.). After 30 minutes of incubation
at 40.degree. C., the PBMC were washed twice in RPMI containing 10%
Fetal Calf Serum (FCS). Paramagnetic beads coated with goat
antibodies to mouse IgG (Dynabeads M450, Dynal, Oslo, Norway) were
added at a final concentration of 5 beads/cell to be depleted.
Unwanted cells were subsequently removed by 3 passages on a magnet.
The remaining cells were CD4+ cells at 87% purity which were
diluted to 10.sup.7 cells/ml in DMEM F12 (Gibco, Gaithersburg, Md.)
containing 10% FCS, 10 ng/ml PMA (Sigma, St. Louis, Mo.) and 500
ng/ml ionomycin (Sigma, St. Louis, Mo.). After incubation for 4
hours at 37.degree. C. in 5% CO.sub.2, the medium was changed to
methionine and cysteine free DMEM (ICN Biomedicals, Costa Mesa,
Calif.), supplemented with 1% dialyzed FCS, 10 ng/ml PMA and 500
ng/ml ionomycin, and incubated for 1 hour at 37.degree. C. in 5%
CO.sub.2. 100 .mu.Ci/ml of .sup.35S-methionine and
.sup.35S-cysteine (Amersham) was added, and metabolic labeling was
carried out for 18 hours at 37.degree. C. in 5% CO.sub.2. Following
preclearing of the supernatants with anti-IFN-.gamma. Mab B27 and
0.5 ml of Protein-G Sepharose (Sigma St. Louis, Mo.), the
supernatants were immunoprecipated using monoclonal antibodies to
human CTLA-8. Immunoprecipated proteins were analyzed on SDS-PAGE.
CD4+ T cells and transfected NSO cells reveal two bands at 28,000
and 33,000 daltons corresponding respectively to non N-glycosylated
and N-glycosylated forms of human CTLA-8 dimers. Therefore, human
CTLA-8 derived from transfected NSO cells and CTLA-8. isolated from
activated T cells display the same biological characteristics.
[0190] V. Large Scale Production of Human CTLA-8
[0191] For biological assays, human CTLA-8 and human CTLA-8-FLAG
were produced in large amounts with transfected COS-7 cells grown
in RPMI medium supplemented with 1% Nutridoma HU (Boeringer
Mannheim, Mannheim, Germany) and subsequently purified.
[0192] In order to produce larger quantities of native human CTLA-8
or human CTLA-8-FLAG, stable transformants of NSO cells were
prepared according to the methodology developed by Celltech
(Slough, Berkshire, UK; International Patent Applications
WO86/05807, WO87/04462, WO89/01036, and WO89/10404). Both CTLA-8
and CTLA-8-FLAG were subcloned into pEE12 and subsequently
transfected into NSO cells by electroporation. Transfected NSO
cells were seeded in selective glutamine-free DMEM supplemented
with 10% Fetal Calf Serum as described in Celltech's protocol.
Supernatants from the best producing lines were used in biological
assays and purification of human CTLA-8 and human CTLA-8-FLAG.
[0193] Purification of Human CTLA-8 Protein
[0194] Typically, 1 liter of supernatant containing human CTLA-8 or
CTLA-8-FLAG was passed on a 60 ml column of Zn.sup.++ ions grafted
to a Chelating Sepharose Fast Flow matrix (Pharmacia, Upsalla,
Sweden). After washing with 10 volumes of binding buffer (His-Bind
Buffer kit, Novagen, Madison, Wis.), the proteins retained by the
metal ions were eluted with a gradient of 20-100 mM Imidazole. The
content of human CTLA-8-FLAG in the eluted fractions was determined
by dot blot using the anti-FLAG monoclonal antibody M2 (Eastman
Kodak, New-Haven, Conn.), whereas the content of human CTLA-8 was
assessed by silver staining of non-reducing SDS-PAGE. The CTLA-8
containing fractions were then pooled and dialyzed against PBS, and
were either used in biological assays or further purified by anion
exchange HPLC on a DEAE column. A third step of gle filtration
chromatograph was performed on a SUPERDEX G-75 HRD30 column
(Pharmacia Uppsala, Sweden) and yielded practically pure human
CTLA-8-8 as analyzed by silver stained SDS-PAGE.
[0195] Preparation of Antibodies Specific for CTLA-8
[0196] Inbred Balb/c mice were immunized intraperitoneally with 1
ml of purified human CTLA-8-FLAG emulsified in Freund's complete
adjuvant on day 0, and in Freund's incomplete adjuvant on days 15
and 22. The mice were boosted with 0.5 ml of purified human
CTLA-8-8 administered intravenously.
[0197] Hybridomas were created using the non-secreting myeloma
cells line SP2/0-Ag8 and polyethylene glycol 1000 (Sigma, St.
