U.S. patent application number 10/777524 was filed with the patent office on 2004-07-22 for isolated mammalian monocyte cell genes; related reagents.
This patent application is currently assigned to Schering Corporation, a New Jersey corporation. Invention is credited to Adema, Gosse Jan, Gorman, Daniel M., Lanier, Lewis L., McClanahan, Terrill K., Meyaard, Linde, Phillips, Joseph H. JR., Zurawski, Gerard, Zurawski, Sandra M..
Application Number | 20040143858 10/777524 |
Document ID | / |
Family ID | 27487979 |
Filed Date | 2004-07-22 |
United States Patent
Application |
20040143858 |
Kind Code |
A1 |
Adema, Gosse Jan ; et
al. |
July 22, 2004 |
Isolated mammalian monocyte cell genes; related reagents
Abstract
Nucleic acids encoding various monocyte cell proteins from a
primate, reagents related thereto, including specific antibodies,
and purified proteins are described. Methods of using said reagents
and related diagnostic kits are also provided.
Inventors: |
Adema, Gosse Jan;
(Groesbeek, NL) ; Meyaard, Linde; (Amsterdam,
NL) ; Gorman, Daniel M.; (Newark, CA) ;
McClanahan, Terrill K.; (Sunnyvale, CA) ; Zurawski,
Sandra M.; (San Juan Bautista, CA) ; Zurawski,
Gerard; (San Juan Bautista, CA) ; Lanier, Lewis
L.; (Los Altos, CA) ; Phillips, Joseph H. JR.;
(Palo Alto, CA) |
Correspondence
Address: |
DNAX RESEARCH, INC.
LEGAL DEPARTMENT
901 CALIFORNIA AVENUE
PALO ALTO
CA
94304
US
|
Assignee: |
Schering Corporation, a New Jersey
corporation
|
Family ID: |
27487979 |
Appl. No.: |
10/777524 |
Filed: |
February 11, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10777524 |
Feb 11, 2004 |
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10290631 |
Nov 8, 2002 |
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Current U.S.
Class: |
800/8 ; 435/196;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
Y10S 435/81 20130101;
C07K 2319/00 20130101; C07K 16/28 20130101; C07K 16/18 20130101;
C07K 14/47 20130101; Y10S 435/975 20130101; C12Q 1/6876 20130101;
C07K 14/70503 20130101; G01N 33/68 20130101 |
Class at
Publication: |
800/008 ;
435/069.1; 435/196; 435/320.1; 435/325; 536/023.2 |
International
Class: |
A01K 067/00; C07H
021/04; C12N 009/16 |
Claims
What is claimed is:
1. A composition of matter selected from the group consisting of:
a) a substantially pure or recombinant FDF03 protein or peptide
exhibiting at least about 85% sequence identity over a length of at
least about 12 amino acids to a mature polypeptide from SEQ ID NO:
2 or 4; b) a natural sequence FDF03 of SEQ ID NO: 2 or 4; c) a
fusion protein comprising FDF03 sequence; d) a substantially pure
or recombinant YE01 protein or peptide exhibiting at least about
85% sequence identity over a length of at least about 12 amino
acids to a mature polypeptide from SEQ ID NO: 6, 8, or 10; e) a
natural sequence YE01 of SEQ ID NO: 6, 8, or 10; f) a fusion
protein comprising YE01 sequence; g) a substantially pure or
recombinant KTE03 protein or peptide exhibiting at least about 85%
sequence identity over a length of at least about 12 amino acids to
SEQ ID NO: 12, 14, 16, 18, 20, or 22; h) a natural sequence KTE03
of SEQ ID NO: 12, 14, 16, 18, 20, or 22; and i) a fusion protein
comprising KTE03 sequence.
2. A substantially pure or isolated protein comprising a segment
exhibiting sequence identity to a corresponding portion of a FDF03,
YE01, or KTE03 of claim 1, wherein: a) said homology is at least
about 90% identity and said portion is at least about 9 amino
acids; b) said homology is at least about 80% identity and said
portion is at least about 17 amino acids; or c) said homology is at
least about 70% identity and said portion is at least about 25
amino acids.
3. The composition of matter of claim 1, wherein said: a) FDF03
comprises a mature sequence of Table 1; b) YE01 comprises a mature
sequence of Table 2; c) KTE03 comprises a mature sequence of Table
3; d) protein or peptide: i) is from a warm blooded animal selected
from a mammal, including a primate or rodent; ii) comprises at
least one polypeptide segment of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, or 22; iii) exhibits a plurality of portions exhibiting
said identity; iv) is a natural allelic variant of FDF03, YE01, or
KTE03; v) has a length at least about 30 amino acids; vi) exhibits
at least two non-overlapping epitopes which are specific for a
mammalian FDF03, YE01, or KTE03; vii) exhibits a sequence identity
at least about 90% over a length of at least about 20 amino acids
to a rodent FDF03, YE01, or KTE03; viii) exhibits at least two
non-overlapping epitopes which are specific for a primate FDF03,
YE01, or KTE03; ix) exhibits a sequence identity at least about 90%
over a length of at least about 20 amino acids to a primate FDF03,
YE01, or KTE03; x) is glycosylated; xi) has a molecular weight of
at least 7 kD with natural glycosylation; xii) is a synthetic
polypeptide; xiii) is attached to a solid substrate; xiv) is
conjugated to another chemical moiety; xv) is a 5-fold or less
substitution from natural sequence; or xvi) is a deletion or
insertion variant from a natural sequence.
4. A composition comprising: a) a sterile FDF03 protein or peptide
of claim 1; b) said FDF03 protein or peptide of claim 1 and a
carrier, wherein said carrier is: i) an aqueous compound, including
water, saline, and/or buffer; and/or ii) formulated for oral,
rectal, nasal, topical, or parenteral administration; c) a sterile
YE01 protein or peptide of claim 1; d) said YE01 protein or peptide
of claim 1 and a carrier, wherein said carrier is: i) an aqueous
compound, including water, saline, and/or buffer; and/or ii)
formulated for oral, rectal, nasal, topical, or parenteral
administration; e) a sterile KTE03 protein or peptide of claim 1;
or f) said KTE03 protein or peptide of claim 1 and a carrier,
wherein said carrier is: i) an aqueous compound, including water,
saline, and/or buffer; and/or ii) formulated for oral, rectal,
nasal, topical, or parenteral administration.
5. The fusion protein of claim 1, comprising: a) mature protein
sequence of Table 1, 2, or 3; b) a detection or purification tag,
including a FLAG, His6, or Ig sequence; or c) sequence of another
cell surface protein.
6. A kit comprising a protein or polypeptide of claim 1, and: a) a
compartment comprising said protein or polypeptide; and/or b)
instructions for use or disposal of reagents in said kit.
7. A binding compound comprising an antigen binding portion from an
antibody, which specifically binds to a natural FDF03, YE01, or
KTE03 protein of claim 1, wherein: a) said protein is a rodent
protein; b) said binding compound is an Fv, Fab, or Fab2 fragment;
c) said binding compound is conjugated to another chemical moiety;
or d) said antibody: i) is raised against a peptide sequence of a
mature polypeptide of Table 1, 2, or 3; ii) is raised against a
mature FDF03, YE01, or KTE03; iii) is raised to a purified FDF03,
YE01, or KTE03; iv) is immunoselected; v) is a polyclonal antibody;
vi) binds to a denatured FDF03, YE01, or KTE03; vii) exhibits a Kd
to antigen of at least 30 .mu.M; viii) is attached to a solid
substrate, including a bead or plastic membrane; ix) is in a
sterile composition; or x) is detectably labeled, including a
radioactive or fluorescent label.
8. A kit comprising said binding compound of claim 7, and: a) a
compartment comprising said binding compound; and/or b)
instructions for use or disposal of reagents in said kit.
9. The kit of claim 8 capable of making a qualitative or
quantitative analysis.
10. A composition comprising: a) a sterile binding compound of
claim 7; or b) said binding compound of claim 7 and a carrier,
wherein said carrier is: i) an aqueous compound, including water,
saline, and/or buffer; and/or ii) formulated for oral, rectal,
nasal, topical, or parenteral administration.
11. An isolated or recombinant nucleic acid encoding a protein or
peptide or fusion protein of claim 1, wherein: a) said protein is
from a mammal, including a primate; or b) said nucleic acid: i)
encodes an antigenic peptide sequence of Table 1, 2, or 3; ii)
encodes a plurality of antigenic peptide sequences of Table 1, 2,
or 3; iii) exhibits at least about 80% identity to a natural cDNA
encoding said segment; iv) is an expression vector; v) further
comprises an origin of replication; vi) is from a natural source;
vii) comprises a detectable label; viii) comprises synthetic
nucleotide sequence; ix) is less than 6 kb, preferably less than 3
kb; x) is from a mammal, including a primate; xi) comprises a
natural full length coding sequence; xii) is a hybridization probe
for a gene encoding said protein; or xiii) is a PCR primer, PCR
product, or mutagenesis primer.
12. A cell or tissue comprising a recombinant nucleic acid of claim
11.
13. The cell of claim 12, wherein said cell is: a) a prokaryotic
cell; b) a eukaryotic cell; c) a bacterial cell; d) a yeast cell;
e) an insect cell; f) a mammalian cell; g) a mouse cell; h) a
primate cell; or i) a human cell.
14. A kit comprising said nucleic acid of claim 11, and: a) a
compartment comprising said nucleic acid; b) a compartment further
comprising a FDF03, YE01, or KTE03 protein or polypeptide; and/or
b) instructions for use or disposal of reagents in said kit.
15. The kit of claim 14 capable of making a qualitative or
quantitative analysis.
16. A nucleic acid which: a) hybridizes under wash conditions of
30.degree. C. and less than 2M salt to the coding portion from SEQ
ID NO: 1 or 3; b) hybridizes under wash conditions of 30.degree. C.
and less than 2 M salt to the coding portion from SEQ ID NO: 5, 7,
or 9; c) hybridizes under wash conditions of 30.degree. C. and less
than 2M salt to the coding portion from SEQ ID NO: 11, 13, 15, 17,
19, or 21; d) exhibits at least about 85% identity over a stretch
of at least about 30 nucleotides to a primate FDF03; e) exhibits at
least about 85% identity over a stretch of at least about 30
nucleotides to a primate YE01; or f) exhibits at least about 85%
identity over a stretch of at least about 30 nucleotides to a
primate KTE03.
17. The nucleic acid of claim 16, wherein: a) said wash conditions
are at 45.degree. C. and/or 500 mM salt; or b) said identity is at
least 90% and/or said stretch is at least 55 nucleotides.
18. The nucleic acid of claim 17, wherein: a) said wash conditions
are at 55.degree. C. and/or 150 mM salt; or b) said identity is at
least 95% and/or said stretch is at least 75 nucleotides.
19. A method of modulating physiology or development of a cell or
tissue culture cell comprising contacting said cell with an agonist
or antagonist of a FDF03, YE01, or KTE03.
20. The method of claim 19, wherein the cell is a leukocyte, and
the antagonist is to YE01 and is a monoclonal antibody which binds
to DLAIR-1.
Description
[0001] This filing is a conversion of, and claims benefit of
priority to, provisional U.S. Patent Applications U.S. S No.
60/032,252, filed Dec. 6, 1996; U.S. S No. 60/033,181, filed Dec.
16, 1996; and U.S. S No. 60/041,279, filed Mar. 21, 1997, each of
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention contemplates compositions related to
genes found in monocyte cells, cells which function in the immune
system. These genes function in controlling development,
differentiation, and/or physiology of the mammalian immune system.
In particular, the application provides nucleic acids, proteins,
antibodies, and methods of using them.
BACKGROUND OF THE INVENTION
[0003] The circulating component of the mammalian circulatory
system comprises various cell types, including red and white blood
cells of the erythroid and myeloid cell lineages. See, e.g.,
Rapaport (1987) Introduction to Hematology (2d ed.) Lippincott,
Philadelphia, Pa.; Jandl (1987) Blood: Textbook of Hematology,
Little, Brown and Co., Boston, Mass.; and Paul (ed.) (1993)
Fundamental Immunology (3d ed.) Raven Press, NY
[0004] Monocytes are phagocytic cells that belong to the
mononuclear phagocyte system and reside in the circulation. See
Roitt (ed) Encyclopedia of Immunology Academic Press, San Diego.
These cells originate in the bone marrow and remain only a short
time in the marrow compartment once they differentiate. They then
enter the circulation and can remain there for a relatively long
period of time, e.g., a few days. The monocytes can enter the
tissues and body cavities by the process designated diapedesis,
where they differentiate into macrophages and possibly into
dendritic cells. In an inflammatory response, the number of
monocytes in the circulation may double, and many of the increased
number of monocytes diapedese to the site of inflammation.
[0005] Antigen presentation refers to the cellular events in which
a proteinaceous antigen is taken up, processed by antigen
presenting cells (APC), and then recognized to initiate an immune
response. The most active antigen presenting cells have been
characterized as the macrophages, which are direct developmental
products from monocytes; dendritic cells; and certain B cells.
[0006] Macrophages are found in most tissues and are highly active
in internalization of a wide variety of protein antigens and
microorganisms. They have a highly developed endocytic activity,
and secrete many products important in the initiation of an immune
response. For this reason, it is believed that many genes expressed
by monocytes or induced by monocyte activation are likely to be
important in antigen uptake, processing, presentation, or
regulation of the resulting immune response.
[0007] However, monocytes are poorly characterized, both in terms
of proteins they express, and many of their functions and
mechanisms of action, including their activated states. In
particular, the processes and mechanisms related to the initiation
of an immune response, including antigen processing and
presentation, remain unclear. The absence of knowledge about the
structural, biological, and physiological properties of these cells
limits their understanding. Thus, medical conditions where
regulation, development, or physiology of antigen presenting cells
is unusual remain unmanageable.
SUMMARY OF THE INVENTION
[0008] The present invention is based, in part, upon the discovery
of various genes isolated from activated monocytes. These molecules
have been designated FDF03 (a type I transmembrane protein with
Ig-like extracellular portion); YE01 (an Fc gamma/alpha-like
receptor); and KTE03 class (cell surface receptors exhibiting
Ig-like domains), represented by YYB01, YYB04 related, KLM63,
KLM66, and KLM67 embodiments.
[0009] The invention provides various compositions of matter
selected from: a substantially pure or recombinant FDF03 protein or
peptide exhibiting at least about 85% sequence identity over a
length of at least about 12 amino acids to mature SEQ ID NO: 2 or
4; a natural sequence FDF03 of SEQ ID NO: 2 or 4; a fusion protein
comprising FDF03 sequence; a substantially pure or recombinant YE01
protein or peptide exhibiting at least about 85% sequence identity
over a length of at least about 12 amino acids to mature SEQ ID NO:
6, 8, or 10; a natural sequence YE01 of SEQ ID NO: 6, 8, or 10; a
fusion protein comprising YE01 sequence; a substantially pure or
recombinant KTE03 protein or peptide exhibiting at least about 85%
sequence identity over a length of at least about 12 amino acids to
SEQ ID NO: 12, 14, 16, 18, 20, or 22; a natural sequence KTE03 of
SEQ ID NO: 12, 14, 16, 18, 20, or 22; or a fusion protein
comprising KTE03 sequence. Preferably, the substantially pure or
isolated protein comprises a segment exhibiting sequence identity
to a corresponding portion of a FDF03, YE01, or KTE03, wherein: the
homology is at least about 90% identity and the portion is at least
about 9 amino acids; the homology is at least about 80% identity
and the portion is at least about 17 amino acids; or the homology
is at least about 70% identity and the portion is at least about 25
amino acids. In other forms, the invention provides such
composition of matter, wherein the: FDF03 comprises a mature
sequence of Table 1; YE01 comprises a mature sequence of Table 2;
KTE03 comprises a mature sequence of Table 3; or the protein or
peptide: is from a warm blooded animal selected from a mammal,
including a primate or rodent; comprises at least one polypeptide
segment of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22;
exhibits a plurality of portions exhibiting the identity; is a
natural allelic variant of FDF03, YE01, or KTE03; has a length at
least about 30 amino acids; exhibits at least two non-overlapping
epitopes which are specific for a mammalian FDF03, YE01, or KTE03;
exhibits a sequence identity at least about 90% over a length of at
least about 20 amino acids to a rodent FDF03, YE01, or KTE03;
exhibits at least two non-overlapping epitopes which are specific
for a primate FDF03, YE01, or KTE03; exhibits a sequence identity
at least about 90% over a length of at least about 20 amino acids
to a primate FDF03, YE01, or KTE03; is glycosylated; has a
molecular weight of at least 7 kD with natural glycosylation; is a
synthetic polypeptide; is attached to a solid substrate; is
conjugated to another chemical moiety; is a 5-fold or less
substitution from natural sequence; or is a deletion or insertion
variant from a natural sequence.
[0010] Other compositions include those comprising: a sterile FDF03
protein or peptide; the FDF03 protein or peptide and a carrier,
wherein the carrier is: an aqueous compound, including water,
saline, and/or buffer; and/or formulated for oral, rectal, nasal,
topical, or parenteral administration; a sterile YE01 protein or
peptide; the YE01 protein or peptide and a carrier, wherein the
carrier is: an aqueous compound, including water, saline, and/or
buffer; and/or formulated for oral, rectal, nasal, topical, or
parenteral administration; a sterile KTE03 protein or peptide; or
the KTE03 protein or peptide and a carrier, wherein the carrier is:
an aqueous compound, including water, saline, and/or buffer; and/or
formulated for oral, rectal, nasal, topical, or parenteral
administration.
[0011] In fusion protein embodiments, the invention provides those
which comprise: mature protein sequence of Table 1, 2, or 3; a
detection or purification tag, including a FLAG, His6, or Ig
sequence; or sequence of another cell surface protein.
[0012] Various kits include those comprising a protein or
polypeptide, and: a compartment comprising the protein or
polypeptide; and/or instructions for use or disposal of reagents in
the kit.
[0013] Antibodies and binding compounds include those comprising an
antigen binding portion from an antibody, which specifically binds
to a natural FDF03, YE01, or KTE03 protein, wherein: the protein is
a primate protein; the binding compound is an Fv, Fab, or Fab2
fragment; the binding compound is conjugated to another chemical
moiety; or the antibody: is raised against a peptide sequence of a
mature polypeptide of Table 1, 2, or 3; is raised against a mature
FDF03, YE01, or KTE03; is raised to a purified FDF03, YE01, or
KTE03; is immunoselected; is a polyclonal antibody; binds to a
denatured FDF03, YE01, or KTE03; exhibits a Kd to antigen of at
least 30 .mu.M; is attached to a solid substrate, including a bead
or plastic membrane; is in a sterile composition; or is detectably
labeled, including a radioactive or fluorescent label. A kit
comprising the binding compound is provided including, e.g., the
binding compound and: a compartment comprising the binding
compound; and/or instructions for use or disposal of reagents in
the kit. Preferably, the kit is capable of making a qualitative or
quantitative analysis.
[0014] Various other compositions include those comprising: a
sterile binding compound; or the binding compound and a carrier,
wherein the carrier is: an aqueous compound, including water,
saline, and/or buffer; and/or formulated for oral, rectal, nasal,
topical, or parenteral administration.
[0015] Nucleic acid embodiments include an isolated or recombinant
nucleic acid encoding a protein or peptide or fusion protein as
described, wherein: the protein is from a mammal, including a
primate; or the nucleic acid: encodes an antigenic peptide sequence
of Table 1, 2, or 3; encodes a plurality of antigenic peptide
sequences of Table 1, 2, or 3; exhibits at least about 80% identity
to a natural cDNA encoding the segment; is an expression vector;
further comprises an origin of replication; is from a natural
source; comprises a detectable label; comprises synthetic
nucleotide sequence; is less than 6 kb, preferably less than 3 kb;
is from a mammal, including a primate; comprises a natural full
length coding sequence; is a hybridization probe for a gene
encoding the protein; or is a PCR primer, PCR product, or
mutagenesis primer.
[0016] Various cells are provided, including those comprising a
described recombinant nucleic acid. Preferably, the cell is: a
prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast
cell; an insect cell; a mammalian cell; a mouse cell; a primate
cell; or a human cell. Kits with such nucleic acids include those
with the nucleic acid and: a compartment comprising the nucleic
acid; a compartment further comprising a FDF03, YE01, or KTE03
protein or polypeptide; and/or instructions for use or disposal of
reagents in the kit. Preferably, the kit is capable of making a
qualitative or quantitative analysis.
[0017] Other nucleic acids include those which: hybridize under
wash conditions of 300.degree. C. and less than 2M salt to the
coding portions of SEQ ID NO: 1 or 3; hybridize under wash
conditions of 30.degree. C. and less than 2 M salt to the coding
portions of SEQ ID NO: 5, 7, or 9; hybridize under wash conditions
of 300.degree. C. and less than 2M salt to the coding portions of
SEQ ID NO: 11, 13, 15, 17, 19, or 21; exhibit at least about 85%
identity over a stretch of at least about 30 nucleotides to a
primate FDF03; exhibit at least about 85% identity over a stretch
of at least about 30 nucleotides to a primate YE01; or exhibit at
least about 85% identity over a stretch of at least about 30
nucleotides to a primate KTE03. In preferred embodiments, the wash
conditions are at 450.degree. C. and/or 500 mm salt; or at
550.degree. C. and/or 150 mM salt; or the identity is at least 90%
and/or the stretch is at least 55 nucleotides; or the identity is
at least 95% and/or the stretch is at least 75 nucleotides.
