U.S. patent application number 11/529598 was filed with the patent office on 2007-04-05 for receptor on the surface of activated t-cells: act-4.
Invention is credited to David Buck, Edgar G. Engleman, Wayne Godfrey.
Application Number | 20070077247 11/529598 |
Document ID | / |
Family ID | 22522890 |
Filed Date | 2007-04-05 |
United States Patent
Application |
20070077247 |
Kind Code |
A1 |
Godfrey; Wayne ; et
al. |
April 5, 2007 |
Receptor on the surface of activated T-cells: ACT-4
Abstract
The invention provides purified ACT-4 receptor polypeptides,
antibodies against these polypeptides and nucleic acids encoding
ACT-4 receptor polypeptides. Also provided are methods of diagnosis
and treatment using the same. ACT-4 receptors are preferentially
expressed on the surface of activated CD4.sup.+ T-cells. ACT-4
receptors are usually expressed at low levels on the surface of
activated CD8.sup.+ cells, and are usually substantially absent on
resting T-cells, and on monocytes and B-cells (resting or
activated). An exemplary ACT-4 receptor, termed ACT-4-h-1, has a
signal sequence, an extracellular domain comprising three
disulfide-bonded intrachain loops, a transmembrane domain, and an
intracellular domain.
Inventors: |
Godfrey; Wayne; (Woodside,
CA) ; Buck; David; (Half Moon Bay, CA) ;
Engleman; Edgar G.; (Atherton, CA) |
Correspondence
Address: |
ARNOLD & PORTER LLP;ATTN: IP DOCKETING DEPT.
555 TWELFTH STREET, N.W.
WASHINGTON
DC
20004-1206
US
|
Family ID: |
22522890 |
Appl. No.: |
11/529598 |
Filed: |
September 29, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10805377 |
Mar 22, 2004 |
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11529598 |
Sep 29, 2006 |
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09852845 |
May 11, 2001 |
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10805377 |
Mar 22, 2004 |
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08472940 |
Jun 6, 1995 |
6277962 |
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09852845 |
May 11, 2001 |
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08147784 |
Nov 3, 1993 |
5821332 |
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08472940 |
Jun 6, 1995 |
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Current U.S.
Class: |
424/144.1 ;
435/320.1; 435/325; 435/6.17; 435/69.1; 435/7.2; 530/350;
530/388.22; 536/23.5 |
Current CPC
Class: |
A61P 31/04 20180101;
C07K 14/70578 20130101; A61P 37/06 20180101; C07K 16/2878 20130101;
G01N 33/9493 20130101; G01N 33/56972 20130101; A61P 37/00 20180101;
A61K 38/00 20130101; C07K 2319/00 20130101; G01N 2500/04 20130101;
C07K 14/70514 20130101; A61P 29/00 20180101 |
Class at
Publication: |
424/144.1 ;
435/006; 435/069.1; 435/007.2; 435/320.1; 435/325; 530/350;
530/388.22; 536/023.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/567 20060101 G01N033/567; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; A61K 39/395 20060101
A61K039/395; C07K 14/705 20060101 C07K014/705; C07K 16/28 20060101
C07K016/28 |
Claims
1. A purified ACT-4 receptor polypeptide that comprises at least
five contiguous amino acids from an amino acid sequence shown in
FIG. 5.
2. The polypeptide of claim 1 that exhibits at least eighty percent
sequence identity to the amino acid sequence of FIG. 5.
3. The polypeptide of claim 2 having an antigenic determinant
common to a protein comprising the amino acid sequence shown in
FIG. 5.
4. The polypeptide of claim 3 that is a full-length
polypeptide.
5. The polypeptide of claim 4 comprising a domain selected from a
group of domains consisting of a signal sequence, an intracellular
domain, a transmembrane domain, and an extracellular domain.
6. The polypeptide of claim 5 that comprises an extracellular
domain.
7. The polypeptide of claim 6, wherein said extracellular domain
comprises an intrachain loop formed by disulfide bonding of two
cysteine residues.
8. The polypeptide of claim 7, wherein said extracellular domain
comprises three intrachain loops, each formed by disulfide bonding
of two cysteine residues.
9. The polypeptide of claim 8, wherein said polypeptide is present
on the surface of activated CD4.sup.+ T-cells, and is substantially
absent on activated CD8.sup.+ T cells and resting T-cells.
10. The polypeptide of claim 9 that is naturally occurring.
11. The polypeptide of claim 10 that is human.
12. The polypeptide of claim 11 that has a molecular weight of
about 50 kDa before deglycosylation and about 27 kDa
thereafter.
13. The polypeptide of claim 12 comprising the amino acid sequence
of FIG. 5.
14. An extracellular domain of a polypeptide of claim 3.
15. The extracellular domain of claim 14 comprising an intrachain
loop formed by disulfide bonding of two cysteine residues.
16. The extracellular domain of claim 15 comprising three
intrachain loops, each formed by disulfide bonding of two cysteine
residues.
17. The extracellular domain of claim 16 that is soluble.
18. The extracellular domain of claim 17 that is capable of
specifically binding to an ACT-4 ligand.
19. The extracellular domain of claim 18 that is immunogenic.
20. The extracellular domain of claim 19 that competes with an
ACT-4-h-1 receptor polypeptide for specific binding to an
antibody.
21. The extracellular domain of claim 20 that is fused to a second
polypeptide.
22. The extracellular domain of claim 21, wherein the second
polypeptide is a constant region of an immunoglobulin heavy
chain.
23. A polypeptide consisting essentially of an epitope specifically
bound by an antibody designated L106.
24. An antibody that specifically binds to an ACT-4-h-1 receptor
polypeptide.
25. The antibody of claim 24 that is a monoclonal antibody.
26. The antibody of claim 25 that inhibits activation of CD4.sup.+
T-cells.
27. The monoclonal antibody of claim 25 that stimulates activation
of CD4.sup.+ T-cells.
28. The antibody of claim 25 that competes with an antibody
designated L106 for specific binding to an ACT-4-h-1 receptor
polypeptide.
29. The antibody of claim 25 that competes with an antibody
designated L106 for specific binding to activated CD4.sup.+
T-cells.
30. The antibody of claim 25 that is fused to a coat protein of a
filamentous phage.
31. The antibody of claim 29 that is L106.
32. A humanized antibody comprising a humanized heavy chain and a
humanized light chain: (1) the humanized light chain comprising
three complementarity determining regions (CDR1, CDR2 and CDR3)
having amino acid sequences from the corresponding complementarity
determining regions of a L106 antibody light chain, and having a
variable region framework sequence substantially identical to a
human light chain variable region framework sequence; and (2) the
humanized heavy chain comprising three complementarity determining
regions (CDR1, CDR2 and CDR3) having amino acid sequences from the
corresponding complementarity determining regions of a L106
antibody heavy chain, and having a variable region framework
sequence substantially identical to a human heavy chain variable
region framework sequence; wherein the humanized antibody
specifically binds to an ACT-4-h-1 receptor polypeptide with a
binding affinity that is within three-fold of the binding affinity
of a L106 antibody.
33. An immunotoxin comprising the antibody of claim 31 fused to a
toxin polypeptide.
34. The antibody of claim 25 that specifically binds to a different
epitope on an ACT-4-h-1 receptor polypeptide than that specifically
bound by an L106 antibody.
35. A fragment of the antibody of claim 31 that specifically binds
to an ACT-4-h-1 receptor polypeptide.
36. A hybridoma producing antibody L106, said hybridoma deposited
as ATCC______.
37. A method of screening an antibody for specific binding to the
same epitope as that bound by an L106 antibody, said method
comprising: providing a solution comprising an antibody to be
screened, said L106 antibody, and an ACT-4-h-1 receptor
polypeptide, said polypeptide specifically binding to said L106
antibody; and measuring specific binding between said polypeptide
and said L106 antibody, or between said polypeptide and said
antibody to be screened, to indicate whether said antibody to be
screened reacts with the same epitope as said L106 antibody.
38. A method of localizing an epitope specifically bound by an L106
antibody, said method comprising: providing a family of ACT-4-h-1
receptor polypeptides, each member of said family comprising a
contiguous segment of at least four amino acids; and measuring
specific binding between a polypeptide from said family and said
L106 antibody to indicate the presence of said epitope within said
polypeptide.
39. A nucleic acid fragment encoding a heavy chain of an antibody
of claim 31.
40. A nucleic acid fragment encoding a light chain of an antibody
of claim 31.
41. A nucleic acid fragment encoding an ACT-4 polypeptide of claim
1.
42. The nucleic acid fragment of claim 41 that exhibits at least
eighty percent sequence identity with the nucleic acid sequence
shown in FIG. 5.