Louis, Mo.) as the fusing agent. Hybridoma cells were placed in a
96-well Falcon tissue culture plate (Becton Dickinson, N.J.) and
fed with DMEM F12 (Gibco, Gaithersburg, Md.) supplemented with 80
.mu.g/ml gentamycin, 2 mM glutamine, 10% horse serum (Gibco,
Gaithersburg, Md.), 1% ADCM (CRTS, Lyon, France:) 10.sup.-5 M
azaserine (Sigma, St. Louis, Mo.) and 5.times.10.sup.-5 M
hypoxanthine. Hybridoma supernatants were screened for antibody
production against human CTLA-8 by immunocytochemistry (ICC) using
acetone fixed human CTLA-8 transfected COS-7 cells and by ELISA
using human CTLA-8-FLAG purified from COS-7 supernatants as a
coating antigen. Aliquots of positive cell clones were expanded for
6 days and cryopreserved as well as propagated in ascites from
pristane (2,6,10,14-teramethylpentadecane, Sigma, St. Louis, Mo.)
treated Balb/c mice who had received on intraperitoneal injection
of pristane 15 days before. About 10.sup.5 hybridoma cells in 1 ml
of PBS were given intraperitoneally, and 10 days later, ascites
were collected from each mouse.
[0198] After centrifugation of the ascites, the antibody fraction
was isolated by ammonium sulfate precipitation and anion-exchange
chromatography on a zephyr-D silicium column (IBF Sepracor)
equilabrated with 20 mM Tris pH 8.0. Proteins were eluted with a
NaCl gradient (ranging from 0 to 1 M NaCl). 2 ml fractions were
collected and tested by ELISA for the presence of anti-CTLA-8
antibody. The fractions containing specific anti-CTLA-8 activity
were pooled, dialyzed, and frozen. Aliquots of the purified
monoclonal antibodies were peroxydase labeled.
[0199] Quantification of Human CTLA-8
[0200] Among the antibodies specific for CTLA-8, Ab25, and
peroxydase labeled Ab16 were selected to quantitate levels of human
CTLA-8 using a sandwich assay. Purified Ab25 was diluted at 2
.mu.g/ml in coating buffer (carbonate buffer, pH 9.6. 15 mM
Na.sub.2CO.sub.3, 35 mM NaHCO.sub.3). This diluted solution was
coated onto the wells of a 96-well ELISA plate (Immunoplate
Maxisorp F96 certified, NUNC, Denmark) overnight at room
temperature. The plates were then washed manually one with a
washing buffer consisting of Phosphate Buffered Saline and 0.05%
Tween 20 (Technicon Diagnositics, USA). 110 .mu.l of purified human
CTLA-8 diluted in TBS-B-T buffer [20 mM Tris, 150 mM NaCl, 1% BSA
(Sigma, St. Louis, Mo.), and 0.05% Tween 20] was added to each
well. After 3 hours of incubation at 37.degree. C., the plates were
washed once. 100 .mu.l of peroxydase labeled Ab16 diluted to 5
.mu.g/ml in TBS-B-T buffer was added to each well, and incubated
for 2 hours at 37.degree. C. The wells were then washed three times
in washing buffer. 100 .mu.l of peroxydase substrate, 2.2'
Azino-bis(3 ethylbenzthiazoine-6-sulfonic acid) (ABTS), diluted to
1 mg/ml in citrate/phosphate buffer, was added to each well, and
the colorimetric reaction was read at 405 nm. The lowest
concentration of human CTLA-8 detected was 0.015 ng/ml.
[0201] V. Induction of IL-6 Secretion by Treatment of Various Cell
Types with CTLA-8
[0202] Synoviocytes from normal and rheumatoid arthritic patients
(10.sup.4 cells/well) were incubated with increasing concentrations
of human CTLA-8-8. After 48 hours, concentrations of IL-6 were
measured by standard ELISA techniques. Secretion of IL-6 was
increased in both types of cells in a dose dependent manner.
[0203] Kidney epithelial carcinoma cell lines TUMT and CHA were
also cultured in complete RPMI 1640 medium (Gibco BRL, Grand
Island, N.Y.), supplemented with 2 mM L-glutamine, 100 U/ml
penicillin, 50 .mu.g/ml gentamycin, 20 mM Hepes buffer and
heat-inactivated 10% FCS. Cells (104 cells/well) were incubated in
96-well plates (Falcon) in a final volume of 250 .mu.l of complete
culture medium. Increasing concentrations of human CTLA-8-8 were
added at the onset of the culture. Cell-free supernatants were
collected after 48 hours, and stored at -20.degree. C. until
cytokine assays. IL-6 levels were measured by two-site sandwich
ELISA as described in Abrams, et al. (1992). Immunol. Rev.
127:5-24. Both cell lines exhibited dose dependent increases in
IL-6 secretion with increasing concentrations of CTLA-8. In view of
these results, other cell lines will also be screened for responses
to other species of CTLA-8 variants.