[0018] The invention further provides a method of modulating
physiology or development of a cell or tissue culture cell
comprising contacting the cell with an agonist or antagonist of a
FDF03, YE01, or KTE03. In preferred embodiments, the cell is a
leukocyte, and the antagonist is to YE01 and is a monoclonal
antibody which binds to DLAIR-1.
DETAILED DESCRIPTION
[0019] Outline
[0020] I. General
[0021] II. Definitions
[0022] III. Nucleic Acids
[0023] IV. Making Proteins
[0024] V. Antibodies
[0025] VI. Purified Proteins
[0026] VII. Physical Variants
[0027] VIII. Binding Agent:Monocyte Protein Complexes
[0028] IX. Uses
[0029] X. Kits
[0030] XI. Binding Partner Isolation
[0031] I. General
[0032] The present invention provides DNA sequences encoding
mammalian proteins expressed on monocytes. For a review of
monocytes and their functions, see, e.g., Gallin, et al. (eds.
1988) Inflammation: Basic Principles and Clinical Correlates Raven
Press, NY; van Furth (ed. 1985) Mononuclear Phagocytes:
Characteristics. Physiology and Function Martinus Nijhoff,
Dordrecht, Netherlands.
[0033] Specific human embodiments of these proteins are provided
below. The descriptions below are directed, for exemplary purposes,
to human monocyte genes, but are likewise applicable to
structurally, e.g., sequence, related embodiments from other
sources or mammalian species, including polymorphic or individual
variants. These will include, e.g., proteins which exhibit a
relatively few changes in sequence, e.g., less than about 5%, and
number, e.g., less than 20 residue substitutions, typically less
than 15, preferably less than 10, and more preferably less than 5
substitutions. These will also include versions which are truncated
from full length, as described, and fusion proteins containing
substantial segments of these sequences.
[0034] II. Definitions
[0035] The term "binding composition" refers to molecules that bind
with specificity to a these monocyte proteins, e.g., in an
antibody-antigen interaction, or compounds, e.g., proteins, which
specifically associate with the respective protein. Typically, the
association will be in a natural physiologically relevant
protein-protein interaction, either covalent or non-covalent, and
may include members of a multiprotein complex, including carrier
compounds or dimerization partners. The molecule may be a polymer,
or chemical reagent. 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 interacting determinants. The variants may serve as
agonists or antagonists of the protein, see, e.g., Goodman, et al.
(eds.) (1990) Goodman & Gilman's: The Pharmacological Bases of
Therapeutics (8th ed.) Pergamon Press, Tarrytown, N.Y.
[0036] The term "binding agent:monocyte protein complex", as used
herein, refers to a complex of a binding agent and the monocyte
protein. Specific binding of the binding agent means that the
binding agent has a specific binding site that recognizes a site on
the respective monocyte protein. For example, antibodies raised to
the monocyte protein and recognizing an epitope on the monocyte
protein are capable of forming a binding agent:monocyte protein
complex by specific binding. Typically, the formation of a binding
agent:monocyte protein complex allows the measurement of monocyte
protein in a mixture of other proteins and biologics. The term
"antibody:monocyte protein complex" refers to a binding
agent:monocyte protein complex in which the binding agent is an
antibody. The antibody may be monoclonal, polyclonal or even an
antigen binding fragment of an antibody.
[0037] "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 and/or phylogenetic relationship, or based upon
hybridization conditions. Hybridization conditions are described in
greater detail below.
[0038] 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.,
proteins 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. 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.
[0039] As used herein, the term "monocyte protein" shall encompass,
when used in a protein context, a protein having amino acid
sequences as shown in SEQ ID NO: 2 or 4; 6, 8, or 10; or 12, 14,
16, 18, 20, or 22, or a significant fragment of such a protein. It
refers to a polypeptide which interacts with the respective
monocyte protein specific binding components. These binding
components, e.g., antibodies, typically bind to the monocyte
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.
[0040] The term "polypeptide" or "protein" as used herein includes
a significant fragment or segment of said monocyte protein, 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. Fragment or size limitations applicable for comparison to
one group, e.g., to the FDF03, do not necessarily imply similar
size limitations on fragments for the others.
[0041] 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 non-naturally
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.
[0042] "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 & Co., San Francisco, Calif.; and Cantor and
Schimmel (1980) Biophysical Chemistry parts 1-3, W.H. Freeman &
Co., San Francisco, Calif. 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 30S, more
typically less than about 15S, usually less than about 10S, more
usually less than about 6S, and, in particular embodiments,
preferably less than about 4S, and more preferably less than about
3S. 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.
[0043] 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.
[0044] 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 protein.
[0045] "Substantially pure" typically means that the protein is
isolated from other contaminating proteins, nucleic acids, and
other biologicals derived from the original source organism.
Purity, or "isolation" may be assayed by standard methods, and will
ordinarily be at least about 50% pure, more ordinarily at least
about 60% pure, generally at least about 70% pure, more generally
at least about 80% pure, often at least about 85% pure, more often
at least about 90% pure, preferably at least about 95% pure, more
preferably at least about 98% pure, and in most preferred
embodiments, at least 99% pure.
[0046] "Substantial similarity" 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 similarity exists when the segments will
hybridize under selective hybridization conditions, to a strand, or
its complement, typically using a sequence derived from SEQ ID NO:
1 or 3; 5, 7, or 9; or 11, 13, 15, 17, 19, or 21. Typically,
selective hybridization will occur when there is at least about 55%
similarity over a stretch of at least about 30 nucleotides,
preferably at least about 65% over a stretch of at least about 25
nucleotides, more preferably at least about 75%, and most
preferably at least about 90% over about 20 nucleotides. See, e.g.,
Kanehisa (1984) Nucl. Acids Res. 12:203-213. The length of
similarity 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.
[0047] "Stringent conditions", in referring to homology or
substantial similarity in the hybridization context, will be
stringent combined conditions of salt, temperature, organic
solvents, and other parameters, typically those controlled in
hybridization reactions. The combination of parameters is more
important than the measure of any single parameter. See, e.g.,
Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370. A nucleic acid
probe which binds to a target nucleic acid under stringent
conditions is specific for said target nucleic acid. Such a probe
is typically more than 11 nucleotides in length, and is
sufficiently identical or complementary to a target nucleic acid
over the region specified by the sequence of the probe to bind the
target under stringent hybridization conditions.
[0048] Counterpart monocyte proteins from other mammalian species
can be cloned and isolated by cross-species hybridization of
closely related species. See, e.g., below. Similarity 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.
[0049] The phrase "specifically binds to an antibody" or
"specifically immunoreactive with", when referring to a protein or
peptide, refers to a binding reaction which is determinative of the
presence of the protein in the presence of a heterogeneous
population of proteins and other biological components. Thus, under
designated immunoassay conditions, the specified antibodies bind to
a particular protein and do not significantly bind other proteins
present in the sample. Specific binding to an antibody under such
conditions may require an antibody that is selected for its
specificity for a particular protein. For example, antibodies
raised to the human monocyte protein immunogen with the amino acid
sequence depicted in SEQ ID NO: 2 can be selected to obtain
antibodies specifically immunoreactive with that monocyte protein
and not with other proteins. These antibodies recognize proteins
highly similar to the homologous human monocyte protein.
[0050] III. Nucleic Acids
[0051] These monocyte genes are specifically expressed on dendritic
cells. The preferred embodiments, as disclosed, will be useful in
standard procedures to isolate genes from other species, e.g., warm
blooded animals, such as birds and mammals. Cross hybridization
will allow isolation of related proteins from individuals, strains,
or species. A number of different approaches are available
successfully to isolate a suitable nucleic acid clone based upon
the information provided herein. Southern blot hybridization
studies should identify homologous genes in other species under
appropriate hybridization conditions.
[0052] Purified protein or defined peptides, are useful for
generating antibodies by standard methods, as described below.
Synthetic peptides or purified protein can be presented to an
immune system to generate polyclonal and monoclonal antibodies.
See, e.g., Coligan (1991) Current Protocols in Immunology
Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A
Laboratory Manual Cold Spring Harbor Press, NY, which are
incorporated herein by reference. Alternatively, a CD protein
binding composition can be useful as a specific binding reagent,
and advantage can be taken of its specificity of binding, for,
e.g., purification of a monocyte protein.
[0053] The specific binding composition can be used for screening
an expression library made from a cell line which expresses the
respective monocyte protein. Many methods for screening are
available, e.g., standard staining of surface expressed ligand, 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 ligand.
1TABLE 1 Sequence encoding a human FDF03 protein, containing Ig
domains. The putative coding region runs from about 154 to 1062.
See SEQ ID NO: 1 and 2. This 1249 bp clone was isolated from a
monocyte cell library. A putative (hydrophobic) signal sequence
runs from -19 to about -1; a putative transmembrane (hydrophobic)
segment runs from about 178 to 199. The extracellular region is
probably about 170 amino acids, with a potential Ig-like domain
structure; the intracellular region is about 80 residues. Sequence
analysis indicates similarity to GenBank clones H26010 and R50327
from humans. GTTTGGGGAA GGCTCCTGGC CCCCACAGCC CTCTTCGGAG CCTGAGCCCG
GCTCTCCTCA 60 CTCACCTCAA CCCCCAGGCG GCCCCTCCAC AGGGCCCCTC
TCCTGCCTGG ACGGCTCTGC 120 TGGTCTCCCC GTCCCCTGGA GAAGAACAAG GCC ATG
GGT CGG CCC CTG CTG CTG 174 Met Gly Arg Pro Leu Leu Leu -19 -15 CCC
CTA CTG CCC CTG CTG CTG CCG CCA GCA TTT CTG CAG CCT AGT GGC 222 Pro
Leu Leu Pro Leu Leu Leu Pro Pro Ala Phe Leu Gln Pro Ser Gly -10 -5
1 TCC ACA GGA TCT GGT CCA AGC TAC CTT TAT GGG GTC ACT CAA CCA AAA
270 Ser Thr Gly Ser Gly Pro Ser Tyr Leu Tyr Gly Val Thr Gln Pro Lys
5 10 15 20 CAC CTC TCA GCC TCC ATG GGT GGC TCT GTG GAA ATC CCC TTC
TCC TTC 318 His Leu Ser Ala Ser Met Gly Gly Ser Val Glu Ile Pro Phe
Ser Phe 25 30 35 TAT TAC CCC TGG GAG TTA GCC ACA GCT CCC GAC GTG
AGA ATA TCC TGG 366 Tyr Tyr Pro Trp Glu Leu Ala Thr Ala Pro Asp Val
Arg Ile Ser Trp 40 45 50 AGA CGG GGC CAC TTC CAC GGG CAG TCC TTC
TAC AGC ACA AGG CCG CCT 414 Arg Arg Gly His Phe His Gly Gln Ser Phe
Tyr Ser Thr Arg Pro Pro 55 60 65 TCC ATT CAC AAG GAT TAT GTG AAC
CGG CTC TTT CTG AAC TGG ACA GAG 462 Ser Ile His Lys Asp Tyr Val Asn
Arg Leu Phe Leu Asn Trp Thr Glu 70 75 80 GGT CAG AAG AGC GGC TTC
CTC AGG ATC TCC AAC CTG CAG AAG CAG GAC 510 Gly Gln Lys Ser Gly Phe
Leu Arg Ile Ser Asn Leu Gln Lys Gln Asp 85 90 95 100 CAG TCT GTG
TAT TTC TGC CGA GTT GAG CTG GAC ACA CGG AGC TCA GGG 558 Gln Ser Val
Tyr Phe Cys Arg Val Glu Leu Asp Thr Arg Ser Ser Gly 105 110 115 AGG
CAG CAG TGG CAG TCC ATC GAG GGG ACC AAA CTC TCC ATC ACC CAG 606 Arg
Gln Gln Trp Gln Ser Ile Glu Gly Thr Lys Leu Ser Ile Thr Gln 120 125
130 GCT GTC ACG ACC ACC ACC CAG AGG CCC AGC AGC ATG ACT ACC ACC TGG
654 Ala Val Thr Thr Thr Thr Gln Arg Pro Ser Ser Met Thr Thr Thr Trp
135 140 145 AGG CTC AGT AGC ACA ACC ACC ACA ACC GGC CTC AGG GTC ACA
CAG GGC 702 Arg Leu Ser Ser Thr Thr Thr Thr Thr Gly Leu Arg Val Thr
Gln Gly 150 155 160 AAA CGA CGC TCA GAC TCT TGG CAC ATA AGT CTG GAG
ACT GCT GTG GGG 750 Lys Arg Arg Ser Asp Ser Trp His Ile Ser Leu Glu
Thr Ala Val Gly 165 170 175 180 GTG GCA GTG GCT GTC ACT GTG CTC GGA
ATC ATG ATT TTG GGA CTG ATC 798 Val Ala Val Ala Val Thr Val Leu Gly
Ile Met Ile Leu Gly Leu Ile 185 190 195 TGC CTC CTC AGG TGG AGG AGA
AGG AAA GGT CAG CAG CGG ACT AAA GCC 846 Cys Leu Leu Arg Trp Arg Arg
Arg Lys Gly Gln Gln Arg Thr Lys Ala 200 205 210 ACA ACC CCA GCC AGG
GAA CCC TTC CAA AAC ACA GAG GAG CCA TAT GAG 894 Thr Thr Pro Ala Arg
Glu Pro Phe Gln Asn Thr Glu Glu Pro Tyr Glu 215 220 225 AAT ATC AGG
AAT GAA GGA CAA AAT ACA GAT CCC AAG CTA AAT CCC AAG 942 Asn Ile Arg
Asn Glu Gly Gln Asn Thr Asp Pro Lys Leu Asn Pro Lys 230 235 240 GAT
GAC GGC ATC GTA TAT GCT TCC CTT GCC CTC TCC AGC TCC ACC TCA 990 Asp
Asp Gly Ile Val Tyr Ala Ser Leu Ala Leu Ser Ser Ser Thr Ser 245 250
255 260 CCC AGA GCA CCT CCC AGC CAC CGT CCC CTC AAG AGC CCC CAG AAC
GAG 1038 Pro Arg Ala Pro Pro Ser His Arg Pro Leu Lys Ser Pro Gln
Asn Glu 265 270 275 ACC CTG TAC TCT GTC TTA AAG GCC TAACCAATGG
ACAGCCCTCT CAAGACTGAA 1092 Thr Leu Tyr Ser Val Leu Lys Ala 280
TGGTGAGGCC AGGTACAGTG GCGCACACCT GTAATCCCAG CTACTCTGAA GCCTGAGGCA
1152 GAATCAAGTG AGCCCAGGAG TTCAGGGCCA GCTTTGATAA TGGAGCGAGA
TGCCATCTCT 1212 AGTTAAAAAT ATATATTAAC AATAAAGTAA CAAATTT 1249 A
mouse counterpart partial sequence is (SEQ ID NO: 3 and 4):
CCCCAGTGTC CCTAGACAGA GCATCCTTGC CTTCCTGATG GCTTTGCTGA TCTCGCTTCC
60 CTGGAGGGAC TCCAGCC ATG GCT CAG GTC CTG CTT CTG CTC TCA TCA GGC
110 Met Ala Gln Val Leu Leu Leu Leu Ser Ser Gly 1 5 10 TGT CTG CAT
GCT GGA AAT TCA GAA AGA TAC AAC AGA AAA AAT GGC TTT 158 Cys Leu His
Ala Gly Asn Ser Glu Arg Tyr Asn Arg Lys Asn Gly Phe 15 20 25 GGG
GTC AAC CAA CCT GAA CGC TGC TCT GGA GTC CAG GGT GGC TCC ATC 206 Gly
Val Asn Gln Pro Glu Arg Cys Ser Gly Val Gln Gly Gly Ser Ile 30 35
40 GAC ATC CCC TTC TCC TTC TAT TTC CCC TGG AAG TTG GCC AAG GAT CCA
254 Asp Ile Pro Phe Ser Phe Tyr Phe Pro Trp Lys Leu Ala Lys Asp Pro
45 50 55 CAG ATG AGC ATA GCC TGG AAA TGG AAG GAT TTC CAT GGG GAA
GTC ATC 302 Gln Met Ser Ile Ala Trp Lys Trp Lys Asp Phe His Gly Glu
Val Ile 60 65 70 75 TAC AAC TCC TCC CTG CCT TTC ATA CAT GAG CAC TTC
AAG GGC CGG CTC 350 Tyr Asn Ser Ser Leu Pro Phe Ile His Glu His Phe
Lys Gly Arg Leu 80 85 90 ATC CTG AAC TGG ACA CAG GGT CAG AC 376 Ile
Leu Asn Trp Thr Gln Gly Gln 95 partial human/mouse alignment: hu
MGRPLLLPLLPLLLPPAFLQPSGSTGSGPSYLYGVTQPKHLSASMGGSVEI- PFSFYYPWE mo
MAQVLLLLSSGCLHAGNSERYNRKNG------FGVNQPERCSGVQGGSIDIPF- SFYFPWK hu
LATAPDVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQKS- GFLRISNLQK... mo
LAKDPQMSIAWKWKDFHGEVIYNSSLPFIHEHFKGRLILNWTQGQ...
[0054]
2TABLE 2 Sequence encoding a protein related to Ig family members,
designated YE01, isolated from an activated monocyte cell library.