43. The nucleic acid fragment of claim 42 that encodes a
full-length ACT-4 polypeptide.
44. The nucleic acid fragment of claim 43 comprising the translated
region of the nucleic acid sequence shown in FIG. 5.
45. An isolated cell line containing a nucleic acid fragment of
claim 41.
46. The isolated cell line of claim 45, wherein the ACT-4 receptor
polypeptide is expressed on the surface of said cell.
47. The isolated cell line of claim 46 that is stable.
48. The isolated cell line of claim 47, wherein said nucleic acid
fragment is incorporated in the genome of said cell line.
49. The isolated cell line of claim 48, wherein said cell line is
COS-7.
50. A method of screening for immunosuppressive agents, said method
comprising: contacting an ACT-4-h-1 receptor polypeptide with a
potential immunosuppressive agent; and detecting specific binding
between said ACT-4-h-1 receptor polypeptide and said agent, said
specific binding indicative of immunosuppressive activity.
51. The method of claim 50, wherein said ACT-4 receptor polypeptide
is immobilized to a solid surface.
52. A method for screening for an ACT-4 ligand, said method
comprising: contacting a biological sample containing said ACT-4
ligand with an ACT-4-h-1 receptor polypeptide; isolating a complex
formed between said ligand and said ACT-4-h-1 receptor polypeptide;
and dissociating said complex to obtain said ligand.
53. A method of suppressing an immune response in a patient
suffering from an immune disease or condition, said method
comprising administering to a patient a therapeutically effective
dose of a pharmaceutical composition comprising a pharmaceutically
active carrier and a monoclonal antibody of claim 26.
54. A method of inducing an immune response to a selected antigen
comprising: administering a monoclonal antibody of claim 27 to a
patient; and exposing said patient to said selected antigen.
55. A method of detecting activated CD4.sup.+ T-cells, said method
comprising: contacting a tissue sample from a patient with a
monoclonal antibody of claim 24 and detecting specific binding
between said monoclonal antibody and said tissue sample to reveal
the presence of said activated CD4.sup.+ T-cells.
56. The method of claim 55, wherein the presence of activated
T-cells revealed by said specific binding is diagnostic of a
disease or condition of the immune system.
57. A pharmaceutical composition comprising a monoclonal antibody
of claim 26 and a pharmaceutically acceptable excipient.
58. An ACT-4 ligand that specifically binds to an ACT-4-h-1
receptor polypeptide.
Description
TECHNICAL FIELD
[0001] This invention relates generally to the isolation and
characterization of a cell-surface receptor, termed ACT-4, and
antibodies thereto, and the use of the antigen and antibodies for
monitoring and/or modulating immune responses.
BACKGROUND OF THE INVENTION
[0002] Immune responses are largely mediated by a diverse
collection of peripheral blood cells termed leukocytes. The
leukocytes include lymphocytes, granulocytes and monocytes.
Granulocytes are further subdivided into neutrophils, eosinophils
and basophils. Lymphocytes are further subdivided into T and B
lymphocytes. T-lymphocytes originate from lymphocytic-committed
stem cells of the embryo. Differentiation occurs in the thymus and
proceeds through prothymocyte, cortical thymocyte and medullary
thymocyte intermediate stages, to produce various types of mature
T-cells. These subtypes include CD8.sup.+ T cells (also known as
cytotoxic/suppressor T cells), which, when activated, have the
capacity to lyse target cells, and CD4.sup.+ T cells (also known as
T helper and T inducer cells), which, when activated, have the
capacity to stimulate other immune system cell types.
[0003] Immune system responses are elicited in several differing
situations. The most frequent response is as a desirable protection
against infectious microorganisms. However, undesired immune
response can occur following transplantation of foreign tissue, or
in an autoimmune disease, in which one of a body's own antigens is
the target for the immune response. Immune responses can also be
initiated in vitro by mitogens or antibodies against certain
receptors. In each of these situations, an immune response is
transduced from a stimulating event via a complex interaction of
leukocytic cell types. However, the participating cell types and
nature of the interaction between cell types may vary for different
stimulating events. For example, immune responses against invading
bacteria are often transduced by formation of complexes between an
MHC Class II receptor and a bacterial antigen, which then activate
CD4.sup.+ T-cells. By contrast, immune responses against viral
infections are principally transduced by formation of MHC Class
I/viral antigen complexes and subsequent activation of CD8.sup.+
cells.
[0004] Over recent years, many leukocyte cell surface antigens have
been identified, some of which have been shown to have a role in
signal transduction. It has been found that signals may be
transduced between a cell-surface receptor and either a soluble
ligand or a cell-surface-bound ligand. The amino acid sequences of
leukocyte surface molecules comprise a number of characteristic
recurring sequences or motifs. These motifs are predicted to be
related in evolution, have similar folding patterns and mediate
similar types of interactions. A number of superfamilies, including
the immunoglobulin and nerve growth factor receptor superfamilies,
have been described. Members of the nerve growth factor receptor
family include NGFR, found on neural cells; the B-cell antigen
CD40; the rat OX-40 antigen, found on activated CD4.sup.+ cells
(Mallet et al., EMBO J. 9:1063-1068 (1990) (hereby incorporated by
reference for all purposes); two receptors for tumor necrosis
factor (TNF), LTNFR-1 and TNFR-II, found on a variety of cell
types; 4-1BB found on T-cells; SFV-T2, an open reading frame in
Shope fibroma virus; and possibly fas, CD27 and CD30. See generally
Mallet & Barclay, Immunology Today 12:220-222 (1990) (hereby
incorporated by reference for all purposes).
[0005] The identification of cell-surface receptors has suggested
new agents for suppressing undesirable immune responses such as
transplant rejection, autoimmune disease and inflammation. Agents,
particularly antibodies, that block receptors of immune cells from
binding to soluble molecules or cell-bound receptors can impair
immune responses. Ideally, an agent should block only undesired
immune responses (e.g., transplant rejection) while leaving a
residual capacity to effect desirable responses (e.g., responsive
to pathogenic microorganisms). The immunosuppressive action of some
agents, for example, antibodies against the CD3 receptor and the
IL-2 receptor have already been tested in clinical trials. Although
some trials have shown encouraging results, significant problems
remain. First, a patient may develop an immune response toward the
blocking agent preventing continued immunosuppressive effects
unless different agents are available. Second, cells expressing the
target antigen may be able to adapt to the presence of the blocking
agent by ceasing to express the antigen, while retaining immune
functions. In this situation, continued treatment with a single
immunosuppressive agent is ineffective. Third, many targets for
therapeutic agents are located on more than one leukocyte subtype,
with the result that it is generally not possible to selectively
block or eliminate the response of only specific cellular subtypes
and thereby leave unimpaired a residual immune capacity for
combating infectious microorganisms.
[0006] Based on the foregoing it is apparent that a need exists for
additional and improved agents capable of suppressing immune
responses, particularly agents capable of selective suppression.
The present invention fulfills these and other needs, in part, by
providing a cellular receptor localized on activated human
CD4.sup.+ T-lymphocytes.
SUMMARY OF THE INVENTION
[0007] In one embodiment of the invention, purified ACT-4 receptor
polypeptides are provided. The amino acid sequence of one such
polypeptide, termed ACT-4-h-1, is shown in FIG. 5. ACT-4 receptor
polypeptides typically exhibit at least 80% amino acid sequence
identity to the ACT-4-h-1 amino acid sequence. The polypeptides
usually comprise at least one, and sometimes all of the following
domains: a signal sequence, an intracellular domain, a
transmembrane domain and an extracellular domain. Many polypeptides
are characterized by their presence on activated CD4.sup.+ T-cells
and their substantial absence on resting T-cells. Some full-length
polypeptides have a molecular weight of about 50 kDa before
deglycosylation and about 27 kDa thereafter.
[0008] The invention also provides extracellular domains of ACT-4
receptor polypeptides. The extracellular domains typically comprise
at least one disulfide-bonded loop and sometimes three such loops.
The extracellular domains are usually soluble and capable of
specific binding to an ACT-4 ligand. Sometimes an extracellular
domain is fused to a second polypeptide such as a constant region
of an immunoglobulin heavy chain. Some extracellular domains
consist essentially of an epitope specifically bound by an antibody
designated L106.
[0009] In another aspect of the invention, antibodies that
specifically bind to an ACT-4-h-1 receptor polypeptide are
provided. The antibodies are usually monoclonal antibodies. One
example of such an antibody is designated L106. Some antibodies
inhibit activation of CD4.sup.+ T-cells, whereas other antibodies
stimulate activation of these cells. Some antibodies of the
invention compete with the L106 antibody for specific binding to an
ACT-4-h-1 receptor polypeptide, and most of these antibodies also
compete with L106 for specific binding to activated CD4.sup.+
T-cells. Other antibodies of the invention specifically bind to a
different epitope than that bound by the L106 antibody. Also
provided are fragments of the L106 antibody that specifically bind
to an ACT-4-h-1 receptor polypeptide.