[0204] MRC-5 human lung fibroblasts were obtained from the ATCC
(Rockville, Md.) and were cultured in complete RPMI 1640 medium
(Gibco BRL, Grand Island, N.Y.), supplemented with 2 mM
L-glutamine, 100 U/ml penicillin, 50 mg/ml gentamycin, 20 mM Hepes
buffer and heat-inactivated 10% FCS. Cells (10.sup.4 cells/well)
were incubated in 96-well plates (Falcon) in a final volume of 250
ml of complete culture medium. Increasing concentrations of human
CTLA-8-8 was added at the onset of the culture. Cell-free
supernatants were collected after 48 hours, and stored at
-20.degree. C. until cytokine assays. IL-6 levels, measured by
ELISA. Dose dependent induction of IL-6 was observed.
[0205] Similar results were obtained using adult and child dermal
fibroblasts, human brain epithelial cells, and human bronchus
epithelial cells. Kidney mesangium cells are also expected to
respond similarly.
[0206] VI. Isolating CTLA-8 Homologues
[0207] The binding composition is used for screening of an
expression library made from a cell line which expresses a CTLA-8
protein. Standard staining techniques are used to detect or sort
intracellular or surface expressed antigen, or surface expressing
transformed cells are screened by panning. Screening of
intracellular expression is performed by various staining or
immunofluorescence procedures. See also McMahan, et al. (1991) EMBO
J. 10:2821-2832.
[0208] For example, on day 0, precoat 2-chamber permanox slides
with 1 ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min
at room temperature. Rinse once with PBS. Then plate COS cells at
2-3.times.10.sup.5 cells per chamber in 1.5 ml of growth media.
Incubate overnight at 37.degree. C.
[0209] On day 1 for each sample, prepare 0.5 ml of a solution of 66
.mu.g/ml DEAE-dextran, 66 .mu.M chloroquine, and 4 .mu.g DNA in
serum free DME. For each set, a positive control is prepared, e.g.,
of huIL-10-FLAG cDNA at 1 and 1/200 dilution, and a negative mock.
Rinse cells with serum free DME. Add the DNA solution and incubate
5 hr at 37.degree. C. Remove the medium and add 0.5 ml 10% DMSO in
DME for 2.5 min. Remove and wash once with DME. Add 1.5 ml growth
medium and incubate overnight.
[0210] On day 2, change the medium. On days 3 or 4, the cells are
fixed and stained. Rinse the cells twice with Hank's Buffered
Saline Solution (HBSS) and fix in 4% paraformaldehyde (PFA)/glucose
for 5 min. Wash 3.times. with HBSS. The slides may be stored at
-80.degree. C. after all liquid is removed. For each chamber, 0.5
ml incubations are performed as follows. Add HBSS/saponin (0.1%)
with 32 .mu.l/ml of 1 M NaN.sub.3 for 20 min. Cells are then washed
with HBSS/saponin 1.times.. Soluble antibody is added to cells and
incubate for 30 min. Wash cells twice with HBSS/saponin. Add second
antibody, e.g., Vector anti-mouse antibody, at 1/200 dilution, and
incubate for 30 min. Prepare ELISA solution, e.g., Vector Elite ABC
horseradish peroxidase solution, and preincubate for 30 min. Use,
e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin)
per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add
ABC HRP solution and incubate for 30 min. Wash cells twice with
HBSS, second wash for 2 min, which closes cells. Then add Vector
diaminobenzoic acid (DAB) for 5 to 10 min. Use 2 drops of buffer
plus 4 drops DAB plus 2 drops of H.sub.2O.sub.2 per 5 ml of glass
distilled water. Carefully remove chamber and rinse slide in water.
Air dry for a few minutes, then add 1 drop of Crystal Mount and a
cover slip. Bake for 5 min at 85-90.degree. C.
[0211] Alternatively, the binding compositions are used to affinity
purify or sort out cells expressing the antigen. See, e.g.,
Sambrook, et al. or Ausubel, et al.
[0212] Similar methods are applicable to isolate either species or
allelic variants. Species variants are isolated using cross-species
hybridization techniques based upon a full length isolate or
fragment from one species as a probe, or appropriate species.
[0213] All references cited herein are incorporated herein by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference.
[0214] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
SEQUENCE SUBMISSION
[0215] SEQ ID NO: 1 is murine CTLA-8 cDNA nucleic acid
sequence.
[0216] SEQ ID NO: 2 is murine CTLA-8 peptide amino acid
sequence.
[0217] SEQ ID NO: 3 is herpesvirus ORF13 nucleic acid sequence.
[0218] SEQ ID NO: 4 is predicted ORF13 amino acid sequence.
[0219] SEQ ID NO: 5 is human CTLA-8 cDNA nucleic acid sequence.
[0220] SEQ ID NO: 6 is predicted human CTLA-8 amino acid
sequence.
[0221] SEQ ID NO: 7 is human CTLA-8 cDNA nucleic acid sequence.
[0222] SEQ ID NO: 8 is predicted human CTLA-8 amino acid
sequence.
[0223] SEQ ID NO: 9 is mouse CTLA-8 cDNA nucleic acid sequence.
[0224] SEQ ID NO: 10 is mouse CTLA-8 predicted amino acid sequence.
Sequence CWU 1
1
* * * * *