See SEQ ID NO: 5 and 6. Signal sequence is indicated. Nucleotide
1247 may be C or T. Sequence analysis suggests YE01 is a member of
the Ig superfamily of receptors, and is closely related to the CD8
family, which contain a V1J-type fold, particularly the Fc
receptors alpha and/or gamma. Because it contains an ITAM-like
motif, the protein may well be a monocyte version of the KIR
proteins, the Killer Inhibitory Receptors, which send a negative
signal to inhibit killer cell function. This protein may share
similar function in inhibiting monocyte effector function, e.g.,
antigen presentation or subsequent response initiation. A mouse
counterpart is probably encoded in the EST W55567. ACCGGTCCGG
AATTCCCGGG TCGACCCACG CGTCCGGGAA GCCCCATAGG CAGGAGGCCC 60
CCGGGCAGCA CATCCTGTCT GCTTGTGTCT GCTGCAGAGT TCTGTCCTTG CATTGGTGCG
120 CCTCAGGCCA GGCTGCACTG CTGGGACCTG GGCC ATG TCT CCC CAC CCC ACC
172 Met Ser Pro His Pro Thr -21 -20 GCC CTC CTG GGC CTA GTG CTC TGC
CTG GCC CAG ACC ATC CAC ACG CAG 220 Ala Leu Leu Gly Leu Val Leu Cys
Leu Ala Gln Thr Ile His Thr Gln -15 -10 -5 1 GAG GAA GAT CTG CCC
AGA CCC TCC ATC TCG GCT GAG CCA GGC ACC GTG 268 Glu Glu Asp Leu Pro
Arg Pro Ser Ile Ser Ala Glu Pro Gly Thr Val 5 10 15 ATC CCC CTG GGG
AGC CAT GTG ACT TTC GTG TGC CGG GGC CCG GTT GGG 316 Ile Pro Leu Gly
Ser His Val Thr Phe Val Cys Arg Gly Pro Val Gly 20 25 30 GTT CAA
ACA TTC CGC CTG GAG AGG GAG AGT AGA TCC ACA TAC AAT GAT 364 Val Gln
Thr Phe Arg Leu Glu Arg Glu Ser Arg Ser Thr Tyr Asn Asp 35 40 45
ACT GAA GAT GTG TCT CAA GCT AGT CCA TCT GAG TCA GAG GCC AGA TTC 412
Thr Glu Asp Val Ser Gln Ala Ser Pro Ser Glu Ser Glu Ala Arg Phe 50
55 60 65 CGC ATT GAC TCA GTA AGT GAA GGA AAT GCC GGG CCT TAT CGC
TGC ATC 460 Arg Ile Asp Ser Val Ser Glu Gly Asn Ala Gly Pro Tyr Arg
Cys Ile 70 75 80 TAT TAT AAG CCC CCT AAA TGG TCT GAG CAG AGT GAC
TAC CTG GAG CTG 508 Tyr Tyr Lys Pro Pro Lys Trp Ser Glu Gln Ser Asp
Tyr Leu Glu Leu 85 90 95 CTG GTG AAA GAA ACC TCT GGA GGC CCG GAC
TCC CCG GAC ACA GAG CCC 556 Leu Val Lys Glu Thr Ser Gly Gly Pro Asp
Ser Pro Asp Thr Glu Pro 100 105 110 GGC TCC TCA GCT GGA CCC ACG CAG
AGG CCC TCG GAC AAC AGT CAC AAT 604 Gly Ser Ser Ala Gly Pro Thr Gln
Arg Pro Ser Asp Asn Ser His Asn 115 120 125 GAG CAT GCA CCT GCT TCC
CAA GGC CTG AAA GCT GAG CAT CTG TAT ATT 652 Glu His Ala Pro Ala Ser
Gln Gly Leu Lys Ala Glu His Leu Tyr Ile 130 135 140 145 CTC ATC GGG
GTC TCA GTG GTC TTC CTC TTC TGT CTC CTC CTC CTG GTC 700 Leu Ile Gly
Val Ser Val Val Phe Leu Phe Cys Leu Leu Leu Leu Val 150 155 160 CTC
TTC TGC CTC CAT CGC CAG AAT CAG ATA AAG CAG GGG CCC CCC AGA 748 Leu
Phe Cys Leu His Arg Gln Asn Gln Ile Lys Gln Gly Pro Pro Arg 165 170
175 AGC AAG GAC GAG GAG CAG AAG CCA CAG CAG AGG CCT GAC CTG GCT GTT
796 Ser Lys Asp Glu Glu Gln Lys Pro Gln Gln Arg Pro Asp Leu Ala Val
180 185 190 GAT GTT CTA GAG AGG ACA GCA GAC AAG GCC ACA GTC AAT GGA
CTT CCT 844 Asp Val Leu Glu Arg Thr Ala Asp Lys Ala Thr Val Asn Gly
Leu Pro 195 200 205 GAG AAG GAC AGA GAG ACG GAC ACC TCG GCC CTG GCT
GCA GGG AGT TCC 892 Glu Lys Asp Arg Glu Thr Asp Thr Ser Ala Leu Ala
Ala Gly Ser Ser 210 215 220 225 CAG GAG GTG ACG TAT GCT CAG CTG GAC
CAC TGG GCC CTC ACA CAG AGG 940 Gln Glu Val Thr Tyr Ala Gln Leu Asp
His Trp Ala Leu Thr Gln Arg 230 235 240 ACA GCC CGG GCT GTG TCC CCA
CAG TCC ACA AAG CCC ATG GCC GAG TCC 988 Thr Ala Arg Ala Val Ser Pro
Gln Ser Thr Lys Pro Met Ala Glu Ser 245 250 255 ATC ACG TAT GCA GCC
GTT GCC AGA CAC TGACCCCATA CCCACCTGGC 1035 Ile Thr Tyr Ala Ala Val
Ala Arg His 260 265 CTCTGCACCT GAGGGTAGAA AGTCACTCTA GGAAAAGCCT
GAAGCAGCCA TTTGGAAGGC 1095 TTCCTGTTGG ATTCCTCTTC ATCTAGAAAG
CCAGCCAGGC AGCTGTCCTG GAGACAAGAG 1155 CTGGAGACTG GAGGTTTCTA
ACCAGCATCC AGAAGGTTCG TTAGCCAGGT GGTCCCTTCT 1215 ACAATCGGAC
AGCTCCTTGG ACAGACTGTT TCTCAGTTAT TTCCAAAAAC CCAGCTACAG 1275 TTCC
1279 A similar gene was cloned by expressing cloning using a
monoclonal antibody DX26, which was raised against the immunogen of
human NK cell clone NK681.D5, and selected for inhibiting killing
by NK cell clones of Fc receptor bearing target cells (SP2/0). SEQ
ID NO: 7 and 8. AAAGGCTGCA GAGTTCTGTC CTTGCATTGG TGCGCCTCAG
GCCAGGCTGC ACTGCTGGGA 60 CCTGGGCG ATG TCT CCC CAC CCC ACC GCC CTC
CTG GGC CTA GTG CTC TGC 110 Met Ser Pro His Pro Thr Ala Leu Leu Gly
Leu Val Leu Cys -21 -20 -15 -10 CTG GCC CAG ACC ATC CAC ACG CAG GAG
GAA GAT CTG CCC AGA CCC TCC 158 Leu Ala Gln Thr Ile His Thr Gln Glu
Glu Asp Leu Pro Arg Pro Ser -5 1 5 ATC TCG GCT GAG CCA GGC ACC GTG
ATC CCC CTG GGG AGC CAT GTG ACT 206 Ile Ser Ala Glu Pro Gly Thr Val
Ile Pro Leu Gly Ser His Val Thr 10 15 20 25 TTC GTG TGC CGG GGC CCG
GTT GGG GTT CAA ACA TTC CGC CTG GAG AGG 254 Phe Val Cys Arg Gly Pro
Val Gly Val Gln Thr Phe Arg Leu Glu Arg 30 35 40 GAG AGT AGA TCC
ACA TAC AAT GAT ACT GAA GAT GTG TCT CAA GCT AGT 302 Glu Ser Arg Ser
Thr Tyr Asn Asp Thr Glu Asp Val Ser Gln Ala Ser 45 50 55 CCA TCT
GAG TCA GAG GCC AGA TTC CGC ATT GAC TCA GTA AGT GAA GGA 350 Pro Ser
Glu Ser Glu Ala Arg Phe Arg Ile Asp Ser Val Ser Glu Gly 60 65 70
AAT GCC GGG CCT TAT CGC TGC ATC TAT TAT AAG CCC CCT AAA TGG TCT 398
Asn Ala Gly Pro Tyr Arg Cys Ile Tyr Tyr Lys Pro Pro Lys Trp Ser 75
80 85 GAG CAG AGT GAC TAC CTG GAG CTG CTG GTG AAA GAA ACC TCT GGA
GGC 446 Glu Gln Ser Asp Tyr Leu Glu Leu Leu Val Lys Glu Thr Ser Gly
Gly 90 95 100 105 CCG GAC TCC CCG GAC ACA GAG CCC GGC TCC TCA GCT
GGA CCC ACG CAG 494 Pro Asp Ser Pro Asp Thr Glu Pro Gly Ser Ser Ala
Gly Pro Thr Gln 110 115 120 AGG CCG TCG GAC AAC AGT CAC AAT GAG CAT
GCA CCT GCT TCC CAA GGC 542 Arg Pro Ser Asp Asn Ser His Asn Glu His
Ala Pro Ala Ser Gln Gly 125 130 135 CTG AAA GCT GAG CAT CTG TAT ATT
CTC ATC GGG GTC TCA GTG GTC TTC 590 Leu Lys Ala Glu His Leu Tyr Ile
Leu Ile Gly Val Ser Val Val Phe 140 145 150 CTC TTC TGT CTC CTC CTC
CTG GTC CTC TTC TGC CTC CAT CGC CAG AAT 638 Leu Phe Cys Leu Leu Leu
Leu Val Leu Phe Cys Leu His Arg Gln Asn 155 160 165 CAG ATA AAG CAG
GGG CCC CCC AGA AGC AAG GAC GAG GAG CAG AAG CCA 686 Gln Ile Lys Gln
Gly Pro Pro Arg Ser Lys Asp Glu Glu Gln Lys Pro 170 175 180 185 CAG
CAG AGG CCT GAC CTG GCT GTT GAT GTT CTA GAG AGG ACA GCA GAC 734 Gln
Gln Arg Pro Asp Leu Ala Val Asp Val Leu Glu Arg Thr Ala Asp 190 195
200 AAG GCC ACA GTC AAT GGA CTT CCT GAG AAG GAC AGA GAG ACG GAC ACC
782 Lys Ala Thr Val Asn Gly Leu Pro Glu Lys Asp Arg Glu Thr Asp Thr
205 210 215 TCG GCC CTG GCT GCA GGG AGT TCC CAG GAG GTG ACG TAT GCT
CAG CTG 830 Ser Ala Leu Ala Ala Gly Ser Ser Gln Glu Val Thr Tyr Ala
Gln Leu 220 225 230 GAC CAC TGG GCC CTC ACA CAG AGG ACA GCC CGG GCT
GTG TCC CCA CAG 878 Asp His Trp Ala Leu Thr Gln Arg Thr Ala Arg Ala
Val Ser Pro Gln 235 240 245 TCC ACA AAG CCC ATG GCC GAG TCC ATC ACG
TAT GCA GCC GTT GCC AGA 926 Ser Thr Lys Pro Met Ala Glu Ser Ile Thr
Tyr Ala Ala Val Ala Arg 250 255 260 265 CAC TGACCCCATA CCCACCTGGC
CTCTGCACCT GAGGGTAGAA AGTCACTCTA 979 His GGAAAAGCCT GAAGCAGCCA
TTTGGAAGGC TTCCTGTTGG ATTCCTCTTC ATCTAGAAAG 1039 CCAGCCAGGC
AGCTGTCCTG GAGACAAGAG CTGGAGACTG GAGGTTTCTA ACCAGCATCC 1099
AGAAGGTTCG TTAGCCAGGT GGTCCCTTCT ACAATCGAGC AGCTCCTTGG ACAGACTGTT
1159 TCTCAGTTAT TTCCAGAGAC CCAGCTACAG TTCCCTGGCT GTTTCTAGAG
ACCCAGCTTT 1219 ATTCACCTGA CTGTTTCCAG AGACCCAGCT AAAGTCACCT
GCCTGTTCTA AAGGCCCAGC 1279 TACAGCCAAT CAGCCGATTT CCTGAGCAGT
GATGCCACCT CCAAGCTTGT CCTAGGTGTC 1339 TGCTGTGAAC CTCCAGTGAC
CCCAGAGACT TTGCTGTAAT TATCTGCCCT GCTGACCCTA 1399 AAGACCTTCC
TAGAAGTCAA GAGCTAGCCT TGAGACTGTG CTATACACAC ACAGCTGAGA 1459
GCCAAGCCCA GTTCTCTGGG TTGTGCTTTA CTCCACGCAT CAATAAATAA TTTTGAAGGC
1519 CTCACATCTG GCAGCCCCAG GCCTGGTCCT GGGTGCATAG GTCTCTCGGA
CCCACTCTCT 1579 GCCTTCACAG TTGTTCAAAG CTGAGTGAGG GAAACAGGAC
TTACGAAAAC GTGTCAGCGT 1639 TTTCTTTTTA AAATTTAATT GATCAGGATT
GTACGTAAAA AAAAAAAAAA AAAAAAAAAA 1699 AAAAAAAAAA AAAAAAAAAA
AAAAAAAGG 1728 Nucleic acid and putative amino acid sequence of
soluble DLAIR-2. The signal sequence runs from about Met(-21) to
Thr(-1) (SEQ ID NO: 9 and 10). CCACGCGTCC GGGGACCGGG GCC ATG TCT
CCA CAC CTC ACT GCT CTC CTG 50 Met Ser Pro His Leu Thr Ala Leu Leu
-21 -20 -15 GGC CTA GTG CTC TGC CTG GCC CAG ACC ATC CAC ACG CAG GAG
GGG GCC 98 Gly Leu Val Leu Cys Leu Ala Gln Thr Ile His Thr Gln Glu
Gly Ala -10 -5 1 CTT CCC AGA CCC TCC ATC TCG GCT GAG CCA GGC ACT
GTG ATC TCC CCG 146 Leu Pro Arg Pro Ser Ile Ser Ala Glu Pro Gly Thr
Val Ile Ser Pro 5 10 15 20 GGG AGC CAT GTG ACT TTC ATG TGC CGG GGC
CCG GTT GGG GTT CAA ACA 194 Gly Ser His Val Thr Phe Met Cys Arg Gly
Pro Val Gly Val Gln Thr 25 30 35 TTC CGC CTG GAG AGG GAG GAT AGA
GCC AAG TAC AAA GAT AGT TAT AAT 242 Phe Arg Leu Glu Arg Glu Asp Arg
Ala Lys Tyr Lys Asp Ser Tyr Asn 40 45 50 GTG TTT CGA CTT GGT CCA
TCT GAG TCA GAG GCC AGA TTC CAC ATT GAC 290 Val Phe Arg Leu Gly Pro
Ser Glu Ser Glu Ala Arg Phe His Ile Asp 55 60 65 TCA GTA AGT GAA
GGA AAT GCC GGG CTT TAT CGC TGC CTC TAT TAT AAG 338 Ser Val Ser Glu
Gly Asn Ala Gly Leu Tyr Arg Cys Leu Tyr Tyr Lys 70 75 80 CCC CCT
GGA TGG TCT GAG CAC AGT GAC TTC CTG GAG CTG CTG GTG AAA 386 Pro Pro
Gly Trp Ser Glu His Ser Asp Phe Leu Glu Leu Leu Val Lys 85 90 95
100 GGG ACT GTG CCA GGC ACT GAA GCC TCC GGA TTT GAT GCA CCA 428 Gly
Thr Val Pro Gly Thr Glu Ala Ser Gly Phe Asp Ala Pro 105 110
TGAATGAGGA GAAATGGCCT CCCGTCTTGT GAACTTCAAT GGGGAGAAAT AATTAGAATG
488 AGCAATAGAA ATGCACAGAT GCCTATACAT ACATATACAA ATAAAAAGAT
ACGATTCGCA 548 AAAAAAAAAA AAAAAAGGGC 568
[0055]
3TABLE 3 Human KTE03 sequences, e.g., alternative splicing,
encoding related proteins with homology to several NK KIR surface
molecules, and to the Fc receptors gamma and alpha. YYB01 coding
sequence appears to run from about 81 to 1397. The message appears
to be IL- 10 upregulated. See SEQ ID NO: 11 and 12. Because of
significant identity of sequence which ends at specific locations,
it appears that there may be splice junctions around nucleotide 36,
1264, and 1587. The YYB04 sequence provided below indicates that
certain insertions of sequence lead to a frameshift and alternative
carboxy terminal sequence. Moreover, certain peculiar differences
in sequence suggest either sequencing errors, or a mechanism of
variability generated by a mechanism perhaps analogous to
hypervariable region combinations. GTCGACCCAC GCGTCCGCCT CTGTCCTGCC
AGCACCGAGG GCTCATCCAT CCACAGAGCA 60 GTGCAGTGGG AGGAGACGCC ATG ACC
CCC ATC CTC ACG GTC CTG ATC TGT 110 Met Thr Pro Ile Leu Thr Val Leu
Ile Cys 1 5 10 CTC GGG CTG AGC CTG GAC CCC AGG ACC CAC GTG CAG GCA
GGG CCC CTC 158 Leu Gly Leu Ser Leu Asp Pro Arg Thr His Val Gln Ala
Gly Pro Leu 15 20 25 CCC AAG CCC ACC CTC TGG GCT GAG CCA GGC TCT
GTG ATC ACC CAA GGG 206 Pro Lys Pro Thr Leu Trp Ala Glu Pro Gly Ser
Val Ile Thr Gln Gly 30 35 40 AGT CCT GTG ACC CTC AGG TGT CAG GGG
AGC CTG GAG ACG CAG GAG TAC 254 Ser Pro Val Thr Leu Arg Cys Gln Gly
Ser Leu Glu Thr Gln Glu Tyr 45 50 55 CAT CTA TAT AGA GAA AAG AAA
ACA GCA CTC TGG ATT ACA CGG ATC CCA 302 His Leu Tyr Arg Glu Lys Lys
Thr Ala Leu Trp Ile Thr Arg Ile Pro 60 65 70 CAG GAG CTT GTG AAG
AAG GGC CAG TTC CCC ATC CTA TCC ATC ACC TGG 350 Gln Glu Leu Val Lys
Lys Gly Gln Phe Pro Ile Leu Ser Ile Thr Trp 75 80 85 90 GAA CAT GCA
GGG CGG TAT TGC TGT ATC TAT GGC AGC CAC ACT GCA GGC 398 Glu His Ala
Gly Arg Tyr Cys Cys Ile Tyr Gly Ser His Thr Ala Gly 95 100 105 CTC
TCA GAG AGC AGT GAC CCC CTG GAG CTG GTG GTG ACA GGA GCC TAC 446 Leu
Ser Glu Ser Ser Asp Pro Leu Glu Leu Val Val Thr Gly Ala Tyr 110 115
120 AGC AAA CCC ACC CTC TCA GCT CTG CCC AGC CCT GTG GTG ACC TCA GGA
494 Ser Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Val Val Thr Ser Gly
125 130 135 GGG AAT GTG ACC ATC CAG TGT GAC TCA CAG GTG GCA TTT GAT
GGC TTC 542 Gly Asn Val Thr Ile Gln Cys Asp Ser Gln Val Ala Phe Asp
Gly Phe 140 145 150 ATT CTG TGT AAG GAA GGA GAA GAT GAA CAC CCA CAA
TGC CTG AAC TCC 590 Ile Leu Cys Lys Glu Gly Glu Asp Glu His Pro Gln
Cys Leu Asn Ser 155 160 165 170 CAT TCC CAT GCC CGT GGG TCA TCC CGG
GCC ATC TTC TCC GTG GGC CCC 638 His Ser His Ala Arg Gly Ser Ser Arg
Ala Ile Phe Ser Val Gly Pro 175 180 185 GTG AGC CCA AGT CGC AGG TGG
TCG TAC AGG TGC TAT GGT TAT GAC TCG 686 Val Ser Pro Ser Arg Arg Trp
Ser Tyr Arg Cys Tyr Gly Tyr Asp Ser 190 195 200 CGC GCT CCC TAT GTG
TGG TCT CTA CCC AGT GAT CTC CTG GGG CTC CTG 734 Arg Ala Pro Tyr Val
Trp Ser Leu Pro Ser Asp Leu Leu Gly Leu Leu 205 210 215 GTC CCA GGT
GTT TCT AAG AAG CCA TCA CTC TCA GTG CAG CCG GGT CCT 782 Val Pro Gly
Val Ser Lys Lys Pro Ser Leu Ser Val Gln Pro Gly Pro 220 225 230 GTC
GTG GCC CCT GGG GAG AAG CTG ACC TTC CAG TGT GGC TCT GAT GCC 830 Val
Val Ala Pro Gly Glu Lys Leu Thr Phe Gln Cys Gly Ser Asp Ala 235 240
245 250 GGC TAC GAC AGA TTT GTT CTG TAC AAG GAG TGG GGA CGT GAC TTC
CTC 878 Gly Tyr Asp Arg Phe Val Leu Tyr Lys Glu Trp Gly Arg Asp Phe
Leu 255 260 265 CAG CGC CCT GGC CGG CAC CCC CAG GCT GGG CTC TCC CAG
GCC AAC TTC 926 Gln Arg Pro Gly Arg Gln Pro Gln Ala Gly Leu Ser Gln
Ala Asn Phe 270 275 280 ACC CTG GGC CCT GTG AGC CGC TCC TAC GGG GGC
CAG TAC ACA TGC TCC 974 Thr Leu Gly Pro Val Ser Arg Ser Tyr Gly Gly
Gln Tyr Thr Cys Ser 285 290 295 GGT GCA TAC AAC CTC TCC TCC GAG TGG
TCG GCC CCC AGC GAC CCC CTG 1022 Gly Ala Tyr Asn Leu Ser Ser Glu
Trp Ser Ala Pro Ser Asp Pro Leu 300 305 310 GAC ATC CTG ATC ACA GGA
CAG ATC CGT GCC AGA CCC TTC CTC TCC GTG 1070 Asp Ile Leu Ile Thr
Gly Gln Ile Arg Ala Arg Pro Phe Leu Ser Val 315 320 325 330 CGG CCG
GGC CCC ACA GTG GCC TCA GGA GAG AAC GTG ACC CTG CTG TGT 1118 Arg
Pro Gly Pro Thr Val Ala Ser Gly Glu Asn Val Thr Leu Leu Cys 335 340
345 CAG TCA CAG GGA GGG ATG CAC ACT TTC CTT TTG ACC AAG GAG GGG GCA
1166 Gln Ser Gln Gly Gly Met His Thr Phe Leu Leu Thr Lys Glu Gly
Ala 350 355 360 GCT GAT TCC CCG CTG CGT CTA AAA TCA AAG CGC CAA TCT
CAT AAG TAC 1214 Ala Asp Ser Pro Leu Arg Leu Lys Ser Lys Arg Gln
Ser His Lys Tyr 365 370 375 CAG GCT GAA TTC CCC ATG AGT CCT GTG ACC
TCG GCC CAC GCG GGG ACC 1262 Gln Ala Glu Phe Pro Met Ser Pro Val
Thr Ser Ala His Ala Gly Thr 380 385 390 TAC AGG TGC TAC GGC TCA CTC
AGC TGG AAC CCC TAC CTG CTG ACT CAC 1310 Tyr Arg Cys Tyr Gly Ser
Leu Ser Ser Asn Pro Tyr Leu Leu Thr His 395 400 405 410 CCC AGT GAC
CCC CTG GAG CTC GTG GTC TCA GGA GCA GCT GAG ACC CTC 1358 Pro Ser
Asp Pro Leu Glu Leu Val Val Ser Gly Ala Ala Glu Thr Leu 415 420 425
AGC CCA CCA CAA AAC AAG TCC GAC TCC AAG GCT GGT GAG TGAGGAGATG 1407
Ser Pro Pro Gln Asn Lys Ser Asp Ser Lys Ala Gly Glu 430 435
CTTGCCGTGA TGACGGTGGG CACAGAGGGT CAGGTCCTGT CAAGAGGAGC TGGGTGTCCT
1467 GGGTGGACAT TTGAAGAATT ATATTCATTC CAACTTGAAG AATTATTCAA
CACCTTTAAC 1527 AATGTATATG TGAAGTACTT TATTCTTTCA TATTTTAAAA
ATAAAAGATA ATTATCCATG 1587 AAAAAAAAAA AAAAAAAAAA AAAGGGCGGC CGC
1620 YYB04: Related to YYB01, apparently through alternative
splicing from the same or a very highly related gene. The coding