[0010] Also provided are humanized antibodies comprising a
humanized heavy chain and a humanized light chain. The humanized
light chain comprises three complementarity determining regions
(CDR1, CDR2 and CDR3) having amino acid sequences from the
corresponding complementarity determining regions of a L106
antibody light chain, and having a variable region framework
sequence substantially identical to a human light chain variable
region framework sequence. The humanized heavy chain comprises
three complementarity determining regions (CDR1, CDR2 and CDR3)
having amino acid sequences from the corresponding complementarity
determining regions of an L106 antibody heavy chain, and having a
variable region framework sequence substantially identical to a
human heavy chain variable region framework sequence. The humanized
antibodies specifically bind to an ACT-4-h-1 receptor polypeptide
with a binding affinity that is within three-fold of the binding
affinity of the L106 antibody.
[0011] In another aspect, the invention provides nucleic acids
fragments encoding the ACT-4 receptor polypeptides discussed supra.
An example of such a nucleic acid fragment comprises the nucleotide
sequence encoding the ACT-4-h-1 receptor shown in FIG. 5. The
nucleic acid fragments typically exhibit at least eighty percent
sequence identity to the nucleic acid sequence of FIG. 5.
[0012] The invention also provides isolated cell lines containing
the nucleic acid fragments discussed supra. The cell lines usually
express an ACT-4 receptor polypeptide on their cell surface. Some
of the cell lines are stable, as when the nucleic acid fragment is
incorporated in the genome of the cell line.
[0013] The invention also provides methods of screening for
immunosuppressive agents. An ACT-4-h-1 receptor polypeptide is
contacted with a potential immunosuppressive agent. Specific
binding between the ACT-4-h-1 receptor polypeptide or fragment and
the agent is then detected. The existence of specific binding is
indicative of immunosuppressive activity.
[0014] The invention also provides methods of screening for an
ACT-4 ligand. A biological sample containing the ACT-4 ligand is
contacted with an ACT-4-h-1 receptor polypeptide. A complex is
formed between the ligand and the ACT-4-h-1 receptor polypeptide.
The complex is then dissociated to obtain the ligand.
[0015] In another aspect, the invention provides methods of
suppressing an immune response in a patient suffering from an
immune disease or condition. A therapeutically effective dose of a
pharmaceutical composition is administered to the patient. The
pharmaceutical composition comprises a pharmaceutically active
carrier and a monoclonal antibody that specifically binds to an
ACT-4-h-1 receptor polypeptide.
[0016] Also provided are methods of detecting activated CD4.sup.+
T-cells. A tissue sample from a patient is contacted with a
monoclonal antibody that specifically binds to an ACT-4-h-1
receptor polypeptide. Specific binding between the monoclonal
antibody and the tissue sample is detected. The existence of
specific binding reveals the presence of activated CD4.sup.+
T-cells. The presence of activated CD4.sup.+ T-cells is often
diagnostic of a disease or condition of the immune system.
[0017] Also provided are methods of inducing an immune response to
a selected antigen. A monoclonal antibody that specifically binds
to an ACT-4-h-1 receptor polypeptide and that stimulates activation
of CD4.sup.+ T-cells is administered to a patient. The patient is
exposed to the selected antigen.
[0018] The invention also provides ACT-4 ligands that specifically
bind to an ACT-4-h-1 receptor polypeptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1: Two-color staining of peripheral blood lymphocytes
to analyze expression of ACT-4-h-1 on different cell types.
[0020] FIG. 2: Kinetics of ACT-4-h-1 expression on
alloantigen-activated CD4.sup.+ T-cells. MCF=Mean channel
fluorescence.
[0021] FIG. 3: Kinetics of ACT-4-h-1 expression on
tetanus-toxoid-activated CD4+ T-cells.
[0022] FIG. 4: Kinetics of ACT-4-h-1 expression on PHA-activated
CD4.sup.+ T-cells.
[0023] FIG. 5: cDNA (upper) and deduced amino acid sequence (lower)
of ACT-4-h-1. The Figure indicates the locations of an N-terminal
signal sequence, two possible signal cleavage sites (vertical
arrows), two glycosylation sites (gly), a transmembrane domain
(TM), a stop codon and a poly-A signal sequence.
[0024] FIG. 6: Construction of expression vector for production of
stable transfectants expressing ACT-4-h-1.
[0025] FIG. 7: FACS.TM. analysis showing expression of ACT-4-h-1 on
stable transfectants of COS-7, Jurkat and SP2/O cell lines.
[0026] FIG. 8: Fusion of an ACT-4-h-1 extracellular domain with an
immunoglobulin heavy chain constant region to form a recombinant
globulin.
[0027] FIG. 9: Schematic topographical representation of
recombinant globulin formed from fusion of an ACT-4-h-1
extracellular domain with an immunoglobulin heavy chain constant
region to form a recombinant globulin.
DEFINITIONS
[0028] Abbreviations for the twenty naturally occurring amino acids
follow conventional usage (Immunology--A Synthesis, (E. S. Golub
& D. R. Gren, eds., Sinauer Associates, Sunderland, MA, 2nd
ed., 1991) (hereby incorporated by reference for all purposes).
Stereoisomers (e.g., D-amino acids) of the twenty conventional
amino acids, unnatural amino acids such as
.alpha.,.alpha.-disubstituted amino acids, N-alkyl amino acids,
lactic acid, and other unconventional amino acids may also be
suitable components for polypeptides of the present invention.
Examples of unconventional amino acids include: 4-hydroxyproline,
.gamma.-carboxyglutamate, .epsilon.-N,N,N-trimethyllysine,
.epsilon.-N-acetyllysine, O-phosphoserine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
.omega.-N-methylarginine, and other similar amino acids and imino
acids (e.g., 4-hydroxyproline). In the polypeptide notation used
herein, the left-hand direction is the amino terminal direction and
the right-hand direction is the carboxy-terminal direction, in
accordance with standard usage and convention. Similarly, unless
specified otherwise, the lefthand end of single-stranded
polynucleotide sequences is the 5' end; the lefthand direction of
double-stranded polynucleotide sequences is referred to as the 5'
direction. The direction of 5' to 3' addition of nascent RNA
transcripts is referred to as the transcription direction; sequence
regions on the DNA strand having the same sequence as the RNA and
which are 5' to the 5' end of the RNA transcript are referred to as
"upstream sequences"; sequence regions on the DNA strand having the
same sequence as the RNA and which are 3' to the 3' end of the RNA
transcript are referred to as "downstream sequences".
[0029] The phrase "polynucleotide sequence" refers to a single or
double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. It includes self-replicating
plasmids, infectious polymers of DNA or RNA and non-functional DNA
or RNA.
[0030] The following terms are used to describe the sequence
relationships between two or more polynucleotides: "reference
sequence", "comparison window", "sequence identity", "percentage of
sequence identity", and "substantial identity". A "reference
sequence" is a defined sequence used as a basis for a sequence
comparison; a reference sequence may be a subset of a larger
sequence, for example, as a segment of a full-length cDNA or gene
sequence given in a sequence listing, such as a polynucleotide
sequence shown in FIG. 5, or may comprise a complete cDNA or gene
sequence. Generally, a reference sequence is at least 20
nucleotides in length, frequently at least 25 nucleotides in
length, and often at least 50 nucleotides in length. Since two
polynucleotides may each (1) comprise a sequence (i.e., a portion
of the complete polynucleotide sequence) that is similar between
the two polynucleotides, and (2) may further comprise a sequence
that is divergent between the two polynucleotides, sequence
comparisons between two (or more) polynucleotides are typically
performed by comparing sequences of the two polynucleotides over a
"comparison window" to identify and compare local regions of
sequence similarity. A "comparison window", as used herein, refers
to a conceptual segment of at least 20 contiguous nucleotide
positions wherein a polynucleotide sequence may be compared to a
reference sequence of at least 20 contiguous nucleotides and
wherein the portion of the polynucleotide sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
of 20 percent or less as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. Optimal alignment of sequences for aligning a
comparison window may be conducted by the local homology algorithm
of Smith & Waterman, Appl. Math. 2:482 (1981), by the homology
alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443
(1970), by the search for similarity method of Pearson &
Lipman, Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), by
computerized implementations of these algorithms (FASTDB
(Intelligenetics), BLAST (National Center for Biomedical
Information) or GAP, BESTFIT, FASTA, and TFASTA (Wisconsin Genetics
Software Package Release 7.0, Genetics Computer Group, 575 Science
Dr., Madison, Wis.)), or by inspection, and the best alignment
(i.e., resulting in the highest percentage of sequence similarity
over the comparison window) generated by the various methods is
selected. The term "sequence identity" means that two
polynucleotide sequences are identical (i.e., on a
nucleotide-by-nucleotide basis) over the window of comparison. The
term "percentage of sequence identity" is calculated by comparing
two optimally aligned sequences over the window of comparison,
determining the number of positions at which the identical nucleic
acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to
yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the window of
comparison (i.e., the window size), and multiplying the result by
100 to yield the percentage of sequence identity. The terms
"substantial identity" as used herein denotes a characteristic of a
polynucleotide sequence, wherein the polynucleotide comprises a
sequence that has at least 70, 80 or 85 percent sequence identity,
preferably at least 90 to 95 percent sequence identity, more
usually at least 99 percent sequence identity as compared to a
reference sequence over a comparison window of at least 20
nucleotide positions, frequently over a window of at least 25-50
nucleotides, wherein the percentage of sequence identity is
calculated by comparing the reference sequence to the
polynucleotide sequence which may include deletions or additions
which total 20 percent or less of the reference sequence over the
window of comparison. The reference sequence may be a subset of a
larger sequence, for example, as a segment of the full-length
ACT-4-h-1 sequence shown in FIG. 5.