region runs from about 191 to 1493, but the initiation methionine
may actually be at the numbered Met at 18. See SEQ ID NO: 13 and
14. Another transcript was isolated which contains evidence for
existence of an insert of sequence TGCTACGGCT CACTCAACTC CGACCCCTAC
CTGCTGTCTC ACCCCAGTGA GCCCCTGGAG CTCGTGGTCT CAGG between residues
1426 and 1427, which changes the downstream reading frame of the
subsequent sequence, to encode, from residue 413, CYG SLNSD PYLLS
HPSEP LELVV SGPSM GSSPP PTGPI STPAG PEDQP LTPTG SDPQS GLGRH LGVVI
GILVA VVLLL LLLLL LFLIL RHRRQ GKHWT STQRK ADFQH PAGAV GPEPT DRGLQ
WRSSP AADAQ EENLY AAVKD TQPED GVEMD TRAAA SEAPQ DVTYA QLHSL TLRRK
ATEPP PSQER EPPAE PSIYA TLAIH. (SEQ ID NO: 15 and 16.) This
alternative sequence contains a transmembrane segment from about
478 to 500. GTCGACCCAC GCGTCCGGTC AACTTTTCTT CCCCTACTTC CCTGCATTTC
TCCTCTGTGC 60 TCACTGCCAC ACGCAGCTCA ACCTGGACGG CACAGCCAGA
TGCGAGATGC GTCTCTGCTG 120 ATCTGAGTCT GCCTGCAGCA TGGACCTGGG
TCTTCCCTGA AGCATCTCCA GGGCTGGAGG 180 GACGACTGCC ATG CAC CGA GGG CTC
ATC CAT CCG CAG AGC AGG GCA GTG 229 Met His Arg Gly Leu Ile His Pro
Gln Ser Arg Ala Val 1 5 10 GGA GGA GAC GCC ATG ACC CCC ATC GTC ACA
GTC CTG ATC TGT CTC GGG 277 Gly Gly Asp Ala Met Thr Pro Ile Val Thr
Val Leu Ile Cys Leu Gly 15 20 25 CTG AGT CTG GGC CCC AGG ACC CAC
GTG CAG ACA GGG ACC ATC CCC AAG 325 Leu Ser Leu Gly Pro Arg Thr His
Val Gln Thr Gly Thr Ile Pro Lys 30 35 40 45 CCC ACC CTG TGG GCT GAG
CCA GAC TCT GTG ATC ACC CAG GGG AGT CCC 373 Pro Thr Leu Trp Ala Glu
Pro Asp Ser Val Ile Thr Gln Gly Ser Pro 50 55 60 GTC ACC CTC AGT
TGT CAG GGG AGC CTT GAA GCC CAG GAG TAC CGT CTA 421 Val Thr Leu Ser
Cys Gln Gly Ser Leu Glu Ala Gln Glu Tyr Arg Leu 65 70 75 TAT AGG
GAG AAA AAA TCA GCA TCT TGG ATT ACA CGG ATA CGA CCA GAG 469 Tyr Arg
Glu Lys Lys Ser Ala Ser Trp Ile Thr Arg Ile Arg Pro Glu 80 85 90
CTT GTG AAG AAC GGC CAG TTC CAC ATC CCA TCC ATC ACC TGG GAA CAC 517
Leu Val Lys Asn Gly Gln Phe His Ile Pro Ser Ile Thr Trp Glu His 95
100 105 ACA GGG CGA TAT GGC TGT CAG TAT TAC AGC CGC GCT CGG TGG TCT
GAG 565 Thr Gly Arg Tyr Gly Cys Gln Tyr Tyr Ser Arg Ala Arg Trp Ser
Glu 110 115 120 125 CTC AGT GAC CCC CTG GTG CTG GTG ATG ACA GGA GCC
TAC CCA AAA CCC 613 Leu Ser Asp Pro Leu Val Leu Val Met Thr Gly Ala
Tyr Pro Lys Pro 130 135 140 ACC CTC TCA GCC CAG CCC AGC CCT GTG GTG
ACC TCA GGA GGA AGG GTG 661 Thr Leu Ser Ala Gln Pro Ser Pro Val Val
Thr Ser Gly Gly Arg Val 145 150 155 ACC CTC CAG TGT GAG TCA CAG GTG
GCA TTT GGC GGC TTC ATT CTG TGT 709 Thr Leu Gln Cys Glu Ser Gln Val
Ala Phe Gly Gly Phe Ile Leu Cys 160 165 170 AAG GAA GGA GAA GAT GAA
CAC CCA CAA TGC CTG AAC TCC CAG CCC CAT 757 Lys Glu Gly Glu Asp Glu
His Pro Gln Cys Leu Asn Ser Gln Pro His 175 180 185 GCC CGT GGG TCG
TCC CGC GCC ATC TTC TCC GTG GGC CCC GTG AGC CCG 805 Ala Arg Gly Ser
Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro 190 195 200 205 AAT
CGC AGG TGG TCG CAC AGG TGC TAT GGT TAT GAC TTG AAC TCT CCC 853 Asn
Arg Arg Trp Ser His Arg Cys Tyr Gly Tyr Asp Leu Asn Ser Pro 210 215
220 TAT GTG TGG TCT TCA CCC AGT GAT CTC CTG GAG CTC CTG GTC CCA GGT
901 Tyr Val Trp Ser Ser Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly
225 230 235 GTT TCT AAG AAG CCA TCA CTC TCA GTG CAG CCG GGT CCT GTC
GTG GCC 949 Val Ser Lys Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Val
Val Ala 240 245 250 CCT GGG GAA AGC CTG ACC CTC CAG TGT GTC TCT GAT
GTC GGC TAT GAC 997 Pro Gly Glu Ser Leu Thr Leu Gln Cys Val Ser Asp
Val Gly Tyr Asp 255 260 265 AGA TTT GTT CTG TAC AAG GAG GGG GAA CGT
GAC CTT CGC CAG CTC CCT 1045 Arg Phe Val Leu Tyr Lys Glu Gly Glu
Arg Asp Leu Arg Gln Leu Pro 270 275 280 285 GGC CGG CAG CCC CAG GCT
GGG CTC TCC CAG GCC AAC TTC ACC CTG GGC 1093 Gly Arg Gln Pro Gln
Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly 290 295 300 CCT GTG AGC
CGC TCC TAC GGG GGC CAG TAC AGA TGC TAC GGT GCA TAC 1141 Pro Val
Ser Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala Tyr 305 310 315
AAC CTC TCC TCC GAG TGG TCG GCC CCC AGC GAC CCC CTG GAC ATC CTG
1189 Asn Leu Ser Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile
Leu 320 325 330 ATC ACA GGA CAG ATC CAT GGC ACA CCC TTC ATC TCA GTG
CAG CCA GGC 1237 Ile Thr Gly Gln Ile His Gly Thr Pro Phe Ile Ser
Val Gln Pro Gly 335 340 345 CCC ACA GTG GCC TCA GGA GAG AAC GTG ACC
CTG CTG TGT CAG TCA TGG 1285 Pro Thr Val Ala Ser Gly Glu Asn Val
Thr Leu Leu Gys Gln Ser Trp 350 355 360 365 CGG CAG TTC CAC ACT TTC
CTT CTG ACC AAG GCG GGA GCA GCT GAT GCC 1333 Arg Gln Phe His Thr
Phe Leu Leu Thr Lys Ala Gly Ala Ala Asp Ala 370 375 380 CCA CTC CGT
CTA AGA TCA ATA CAC GAA TAT CCT AAG TAC CAG GCT GAA 1381 Pro Leu
Arg Leu Arg Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala Glu 385 390 395
TTC CCC ATG AGT CCT GTG ACC TCA GCC CAC GCG GGG ACC TAC AGG ACC
1429 Phe Pro Met Ser Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg
Thr 400 405 410 CTC CAT GGG TTC CAG CCC CCC ACC CAC CGG TCC CAT CTC
CAC ACC TGC 1477 Leu His Gly Phe Gln Pro Pro Thr His Arg Ser His
Leu His Thr Cys 415 420 425 AGG CCC TGAGGACCAG CCCCTCACCC
CCACTGGGTC GGATCCCCAA AGTGGTCTGG 1533 Arg Pro 430 GAAGGCACCT
GGGGGTTGTG ATCGGCATCT TGGTGGCCGT CGTCCTACTG CTCCTCCTCC 1593
TCCTCCTCCT CTTCCTCATC CTCCGACATC GACGTCAGGG CAAACACTGG ACATCGACCC
1653 AGAGAAAGGC TGATTTCCAA CATCCTGCAG GGGCTGTGGG GCCAGAGCCC
ACAGACAGAG 1713 GCCTGCAGTG GAGGTCCAGC CCAGCTGCCG ACGCCCAGGA
AGAAAACCTC TATGCTGCCG 1773 TGAAGGACAC ACAGCCTGAA GATGGGGTGG
AGATGGACAC TCGGGCTGCT GGATCTGAAG 1833 CCCCCCAGGA TGTGACCTAC
GCCCAGCTGC ACAGCTTGAC CCTCAGACGG AAGGCAACTG 1893 AGCCTCCTCC
ATCCCAGGAA AGGGAACCTC CAGCTGAGCC CAGCATTTAC GCCACCCTGG 1953
CCATCCACTA GCCCGGAGGG TACGCAGACT CCACACTCAG TAGAAGGAGA CTCAGGACTG
2013 CTGAAGGCAC GGGAGCTGCC CCCAGTGGAC ACCAATGAAC CCCAGTCAGC
CTGGACCCCT 2073 AACAAAGACC ATGAGGAGAT GCTGGGAACT TTGGGACTCA
CTTGATTCTG CAGTGGAAAT 2133 AACTAATATC CCTACATTTT TTAATTAAAG
CAACAGACTT CTCAATAATC AATGAGTTAA 2193 CCGA 2197 A KTE03 embodiment
designated KLM63 (SEQ ID NO: 17 and 18): AAAGAAGTCA ACTTTTCTTC
CCCTACTTCC CTGCATTTCT CCTCTGTGCT CACTGCCACA 60 CGCAGCTCAA
CCTGGACGGC ACAGCCAGAT GCGAGATGCG TCTCTGCTGA TCTGAGTCTG 120
CCTGCAGCAT GGACCTGGGT CTTCCCTGAA GCATCTCCAG GGCTGGAGGG ACGACTGCC
179 ATG CAC CGA GGG CTC ATC CAT CCG CAG AGC AGG GCA GTG GGA GGA GAC
227 Met His Arg Gly Leu Ile His Pro Gln Ser Arg Ala Val Gly Gly Asp
1 5 10 15 GCC ATG ACC CCC ATC GTC ACA GTC CTG ATC TGT CTC GGG CTG
AGT CTG 275 Ala Met Thr Pro Ile Val Thr Val Leu Ile Cys Leu Gly Leu
Ser Leu 20 25 30 GGC CCC AGG ACC CAC GTG CAG ACA GGG ACC ATC CCC
AAG CCC ACC CTG 323 Gly Pro Arg Thr His Val Gln Thr Gly Thr Ile Pro
Lys Pro Thr Leu 35 40 45 TGG GCT GAG CCA GAC TCT GTG ATC ACC CAG
GGG AGT CCC GTC ACC CTC 371 Trp Ala Glu Pro Asp Ser Val Ile Thr Gln
Gly Ser Pro Val Thr Leu 50 55 60 AGT TGT CAG GGG AGC CTT GAA GCC
CAG GAG TAC CGT CTA TAT AGG GAG 419 Ser Cys Gln Gly Ser Leu Glu Ala
Gln Glu Tyr Arg Leu Tyr Arg Glu 65 70 75 80 AAA AAA TCA GCA TCT TGG
ATT ACA CGG ATA CGA CCA GAG CTT GTG AAG 467 Lys Lys Ser Ala Ser Trp
Ile Thr Arg Ile Arg Pro Glu Leu Val Lys 85 90 95 AAC GGC CAG TTC
CAC ATC CCA TCC ATC ACC TGG GAA CAC ACA GGG CGA 515 Asn Gly Gln Phe
His Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg 100 105 110 TAT GGC
TGT CAG TAT TAC AGC CGC GCT CGG TGG TCT GAG CTC AGT GAC 563 Tyr Gly
Cys Gln Tyr Tyr Ser Arg Ala Arg Trp Ser Glu Leu Ser Asp 115 120 125
CCC CTG GTG CTG GTG ATG ACA GGA GCC TAC CCA AAA CCC ACC CTC TCA 611
Pro Leu Val Leu Val Met Thr Gly Ala Tyr Pro Lys Pro Thr Leu Ser 130
135 140 GCC CAG CCC AGC CCT GTG GTG ACC TCA GGA GGA AGG GTG ACC CTC
CAG 659 Ala Gln Pro Ser Pro Val Val Thr Ser Gly Gly Arg Val Thr Leu
Gln 145 150 155
160 TGT GAG TCA CAG GTG GCA TTT GGC GGC TTC ATT CTG TGT AAG GAA GGA
707 Cys Glu Ser Gln Val Ala Phe Gly Gly Phe Ile Leu Cys Lys Glu Gly
165 170 175 GAA GAT GAA CAC CCA CAA TGG CTG AAC TCC CAG CCC CAT GCC
CGT GGG 755 Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala
Arg Gly 180 185 190 TCG TCC CGC GCC ATC TTC TCC GTG GGC CCC GTG AGC
CCG AAT CGC AGG 803 Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser
Pro Asn Arg Arg 195 200 205 TGG TCG CAC AGG TGC TAT GGT TAT GAC TTG
AAC TCT CCC TAT GTG TGG 851 Trp Ser His Arg Cys Tyr Gly Tyr Asp Leu
Asn Ser Pro Tyr Val Trp 210 215 220 TCT TCA CCC AGT GAT CTC CTG GAG
CTC CTG GTC CCA GGT GTT TCT AAG 899 Ser Ser Pro Ser Asp Leu Leu Glu
Leu Leu Val Pro Gly Val Ser Lys 225 230 235 240 AAG CCA TCA CTC TCA
GTG CAG CCG GGT CCT GTC GTG GCC CCT GGG GAA 947 Lys Pro Ser Leu Ser
Val Gln Pro Gly Pro Val Val Ala Pro Gly Glu 245 250 255 AGC CTG ACC
CTC CAG TGT GTC TCT GAT GTC GGC TAT GAC AGA TTT GTT 995 Ser Leu Thr
Leu Gln Cys Val Ser Asp Val Gly Tyr Asp Arg Phe Val 260 265 270 CTG
TAC AAG GAG GGG GAA CGT GAC CTT CGC CAG CTC CCT GGC CGG CAG 1043
Leu Tyr Lys Glu Gly Glu Arg Asp Leu Arg Gln Leu Pro Gly Arg Gln 275
280 285 CCC CAG GCT GGG CTC TCC CAG GCC AAC TTC ACC CTG GGC CCT GTG
AGC 1091 Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro
Val Ser 290 295 300 CGC TCC TAG GGG GGC CAG TAC AGA TGC TAC GGT GCA
TAC AAC CTC TCC 1139 Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly
Ala Tyr Asn Leu Ser 305 310 315 320 TCC GAG TGG TCG GCC CCC AGC GAC
CCC CTG GAC ATC CTG ATC ACA GGA 1187 Ser Glu Trp Ser Ala Pro Ser
Asp Pro Leu Asp Ile Leu Ile Thr Gly 325 330 335 CAG ATC CAT GGC ACA
CCC TTC ATC TCA GTG CAG CCA GGC CCC ACA GTG 1235 Gln Ile His Gly
Thr Pro Phe Ile Ser Val Gln Pro Gly Pro Thr Val 340 345 350 GCC TCA
GGA GAG AAC GTG ACC CTG CTG TGT CAG TCA TGG CGG CAG TTC 1283 Ala
Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp Arg Gln Phe 355 360
365 CAC ACT TTC CTT CTG ACC AAG GCG GGA GCA GCT GAT GCC CCA CTC CGT
1331 His Thr Phe Leu Leu Thr Lys Ala Gly Ala Ala Asp Ala Pro Leu
Arg 370 375 380 CTA AGA TCA ATA CAC GAA TAT CCT AAG TAC CAG GCT GAA
TTC CCC ATG 1379 Leu Arg Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala
Glu Phe Pro Met 385 390 395 400 AGT CCC GTG ACC TCA GCC CAC GCG GGG
ACC TAC AGG TGC TAC GGC TCA 1427 Ser Pro Val Thr Ser Ala His Ala
Gly Thr Tyr Arg Cys Tyr Gly Ser 405 410 415 CTC AAC TCC GAC CCC TAC
CTG CTG TCT CAC CCC AGT GAG CCC CTG GAG 1475 Leu Asn Ser Asp Pro
Tyr Leu Leu Ser His Pro Ser Glu Pro Leu Glu 420 425 430 CTC GTG GTC
TCA GGA CCC TCC ATG GGT TCC AGC CCC CCA CCC ACC GGT 1523 Leu Val
Val Ser Gly Pro Ser Met Gly Ser Ser Pro Pro Pro Thr Gly 435 440 445
CCC ATC TCC ACA CCT GCA GGC CCT GAG GAC CAG CCC CTC ACC CCC ACT
1571 Pro Ile Ser Thr Pro Ala Gly Pro Glu Asp Gln Pro Leu Thr Pro
Thr 450 455 460 GGG TCG GAT CCC CAA AGT GGT CTG GGA AGG CAC CTG GGG
GTT GTG ATC 1619 Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu
Gly Val Val Ile 465 470 475 480 GGC ATC TTG GTG GCC GTC GTC CTA CTG
CTC CTC CTC CTC CTC CTC CTC 1667 Gly Ile Leu Val Ala Val Val Leu
Leu Leu Leu Leu Leu Leu Leu Leu 485 490 495 TTC CTC ATC CTC CGA CAT
CGA CGT CAG GGC AAA CAC TGG ACA TCG ACC 1715 Phe Leu Ile Leu Arg
His Arg Arg Gln Gly Lys His Trp Thr Ser Thr 500 505 510 CAG AGA AAG
GCT GAT TTC CAA CAT CCT GCA CCC GCT GTG GGG CCA GAG 1763 Gln Arg
Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu 515 520 525
CCC ACA GAC AGA GGC CTG CAG TGG AGG TCC AGC CCA GCT GCC GAC GCC
1811 Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp
Ala 530 535 540 CAG GAA GAA AAC CTC TAT GCT GCC GTG AAG GAC ACA CAG
CCT GAA GAT 1859 Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys Asp Thr
Gln Pro Glu Asp 545 550 555 560 GGG GTG GAG ATG GAC ACT CGG GCT GCT
GCA TCT GAA GCC CCC CAG GAT 1907 Gly Val Glu Met Asp Thr Arg Ala
Ala Ala Ser Glu Ala Pro Gln Asp 565 570 575 GTG ACC TAC GCC CAG CTG
CAC AGC TTG ACC CTC AGA CGG AAG GCA ACT 1955 Val Thr Tyr Ala Gln
Leu His Ser Leu Thr Leu Arg Arg Lys Ala Thr 580 585 590 GAG CCT CCT
CCA TCC CAG GAA AGG GAA CCT CCA GCT GAG CCC AGC ATC 2003 Glu Pro
Pro Pro Ser Gln Glu Arg Glu Pro Pro Ala Glu Pro Ser Ile 595 600 605
TAC GCC ACC CTG GCC ATC CAC TAGCCCGGAG GGTACGCAGA CTCCACACTC 2054
Tyr Ala Thr Leu Ala Ile His 610 615 AGTAGAAGGA GACTCAGGAC
TGCTGAAGGC ACGGGAGCTG CCCCCAGTGG ACACCAATGA 2114 ACCCCAGTCA
GCCTGGACCC CTAACAAAGA CCATGAGGAG ATGCTGGGAA CTTTGGGACT 2174
CACTTGATTC TGCAGTCGAA ATAACTAATA TCCCTACATT TTTTAATTAA AGCAACAGAC
2234 TTCTCAATAA TCAATGAGTT AACCGAGAAA ACTAAAATCA GAAGTAAGAA
TGTGCTTTAA 2294 ACTGAATCAC AATATAAATA TTACACATCA CACAATGAAA
TTGAAAAAGT ACAAACCACA 2354 AATGAAAAAA GTAGAAACGA AAAAAAAAAA AAAA
2388 A KTE03 embodiment designated KLM66 (SEQ IDS NO: 19 and 20):
GTCAACTTTT CTTCCCCTAC TTCCCTGCAT TTCTCCTCTG TGCTCACTGC CACACGCAGC
60 TCAACCTGGA CGGCACAGCC AGATGCGAGA TGCGTCTCTG CTGATCTGAG
TCTGCCTGCA 120 GCATGGACCT GGGTCTTCCC TGAAGCATCT CCAGGGCTGG
AGGGACGACT GCC ATG 176 Met 1 CAC CGA GGG CTC ATC CAT CCG CAG AGC
AGG GCA GTG GGA GGA GAC GCC 224 His Arg Gly Leu Ile His Pro Gln Ser
Arg Ala Val Gly Gly Asp Ala 5 10 15 ATG ACC CCC ATC GTC ACA GTC CTG
ATC TGT CTC GGG CTG AGT CTG GGC 272 Met Thr Pro Ile Val Thr Val Leu
Ile Cys Leu Gly Leu Ser Leu Gly 20 25 30 CCC AGG ACC CAC GTG CAG
ACA GGG ACC ATC CCC AAG CCC ACC CTG TGG 320 Pro Arg Thr His Val Gln
Thr Gly Thr Ile Pro Lys Pro Thr Leu Trp 35 40 45 GCT GAG CCA GAC
TCT GTG ATC ACC CAG GGG AGT CCC GTC ACC CTC AGT 368 Ala Glu Pro Asp
Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Ser 50 55 60 65 TGT CAG
GGG AGC CTT GAA GCC CAG GAG TAC CGT CTA TAT AGG GAG AAA 416 Cys Gln
Gly Ser Leu Glu Ala Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 70 75 80
AAA TCA GCA TCT TGG ATT ACA CGG ATA CGA CCA GAG CTT GTG AAG AAC 464
Lys Ser Ala Ser Trp Ile Thr Arg Ile Arg Pro Glu Leu Val Lys Asn 85
90 95 GGC CAG TTC CAC ATC CCA TCC ATC ACC TGG GAA CAC ACA GGG CGA
TAT 512 Gly Gln Phe His Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg
Tyr 100 105 110 GGC TGT CAG TAT TAC AGC CGC GCT CGG TGG TCT GAG CTC
AGT GAC CCC 560 Gly Cys Gln Tyr Tyr Ser Arg Ala Arg Trp Ser Glu Leu
Ser Asp Pro 115 120 125 CTG GTG CTG GTG ATG ACA GGA GCC TAC CCA AAA
CCC ACC CTC TCA GCC 608 Leu Val Leu Val Met Thr Gly Ala Tyr Pro Lys
Pro Thr Leu Ser Ala 130 135 140 145 CAG CCC AGC CCT GTG GTG ACC TCA
GGA GGA AGG GTG ACC CTC CAG TGT 656 Gln Pro Ser Pro Val Val Thr Ser
Gly Gly Arg Val Thr Leu Gln Cys 150 155 160 GAG TCA CAG GTG GCA TTT
GGC GGC TTC ATT CTG TGT AAG GAA GGA GAA 704 Glu Ser Gln Val Ala Phe
Gly Gly Phe Ile Leu Cys Lys Glu Gly Glu 165 170 175 GAT GAA CAC CCA
CAA TGC CTG AAC TCC CAG CCC CAT GCC CGT GGG TCG 752 Asp Glu His Pro
Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly Ser 180 185 190 TCC CGC
GCC ATC TTC TCC GTG GGC CCC GTG AGC CCG AAT CGC AGG TGG 800 Ser Arg
Ala Ile Phe Ser Val Gly Pro Val Ser Pro Asn Arg Arg Trp 195 200 205
TCG CAC AGG TGC TAT GGT TAT GAC TTG AAC TCT CCC TAT GTG TGG TCT 848
Ser His Arg Cys Tyr Gly Tyr Asp Leu Asn Ser Pro Tyr Val Trp Ser 210
215 220 225 TCA CCC AGT GAT CTC CTG GAG CTC CTG GTC CCA GGT GTT TCT
AAG AAG 896 Ser Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly Val Ser
Lys Lys 230 235 240 CCA TCA CTC TCA GTG CAG CCG GGT CCT GTC GTG GCC
CCT GGG GAA AGC 944 Pro Ser Leu Ser Val Gln Pro Gly Pro Val Val Ala
Pro Gly Glu Ser 245 250 255 CTG ACC CTC CAG TGT GTC TCT GAT GTC GGC
TAT GAC AGA TTT GTT CTG 992 Leu Thr Leu Gln Cys Val Ser Asp Val Gly
Tyr Asp Arg Phe Val Leu 260 265 270 TAC AAG GAG GGG GAA CGT GAC CTT
CGC CAG CTC CCT GGC CGG CAG CCC 1040 Tyr Lys Glu Gly Glu Arg Asp
Leu Arg Gln Leu Pro Gly Arg Gln Pro 275 280 285 CAG GCT GGG CTC TCC
CAG GCC AAC TTC ACC CTG GGC CCT GTG AGC CGC 1088 Gln Ala Gly Leu
Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Arg 290 295 300 305 TCC
TAC GGG GGC CAG TAC AGA TGC TAC GGT GCA TAC AAC CTC TCC TCC 1136
Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala Tyr Asn Leu Ser Ser 310
315 320 GAG TGG TCG GCC CCC AGC GAC CCC CTG GAC ATC CTG ATC ACA GGA
CAG 1184 Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr
Gly Gln 325 330 335 ATC CAT GGC ACA CCC TTC ATC TCA GTG CAG CCA GGC
CCC ACA GTG GCC 1232 Ile His Gly Thr Pro Phe Ile Ser Val Gln Pro
Gly Pro Thr Val Ala 340 345 350 TCA GGA GAG AAC GTG ACC CTG CTG TGT
CAG TCA TGG CGG CAG TTC CAC 1280 Ser Gly Glu Asn Val Thr Leu Leu
Cys Gln Ser Trp Arg Gln Phe His 355 360 365 ACT TTC GTT CTG ACC AAG
GCG GGA GCA GCT GAT GCC CCA CTC CGT CTA 1328 Thr Phe Leu Leu Thr
Lys Ala Gly Ala Ala Asp Ala Pro Leu Arg Leu 370 375 380 385 AGA TCA
ATA CAC GAA TAT CCT AAG TAC CAG GCT GAA TTC CCC ATG AGT 1376 Arg
Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala Glu Phe Pro Met Ser 390 395
400 CCT GTG ACC TCA GCC CAC GCG GGG ACC TAC AGG ACC CTC CAT GGG TTC
1424 Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Thr Leu His Gly
Phe 405 410 415 CAG CCC CCC ACC CAC CGG TCC CAT CTC CAC ACC TGC AGG
CCC 1466 Gln Pro Pro Thr His Arg Ser His Leu His Thr Cys Arg Pro
420 425 430 TGAGGACCAG CCCCTCACCC CCACTGGGTC GGATCCCCAA AGTGGTCTGG
GAAGGCACCT 1526 GGGGGTTGTG ATCGGCATCT TGGTGGCCGT CGTCCTACTG
CTCCTCCTCC TCCTCCTCCT 1586 CTTCCTCATC CTCCGACATC GACGTCAGGG
CAAACACTGG ACATCGACCC AGAGAAAGGC 1646 TGATTTCCAA CATCCTGCAG
GGGCTGTGGG GCCAGAGCCC ACAGACAGAG GCCTGCAGTG 1706 GAGGTCCAGC
CCAGCTGCCG ACGCCCAGGA AGAAAACCTC TATGCTGCCG TGAAGGACAC 1766
ACAGCCTGAA GATGGGGTGG AGATGGACAC TCGGGCTGCT GCATCTGAAG CCCCCCAGGA
1826 TGTGACCTAC GCCCAGCTGC ACAGCTTGAC CCTCAGACGG AAGGCAACTG
AGCCTCCTCC 1886 ATCCCAGGAA AGGGAACCTC CAGCTGAGCC CAGCATCTAC
GCCACCCTGG CCATCCACTA 1946 GCCCGGAGGG TACGCAGACT CCACACTCAG
TAGAAGGAGA CTCAGGACTG CTGAAGGCAC 2006 GGGAGCTGCC CCCAGTGGAC
ACCAATGAAC CCCAGTCAGC CTGGACCCCT AACAAAGACC 2066 ATGAGGAGAT
GCTGGGAACT TTGGGACTCA CTTGATTCTG CAGTCGAAAT AACTAATATC 2126
CCTACATTTT TTAATTAAAG CAACAGACTT CTCAATAATC AATGAGTTAA CCGAGAAAAC
2186 TAAAAAAAAA AAAA 2200 A KTE03 embodiment designated KLM67 (SEQ
ID NO: 21 and 22): GCCACACGCA GCTCAGCCTG GGCGGCACAG CCAGATGCGA
GATGCGTCTC TGCTGATCTG 60 AGTCTGCCTG CAGCATGGAC CTGGGTCTTC
CCTGAAGCAT CTCCAGGGCT GGAGGGACGA 120 CTGCCATGCA CCGAGGGCTC
ATCCATCCAC AGAGCAGGGC AGTGGGAGGA GACGGC 176 ATG ACC CCC ATC CTC ACG
GTC CTG ATC TGT CTC GGG CTG AGT CTG GGC 224 Met Thr Pro Ile Leu Thr
Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 CCC CGG ACC CAC
GTG CAG GCA GGG CAC CTC CCC AAG CCC ACC CTC TGG 272 Pro Arg Thr His
Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp 20 25 30 GCT GAA
CCA GGC TCT GTG ATC ACC CAG GGG AGT CCT GTG ACC CTC AGG 320 Ala Glu
Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg 35 40 45
TGT CAG GGG GGC CAG GAG ACC CAG GAG TAC CGT CTA TAT AGA GAA AAG 368
Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50
55 60 AAA ACA GCA CCC TGG ATT ACA CGG ATC CCA CAG GAG CTT GTG AAG
AAG 416 Lys Thr Ala Pro Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys
Lys 65 70 75 80 GGC CAG TTC CCC ATC CCA TCC ATC ACC TGG GAA CAT GCA
GGG CGG TAT 464 Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala
Gly Arg Tyr 85 90 95 CGC TGT TAC TAT GGT AGC GAC ACT GCA GGC CGC
TCA GAG AGC AGT GAC 512 Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg
Ser Glu Ser Ser Asp 100 105 110 CCC CTG GAG CTG GTG GTG ACA GGA GCC
TAC ATC AAA CCC ACC CTC TCA 560 Pro Leu Glu Leu Val Val Thr Gly Ala
Tyr Ile Lys Pro Thr Leu Ser 115 120 125 GCC CAG CCC AGC CCC GTG GTG
AAC TCA GGA GGG AAT GTA ACC CTC CAG 608 Ala Gln Pro Ser Pro Val Val
Asn Ser Gly Gly Asn Val Thr Leu Gln 130 135 140 TGT GAC TCA CAG GTG
GCA TTT GAT GGC TTC ATT CTG TGT AAG GAA GGA 656 Cys Asp Ser Gln Val
Ala Phe Asp Gly Phe Ile Leu Cys Lys Glu Gly 145 150 155 160 GAA GAT
GAA CAC CCA CAA TGC CTG AAC TCC CAG CCC CAT GCC CGT GGG 704 Glu Asp
Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly 165 170 175
TCG TCC CGC GCC ATC TTC TCC GTG GGC CCC GTG AGC CCG AGT CGC AGG 752
Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg 180
185 190 TGG TGG TAC AGG TGC TAT GCT TAT GAC TCG AAC TCT CCC TAT GAG
TGG 800 Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu
Trp 195 200 205 TCT CTA CCC AGT GAT CTC CTG GAG CTC CTG GTC CTA GGT
GTT TCT AAG 848 Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly
Val Ser Lys 210 215 220 AAC CCA TCA CTC TCA GTG CAG CCA GGT CCT ATC
GTG GCC CCT GAG GAG 896 Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile
Val Ala Pro Glu Glu 225 230 235 240 ACC CTG ACT CTG CAG TGT GGC TCT
GAT GCT GGC TAC AAC AGA TTT GTT 944 Thr Leu Thr Leu Gln Cys Gly Ser
Asp Ala Gly Tyr Asn Arg Phe Val 245 250 255 CTG TAT AAG GAC GGG GAA
CGT GAC TTC CTT CAG CTC GCT GGC GCA CAG 992 Leu Tyr Lys Asp Gly Glu
Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln
260 265 270 CCC CAG GCT GGG CTC TCC CAG GCC AAC TTC ACC CTG GGC CCT
GTG AGC 1040 Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly
Pro Val Ser 275 280 285 CGC TCC TAC GGG GGC CAG TAC AGA TGC TAC GGT
GCA CAC AAC CTC TCC 1088 Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr
Gly Ala His Asn Leu Ser 290 295 300 TCC GAG TGG TCG GCC CCC AGC GAC
CCC CTG GAC ATC CTG ATC GCA GGA 1136 Ser Glu Trp Ser Ala Pro Ser
Asp Pro Leu Asp Ile Leu Ile Ala Gly 305 310 315 320 CAG TTC TAT GAC
AGA GTC TCC CTC TCG GTG CAG CCG GGC CCC ACG GTG 1184 Gln Phe Tyr
Asp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val 325 330 335 GCC
TCA GGA GAG AAC GTG ACC CTG CTG TGT CAG TCA CAG GGA TGG ATG 1232
Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met 340
345 350 CAA ACT TTC CTT CTG ACC AAG GAG GGG GCA GCT GAT GAC CCA TGG
GGT 1280 Gln Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro
Trp Arg 355 360 365 CTA AGA TCA ACG TAC CAA TCT CAA AAA TAC CAG GCT
GAA TTC CCC ATG 1328 Leu Arg Ser Thr Tyr Gln Ser Gln Lys Tyr Gln
Ala Glu Phe Pro Met 370 375 380 GGT CCT GTG ACC TCA GCC CAT GCG GGG
ACC TAC AGG TGC TAC GGC TCA 1376 Gly Pro Val Thr Ser Ala His Ala
Gly Thr Tyr Arg Cys Tyr Gly Ser 385 390 395 400 CAG AGC TCC AAA CCC
TAC CTG CTG ACT CAC CCC AGT GAC CCC CTG GAG 1424 Gln Ser Ser Lys
Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu 405 410 415 CTC GTG
GTC TCA GGA CCG TCT GGG GGC CCC AGC TCC CCG ACA ACA GGC 1472 Leu
Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly 420 425
430 CCC ACC TCC ACA TCT GGC CCT GAG GAC CAG CCC CTC ACC CCC ACC GGG
1520 Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr
Gly 435 440 445 TCG GAT CCC CAG AGT GGT CTG GGA AGG CAC CTG GGG GTT
GTG ATC GGC 1568 Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly
Val Val Ile Gly 450 455 460 ATC TTG GTG GCC GTC ATC CTA CTG CTC CTC
CTC CTC CTC CTG CTG TTC 1616 Ile Leu Val Ala Val Ile Leu Leu Leu
Leu Leu Leu Leu Leu Leu Phe 465 470 475 480 CTC ATC CTC CGA CAT CGA
CGT CAG GGC AAA CAC TGG ACA TCG ACC CAG 1664 Leu Ile Leu Arg His
Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln 485 490 495 AGA AAG GCT
GAT TTC CAA CAT CCT GCA GGG GCT GTG GGG CCA GAG CCC 1712 Arg Lys
Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro 500 505 510
ACA GAC AGA CGC CTG CAG TGG AGG TCC AGC CCA GCT GCC GAT GCC CAG
1760 Thr Asp Arg Arg Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala
Gln 515 520 525 GAA GAA AAC CTC TAT GCT GCC GTG AAG CAC ACA CAG CCT
GAG GAT GGG 1808 Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln
Pro Glu Asp Gly 530 535 540 GTG GAG ATG GAC ACT CGG CAG AGC CCA CAC
GAT GAA GAC CCC CAG GCA 1856 Val Glu Met Asp Thr Arg Gln Ser Pro
His Asp Glu Asp Pro Gln Ala 545 550 555 560 GTG ACG TAT GCC GAG GTG
AAA CAC TCC AGA CCT AGG AGA GAA ATG GCT 1904 Val Thr Tyr Ala Glu
Val Lys His Ser Arg Pro Arg Arg Glu Met Ala 565 570 575 TCT CCT CCT
TCC CCA CTG TCT GGG GAA TTC CTG GAC ACA AAG GAC AGA 1952 Ser Pro
Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg 580 585 590
CAG GCG GAA GAG GAC AGG CAG ATG GAC ACT GAG GCT GCT GCA TCT GAA
2000 Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser
Glu 595 600 605 GCC CCC CAG GAT GTG ACC TAC GCC CAG CTG CAC AGC TTG
ACC CTT AGA 2048 Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser
Leu Thr Leu Arg 610 615 620 CGG AAG GCA ACT GAG CCT CCT CCA TCC CAG
GAA GGG CCC TCT CCA GCT 2096 Arg Lys Ala Thr Glu Pro Pro Pro Ser
Gln Glu Gly Pro Ser Pro Ala 625 630 635 640 GTG CCC AGC ATC TAC GCC
ACT CTG GCC ATC CAC TAG CCCAGGGGGG 2142 Val Pro Ser Ile Tyr Ala Thr
Leu Ala Ile His * 645 650 GACGCAGACC CCACACTCCA TGGAGTCTGG
AATGCATGGG AGCTGCCCCC CCAGTGGACA 2202 CCATTGGACC CCACCCAGCC
TGGATCTACC CCAGGAGACT CTGGGAACTT TTAGGGGTCA 2262 CTCAATTCTG
CAGTATAAAT AACTAATGTC TCTACAATTT TGAAATAAAG CAACAGACTT 2322
CTCAATAATC AATGAAGTAG CTGAGAAAAC TAAGTCAGAA AGTGCATTAA ACTGAATCAC
2382 AATGTAAATA TTACACATCA AGCGATGAAA CTGGAAAACT ACAAGCCACG
AATGAATGAA 2442 TTAGGAAAGA AAAAAAGTAG GAAATGAATG ATCTTGGCTT
TCCTATAAGA AATTTAGGGC 2502 AGGGCACGGT GGCTCACGCC TGTAATTCCA
GCACTTTGGG AGGCCGAGGC GGGCAGATCA 2562 CGAGTTCAGG AGATCGAGAC
CATCTTGGCC AACATGGTGA AACCCTGTCT CTCCTAAAAA 2622 TACAAAAATT
AGCTGGATGT GGTGGCAGTG CCTGTAATCC CAGCTATTTG GGAGGCTGAG 2682
GCAGGAGAAT CGCTTGAACC AGGGAGTCAG AGGTTTCAGT GAGCCAAGAT CGCACCACTG
2742 CTCTCCAGCC TGGCGACAGA GGGAGACTCC ATCTCAAATT AAAAAAAA 2790
[0056] The peptide segments can also be used to produce appropriate
oligonucleotides to screen a library to determine the presence of a
similar gene, e.g., an identical or polymorphic variant, or to
identify a monocyte. The genetic code can be used to select
appropriate oligonucleotides useful as probes for screening. In
combination with polymerase chain reaction (PCR) techniques,
synthetic oligonucleotides will be useful in selecting desired
clones from a library.
[0057] Complementary sequences will also be used as probes or
primers. Based upon identification of the likely amino terminus,
other peptides should be particularly useful, e.g., coupled with
anchored vector or poly-A complementary PCR techniques or with
complementary DNA of other peptides.
[0058] Techniques for nucleic acid manipulation of genes encoding
these monocyte proteins, e.g., subcloning nucleic acid sequences
encoding polypeptides into expression vectors, labeling probes, DNA
hybridization, and the like are described generally in Sambrook, et
al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Vol.
1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,
which is incorporated herein by reference and hereinafter referred
to as "Sambrook, et al." See also, Coligan, et al. (1987 and
periodic supplements) Current Protocols in Molecular Biology
Greene/Wiley, New York, N.Y., referred to as "Coligan, et al."
[0059] There are various methods of isolating the DNA sequences
encoding these monocyte proteins. For example, DNA is isolated from
a genomic or cDNA library using labeled oligonucleotide probes
having sequences identical or complementary to the sequences
disclosed herein. Full-length probes may be used, or
oligonucleotide probes may be generated by comparison of the
sequences disclosed with other proteins and selecting specific
primers. Such probes can be used directly in hybridization assays
to isolate DNA encoding monocyte proteins, or probes can be
designed for use in amplification techniques such as PCR, for the
isolation of DNA encoding monocyte proteins.
[0060] To prepare a cDNA library, mRNA is isolated from cells which
express the monocyte protein. cDNA is prepared from the mRNA and
ligated into a recombinant vector. The vector is transfected into a
recombinant host for propagation, screening and cloning. Methods
for making and screening cDNA libraries are well known. See Gubler
and Hoffman (1983) Gene 25:263-269; Sambrook, et al.; or Coligan,
et al.
[0061] For a genomic library, the DNA can be extracted from tissue
and either mechanically sheared or enzymatically digested to yield
fragments of about 12-20 kb. The fragments are then separated by
gradient centrifugation and cloned in bacteriophage lambda vectors.
These vectors and phage are packaged in vitro, as described, e.g.,
in Sambrook, et al. or Coligan, et al. Recombinant phage are
analyzed by plaque hybridization as described in Benton and Davis
(1977) Science 196:180-182. Colony hybridization is carried out as
generally described in, e.g., Grunstein, et al. (1975) Proc. Natl.
Acad. Sci. USA 72:3961-3965.
[0062] DNA encoding a monocyte protein can be identified in either
cDNA or genomic libraries by its ability to hybridize with the
nucleic acid probes described herein, for example in colony or
plaque hybridization experiments. The corresponding DNA regions are
isolated by standard methods familiar to those of skill in the art.
See Sambrook, et al.
[0063] Various methods of amplifying target sequences, such as the
polymerase chain reaction, can also be used to prepare DNA encoding
monocyte proteins. Polymerase chain reaction (PCR) technology is
used to amplify such nucleic acid sequences directly from mRNA,
from cDNA, and from genomic libraries or cDNA libraries. The
isolated sequences encoding monocyte proteins may also be used as
templates for PCR amplification.
[0064] In PCR techniques, oligonucleotide primers complementary to
two 5' regions in the DNA region to be amplified are synthesized.
The polymerase chain reaction is then carried out using the two
primers. See Innis, et al. (eds.) (1990) PCR Protocols: A Guide to
Methods and Applications Academic Press, San Diego, Calif. Primers
can be selected to amplify the entire regions encoding a selected
full-length monocyte protein or to amplify smaller DNA segments as
desired. Once such regions are PCR-amplified, they can be sequenced
and oligonucleotide probes can be prepared from sequence obtained
using standard techniques. These probes can then be used to isolate
DNAs encoding other forms of the monocyte proteins.