[0031] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs BLAZE (Intelligenetics) GAP or BESTFIT using
default gap weights, share at least 70 percent or 80 percent
sequence identity, preferably at least 90 percent sequence
identity, more preferably at least 95 percent sequence identity or
more (e.g., 99 percent sequence identity). Preferably, residue
positions which are not identical differ by conservative amino acid
substitutions. Conservative amino acid substitutions refer to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Preferred conservative
amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine.
[0032] The term "substantially pure" means an object species is the
predominant species present (i.e., on a molar basis it is more
abundant than any other individual species in the composition), and
preferably a substantially purified fraction is a composition
wherein the object species comprises at least about 50 percent (on
a molar basis) of all macromolecular species present. Generally, a
substantially pure composition will comprise more than about 80 to
90 percent of all macromolecular species present in the
composition. Most preferably, the object species is purified to
essential homogeneity (contaminant species cannot be detected in
the composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species.
[0033] The term "naturally-occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally-occurring.
[0034] The term "epitope" includes any protein determinant capable
of specific binding to an immunoglobulin or T-cell receptor.
Specific binding exists when the dissociation constant for antibody
binding to an antigen is .ltoreq.1 .mu.M, preferably .ltoreq.100 nM
and most preferably .ltoreq.1 nM. Epitopic determinants usually
consist of chemically active surface groupings of molecules such as
amino acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics.
[0035] The term "higher cognate variants" as used herein refers to
a gene sequence that is evolutionarily and functionally related
between humans and higher mammalian species, such as primates,
porcines and bovines. The term does not include gene sequences from
rodents, such as rats. Thus, the cognate primate gene to the
ACT-4-h-1 gene is the primate gene which encodes an expressed
protein which has the greatest degree of sequence identity to the
ACT-4-h-1 receptor protein and which exhibits an expression pattern
similar to that of the ACT-4-h-1 protein (i.e., expressed on
activated CD4.sup.+ cells).
[0036] The term "patient" includes human and veterinary
subjects.
DETAILED DESCRIPTION
I. ACT-4 Receptor Polypeptides
[0037] According to one embodiment of the invention, receptors on
the surface of activated CD4.sup.+ T-cells (referred to as ACT-4
receptors) and fragments thereof are provided. The term ACT-4
receptor polypeptide is used generically to encompass full-length
receptors and fragments thereof. The amino acid sequence of the
first ACT-4 receptor to be characterized [hereinafter ACT-4-h-1] is
shown in FIG. 5. The suffix -h designates human origin and the
suffix -1 indicates that ACT-4-h-1 is the first ACT-4 receptor to
be characterized. The term ACT-4 receptor refers not only to the
protein having the sequence shown in FIG. 5, but also to other
proteins that represent allelic, nonallelic, and higher cognate
variants of ACT-4-h-1, and natural or induced mutants of any of
these. Usually, ACT-4 receptor polypeptides will also show
substantial sequence identity with the ACT-4-h-1 sequence.
Typically, an ACT-4 receptor polypeptide will contain at least 4
and more commonly 5, 6, 7, 10 or 20, 50 or more contiguous amino
acids from the ACT-4-h-1 sequence. It is well known in the art that
functional domains, such as binding domains or epitopes can be
formed from as few as four amino acids residues.
[0038] ACT-4 receptor polypeptides will typically exhibit
substantial amino acid sequence identity with the amino acid
sequence of ACT-4-h-1, and be encoded by nucleotide sequences that
exhibit substantial sequence identity with the nucleotide sequence
encoding ACT-4-h-1 shown in FIG. 5. The nucleotides encoding ACT-4
receptor proteins will also typically hybridize to the ACT-4-h-1
sequence under stringent conditions. However, these nucleotides
will not usually hybridize under stringent conditions to the
nucleic acid encoding OX-40 receptor, as described by Mallet et
al., EMBO J. 9:1063-68 (1990) (hereby incorporated by reference for
all purposes) (See particularly FIG. 2A of the Mallet et al.
reference). Stringent conditions are sequence dependent and will be
different in different circumstances. Generally, stringent
conditions are selected to be about 50.degree. C. lower than the
thermal melting point (Tm) for the specific sequence 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 is at
least about 0.02 molar at Ph 7 and the temperature is at least
about 60.degree. C. As 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 and the extent of base mismatching, the
combination of parameters is more important than the absolute
measure of any one.
[0039] Usually, ACT-4 receptor polypeptides will share at least one
antigenic determinant in common with ACT-4-h-1 but will not be
specifically reactive with antibodies against the rat OX-40
polypeptide. The existence of a common antigenic determinant is
evidenced by cross-reactivity of the variant protein with any
antibody prepared against ACT-4-h-1 (see Section IV).
Cross-reactivity is often tested using polyclonal sera against
ACT-4-h-1, but can also be tested using one or more monoclonal
antibodies against ACT-4-h-1, such as the antibody designated
L106.
[0040] Often ACT-4 receptor polypeptides will contain modified
polypeptide backbones. Modifications include chemical
derivatizations of polypeptides, such as acetylations,
carboxylations and the like. They also include glycosylation
modifications (N- and O-linked) and processing variants of a
typical polypeptide. These processing steps specifically include
enzymatic modifications, such as ubiquitinization and
phosphorylation. See, e.g., Hershko & Ciechanover, Ann. Rev.
Bioch. 51:335-364 (1982). The ACT-4-h-1 protein, for example, is
heavily modified in that the observed molecular weight is about 50
kDa, whereas the predicted molecular weight based on amino acid
sequence is only 27 kDa. Two putative glycosylation sites have been
identified in its extracellular domain.
[0041] ACT-4 receptors likely share some or all of the topological
features found for ACT-4-h-1. The amino acid sequence for ACT-4-h-1
contains a 22 or 24 amino acid putative N-terminal signal sequence.
The 24 amino acid sequence is more probably based on the criteria
of von Heijne, Nucleic Acids Res. 14: 4683-4690 (1986)
(incorporated by reference for all purposes). The ACT-4-h-1
receptor contains a single additional hydrophobic stretch of 27
amino acids spanning residues 213-240. The hydrophobic stretch
probably corresponds to a transmembrane domain and its existence is
consistent with ACT-4-h-1 being a type I integral membrane protein
(i.e., having a single transmembrane domain with the N-terminal
domain comprising the extracellular region and the C-terminus
comprising the intracellular region). The 189 or 191 amino acids
(depending on the exact location of the signal cleavage site) of
ACT-4-h-1 amino-proximal to the transmembrane segment are
designated the extracellular domain, while the 37 amino acids
carboxy-proximal to the transmembrane segment are designated the
intracellular domain. From the amino-terminus, the extracellular
domain has an NH.sub.2-terminal hydrophobic putative signal
sequence, and three intrachain loops formed by disulfide bonding
between paired cysteine residues.
[0042] The topological arrangement of ACT-4 receptor polypeptides
is similar to that of other members of the nerve growth factor
receptor family, particularly to the rat OX-40 receptor. However,
the other members show some divergence in the number of
extracellular disulfide loops and glycosylation sites and in the
size of the intracellular domain. See Mallet & Barclay,
supra.