[0065] Oligonucleotides for use as probes are chemically
synthesized according to the solid phase phosphoramidite triester
method first described by Beaucage and Carruthers (1983)
Tetrahedron Lett. 22(20):1859-1862, or using an automated
synthesizer, as described in Needham-VanDevanter, et al. (1984)
Nucleic Acids Res. 12:6159-6168. Purification of oligonucleotides
is performed e.g., by native acrylamide gel electrophoresis or by
anion-exchange HPLC as described in Pearson and Regnier (1983) J.
Chrom. 255:137-149. The sequence of the synthetic oligonucleotide
can be verified using the chemical degradation method of Maxam and
Gilbert in Grossman and Moldave (eds.) (1980) Methods in Enzymology
65:499-560 Academic Press, New York.
[0066] An isolated nucleic acid encoding a human protein which is a
type I transmembrane protein comprising an extracellular portion
characterized by Ig-like domains, indicating that this gene encodes
a receptor member of the Ig superfamily. This clone has been
designated FDF03. Its nucleotide sequence and corresponding open
reading frame are provided in SEQ ID NO: 1 and 2, respectively. An
N-terminal hydrophobic sequence, e.g., a putative signal sequence,
corresponds to about amino acid residues -19(met) to -1(leu), and a
internal hydrophobic segment, corresponding to a putative
transmembrane segment runs from around ala177 to leu199. Other
mammalian counterparts should become available, e.g., a partial
rodent gene is described in SEQ ID NO: 3 and 4. Standard techniques
will allow isolation of other counterparts, or to extend partial
sequences.
[0067] A second human monocyte cell clone was isolated, designated
YE01, is related to the receptors for Fc gamma and/or Fc alpha.
This has also been referred to as DNAX Leukocyte Associated
Immunoglobulin-like Receptor (DLAIR). See also Meyaard, et al.
(1997) Immunity 7:283-290, which was published by the inventors
after the priority date of this application, and is incorporated
herein by reference. This protein is referred to herein as an Fc
gamma/alpha receptor and is described in SEQ ID NO: 5 and 6.
Another human isolate is described in SEQ ID NO: 7 and 8. A soluble
form of the receptor is encoded in SEQ ID NO: 9 and 10. While the
gene was initially described as a monocyte derived gene, expression
analysis indicates that it is more specific for expression on
lymphocytes. Thus, in the case of YE01, the descriptor "monocyte
gene" may indicate its original identification in a population
enriched for that cell type, though it may have also contained some
other cell types. Sequence analysis suggests YE01 is a member of
the Ig superfamily of receptors, and is closely related to the CD8
family, which contain a V1J-type fold, particularly the Fc
receptors alpha and/or gamma. Because it contains an ITAM-like
motif, the protein may well be a lymphocyte version of the Killer
Inhibitory Receptors (KIR), which send a negative signal to inhibit
killer cell function. This protein exhibits similar function in
inhibiting lymphocyte effector function, e.g., antigen presentation
or subsequent response initiation.
[0068] In particular, signaling through the molecule recognized by
DX26 mAb (designated DNAX Leukocyte Associated Immunoglobulin-like
Receptor (DLAIR)), delivers a negative signal to NK cell clones
that prevents their killing specific target cells. However, the
molecule is expressed on other lymphocytes, including T cells and
monocytes. Thus, the DX26 antibody probably represents an antibody
which both inhibits NK and cytotoxic T cell killing, and the
monocyte distribution suggests that the molecule may inhibit
monocyte-mediated or lymphocyte-mediated effector functions.
[0069] A third monocyte gene was isolated and designated KTE03, and
is represented by six related embodiments, designated YYB01, YYB04
(forms 1 and 2), (KIR-Like Molecule) KLM63, KLM66, and KLM67. See
SEQ ID NO: 11-22. Note that a possible splice variant, which may
encode a variant protein form, has been detected.
[0070] This invention provides isolated DNA or fragments to encode
a monocyte protein, as described. In addition, this invention
provides isolated or recombinant DNA which encodes a biologically
active protein or polypeptide which is capable of hybridizing under
appropriate conditions, e.g., high stringency, with the DNA
sequences described herein. Said biologically active protein or
polypeptide can be a naturally occurring form, or a recombinant
protein or fragment, and have an amino acid sequence as disclosed
in SEQ ID NO: 2 or 4; 6, 8, or 10; or 12, 14, 16, 18, 20, or 22.
Preferred embodiments will be full length natural isolates, e.g.,
from a primate. In glycosylated form, the proteins should exhibit
larger sizes. Further, this invention encompasses the use of
isolated or recombinant DNA, or fragments thereof, which encode
proteins which are homologous to each respective monocyte protein.
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.
[0071] IV. Making Monocyte Gene Products
[0072] DNAs which encode these monocyte proteins 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.
[0073] These DNAs can be expressed in a wide variety of host cells
for the synthesis of a full-length protein or fragments which can,
e.g., be used to generate polyclonal or monoclonal antibodies; for
binding studies; for construction and expression of modified
molecules; and for structure/function studies. Each of these
monocyte proteins or their 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.
[0074] Expression vectors are typically self-replicating DNA or RNA
constructs containing the desired monocyte 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
from the host cell.
[0075] The vectors of this invention contain DNAs which encode the
various monocyte proteins, or a fragment thereof, typically
encoding, e.g., a biologically active polypeptide, or protein. 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 monocyte protein in a prokaryotic or eukaryotic host,
where the vector is compatible with the host and where the
eukaryotic cDNA coding for the protein 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 protein 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 monocyte gene or its fragments into the
host DNA by recombination, or to integrate a promoter which
controls expression of an endogenous gene.
[0076] 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 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. (eds.) (1988) Vectors: A Survey of Molecular
Cloning Vectors and Their Uses Buttersworth, Boston, Mass.
[0077] 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.
[0078] 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 its derivatives. Vectors that can be used to express
monocyte proteins or 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
10:205-236 Buttersworth, Boston, Mass.
[0079] Lower eukaryotes, e.g., yeasts and Dictyostelium, may be
transformed with monocyte gene sequence containing vectors. For
purposes of this invention, the most common lower eukaryotic host
is the baker's yeast, Saccharomyces cerevisiae. It will be used
generically to 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).
[0080] Higher eukaryotic tissue culture cells are the preferred
host cells for expression of the monocyte protein. In principle,
most any higher eukaryotic tissue culture cell line may be used,
e.g., insect baculovirus expression systems, whether from an
invertebrate or vertebrate source. However, mammalian cells are
preferred to achieve proper processing, both cotranslationally and
posttranslationally. Transformation or transfection and propagation
of such cells is routine. 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 (e.g., if genomic DNA is used), a polyadenylation
site, and a transcription termination site. These vectors also may
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; pMC1eo Poly-A, see Thomas, et al. (1987) Cell
51:503-512; and a baculovirus vector such as pAC 373 or pAC
610.
[0081] In certain instances, the monocyte proteins need not be
glycosylated to elicit biological responses in certain assays.
However, it will often be desirable to express a monocyte
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., in
unglycosylated form, to appropriate glycosylating proteins
introduced into a heterologous expression system. For example, a
monocyte gene may be co-transformed with one or more genes encoding
mammalian or other glycosylating enzymes. It is further understood
that over glycosylation may be detrimental to monocyte protein
biological activity, and that one of skill may perform routine
testing to optimize the degree of glycosylation which confers
optimal biological activity.
[0082] A monocyte 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) Biochem. Biophys. Acta 988:427-454; Tse, et al.
(1985) Science 230:1003-1008; Brunner, et al. (1991) J. Cell Biol.
114:1275-1283; and Coligan, et al. (eds.) (1996 and periodic
supplements) Current Protocols in Protein Science, John Wiley &
Sons, New York, N.Y.
[0083] Now that these monocyte proteins have 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, N.Y.; and Bodanszky (1984) The Principles of Peptide
Synthesis Springer-Verlag, New York, N.Y. See also Merrifield
(1986) Science 232:341-347; and Dawson, et al. (1994) Science
266:776-779. 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.
[0084] 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 monocyte proteins of this invention can be obtained in varying
degrees of purity depending upon the desired use. Purification can
be accomplished by use of known protein purification techniques or
by the use of the antibodies or binding partners herein described,
e.g., in immunoabsorbant affinity chromatography. This
immunoabsorbant affinity chromatography is carried out by first
linking the antibodies to a solid support and 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 proteins as a result of DNA
techniques, see below.
[0085] Multiple cell lines may be screened for one which expresses
said protein at a high level compared with other cells. Various
cell lines, e.g., a mouse thymic stromal cell line TA4, is screened
and selected for its favorable handling properties. Natural
monocyte cell proteins can be isolated from natural sources, or by
expression from a transformed cell using an appropriate expression
vector. Purification of the expressed protein is achieved by
standard procedures, or may be combined with engineered means for
effective purification at high efficiency from cell lysates or
supernatants. FLAG or His.sub.6 segments can be used for such
purification features.
[0086] V. Antibodies
[0087] Antibodies can be raised to these various monocyte proteins,
including individual, polymorphic, allelic, strain, or species
variants, and fragments thereof, both in their naturally occurring
(full-length) forms and in their recombinant forms. Additionally,
antibodies can be raised to monocyte proteins in either their
active forms or in their inactive forms. Anti-idiotypic antibodies
may also be used.
[0088] a. Antibody Production
[0089] A number of immunogens may be used to produce antibodies
specifically reactive with these monocyte proteins. Recombinant
protein is the preferred immunogen for the production of monoclonal
or polyclonal antibodies. Naturally occurring protein may also be
used either in pure or impure form. Synthetic peptides made using
the human monocyte protein sequences described herein may also used
as an immunogen for the production of antibodies to the monocyte
protein. Recombinant protein can be expressed in eukaryotic or
prokaryotic cells as described herein, and purified as described.
The product is then injected into an animal capable of producing
antibodies. Either monoclonal or polyclonal antibodies may be
generated for subsequent use in immunoassays to measure the
protein.
[0090] Methods of producing polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen, preferably a
purified protein, is mixed with an adjuvant and animals are
immunized with the mixture. The animals immune response to the
immunogen preparation is monitored by taking test bleeds and
determining the titer of reactivity to the monocyte protein of
interest. When appropriately high titers of antibody to the
immunogen are obtained, blood is collected from the animal and
antisera are prepared. Further fractionation of the antisera to
enrich for antibodies reactive to the protein can be done if
desired. See, e.g., Harlow and Lane.
[0091] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Briefly, spleen cells from an
animal immunized with a desired antigen are immortalized, commonly
by fusion with a myeloma cell. See, e.g., Kohler and Milstein
(1976) Eur. J. Immunol. 6:511-519, which is incorporated herein by
reference. Alternative methods of immortalization include
transformation with Epstein Barr Virus, oncogenes, or retroviruses,
or other methods known in the art. Colonies arising from single
immortalized cells are screened for production of antibodies of the
desired specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells may be enhanced by
various techniques, including injection into the peritoneal cavity
of a vertebrate host. Alternatively, one may isolate DNA sequences
which encode a monoclonal antibody or a binding fragment thereof by
screening a DNA library from human B cells according to the general
protocol outlined by Huse, et al. (1989) Science 246:1275-1281.
[0092] Antibodies, including binding fragments and single chain
versions, against predetermined fragments of these monocyte
proteins can be raised by immunization of animals with conjugates
of the fragments with carrier proteins as described above.
Monoclonal antibodies are prepared from cells secreting the desired
antibody. These antibodies can be screened for binding to normal or
defective monocyte proteins, or screened for agonistic or
antagonistic activity. 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 100 .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. Standard methods
are available for selection of high affinity and selective antibody
preparations.
[0093] 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, N.Y.; and particularly in Kohler and Milstein
(1975) Nature 256:495-497, which discusses one method of generating
monoclonal antibodies. Summarized briefly, this method involves
injecting an animal with an immunogen to initiate a humoral immune
response. 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 secretes 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.
[0094] Other suitable techniques involve 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; and Queen, et al. (1989) Proc. Nat'l Acad. Sci. USA
86:10029-10033.
[0095] The antibodies of this invention can also be used for
affinity chromatography in isolating each monocyte 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
purified monocyte protein will be released.
[0096] 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.
[0097] Antibodies to monocyte proteins may be used for the analysis
or, or identification of specific cell population components which
express the respective protein. By assaying the expression products
of cells expressing monocyte proteins it is possible to diagnose
disease, e.g., immune-compromised conditions, monocyte depleted
conditions, or overproduction of monocyte.
[0098] Antibodies raised against each monocyte 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.
[0099] b. Immunoassays
[0100] A particular protein can be measured by a variety of
immunoassay methods. For a review of immunological and immunoassay
procedures in general, see Stites and Terr (eds.) 1991 Basic and
Clinical Immunology (7th ed.). Moreover, the immunoassays of the
present invention can be performed in any of several
configurations, which are reviewed extensively in Maggio (ed.)
(1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.; Tijan (1985)
"Practice and Theory of Enzyme Immunoassays," Laboratory Techniques
in Biochemistry and Molecular Biology, Elsevier Science Publishers
B.V., Amsterdam; and Harlow and Lane Antibodies, A Laboratory
Manual, supra, each of which is incorporated herein by reference.
See also Chan (ed.) (1987) Immunoassay: A Practical Guide Academic
Press, Orlando, Fla.; Price and Newman (eds.) (1991) Principles and
Practice of Immunoassays Stockton Press, NY; and Ngo (ed.) (1988)
Non-isotopic Immunoassays Plenum Press, NY.
[0101] Immunoassays for measurement of these monocyte proteins can
be performed by a variety of methods known to those skilled in the
art. In brief, immunoassays to measure the protein can be
competitive or noncompetitive binding assays. In competitive
binding assays, the sample to be analyzed competes with a labeled
analyte for specific binding sites on a capture agent bound to a
solid surface. Preferably the capture agent is an antibody
specifically reactive with the monocyte protein produced as
described above. The concentration of labeled analyte bound to the
capture agent is inversely proportional to the amount of free
analyte present in the sample.
[0102] In a competitive binding immunoassay, the monocyte protein
present in the sample competes with labeled protein for binding to
a specific binding agent, for example, an antibody specifically
reactive with the monocyte protein. The binding agent may be bound
to a solid surface to effect separation of bound labeled protein
from the unbound labeled protein. Alternately, the competitive
binding assay may be conducted in liquid phase and any of a variety
of techniques known in the art may be used to separate the bound
labeled protein from the unbound labeled protein. Following
separation, the amount of bound labeled protein is determined. The
amount of protein present in the sample is inversely proportional
to the amount of labeled protein binding.
[0103] Alternatively, a homogeneous immunoassay may be performed in
which a separation step is not needed. In these immunoassays, the
label on the protein is altered by the binding of the protein to
its specific binding agent. This alteration in the labeled protein
results in a decrease or increase in the signal emitted by label,
so that measurement of the label at the end of the immunoassay
allows for detection or quantitation of the protein.
[0104] These monocyte proteins may also be quantitatively
determined by a variety of noncompetitive immunoassay methods. For
example, a two-site, solid phase sandwich immunoassay may be used.
In this type of assay, a binding agent for the protein, for example
an antibody, is attached to a solid support. A second protein
binding agent, which may also be an antibody, and which binds the
protein at a different site, is labeled. After binding at both
sites on the protein has occurred, the unbound labeled binding
agent is removed and the amount of labeled binding agent bound to
the solid phase is measured. The amount of labeled binding agent
bound is directly proportional to the amount of protein in the
sample.
[0105] Western blot analysis can be used to determine the presence
of monocyte proteins in a sample. Electrophoresis is carried out,
e.g., on a tissue sample suspected of containing the protein.
Following electrophoresis to separate the proteins, and transfer of
the proteins to a suitable solid support such as a nitrocellulose
filter, the solid support is incubated with an antibody reactive
with the denatured protein. This antibody may be labeled, or
alternatively may be it may be detected by subsequent incubation
with a second labeled antibody that binds the primary antibody.
[0106] The immunoassay formats described above employ labeled assay
components. The label can be in a variety of forms. The label may
be coupled directly or indirectly to the desired component of the
assay according to methods well known in the art. A wide variety of
labels may be used. The component may be labeled by any one of
several methods. Traditionally a radioactive label incorporating
.sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P is used.
Non-radioactive labels include ligands which bind to labeled
antibodies, fluorophores, chemiluminescent agents, enzymes, and
antibodies which can serve as specific binding pair members for a
labeled protein. The choice of label depends on sensitivity
required, ease of conjugation with the compound, stability
requirements, and available instrumentation. For a review of
various labeling or signal producing systems which may be used, see
U.S. Pat. No. 4,391,904, which is incorporated herein by
reference.
[0107] Antibodies reactive with a particular protein can also be
measured by a variety of immunoassay methods. For reviews of
immunological and immunoassay procedures applicable to the
measurement of antibodies by immunoassay techniques, see, e.g.,
Stites and Terr (eds.) Basic and Clinical Immunology (7th ed.)
supra; Maggio (ed.) Enzyme Immunoassay, supra; and Harlow and Lane
Antibodies, A Laboratory Manual, supra.
[0108] A variety of different immunoassay formats, separation
techniques, and labels can be also be used similar to those
described above for the measurement of specific proteins. Moreover,
many methods are known for evaluating selectivity of binding for
specific protein or closely related proteins.
[0109] VI. Purified Monocyte Proteins
[0110] The human monocyte FDF03 protein amino acid sequence is
provided in SEQ ID NO: 2. Partial mouse sequence is provided in SEQ
ID NO: 4. Human YE01 amino acid and nucleotide sequences for the
Ig-family member are provided in SEQ ID NO: 5-10. The receptor
family members, designated KTE03, including the YYB01, YYB04, and
KLM63, KLM66, and KLM67 embodiments, are described in SEQ ID NO:
11-22.
[0111] The peptide sequences allow preparation of peptides to
generate antibodies to recognize such segments, and allow
preparation of oligonucleotides which encode such sequences.
Moreover, affinity reagents allow detection and purification of
more protein, including full length or recombinant forms. And
oligonucleotide sequences allow detection of cDNAs encoding, or
closely related to, these.
[0112] VII. Physical Variants
[0113] This invention also encompasses proteins or peptides having
substantial amino acid sequence similarity with an amino acid
sequence of SEQ ID NO: 2 or 4; 6, 8, or 10; or 12, 14, 16, 18, 20,
or 22, especially splice variants. Variants exhibiting
substitutions, e.g., 20 or fewer, preferably 10 or fewer, and more
preferably 5 or fewer substitutions, are also enabled. Where the
substitutions are conservative substitutions, the variants will
share immunogenic or antigenic similarity or cross-reactivity with
a corresponding natural sequence protein. Natural variants include
individual, allelic, polymorphic, strain, or species variants.
[0114] Amino acid sequence similarity, 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 include natural allelic and interspecies variations in
each respective protein sequence. Typical homologous proteins or
peptides will have from 50-100% similarity (if gaps can be
introduced), to 75-100% similarity (if conservative substitutions
are included) with the amino acid sequence of the relevant monocyte
protein. Identity measures will be at least about 50%, generally at
least 60%, more generally 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) Time Warps, String Edits, and
Macromolecules: The Theory and Practice of Seauence Comparison
Chapter One, Addison-Wesley, Reading, Mass.; and software packages
from IntelliGenetics, Mountain View, Calif.; and the University of
Wisconsin Genetics Computer Group (GCG), Madison, Wis.
[0115] Nucleic acids encoding the corresponding mammalian monocyte
proteins will typically hybridize, e.g., to SEQ ID NO 1 and/or 3;
5, 7, and/or 9; or 11, 13, 15, 17, 19, and/or 21 under stringent
conditions. For example, nucleic acids encoding the respective
monocyte proteins will typically hybridize to the appropriate
nucleic acid under stringent hybridization conditions, while
providing few false positive hybridization signals. Generally,
stringent conditions are selected to be about 10.degree. C. lower
than the thermal melting point (Tm) for the sequence being
hybridized to at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength and pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe.
Typically, stringent conditions will be those in which the salt
concentration in wash is about 0.02 molar at pH 7 and the
temperature is at least about 50.degree. C. Other factors may
significantly affect the stringency of hybridization, including,
among others, base composition and size of the complementary
strands, the presence of organic solvents such as formamide, and
the extent of base mismatching. A preferred embodiment will include
nucleic acids which will bind to disclosed sequences in 50%
formamide and 20-50 mM NaCl at 42.degree. C. In certain cases, the
stringency may be relaxed to detect other nucleic acids exhibiting
less than complete sequence identity.
[0116] An isolated monocyte gene DNA 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
monocyte antigens, their derivatives, or proteins having highly
similar physiological, immunogenic, or antigenic activity.
[0117] 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 monocyte protein derivatives include
predetermined or site-specific mutations of the respective protein
or its fragments. "Mutant monocyte protein" encompasses a
polypeptide otherwise falling within the homology definition of the
monocyte protein as set forth above, but having an amino acid
sequence which differs from that of the monocyte protein as found
in nature, whether by way of deletion, substitution, or insertion.