[0043] Although not all of the domains discussed above are
necessarily present in all ACT-4 receptor polypeptides, an
extracellular domain is expected to be present in most. Indeed, in
some ACT-4 receptor polypeptides, it is possible that only an
extracellular domain is present, and the natural state of such
proteins is not as cell-surface bound proteins, but as soluble
proteins, for example, dispersed in an extracellular body fluid.
The existence of soluble variant forms has been observed for other
cell surface receptors, including one member of the nerve growth
factor receptor family, SFV-T2. See Mallet & Barclay,
supra.
[0044] Besides substantially full-length polypeptides, the present
invention provides for biologically active fragments of the
polypeptides. Significant biological activities include receptor
binding, antibody binding (e.g., the fragment competes with an
intact ACT-4 receptor for specific binding to an antibody),
immunogenicity (i.e., possession of epitopes that stimulate B or T
cell responses against the fragment), and agonism or antagonism of
the binding of an ACT-4 receptor polypeptide to its ligand. A
segment of an ACT-4 receptor protein or a domain thereof will
ordinarily comprise at least about 5, 7, 9, 11, 13, 16, 20, 40, or
100 contiguous amino acids.
[0045] Segments of ACT-4 receptor polypeptides are often terminated
near boundaries of functional or structural domains. Structural and
functional domains are identified by comparison of nucleotide
and/or amino acid sequence data such as is shown in FIG. 5 to
public or proprietary sequence databases. Preferably, computerized
comparison methods are used to identify sequence motifs or
predicted protein conformation domains that occur in other proteins
of known structure and/or function. Structural domains include an
intracellular domain, transmembrane domain, and extracellular
domain, which is in turn contains three disulfide-bonded loops.
Functional domains include an extracellular binding domain through
which the ACT-4 receptor polypeptide interacts with external
soluble molecules or other cell-bound ligands and an intracellular
signal-transducing domain.
[0046] Some fragments will contain only extracellular domains, such
as one or more disulfide-bonded loops. Such fragments will often
retain the binding specificity of an intact ACT-4 receptor
polypeptide, but will be soluble rather than membrane bound. Such
fragments are useful as competitive inhibitors of ACT-4 receptor
binding.
[0047] ACT-4 receptors are further identified by their status as
members of the nerve growth factor receptor family. The amino acid
sequence of ACT-4-h-l is at least 20% identical to NGF-R, TNF-R,
CD40, 4-1BB, and fas/AP01. ACT-4-h-1 exhibits 62% amino acid
sequence identity with the rat OX-40 gene, which is also
characterized by selective expression on activated CD4.sup.+
cells.
[0048] ACT-4 receptors are also identified by a characteristic
cellular distribution. Most notably, ACT-4 receptors are usually
easily detected on activated CD4.sup.+ T cells (percent cells
expressing usually greater than about 25 or 50% and often about
80%; mean channel fluorescence usually greater than about 10 and
often about 20-25, on a Coulter Profile Flow Cytometer after
immunofluorescence staining). ACT-4 receptors are usually
substantially absent on resting T-cells, B-cells (unless activated
with PMA), NK cells, and monocytes (unless activated with PMA).
Substantially absent means that the percentage of cells expressing
ACT-4 is usually less than about 5%, and more usually less than
about 2%, and that the mean channel is usually less than about 4,
and more usually less than about 2, measured on a Coulter Profile
Flow Cytometer, after immunofluoresence staining of the cells. (See
Example 2) ACT-4 receptors are usually expressed at low levels on
activated CD8.sup.+ cells (percent cells expressing about 4-10%;
mean channel fluorescence about 2-4 on a Coulter Profile Flow
Cytometer after immunofluoresence staining). The low level of
expression observed on CD8.sup.+ cells suggests that expression is
confined to a subpopulation of CD8.sup.+ cells. The expression of
ACT-4 receptors on the surface of activated CD4.sup.+ cells has
been observed for several different mechanisms of activation,
including alloantigenic, tetanus toxoid or mitogenic (e.g., PHA)
stimuli. Expression peaks after about 7 days of allogantigenic or
tetanus toxoid stimulation and after about three days of PHA
stimulation. These data indicate that ACT-4 receptors should be
classified as early activation antigens that are substantially
absent on resting cells. The observation that ACT-4 receptors are
preferentially expressed on activated CD4.sup.+ cells and are
expressed to a much lesser extent on activated CD8.sup.+ cells, but
are substantially absent on most or all other subtypes of lymphoid
cells (except in response to highly nonphysiological stimuli such
as PMA) contrasts with the cell type specificity of other
activation antigens found on human leukocytes.
[0049] The expression of ACT-4 receptors on the surface of
activated CD4.sup.+ T cells suggests that the receptor has a role
in activation of these cells. Such a role is consistent with that
of some other members of the nerve growth factor receptor family.
For example, CD40 stimulates the G1-S phase transition in B
lymphocytes, and nerve growth factor receptor transduces a signal
from the cytokine nerve growth factor., which results in neuronal
differentiation and survival (Barde, Y-A. Neuron 2: 1525-1534
(1989)) (incorporated by reference for all purposes). However,
other roles for ACT-4 receptors can also be envisaged, for example,
interaction with other lymphoid cell types. The existence of such
roles is consistent with the diverse functions of other nerve
growth factor receptor family members, such as tumor necrosis
factor, whose interaction with tumor necrosis factor receptor can
result in inflammation or tumor cell death.
[0050] Fragments or analogs comprising substantially one or more
functional domain (e.g., an extracellular domain) of ACT-4
receptors can be fused to heterologous polypeptide sequences, such
that the resultant fusion protein exhibits the functional
property(ies) conferred by the ACT-4 receptor fragment and/or the
fusion partner. The orientation of the ACT-4 receptor fragment
relative to the fusion partner will depend on experimental
considerations such as ease of construction, stability to
proteolysis, thermal stability, immunological reactivity, amino- or
carboxyl-terminal residue modification, and so forth. Potential
fusion partners include chromogenic enzymes such as
.beta.-galactosidase, protein A or G, a FLAG protein such as
described by Blanar & Rutter, Science 256:1014-1018 (1992),
toxins (e.g., diphtheria toxin, Psuedonomas ectotoxin A, ricin
toxin or phospholipase C) and immunoglobulin components.
[0051] Recombinant globulins (Rg) formed by fusion of ACT-4
receptor fragments and immunoglobulin components often have most or
all of the physiological properties associated with the constant
region of the particular immunoglobulin class used. For example,
the recombinant globulins may be capable of fixing complement,
mediating antibody dependent cell toxicity, stimulating B cells, or
traversing blood vessel walls and entering the interstitial space.
The recombinant globulins are usually formed by fusing the
C-terminus of an ACT-4 receptor extracellular domain to the
N-terminus of the constant region domain of a heavy chain
immunoglobulin, thereby simulating the conformation of an authentic
immunoglobulin chain. The immunoglobulin chain is preferably of
human origin, particularly if the recombinant globulin is intended
for therapeutic use. Recombinant globulins are usually soluble and
have a number of advantageous properties relative to unmodified
ACT-4 receptors. These properties include prolonged serum
half-life, the capacity to lyse target cells for which an ACT-4
receptor has affinity, by effector functions, and the capacity to
bind molecules such as protein A and G, which can be used to
immobilize the recombinant globulin in binding analyses.
II. Methods of Producing Polypeptides
[0052] A. Recombinant Technologies
[0053] The nucleotide and amino acid sequences of ACT-4-h-1 shown
in FIG. 5, and corresponding sequences for other ACT-4 receptor
variants obtained as described in Section III, infra, allow
production of polypeptides of full-length ACT-4 receptor
polypeptides sequences and fragments thereof. Such polypeptides may
be produced in prokaryotic or eukaryotic host cells by expression
of polynucleotides encoding ACT-4 receptor, or fragments and
analogs thereof. The cloned DNA sequences are expressed in hosts
after the sequences have been operably linked to (i.e., positioned
to ensure the functioning of) an expression control sequence in an
expression vector. Expression vectors are typically replicable in
the host organisms either as episomes or as an integral part of the
host chromosomal DNA. Commonly, expression vectors will contain
selection markers, e.g., tetracycline resistance or hygromycin
resistance, to permit detection and/or selection of those cells
transformed with the desired DNA sequences (see, e.g., U.S. Pat.
No. 4,704,362).
[0054] E. coli is one prokaryotic host useful for cloning the DNA
sequences of the present invention. Other microbial hosts suitable
for use include bacilli, such as Bacillus subtilis, and other
Enterobacteriaceae, such as Salmonella, Serratia, and various
Pseudomonas species. In these prokaryotic hosts, one can also make
expression vectors, which will typically contain expression control
sequences compatible with the host cell (e.g., an origin of
replication). In addition, any number of a variety of well-known
promoters will be present, such as the lactose promoter system, a
tryptophan (trp) promoter system, a beta-lactamase promoter system,
or a promoter system from phage lambda. The promoters will
typically control expression, optionally with an operator sequence,
and have ribosome binding site sequences and the like, for
initiating and completing transcription and translation.
[0055] Other microbes, such as yeast, may also be used for
expression. Saccharomyces is a preferred host, with suitable
vectors having expression control sequences, such as promoters,
including 3-phosphoglycerate kinase or other glycolytic enzymes,
and an origin of replication, termination sequences and the like as
desired. Insect cells (e.g., SF9) with appropriate vectors, usually
derived from baculovirus, are also suitable for expressing ACT-4
receptor or ligand polypeptides. See Luckow, et al. Bio/Technology
6:47-55 (1988) (incorporated by reference for all purposes).
[0056] Higher eukaryotic mammalian tissue cell culture may also be
used to express and produce the polypeptides of the present
invention (see Winnacker, From Genes to Clones (VCH Publishers, NY,
N.Y., 1987)) (incorporated by reference for all purposes).
Eukaryotic cells are actually preferred, because a number of
suitable host cell lines capable of secreting and authentically
modifying human proteins have been developed in the art, and
include the CHO cell lines, various COS cell lines, HeLa cells,
myeloma cell lines, Jurkat cells, etc. Expression vectors for these
cells can include expression control sequences, such as an origin
of replication, a promoter (e.g., a HSV tk promoter or pgk
(phosphoglycerate kinase) promoter), an enhancer (Queen et al.,
Immunol. Rev. 89:49 (1986)), and necessary processing information
sites, such as ribosome binding sites, RNA splice sites,
polyadenylation sites (e.g., an SV40 large T Ag poly A addition
site), and transcriptional terminator sequences. Preferred
expression control sequences are promoters derived from
immunoglobulin genes, SV40, adenovirus, bovine papillomavirus, and
the like. The vectors containing the DNA segments of interest
(e.g., polypeptides encoding an ACT-4 receptor) can be transferred
into the host cell by well-known methods, which vary depending on
the type of cellular host. For example, CaCl.sub.2 transfection is
commonly utilized for prokaryotic cells, whereas CaPO.sub.4
treatment or electroporation may be used for other cellular hosts.
Vectors may exist as episome or integrated into the host
chromosome.
[0057] B. Naturally Occurring ACT-4 Receptor Proteins
[0058] Natural ACT-4 receptor polypeptides are isolated by
conventional techniques such as affinity chromatography. For
example, polyclonal or monoclonal antibodies are raised against
previously-purified ACT-4-h-1 and attached to a suitable affinity
column by well known techniques. See, e.g., Hudson & Hay,
Practical Immunology (Blackwell Scientific Publications, Oxford,
UK, 1980), Chapter 8 (incorporated by reference for all purposes).
For example, anti-ACT-4-h-1 can be immobilized to a protein-A
sepharose column via crosslinking of the F.sub.c domain with a
homobifunctional crosslinking agent, such as dimethyl pimelimidate.
Cell extracts are then passed through the column, and ACT-4
receptor protein specifically bound by the column, eluted with, for
example, 0.5 M pyrogenic acid, pH 2.5. Usually, an intact form of
ACT-4 receptor is obtained by such isolation techniques. Peptide
fragments are generated from intact ACT-4 receptors by chemical
(e.g., cyanogen bromide) or enzymatic cleavage (e.g., V8 protease
or trypsin) of the intact molecule.
[0059] C. Other Methods
[0060] Alternatively, ACT-4 receptor polypeptides can be
synthesized by chemical methods or produced by in vitro translation
systems using a polynucleotide template to direct translation.
Methods for chemical synthesis of polypeptides and in vitro
translation are well known in the art, and are described further by
Berger & Kimmel, Methods in Enzymology, Volume 152, Guide to
Molecular Cloning Techniques Academic Press, Inc., San Diego,
Calif., 1987).
III. Nucleic Acids
[0061] A. Cloning ACT-4 Receptor Nucleic Acids
[0062] Example 5 presents nucleic acid sequence data for a cDNA
clone of an ACT-4 receptor designated ACT-4-h-1. The sequence
includes both a translated region and 3' and 5' flanking regions.
This sequence data can be used to design probes with which to
isolate other ACT-4 receptor genes. These genes include the human
genomic gene encoding ACT-4-h-1, and cDNAs and genomic clones from
higher mammalian species, and allelic and nonallelic variants, and
natural and induced mutants of all of these genes. Specifically,
all nucleic acid fragments encoding all ACT-4 receptor polypeptides
disclosed in this application are provided. Genomic libraries of
many species are commercially available (e.g., Clontech, Palo Alto,
Calif.), or can be isolated de novo by conventional procedures.
cDNA libraries are best prepared from activated CD4.sup.+ cells,
which express ACT-4-h-1 in large amounts.
[0063] The probes used for isolating clones typically comprise a
sequence of about at least 24 contiguous nucleotides (or their
complement) of the cDNA sequence shown in FIG. 5. For example, a
full-length polynucleotide corresponding to the sequence shown in
FIG. 5 can be labeled and used as a hybridization probe to isolate
genomic clones from a human genomic clone library in e.g.,
.lamda.EMBL4 or .lamda.GEM11 (Promega Corporation, Madison, Wis.);
typical hybridization conditions for screening plaque lifts (Benton
& Davis, Science 196:180 (1978)) can be: 50% formamide,
5.times.SSC or SSPE, 1-5.times. Denhardt's solution, 0.1-1% SDS,
100-200 .mu.g sheared heterologous DNA or tRNA, 0-10 % dextran
sulfate, 1.times.10.sup.5 to 1.times.10.sup.7 cpm/ml of denatured
probe with a specific activity of about 1.times.10.sup.8 cpm/.mu.g,
and incubation at 42.degree. C. for about 6-36 hours.
Prehybridization conditions are essentially identical except that
probe is not included and incubation time is typically reduced.
Washing conditions are typically 1-3.times.SSC, 0.1-1% SDS,
50-70.degree. C. with change of wash solution at about 5-30
minutes. Hybridization and washing conditions are typically less
stringent for isolation of higher cognate or nonallelic variants
than for e.g., the human genomic clone of ACT-4-h-1.
[0064] Alternatively, probes can be used to clone ACT-4 receptor
genes by methods employing the polymerase chain reaction (PCR).
Methods for PCR amplification are described in e.g., PCR
Technology: Principles and Applications for DNA Amplification (ed.
H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A
Guide to Methods and Applications (eds. Innis, et al., Academic
Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res.
19:4967 (1991); Eckert, K. A. and Kunkel, T. A., PCR Methods and
Applications 1:17 (1991); PCR (eds. McPherson et al., IRL Press,
Oxford); and U.S. Pat. No. 4,683,202 (each of which is incorporated
by reference for all purposes).
[0065] Alternatively, synthetic polynucleotide sequences
corresponding to all or part of the sequences shown in FIG. 5 may
be constructed by chemical synthesis of oligonucleotides.
[0066] Nucleotide substitutions, deletions, and additions can be
incorporated into the polynucleotides of the invention. Nucleotide
sequence variation may result from degeneracy of the genetic code,
from sequence polymorphisms of various ACT-4 receptor alleles,
minor sequencing errors, or may be introduced by random mutagenesis
of the encoding nucleic acids using irradiation or exposure to EMS,
or by changes engineered by site-specific mutagenesis or other
techniques of modern molecular biology. See Sambrook et al.,
Molecular Cloning: A Laboratory Manual (C.S.H.P. Press, NY 2d ed.,
1989) (incorporated by reference for all purposes). For nucleotide
sequence that are capable of being transcribed and translated to
produce a functional polypeptide, degeneracy of the genetic code
results in a number of nucleotide sequences that encode the same
polypeptide. The invention includes all such sequences. Generally,
nucleotide substitutions, deletions, and additions should not
substantially disrupt the ability of an ACT-4 receptor
polynucleotide to hybridize to the sequence of ACT-4-h-1 shown in
FIG. 5 under stringent conditions. Typically, ACT-4 receptor
polynucleotides comprise at least 25 consecutive nucleotides which
are substantially identical to a naturally-occurring ACT-4 receptor
sequence (e.g., FIG. 5), more usually ACT-4 receptor
polynucleotides comprise at least 50 to 100 consecutive
nucleotides, which are substantially identical to a
naturally-occurring ACT-4 receptor sequence.