In particular, "site specific mutant monocyte protein" generally
includes proteins having significant similarity with a protein
having a sequence of SEQ ID NO: 2 or 4; 6, 8, or 10; or 12, 14, 16,
18, 20, or 22. Generally, the variant will share many
physicochemical and biological activities, e.g., antigenic or
immunogenic, with those sequences, and in preferred embodiments
contain most or all of the disclosed sequence. Similar concepts
apply to these various monocyte proteins, particularly those found
in various warm blooded animals, e.g., primates and mammals.
[0118] Although site specific mutation sites are predetermined,
mutants need not be site specific. Monocyte 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 carboxyl-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). 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.
[0119] 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 respective
monocyte 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.
[0120] In addition, new constructs may be made from combining
similar functional domains from other proteins. For example,
domains or other segments may be "swapped" between different new
fusion polypeptides or fragments, typically with related proteins,
e.g., within the Ig family or the Fc receptor family. Preferably,
intact structural domains will be used, e.g., intact Ig portions.
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 protein-binding
specificities and other functional domains. Also, alanine scanning
mutagenesis may be applied, preferably to residues which
structurally are exterior to the secondary structure, which will
avoid most of the critical residues which generally disrupt
tertiary structure.
[0121] "Derivatives" of these monocyte 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 these monocyte protein 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.
[0122] 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, or other moieties, including
ribosyl groups or cross-linking reagents. Also, proteins comprising
substitutions are encompassed, which should retain substantial
immunogenicity, to produce antibodies which recognize a protein of
SEQ ID NO: 2 or 4; 6, 8, or 10; or 12, 14, 16, 18, 20, or 22.
Alternatively, it may be desired to produce antibodies which
recognize all or subsets of SEQ ID NO: 2 and 4; 6, 8, and 10; or
12, 14, 16, 18, 20, and 22. Typically, these proteins will contain
less than 20 residue substitutions from the disclosed sequence,
more typically less than 10 substitutions, preferably less than 5,
and more preferably less than 3. Alternatively, proteins which
begin and end at structural domains will usually retain
antigenicity and cross immunogenicity.
[0123] A major group of derivatives are covalent conjugates of the
monocyte proteins 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 protein derivatization
sites with cross-linking agents are at free amino groups,
carbohydrate moieties, and cysteine residues.
[0124] Fusion polypeptides between these monocyte proteins and
other homologous or heterologous proteins are also provided.
Heterologous polypeptides may be fusions between different surface
markers, resulting in, e.g., a hybrid protein. 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 a protein, e.g., a
receptor-binding segment, so that the presence or location of the
fused protein 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.
[0125] 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.
[0126] This invention also contemplates the use of derivatives of
these monocyte 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 ligands or other binding ligands. For example, a
monocyte protein 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-monocyte protein
antibodies. The monocyte proteins can also be labeled with a
detectable group, e.g., 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 these monocyte proteins may be effected by
immobilized antibodies.
[0127] Isolated monocyte protein genes will allow transformation of
cells lacking expression of a corresponding monocyte protein, e.g.,
either species types or cells which lack corresponding proteins and
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 these monocyte proteins. Subcellular fragments, e.g.,
cytoplasts or membrane fragments, can be isolated and used.
[0128] VIII. Binding Agent: Monocyte Protein Complexes
[0129] A monocyte protein that specifically binds to or that is
specifically immunoreactive with an antibody generated against a
defined immunogen, such as an immunogen consisting of the amino
acid sequence of SEQ ID NO: 2 and/or 4; 6, 8, and/or 10; or 12, 14,
16, 18, 20, and/or 22, is determined in an immunoassay. The
immunoassay uses a polyclonal antiserum which was raised to the
protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22, or
appropriate combination. This antiserum is selected to have low
crossreactivity against other members of the related families, and
any such crossreactivity is, or may be, removed by immunoabsorption
prior to use in the immunoassay.
[0130] In order to produce antisera for use in an immunoassay, the
protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 is
isolated as described herein. For example, recombinant protein may
be produced in a mammalian cell line. An inbred strain of mice such
as Balb/c is immunized with the appropriate protein using a
standard adjuvant, such as Freund's adjuvant, and a standard mouse
immunization protocol (see Harlow and Lane, supra). Alternatively,
a synthetic peptide derived from the sequences disclosed herein and
conjugated to a carrier protein can be used an immunogen.
Polyclonal sera are collected and titered against the immunogen
protein in an immunoassay, e.g., a solid phase immunoassay with the
immunogen immobilized on a solid support. Polyclonal antisera with
a titer of 104 or greater are selected and tested for their cross
reactivity against other related proteins, using a competitive
binding immunoassay such as the one described in Harlow and Lane,
supra, at pages 570-573. See also Hertzenberg, et al. (eds. 1996)
Weir's Handbook of Experimental Immunology vols. 1-4, Blackwell
Science; and Coligan (1991) Current Protocols in Immunology
Wiley/Greene, NY. Preferably two different related proteins are
used in this determination in conjunction with a given monocyte
protein. For example, with the Ig family protein, at least two
other family members are used to absorb out shared epitopes. In
conjunction with the Fc family member, two other members of the
family are used. These other family members can be produced as
recombinant proteins and isolated using standard molecular biology
and protein chemistry techniques as described herein.
[0131] Immunoassays in the competitive binding format can be used
for the crossreactivity determinations. For example, the protein
can be immobilized to a solid support. Proteins added to the assay
compete with the binding of the antisera to the immobilized
antigen. The ability of the above proteins to compete with the
binding of the antisera to the immobilized protein is compared to
the protein of SEQ ID NO 2 and/or 4; 6, 8, and/or 10; or 12, 14,
16, 18, 20, and/or 22. The percent crossreactivity for the above
proteins is calculated, using standard calculations. Those antisera
with less than 10% crossreactivity with each of the proteins listed
above are selected and pooled. The cross-reacting antibodies are
then removed from the pooled antisera by immunoabsorption with the
above-listed proteins.
[0132] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein to the immunogen protein, e.g., the monocyte protein
of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22. In order
to make this comparison, the two proteins are each assayed at a
wide range of concentrations and the amount of each protein
required to inhibit 50% of the binding of the antisera to the
immobilized protein is determined. If the amount of the second
protein required is less than twice the amount of the protein,
e.g., of SEQ ID NO: 2, that is required, then the second protein is
said to specifically bind to an antibody generated to the
immunogen.
[0133] It is understood that monocyte proteins are each a family of
homologous proteins that comprise two or more genes. For a
particular gene product, such as the human Ig family member
protein, the invention encompasses not only the amino acid
sequences disclosed herein, but also to other proteins that are
allelic, polymorphic, non-allelic, or species variants. It is also
understood that the term "human monocyte protein" includes
normatural mutations introduced by deliberate mutation using
conventional recombinant technology such as single site mutation,
or by excising short sections of DNA encoding these proteins or
splice variants from the gene, or by substituting or adding small
numbers of new amino acids. Such minor alterations must
substantially maintain the immunoidentity of the original molecule
and/or its biological activity. Thus, these alterations include
proteins that are specifically immunoreactive with a designated
naturally occurring respective monocyte protein, for example, the
human monocyte protein exhibiting SEQ ID NO: 4. Particular protein
modifications considered minor would include conservative
substitution of amino acids with similar chemical properties, as
described above for each protein family as a whole. By aligning a
protein optimally with the protein of SEQ ID NO 2 and 4; 6, 8, and
10; or 12, 14, 16, 18, 20, and 22, and by using the conventional
immunoassays described herein to determine immunoidentity, one can
determine the protein compositions of the invention.
[0134] IX. Uses
[0135] The present invention provides reagents which will find use
in diagnostic applications as described elsewhere herein, e.g., in
the general description for developmental abnormalities, or below
in the description of kits for diagnosis.
[0136] Monocyte genes, e.g., DNA or RNA may be used as a component
in a forensic assay. For instance, the nucleotide sequences
provided may be labeled using, e.g., .sup.32P or biotin and used to
probe standard restriction fragment polymorphism blots, providing a
measurable character to aid in distinguishing between individuals.
Such probes may be used in well-known forensic techniques such as
genetic fingerprinting. In addition, nucleotide probes made from
monocyte sequences may be used in in situ assays to detect
chromosomal abnormalities.
[0137] Antibodies and other binding agents directed towards
monocyte proteins or nucleic acids may be used to purify the
corresponding monocyte protein molecule. As described in the
Examples below, antibody purification of monocyte proteins is both
possible and practicable. Antibodies and other binding agents may
also be used in a diagnostic fashion to determine whether monocyte
components are present in a tissue sample or cell population using
well-known techniques described herein. The ability to attach a
binding agent to a monocyte protein provides a means to diagnose
disorders associated with expression misregulation. Antibodies and
other monocyte protein binding agents may also be useful as
histological markers. As described in the examples below, the
expression of each of these proteins is limited to specific tissue
types. By directing a probe, such as an antibody or nucleic acid to
the respective monocyte protein, it is possible to use the probe to
distinguish tissue and cell types in situ or in vitro.
[0138] This invention also provides reagents which may exhibit
significant therapeutic value. The monocyte proteins (naturally
occurring or recombinant), fragments thereof, and antibodies
thereto, along with compounds identified as having binding affinity
to the monocyte protein, may 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 monocyte, e.g., as an antigen presenting
cell, is a target for an agonist or antagonist of the protein. The
proteins likely play a role in regulation or development of
hematopoietic cells, e.g., lymphoid cells, which affect
immunological responses, e.g., antigen presentation and the
resulting effector functions.
[0139] For example, the DX26 antibody shows that inhibitory
antibodies will be useful in modulating NK or T cell functions,
e.g., killing. Such modulation will typically be a 20% effect,
either increasing or decreasing, e.g., the killing effect, but in
preferred embodiments will have a 30%, 40%, 50%, or more. Because
the distribution is also in monocytes, the molecule will probably
also affect the regulation of monocyte mediated or initiated
effector functions of the immune system, e.g., autoimmune
responses, transplantation rejection, graft vs. host disease,
inflammatory conditions, etc. These molecules may also affect
elimination of neoplastic conditions, e.g., tumor rejection.
[0140] Other abnormal developmental conditions are known in cell
types shown to possess monocyte protein 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, NY.
Developmental or functional abnormalities, e.g., of the immune
system, cause significant medical abnormalities and conditions
which may be susceptible to prevention or treatment using
compositions provided herein.
[0141] Recombinant monocyte proteins or antibodies might 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. In
particular, these may be useful in a vaccine context, where the
antigen is combined with one of these therapeutic versions of
agonists or antagonists. 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.
[0142] Drug screening using antibodies or receptor or fragments
thereof can identify compounds having binding affinity to these
monocyte proteins, including isolation of associated components.
Subsequent biological assays can then be utilized to determine if
the compound has intrinsic stimulating activity and is therefore a
blocker or antagonist in that it blocks the activity of the
protein. Likewise, a compound having intrinsic stimulating activity
might activate the cell through the protein and is thus an agonist
in that it simulates the cell. This invention further contemplates
the therapeutic use of antibodies to the proteins as
antagonists.
[0143] 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 (1990) Remington's Pharmaceutical Sciences
(17th ed.) 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. 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 .mu.M (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.
[0144] The monocyte proteins, fragments thereof, and antibodies to
it or its fragments, antagonists, and agonists, could 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
many conventional dosage formulations. 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
Pharmacoloaical Bases of Therapeutics (8th ed.) Pergamon Press; and
(1990) Remington's Pharmaceutical Sciences (17th ed.) Mack
Publishing Co., Easton, Pa.; Avis, et al. (eds.) (1993)
Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY;
Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Tablets Dekker, NY; and Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY. The
therapy of this invention may be combined with or used in
association with other chemotherapeutic or chemopreventive
agents.
[0145] Both the naturally occurring and the recombinant form of the
monocyte 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, and other
descriptions of chemical diversity libraries, which describe means
for testing of binding affinity by a plurality of compounds. The
development of suitable assays can be greatly facilitated by the
availability of large amounts of purified, e.g., soluble versions
of, monocyte protein as provided by this invention.
[0146] For example, antagonists can often be found once the protein
has been structurally defined. Testing of potential protein analogs
is now possible upon the development of highly automated assay
methods using a purified surface protein. In particular, new
agonists and antagonists will be discovered by using screening
techniques described herein. Of particular importance are compounds
found to have a combined binding affinity for multiple related cell
surface antigens, e.g., compounds which can serve as antagonists
for species variants of a monocyte protein.
[0147] This invention is particularly useful for screening
compounds by using recombinant monocyte protein in 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 protein from a specific source; (b)
potentially greater number of antigens per cell giving better
signal to noise ratio in assays; and (c) species variant
specificity (theoretically giving greater biological and disease
specificity).
[0148] One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing a monocyte protein. Cells may
be isolated which express that protein in isolation from any
others. Such cells, either in viable or fixed form, can be used for
standard surface 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 monocyte protein) are contacted
and incubated with an antibody having known binding affinity to the
antigen, 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 protein binding. The amount of test compound
bound is inversely proportional to the amount of labeled antibody
binding to the known source. Many techniques can be used to
separate bound from free reagent 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 these monocyte protein mediated functions, e.g., antigen
presentation or helper function.
[0149] Another method utilizes membranes from transformed
eukaryotic or prokaryotic host cells as the source of a monocyte
protein. These cells are stably transformed with DNA vectors
directing the expression of the appropriate protein, e.g., an
engineered membrane bound form. Essentially, the membranes would be
prepared from the cells and used in binding assays such as the
competitive assay set forth above.
[0150] Still another approach is to use solubilized, unpurified or
solubilized, purified monocyte 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.
[0151] Another technique for drug screening involves an approach
which provides high throughput screening for compounds having
suitable binding affinity to the respective monocyte protein 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., supra. Then all the pins are reacted with
solubilized, unpurified or solubilized, purified monocyte protein,
and washed. The next step involves detecting bound reagent, e.g.,
antibody.
[0152] 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, NY.
[0153] X. Kits
[0154] This invention also contemplates use of these monocyte
proteins, fragments thereof, peptides, and their fusion products in
a variety of diagnostic kits and methods for detecting the presence
of a monocyte protein or message. Typically the kit will have a
compartment containing either a defined monocyte peptide or gene
segment or a reagent which recognizes one or the other, e.g.,
antibodies.
[0155] A kit for determining the binding affinity of a test
compound to the respective monocyte protein would typically
comprise a test compound; a labeled compound, for example an
antibody having known binding affinity for the protein; a source of
the monocyte protein (naturally occurring or recombinant); and a
means for separating bound from free labeled compound, such as a
solid phase for immobilizing the monocyte protein. Once compounds
are screened, those having suitable binding affinity to the protein
can be evaluated in suitable biological assays, as are well known
in the art, to determine whether they act as agonists or
antagonists to regulate monocyte function. The availability of
recombinant monocyte polypeptides also provide well defined
standards for calibrating such assays.
[0156] A preferred kit for determining the concentration of, for
example, a monocyte protein in a sample would typically comprise a
labeled compound, e.g., antibody, having known binding affinity for
the monocyte protein, a source of monocyte protein (naturally
occurring or recombinant) and a means for separating the bound from
free labeled compound, for example, a solid phase for immobilizing
the monocyte protein. Compartments containing reagents, and
instructions, will normally be provided.
[0157] Antibodies, including antigen binding fragments, specific
for the respective monocyte or its fragments are useful in
diagnostic applications to detect the presence of elevated levels
of the 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 in serum, or the like. Diagnostic assays may be
homogeneous (without a separation step between free reagent and
antigen-monocyte 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 the monocyte 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 Press, NY; Chan (ed.) (1987) Immunoassay: A
Practical Guide Academic Press, Orlando, Fla.; Price and Newman
(eds.) (1991) Principles and Practice of Immunoassay Stockton
Press, NY; and Ngo (ed.) (1988) Nonisotopic Immunoassay Plenum
Press, NY. In particular, the reagents may be useful for diagnosing
monocyte populations in biological samples, either to detect an
excess or deficiency of monocyte in a sample. The assay may be
directed to histological analysis of a biopsy, or evaluation of
monocyte numbers in a blood or tissue sample.
[0158] Anti-idiotypic antibodies may have similar use to diagnose
presence of antibodies against a monocyte protein, as such may be
diagnostic of various abnormal states. For example, overproduction
of the monocyte protein may result in various immunological
reactions which may be diagnostic of abnormal physiological states,
particularly in proliferative cell conditions such as cancer or
abnormal differentiation.
[0159] 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
receptor, or labeled monocyte 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.
[0160] Many 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 many of these assays, the protein, test compound, monocyte
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.
[0161] There are also numerous methods of separating the bound from
the free protein, or alternatively the bound from the free test
compound. The monocyte protein can be immobilized on various
matrices followed by washing. Suitable matrices include plastic
such as an ELISA plate, filters, and beads. Methods of immobilizing
the monocyte 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/antibody complex by one 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.
[0162] 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.
[0163] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of a respective monocyte protein. These sequences can be used as
probes for detecting levels of the message in samples from patients
suspected of having an abnormal condition, e.g., cancer or immune
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, fluorophores, 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).
[0164] 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.
[0165] XI. Binding Partner Isolation
[0166] Having isolated one member of a binding partner of a
specific interaction, methods exist for isolating the
counter-partner. See, Gearing, et al. (1989) EMBO J. 8:3667-3676.
For example, means to label a monocyte surface protein without
interfering with the binding to its receptor can be determined. For
example, an affinity label can be fused to either the amino- or
carboxyl-terminus of the ligand. An expression library can be
screened for specific binding to the monocyte protein, e.g., by
cell sorting, or other screening to detect subpopulations which
express such a binding component. See, e.g., Ho, et al. (1993)
Proc. Nat'l Acad. Sci. USA 90:11267-11271. Alternatively, a panning
method may be used. See, e.g., Seed and Aruffo (1987) Proc. Nat'l
Acad. Sci. USA 84:3365-3369. A two-hybrid selection system may also
be applied making appropriate constructs with the available
monocyte protein sequences. See, e.g., Fields and Song (1989)
Nature 340:245-246.
[0167] Protein cross-linking techniques with label can be applied
to isolate binding partners of a monocyte protein. This would allow
identification of proteins which specifically interact with the
appropriate monocyte protein.
[0168] 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
[0169] I. General Methods
[0170] Many of the standard methods below are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular Cloning, A
Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor
Press, NY; 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, NY; or Ausubel, et al.
(1987 and Supplements) Current Protocols in Molecular Biology
Wiley/Greene, NY; Innis, et al. (eds.) (1990) PCR Protocols: A
Guide to Methods and Applications Academic Press, NY.
[0171] 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," Methods in Enzmmology vol.
182, and other volumes in this series; Coligan, et al. (1996 and
periodic Supplements) Current Protocols in Protein Science
Wiley/Greene, NY; 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, NY; and Crowe, et al. (1992)
OIAexpress: The High Level Expression & Protein Purification
System QUIAGEN, Inc., Chatsworth, Calif.
[0172] Standard immunological techniques are described, e.g., in
Hertzenberg, et al. (eds. 1996) Weir's Handbook of Experimental
Immunology vols. 1-4, Blackwell Science; Coligan (1991) Current
Protocols in Immunology Wiley/Greene, NY; and Methods in Enzmmology
volumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and
163. See also, e.g., Paul (ed.) (1993) Fundamental Immunology (3d
ed.) Raven Press, NY.
[0173] 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.
[0174] II. Isolation of Human Monocytes
[0175] Healthy donors were subjected to a leukophoresis. Percoll
gradients were used to isolate mononuclear cells which were then
subject to centrifugal elutriation. See, Figdor, et al. (1982)
Blood 60:46-53; and Plas, et al. (1988) Expt'l. Hematol.
16:355-359. This highly enriched monocyte fraction was cultured for
5-7 days in the presence of GM-CSF (800 U/ml) and IL-4 (500 U/ml),
as described in Romani, et al (1994) J. Exp. Med. 180:83-93; and
Sallusto, et al (1994) J. Exp. Med. 179:1109-1118.
[0176] For making dendritic cells, human CD34+ cells were obtained
as follows. See, e.g., Caux, et al. (1995) pages 1-5 in Banchereau
and Schmitt Dendritic Cells in Fundamental and Clinical Immunology
Plenum Press, NY. Peripheral or cord blood cells, sometimes CD34+
selected, were cultured in the presence of Stem Cell Factor (SCF),
GM-CSF, and TNF-.alpha. in endotoxin free RPMI 1640 medium (GIBCO,
Grand Island, N.Y.) supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (FBS; Flow Laboratories, Irvine, Calif.), 10 mM
HEPES, 2 mM L-glutamine, 5.times.10.sup.-5 M 2-mercaptoethanol,
penicillin (100 .mu.g/ml). This is referred to as complete
medium.
[0177] CD34+ cells were seeded for expansion in 25 to 75 cm.sup.2
flasks (Corning, N.Y.) at 2.times.10.sup.4 cells/ml. Optimal
conditions were maintained by splitting these cultures at day 5 and
10 with medium containing fresh GM-CSF and TNF-.alpha. (cell
concentration: 1-3.times.10.sup.5 cells/ml). In certain cases,
cells were FACS sorted for CD1a expression at about day 6.
[0178] In certain situations, cells were routinely collected after
12 days of culture, eventually adherent cells were recovered using
a 5 .mu.M EDTA solution. In other situations, the CD1a+ cells were
activated by resuspension in complete medium at 5.times.10.sup.6
cells/ml and activated for the appropriate time (e.g., 1 or 6 h)
with 1 .mu.g/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and
100 ng/ml ionomycin (Calbiochem, La Jolla, Calif.). These cells
were expanded for another 6 days, and RNA isolated for cDNA library
preparation.