[0067] ACT-4 receptor polynucleotides can be short oligonucleotides
(e.g., about 10, 15, 25, 50 or 100 contiguous bases from the
ACT-h-1 sequence shown in FIG. 5), such as for use as hybridization
probes and PCR (or LCR) primers. ACT-4 receptor polynucleotide
sequences can also comprise part of a larger polynucleotide that
includes sequences that facilitate transcription (expression
sequences) and translation of the coding sequences, such that the
encoded polypeptide product is produced. Construction of such
polynucleotides is well known in the art and is described further
in Sambrook et al., supra (C.S.H.P. Press, NY 2d ed. 1989). The
ACT-4 receptor polynucleotide can be fused in frame with another
polynucleotide sequence encoding a different protein (e.g.,
glutathione S-transferase, .beta.-galactosidase or an
immunoglobulin F.sub.C domain) for encoding expression of a fusion
protein (see, e.g., Byrn et al., Nature, 344:667-670 (1990))
(incorporated by reference for all purposes).
IV. Antibodies and Hybridomas
[0068] In another embodiment of the invention, antibodies against
ACT-4 receptors and to their ligands (see Section V) are
provided.
[0069] A. General Characteristics of Antibodies
[0070] Antibodies or immunoglobulins are typically composed of four
covalently bound peptide chains. For example, an IgG antibody has
two light chains and two heavy chains. Each light chain is
covalently bound to a heavy chain. In turn each heavy chain is
covalently linked to the other to form a "Y" configuration, also
known as an immunoglobulin conformation. Fragments of these
molecules, or even heavy or light chains alone, may bind antigen.
Antibodies, fragments of antibodies, and individual chains are also
referred to herein as immunoglobulins.
[0071] A normal antibody heavy or light chain has an N-terminal
(NH.sub.2) variable (V) region, and a C-terminal (--COOH) constant
(C) region. The heavy chain variable region is referred to as
V.sub.H (including, for example, V.sub..gamma.), and the light
chain variable region is referred to as V.sub.L (including
V.sub..kappa. or V.sub..gamma.). The variable region is the part of
the molecule that binds to the antibody's cognate antigen, while
the Fc region (the second and third domains of the C region)
determines the antibody's effector function (e.g., complement
fixation, opsonization). Full-length immunoglobulin or antibody
"light chains" (generally about 25 kDa, about 214 amino acids) are
encoded by a variable region gene at the N-terminus (generally
about 110 amino acids) and a .kappa. (kappa) or .lamda. (lambda)
constant region gene at the COOH-terminus. Full-length
immunoglobulin or antibody "heavy chains" (generally about 50 Kd,
about 446 amino acids), are similarly encoded by a variable region
gene (generally encoding about 116 amino acids) and one of the
constant region genes, e.g., gamma (encoding about 330 amino
acids). Typically, the "V.sub.L" will include the portion of the
light chain encoded by the V.sub.L and/or J.sub.L (J or joining
region) gene segments, and the "V.sub.H" will include the portion
of the heavy chain encoded by the V.sub.H, and/or D.sub.H (D or
diversity region) and J.sub.H gene segments. See, generally, Roitt
et al., Immunology (2d ed. 1989), Chapter 6 and Paul, Fundamental
Immunology (Raven Press, 2d ed., 1989) (each of which is
incorporated by reference for all purposes).
[0072] An immunoglobulin light or heavy chain variable region
consists of a "framework" region interrupted by three hypervariable
regions, also called complementarity-determining regions or CDRs.
The extent of the framework region and CDRs have been defined (see
Kabat et al. (1987), "Sequences of Proteins of Immunological
Interest," U.S. Department of Health and Human Services; Chothia et
al., J. Mol. Biol. 196:901-917 (1987) (each of which is
incorporated by reference for all purposes). The sequences of the
framework regions of different light or heavy chains are relatively
conserved within a species. The framework region of an antibody,
that is the combined framework regions of the constituent light and
heavy chains, serves to position and align the CDRs in three
dimensional space. The CDRs are primarily responsible for binding
to an epitope of an antigen. The CDRs are typically referred to as
CDR1, CDR2, and CDR3, numbered sequentially starting from the
N-terminus.
[0073] The constant region of the heavy chain molecule, also known
as C.sub.H, determines the isotype of the antibody. Antibodies are
referred to as IgM, IgD, IgG, IgA, and IgE depending on the heavy
chain isotype. The isotypes are encoded in the mu (.mu.), delta
(.DELTA.), gamma (.gamma.), alpha (.alpha.), and epsilon
(.epsilon.) segments of the heavy chain constant region,
respectively. In addition, there are a number of .epsilon.
subtypes. There are two types of light chains, .kappa. and .lamda..
The determinants of these subtypes typically reside in the constant
region of the light chain, also referred to as the C.sub.L in
general, and C.sub..kappa. or C.sub..lamda. in particular.
[0074] The heavy chain isotypes determine different effector
functions of the antibody, such as opsonization or complement
fixation. In addition, the heavy chain isotype determines the
secreted form of the antibody. Secreted IgG, IgD, and IgE isotypes
are typically found in single unit or monomeric form. Secreted IgM
isotype is found in pentameric form; secreted IgA can be found in
both monomeric and dimeric form.
[0075] B. Production of Antibodies
[0076] Antibodies which bind either an ACT-4 receptor, a ligand
thereto, or binding fragments of either, can be produced by a
variety of means. The production of non-human monoclonal
antibodies, e.g., murine, rat and so forth, is well known and may
be accomplished by, for example, immunizing the animal with a
preparation containing an ACT-4 receptor or its ligands, or an
immunogenic fragment of either of these. Particularly, useful as
immunogens are cells stably transfected with recombinant ACT-4
receptor genes and expressing ACT-4 receptors on their cell
surface. Antibody-producing cells obtained from the immunized
animals are immortalized and screened for the production of an
antibody which binds to ACT-4 receptors or their ligands. See
Harlow & Lane, Antibodies, A Laboratory Manual (C.S.H.P. NY,
1988) (incorporated by reference for all purposes).
[0077] Several techniques for generation of human monoclonal
antibodies have also been described but are generally more onerous
than murine techniques and not applicable to all antigens. See,
e.g., Larrick et al., U.S. Pat. No. 5,001,065, for review
(incorporated by reference for all purposes). One technique that
has successfully been used to generate human monoclonal antibodies
against a variety of antigens is the trioma methodology of Ostberg
et al. (1983), Hybridoma 2:361-367, Ostberg, U.S. Pat. No.
4,634,664, and Engleman et al., U.S. Pat. No. 4,634,666
(incorporated by reference for all purposes). The
antibody-producing cell lines obtained by this method are called
triomas, because they are descended from three cells--two human and
one mouse. Triomas have been found to produce antibody more stably
than ordinary hybridomas made from human cells.
[0078] An alternative approach is the generation of humanized
immunoglobulins by linking the CDR regions of non-human antibodies
to human constant regions by recombinant DNA techniques. See Queen
et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO
90/07861 (incorporated by reference for all purposes). The
humanized immunoglobulins have variable region framework residues
substantially from a human immunoglobulin (termed an acceptor
immunoglobulin) and complementarity determining regions
substantially from a mouse immunoglobulin, e.g., the L106 antibody
(referred to as the donor immunoglobulin). The constant region(s),
if present, are also substantially from a human immunoglobulin. The
human variable domains are usually chosen from human antibodies
whose framework sequences exhibit a high degree of sequence
identity with the murine variable region domains from which the
CDRs were derived. The heavy and light chain variable region
framework residues can be derived from the same or different human
antibody sequences. The human antibody sequences can be the
sequences of naturally occurring human antibodies or can be
consensus sequences of several human antibodies. See Carter et al.,
WO 92/22653. Certain amino acids from the human variable region
framework residues are selected for substitution based on their
possible influence on CDR conformation and/or binding to antigen.
Investigation of such possible influences is by modeling,
examination of the characteristics of the amino acids at particular
locations, or empirical observation of the effects of substitution
or mutagenesis of particular amino acids.
[0079] For example, when an amino acid differs between a murine
L106 variable region framework residue and a selected human
variable region framework residue, the human framework amino acid
should usually be substituted by the equivalent framework amino
acid from the mouse antibody when it is reasonably expected that
the amino acid:
[0080] (1) noncovalently binds antigen directly,
[0081] (2) is adjacent to a CDR region,
[0082] (3) otherwise interacts with a CDR region (e.g. is within
about 3A of a CDR region), or
[0083] (3) participates in the VL-VH interface.
[0084] Other candidates for substitution are acceptor human
framework amino acids that are unusual for a human immunoglobulin
at that position. These amino acids can be substituted with amino
acids from the equivalent position of the L106 antibody or from the
equivalent positions of more typical human immunoglobulins.