[0179] III. RNA Isolation and Library Construction
[0180] Total RNA is isolated using, e.g., the guanidine
thiocyanate/CsCl gradient procedure as described by Chirgwin, et
al. (1978) Biochem. 18:5294-5299.
[0181] Alternatively, poly(A)+ RNA is isolated using the OLIGOTEX
mRNA isolation kit (QIAGEN). Double stranded cDNA are generated
using, e.g., the SUPERSCRIPT plasmid system (Gibco BRL,
Gaithersburg, Md.) for cDNA synthesis and plasmid cloning. The
resulting double stranded cDNA is unidirectionally cloned, e.g.,
into pSport1 and transfected by electroporation into ELECTROMAX
DH10BTM Cells (Gibco BRL, Gaithersburg, Md.).
[0182] IV. Sequencing
[0183] DNA isolated from randomly picked clones, or after
subtractive hybridization using unactivated cells, were subjected
to nucleotide sequence analysis using standard techniques. A Taq
DiDeoxy Terminator cycle sequencing kit. (Applied Biosystems,
Foster City, Calif.) can be used. The labeled DNA fragments are
separated using a DNA sequencing gel of an appropriate automated
sequencer. Alternatively, the isolated clone is sequenced as
described, 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. Chemical sequencing
methods are also available, e.g., using Maxam and Gilbert
sequencing techniques.
[0184] V. Isolation of Human Monocyte Protein Genes
[0185] The FDF03, the YE01, and KTE03 (YYB01 and YYB04) clones were
sequenced, and analyzed for open reading frames. The clones were
further analyzed to extend the nucleic acid sequence to a full, or
nearly full, open reading frame.
[0186] mRNA is prepared from appropriate cell populations by the
FastTrack kit (Invitrogen) from which cDNA is generated using,
e.g., SuperScript Plasmid System for cDNA synthesis from GIBCO-BRL
(Gaithersburg, Md.) essentially as described by the manufacturer.
Modification to the procedure may include the substitution of other
cloning adapters for the Sal1 adapters provided with the kit. The
resultant cDNA from these cells is used to generate libraries,
e.g., in the plasmid PCDNA II (Invitrogen). The cDNA is cloned into
the polylinker and is used to transform an appropriate strain,
e.g., DH10B, of E. coli. Plasmid is isolated and purified, e.g.,
with the Qiagen system (Chatsworth, Calif.) which is used to
generate RNA probes from, e.g., the SP6 promoter.
[0187] RNA probes are labeled, e.g., using the Genius System
(Boehringer-Mannheim) as described by the manufacturer. Filter
lifts of the cDNA library can be pre-hybridized, e.g., at
42.degree. C. for 3-6 hours in Church's buffer (50% formamide,
6.times.SSPE, 50 mM NaHPO.sub.4 pH 7.2, 7% SDS, 0.1% N-Lauryl
sarcosine, 2% Boehringer-Mannheim blocking reagent). Filters are
probed, e.g., overnight in the same buffer containing the
appropriate probes. The filters are washed, e.g., as described by
the Genius System. The colonies that hybridize are selected.
[0188] The entire cDNA of human monocyte proteins are sequenced,
e.g., by the dideoxynucleotide chain termination method with T7
polymerase (U.S. Biochemicals, Cleveland, Ohio) using
double-stranded DNA as template. Data base searching and sequence
analysis are performed using IntelliGenetics programs (Mountain
View, Calif.) to determine if homology exists between previously
reported clones.
[0189] Table 1 discloses sequence encoding a human FDF03 gene and
mouse counterpart sequence, and also shows alignment of available
sequence. Likewise, Table 2 discloses three sequences encoding
human YE01 gene products, including a splice variant and a
transcript which encodes a soluble product. Table 3 provides
sequences of embodiments of the KTE03 gene products, and shows
evidence of splice variants.
[0190] VI. Recombinant Monocyte Gene Constructs
[0191] Poly(A).sup.+ RNA is isolated from appropriate cell
populations, e.g., using the FastTrack mRNA kit (Invitrogen, San
Diego, Calif.). Samples are electrophoresed, e.g., in a 1% agarose
gel containing formaldehyde and transferred to a GeneScreen
membrane (NEN Research Products, Boston, Mass.). Hybridization is
performed, e.g., at 65.degree. C. in 0.5 M NaHPO.sub.4 pH 7.2, 7%
SDS, 1 mM EDTA, and 1% BSA (fraction V) with .sup.32P-dCTP labeled
monocyte gene cDNA at 10.sup.7 cpm/ml. After hybridization filters
are washed three times at 50.degree. C. in 0.2.times.SSC, 0.1% SDS,
and exposed to film for 24 h.
[0192] The recombinant gene construct may be used to generate probe
for detecting the message. The insert may be excised and used in
the detection methods described above.
[0193] VII. Expression of Monocyte Gene Protein in E. coli.
[0194] PCR is used to make a construct comprising the open reading
frame, preferably in operable association with proper promoter,
selection, and regulatory sequences. The resulting expression
plasmid is transformed into an appropriate, e.g., the Topp5, E.
coli strain (Stratagene, La Jolla, Calif.). Ampicillin resistant
(50 .mu.g/ml) transformants are grown in Luria Broth (Gibco) at
37.degree. C. until the optical density at 550 nm is 0.7.
Recombinant protein is induced with 0.4 mm
isopropyl-.beta.D-thiogalacto-pyranoside (Sigma, St. Louis, Mo.)
and incubation of the cells continued at 20.degree. C. for a
further 18 hours. Cells from a 1 liter culture are harvested by
centrifugation and resuspended, e.g., in 200 ml of ice cold 30%
sucrose, 50 mM Tris HCl pH 8.0, 1 mM ethylenediamine-tetraacetic
acid. After 10 min on ice, ice cold water is added to a total
volume of 2 liters. After 20 min on ice, cells are removed by
centrifugation and the supernatant is clarified by filtration via a
5 .mu.M Millipak 60 (Millipore Corp., Bedford, Mass.).
[0195] The recombinant protein is purified via standard
purification methods, e.g., various ion exchange chromatography
methods. Immunoaffinity methods using antibodies described below
can also be used. Affinity methods may be used where an epitope tag
is engineered into an expression construct.
[0196] VIII. Mapping of Human Monocyte Genes
[0197] DNA isolation, restriction enzyme digestion, agarose gel
electrophoresis, Southern blot transfer and hybridization are
performed according to standard techniques. See Jenkins, et al.
(1982) J. Virol. 43:26-36. Blots may be prepared with Hybond-N
nylon membrane (Amersham). The probe is labeled with .sup.32P-dCTP;
washing is done to a final stringency, e.g., of 0.1.times.SSC, 0.1%
SDS, 65.degree. C.
[0198] Alternatively, a BIOS Laboratories (New Haven, Conn.) mouse
somatic cell hybrid panel may be combined with PCR methods.
[0199] IX. Analysis of Individual Variation
[0200] From the distribution data, an abundant easily accessible
cell type is selected for sampling from individuals. Using PCR
techniques, a large population of individuals are analyzed for this
gene. cDNA or other PCR methods are used to sequence the
corresponding gene in the different individuals, and their
sequences are compared. This indicates both the extent of
divergence among racial or other populations, as well as
determining which residues are likely to be modifiable without
dramatic effects on function.
[0201] X. Preparation of Antibodies
[0202] Recombinant monocyte proteins are generated by expression in
E. coli as shown above, and tested for biological activity. Active
or denatured proteins may be used for immunization of appropriate
mammals for either polyclonal serum production, or for monoclonal
antibody production. Antibodies are selected for use in Western
blots, against native or denatured antigen, and for those which
modulate a biological activity.
[0203] Antibodies prepared against the FDF03 have confirmed
specific binding on dendritic cells. XI. Isolation of counterpart
primate monocyte genes Human cDNA clones encoding these genes are
used as probes, or to design PCR primers to find counterparts in
various primate species, e.g., chimpanzees.
[0204] XII. Use of Reagents to Analyze Cell Populations
[0205] Detection of the level of monocyte cells present in a sample
is important for diagnosis of certain aberrant disease conditions.
For example, an increase in the number of monocytes in a tissue or
the lymph system can be indicative of the presence of a monocyte
hyperplasia, tissue or graft rejection, or inflammation. A low
monocyte population can indicate an abnormal reaction to, e.g., a
bacterial or viral infection, which may require the appropriate
treat to normalize the monocyte response.
[0206] FACS analysis using a labeled binding agent specific for a
cell surface monocyte protein, see, e.g., 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., is used in determining the number of
monocytes present in a cell mixture, e.g., PBMCs, adherent cells,
etc. The binding agent is also used for histological analysis of
tissue samples, either fresh or fixed, to analyze infiltration of
monocyte. Diverse cell populations may also be evaluated, either in
a cell destructive assay, or in certain assays where cells retain
viability.
[0207] Analysis of the presence of soluble intracellular molecules
is performed, e.g., with a fluorescent binding agent specific for a
monocyte as described in Openshaw, et al. (1995) J. Exp. Med.
182:1357-1367. alternatively, tissue or cell fixation methods may
be used.
[0208] Levels of monocyte transcripts are quantitated, e.g., using
semiquantitative PCR as described in Murphy, et al. (1993) J.
Immunol. Methods 162:211-223. Primers are designed such that
genomic DNA is not detected.
[0209] Distribution of the FDF03 embodiment has been studied using
hybridization and PCR analysis. Northern blot analysis located
transcripts in dendritic cells and the JY cell line. There appear
to be two transcripts of about 700 bp and 1300 bp, which may be
differentially regulated, and an estimated frequency of about 1 in
4000 in resting monocytes or LPS and IFN.gamma. activated
monocytes. The shorter message does not appear to encode a soluble
version of the protein, e.g., lacking the TM and intracellular
segments. Southern blot analysis has detected transcripts in
monocytes, dendritic cells, PBMC, B cells, and splenic B cells. The
message appears to be down-regulated upon monocyte activation.
[0210] Distribution of the YE01 embodiment has also been evaluated.
The message appears to be monocyte specific, and is a low abundance
message. It is detectable in cDNA Southern blots in resting
monocytes, and in activated monocytes. Its highest expression was
found in 6 hour LPS stimulated monocytes. It is also detectable in
anti-CD3 and PMA activated PBMC. It may be faintly detectable in
dendritic cells, but this may be due to contamination of the
dendritic cell population with residual monocytes. At that level of
sensitivity, it is undetectable in NK cells, B or T cells, or any
fetal cells examined. However, the YE01 gene product is
specifically recognized by a monoclonal antibody DX26. This
antibody, when crosslinked, can inhibit NK cell mediated killing of
certain targets. The antibody recognizes protein expressed in T
cells, B cells, NK cells, and monocytes. The gene encoding the
antigen recognized by DX26, which is apparently a polymorphic
variant of the YE01 isolate, has been cloned and has essentially
the sequence:
[0211] The KTE03 expression levels were also investigated. The
message appeared to be up-regulated upon IL-10 exposure when the
monocytes were activated by LPS and IFN.gamma..
[0212] XIII. Isolation of a Binding Counterpart
[0213] A monocyte protein can be used as a specific binding
reagent, by taking advantage of its specificity of binding, much
like an antibody would be used. A binding reagent is either labeled
as described above, e.g., fluorescence or otherwise, or immobilized
to a substrate for panning methods.
[0214] The monocyte protein is used to screen for a cell line which
exhibits binding. Standard staining techniques are used to detect
or sort intracellular or surface expressed ligand, 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.
[0215] 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.
[0216] On day 1 for each sample, prepare 0.5 ml of a solution of 66
mg/ml DEAE-dextran, 66 mM chloroquine, and 4 mg DNA in serum free
DME. For each set, a positive control is prepared, e.g., of human
receptor-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.
[0217] 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 ml/ml of 1M NaN.sub.3 for 20 min. Cells are then washed
with HBSS/saponin 1.times.. Add protein or protein/antibody complex
to cells and incubate for 30 min. Wash cells twice with
HBSS/saponin. If appropriate, add first antibody for 30 min. 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.
[0218] Alternatively, other monocyte protein specific binding
reagents are used to affinity purify or sort out cells expressing a
receptor. See, e.g., Sambrook, et al. or Ausubel, et al.
[0219] Another strategy is to screen for a membrane bound receptor
by panning. The receptor cDNA is constructed as described above.
The ligand can be immobilized and used to immobilize expressing
cells. Immobilization may be achieved by use of appropriate
antibodies which recognize, e.g., a FLAG sequence of a monocyte
protein fusion construct, or by use of antibodies raised against
the first antibodies. Recursive cycles of selection and
amplification lead to enrichment of appropriate clones and eventual
isolation of ligand expressing clones.
[0220] Phage expression libraries can be screened by monocyte
protein. Appropriate label techniques, e.g., anti-FLAG antibodies,
will allow specific labeling of appropriate clones.
[0221] XIV. Isolation of a Soluble YE01
[0222] An additional family member of the previously described
YE01, also designated DNAX Leukocyte Associated Immunoglobulin-like
Receptor (DLAIR; and now designated DLAIR-1) was cloned by
screening a human T cell tumor line cDNA library (TcT). Bacterial
colony lift membranes were hybridized with a DLAIR-1 probe
comprising a BglII-SphI digestion fragment, spanning the Ig loop in
the extracellular domain. Two positives were isolated and
sequenced. Sequence analysis revealed that both clones contained
identical open reading frames of 414 base pairs, encoding a 135
amino acid protein with a predicted 21 amino acid leader sequence
and a predicted molecular weight of 14.7 kDa. This molecule, now
referred to as DLAIR-2, contains one Ig loop. See Table 2. The Ig
loop has 84% homology with DLAIR-1, indicating that it belongs to
the same family, but is encoded by a separate gene. DLAIR-2 lacks a
transmembrane region which suggests that it is a secreted
protein.
[0223] DLAIR-2, as a soluble molecule with similarity to DLAIR-1,
may be used as an antagonist to this inhibitory receptor.
[0224] XV. Preparation of DX26 Monoclonal Antibody
[0225] Mice were immunized with a human NK cell clone and
antibodies were screened for their capacity to inhibit NK
cell-mediated lysis of FcR bearing targets. Alternatively,
antibodies will be raised to purified protein.
[0226] XVI. Cross-Linking DLAIR-1 with mAb Inhibits NK
cell-mediated Killing
[0227] DX26 mAb did not inhibit NK clone killing of the
HLA-negative EBV-transformed B cell line 721.221. However, when
721.221 was transfected with the human Fc.gamma.R-II (CD32) and
used as a target, NK cell-mediated cytolysis was inhibited by DX26
mAb. This indicates that signaling through the molecule recognized
by DX26 mAb (designated DNAX Leukocyte Associated
Immunoglobulin-like Receptor (DLAIR)), delivers a negative signal
to NK cell clones that prevents their killing specific target
cells. In agreement with this, NK cell-mediated cytotoxicity
against Colo-205, PA-1, or FO-1, each an FcR-negative human cell
line, was not inhibited by the addition of DX26 mAb. Moreover,
lysis of P815 cells, an FcR-expressing mouse mastocytoma cell line,
which is killed in vitro by human NK cell clones upon simultaneous
cross linking of CD2, CD16, CD69, or DNAM-1 antigen, was also
inhibited by DX26 mAb. These results lead to a conclusion that
DLAIR delivers a strong inhibitory signal to NK cells, since the
positive signal given by potent inducers of NK cell cytotoxicity
was overruled by DX26 mAb.
[0228] XVII. DLAIR-1 is an Inhibitory Receptor on Resting NK
Cells
[0229] NK cell clones consist of clonally derived populations of
activated NK cells. These cells are potently inhibited by DLAIR
signaling. We set out to study whether DLAIR is also functioning as
an inhibitory receptor on NK cells that had not been previously
activated. Resting NK cells, prepared from peripheral blood by
negative depletion using magnetic beads, were able to lyse P815
target cells when simultaneously activated through CD16. This NK
cell mediated cytotoxicity was inhibited by the addition of DX26
mAb. Thus, DLAIR is functional as an inhibitory receptor on both
activated and resting NK cells.
[0230] XVIII. DLAIR is a Widely Expressed Antigen
[0231] Phenotypic analysis of human peripheral blood lymphocytes
demonstrated that DLAIR is a widely distributed molecule. In
healthy donor PBMC, CD3.sup.+CD4.sup.+ T cells (70-80%),
CD3.sup.+CD8.sup.+T cells (80-90%), CD3.sup.-CD56.sup.+ NK cells
(95-100%), CD3.sup.-CD19.sup.+ B cells (80-90%), and CD3.sup.-CD14+
monocytes (99-100%) all expressed the DLAIR molecule. Human fetal
thymocytes, both the immature CD4+CD8+ cells and mature
CD4+-CD8.sup.+ or CD4.sup.-CD8.sup.+ single positive cells also
expressed DLAIR. Peripheral blood granulocytes, platelets and
erythrocytes did not express DLAIR.
[0232] Human NK cell clones and T cell clones all expressed DLAIR,
with the exception of the long-term cultured NK clones NKL and NK92
(see Table 4). EBV-transformed B cell lines, the B cell tumor
Daudi, and the NK tumor cell line YT and several non-hematopoietic
cell lines did not express DLAIR, whereas human T cell lines did
show DLAIR expression.
4TABLE 4 Expression of DLAIR on human tumor cell lines.sup.1
control IgG1 DX26 mAb cell line type (mean fluorescence intensity)
HUT78 T cell tumor <5 25.8 Peer T cell tumor <5 29.1 Molt4 T
cell tumor <5 30.7 CEM T cell tumor <5 92.7 Jurkat T cell
tumor <5 47.1 HL60 promyeloid tumor <5 46.9 U937 myeloid
tumor <5 49.5 721.221 EBV - B cell <5 <5 JY EBV - B cell
<5 <5 Daudi B cell tumor <5 <5 YT NK cell tumor <5
<5 NKL NK cell clone <5 <5 NK92 NK cell clone <5 <5
Colo205 colon carcinoma <5 <5 293T embryonic kidney <5
<5 PA-1 teratocarcinoma <5 <5 FO-1 melanoma <5 <5
.sup.1cells were stained with control IgG1 or DX26 mAb and
PE-conjugated goat-anti-mouse-IgG as a second step. Cells were
analyzed on a FACScan.
[0233] XIX. Expression Cloning of the DX26 Antigen
[0234] The DX26 antibody was used to expression clone the antigen
the antibody recognizes. The expression cloning was performed using
standard methods. See, e.g., Sambrook, et al. or Coligan, et
al.
[0235] DX26 antigen is expression cloned, e.g., from a polyclonal
human activated NK cell cDNA library in the pJFE14 expression
vector. COS7 cells are transfected with the library and antigen
positive cells were selected using phycoerythrin labeled anti-DX26
mAb. The cDNA sequence was determined and found to match much of
the YE01 sequence. The DX26 antibody specifically binds to the
product of the YE01 gene product.
[0236] 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.
[0237] Moreover, the YE01 gene product is specifically recognized
by a monoclonal antibody DX26. This antibody, when crosslinked, can
inhibit NK cell mediated killing of certain targets. The antibody
recognizes protein expressed in T cells, B cells, NK cells, and
monocytes. The gene encoding the antigen recognized by DX26, which
is apparently a polymorphic variant of the YE01 isolate, has been
cloned and has essentially the sequence (see SEQ ID NO: 7). This
isolate has a different 3' untranslated sequence from the original
YE01 transcript, apparently due to use of an alternative
polyadenylation site. A soluble form of DLAIR has also been
detected (see SEQ ID NO: 9).
[0238] Distribution analysis of the DX26 isolate has determined,
Northern blot analysis, the distribution as follows. Probing of
mRNA of human NK cell clones with DLAIR cDNA, PBMC, the human T
cell line Jurkat, and the human myeloid cell line Jurkat results in
two bands of approximately 1800 bp and 3000-4000 bp. This indicates
that besides the cloned cDNA, another transcript with sequence
similarity to DLAIR exists in these cell lines. Whether this
contains the same open reading frame is at present unknown, but
will be determined upon cloning and sequence analysis of that
transcript. The EBV-transformed human B cell line JY did not show
transcripts that probed with DLAIR cDNA.
[0239] XX. DLAIR-1 Binds SHP-1 and SHP-2
[0240] The existence of two consensus sequences for ITIMs within
the cytoplasmic domain of DLAIR-1, suggested that the generation of
an inhibitory signal in NK cells was manifested by the recruitment
of SHP-1 and/or SHP-2. To determine if DLAIR-1 was capable of
binding protein tyrosine phosphatases, a NK cell clone was
stimulated with pervanadate (an inhibitor of protein tyrosine
phosphatases that induces tyrosine phosphorylation (O'Shea, et al.
(1992) Proc. Natl. Acad. Sci. USA 89:10306-10310), lysed, and
immunoprecipitated with DX26 MAb. Immunoprecipitates were then
analyzed by Western blot using antibodies specific for SHP-1 and
SHP-2. Both SHP-1 and SHP-2 associated with tyrosine phosphorylated
DLAIR-1. These results suggest that recruitment of SHP-1 and SHP-2
may be involved in mediating the negative signal transduced via
engagement of the DLAIR-1 molecule.
[0241] 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 in its entirety for all purposes.
[0242] 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.
* * * * *