[0085] A further approach for isolating DNA sequences which encode
a human monoclonal antibody or a binding fragment thereof is by
screening a DNA library from human B cells according to the general
protocol outlined by Huse et al., Science 246:1275-1281 (1989) and
then cloning and amplifying the sequences which encode the antibody
(or binding fragment) of the desired specificity. The protocol
described by Huse is rendered more efficient in combination with
phage display technology. See, e.g., Dower et al., WO 91/17271 and
McCafferty et al., WO 92/01047. Phage display technology can also
be used to mutagenize CDR regions of antibodies previously shown to
have affinity for ACT-4 receptors or their ligands. Antibodies
having improved binding affinity are selected.
[0086] Anti-ACT-4 receptor antibodies that specifically bind to the
same epitope as the L106 antibody are usually identified by a
competitive binding assay. The assay has three components, an ACT-4
polypeptide (e.g., ACT-4-h-1), L106 antibody, which is usually
labelled, and the antibody under test. Often the ACT-4 receptor
polypeptide is immobilized to a solid support. The test antibody
binds to the same epitope as the L106 antibody if it reduces the
amount of L106 antibody that specifically binds to the ACT-4
receptor polypeptide. The extent of screening necessary to obtain
such antibodies can be reduced by generating antibodies with a
protocol in which the specific epitope bound by L106 is used as an
immunogen. Antibodies binding to the same epitope as L106 may
exhibit a substantially, but not completely, identical amino acid
sequence to the L106 antibody, or may have an unrelated primary
structure to the L106 antibody.
[0087] Anti-ACT-4 receptor antibodies having a different binding
specificity than L106 (i.e., which bind to a different epitope) are
identified by a complementary approach. Test antibodies are
screened for failure to compete with the L106 antibody for binding
to an ACT-4 receptor polypeptide. The extent of screening can be
reduced by generating antibodies with a protocol in which a
fragment lacking a specific epitope bound by L106 is used as an
immunogen.
[0088] Antibodies having the capacity to stimulate or inhibit
activation of CD4.sup.+ cells can be identified by the screening
procedures discussed in Section VI, infra. Some antibodies may
selectively inhibit activation in response to some stimuli (e.g.,
alloantigenic but not mitogenic, or vice versa), and not to others.
Some antibodies' inhibitory capacity may depend on the time after
activation at which the antibody is added. Some antibodies may have
the capacity to activate CD4.sup.+ cells independently of other
stimuli, whereas other anti-ACT-4 receptor antibodies may only have
the capacity to augment the efficacy of another stimulus such as
that provided by PHA.
[0089] Antibodies isolated by the above procedures can be used to
generate anti-idiotypic antibodies by, for example, immunization of
an animal with the primary antibody. For anti-ACT-4 receptor
antibodies, anti-idiotype antibodies whose binding to the primary
antibody is inhibited by ACT-4 receptors or fragments thereof are
selected. Because both the anti-idiotypic antibody and the ACT-4
receptors or fragments thereof bind the primary immunoglobulin, the
anti-idiotypic immunoglobulin may represent the "internal image" of
an epitope and thus may substitute for the ACT-4 ligand.
[0090] C. Epitope Mapping
[0091] The epitope bound by the L106 or any other anti-ACT-4
receptor antibody is determined by providing a family of fragments
containing different amino acid segments from an ACT-4 receptor
polypeptide, such as ACT-4-h-1. Each fragment typically comprises
at least 4, 6, 8, 10, 20, 50 or 100 contiguous amino acids.
Collectively, the family of polypeptide covers much or all of the
amino acid sequence of a full-length ACT-4 receptor polypeptide.
Members of the family are tested individually for binding to e.g.,
the L106 antibody. The smallest fragment that can specifically bind
to the antibody under test delineates the amino acid sequence of
the epitope recognized by the antibody.
[0092] D. Fragments of Antibodies, and Immunotoxins
[0093] In another embodiment of the invention, fragments of
antibodies against ACT-4 receptors or their ligands are provided.
Typically, these fragments exhibit specific binding to the ACT-4
receptor with an affinity of at least 10.sup.7 M, and more
typically 10.sup.8 or 10.sup.9 M. Antibody fragments include
separate heavy chains, light chains Fab, Fab' F(ab').sub.2, Fabc,
and Fv. Fragments are produced by recombinant DNA techniques, or by
enzymic or chemical separation of intact immunoglobulins.
[0094] In another embodiment, immunotoxins are provided. An
immunotoxin is a chimeric compound consisting of a toxin linked to
an antibody having a desired specificity. The antibody serves as a
targeting agent for the toxin. See generally Pastan et al., Cell
47:641-648 (1986). A toxin moiety is couple to an intact antibody
or a fragment thereof by chemical or recombinant DNA techniques.
Preferably, the toxin is linked to an immunoglobulin chain in the
form of a contiguous protein. See, e.g. Chovnick et al., Cancer
Res. 51:465; Chaudhary et al., Nature 339:394 (1989) (incorporated
by reference for all purposes). Examples of suitable toxin
components are listed in Section I, supra, and are reviewed in
e.g., The Specificity and Action of Animal, Bacterial and Plant
Toxins (ed. P. Cuatrecasas, Chapman Hall, London, 1976)
(incorporated by reference for all purposes).
[0095] E. Hybridomas and Other Cell Lines
[0096] All hybridomas, triomas and other cell lines producing the
antibodies and their fragments discussed, supra, are expressly
included in the invention. These include the hybridoma line HBL106,
deposited as ATCC______ , which produces the L106 mouse
antibody.
[0097] F. Uses of Antibodies
[0098] Anti-ACT-4 receptor antibodies and their binding fragments
are useful for screening cDNA expression libraries, preferably
containing human or primate cDNA derived from various tissues and
for identifying clones containing cDNA inserts, which encode
structurally-related, immunocrossreactive proteins. See Aruffo
& Seed, Proc. Natl. Acad. Sci. USA 84:8573-8577 (1987)
(incorporated by reference for all purposes). Antibodies are also
useful to identify and/or purify immunocrossreactive proteins that
are structurally or evolutionarily related to the native ACT-4
receptor polypeptides or to fragments thereof used to generate the
antibody. Antibodies against ACT-4 ligands are analogously useful
in isolating further ligands and variants thereof. Diagnostic and
therapeutic uses of antibodies, binding fragments thereof,
immunotoxins and idiotypic antibodies are described in Section VII,
infra.
V. ACT-4 Ligands
[0099] The term ACT-4 ligand is used to denote a protein that
specifically binds to an ACT-4 receptor polypeptide and that is
capable of forming a complex with such a polypeptide, at least in
part, by noncovalent binding. Ligands can be naturally-occurring or
synthetic molecules, and can be in soluble form or anchored to the
surface of a cell. Multiple different ligands may bind the same
ACT-4 receptor. Conversely, one ligand may bind to more than one
ACT-4 receptor. The term "ACT-4 ligand" does not usually include
antibodies to ACT-4 receptor polypeptides. Usually, binding of a
ligand to an ACT-4 receptor will initiate a signal that alters the
physical and/or functional phenotype of a cell bearing the ACT-4
receptor and/or a cell bearing the ACT-4 ligand. Antibodies against
either ACT-4 or its ligands can have the capacity to block or
stimulate signal transduction. It will, of course, be recognized
the designation of ACT-4 as a receptor and its specific binding
partner as a ligand is somewhat arbitrary and might, in some
circumstances, be reversed.
[0100] ACT-4 ligands are expected to share some of the properties
of other ligands which bind to members of the nerve growth factor
receptor superfamily. These ligands include the cytokines
TNF-.beta., TNF-.beta., CD40-L, CD-27-L and CD30-L. With the
exception of TNF-.beta., these ligands exist both as type II
integral membrane cell surface proteins and as soluble proteins.
The extracellular domains of these ligands consist of about 150
amino acids and form several .beta.-pleated sheets, which assemble
into a slitted cylindrical structure (termed a "jelly role" by
Bazan et al., Current Biology 3:603-606 (1993)) (incorporated by
reference for all purposes).
[0101] Source materials for supplying ACT-4 ligands are identified
by screening different cell types, particularly lymphoid and
hematopoietic cells, bodily fluids and tissue extracts, with
labelled ACT-4 receptor, preferably in soluble form, as a probe.
Often, the ACT-4 receptor or a binding fragment thereof is fused to
a second protein for purposes of screening. Particularly suitable
are recombinant globulins formed by fusing the extracellular
portion of ACT-4 to the constant region of an immunoglobulin heavy
chain.
[0102] ACT-4 ligands are purified from cells or other biological
materials identified by this screening method using techniques of
classical protein chemistry. Such techniques include selective
precipitation with such substances as ammonium sulfate, column
chromatography, immunopurification methods, and others. See, e.g.,
R. Scopes, Protein Purification: Principles and Practice
(Springer-verlag, NY, 1982) (incorporated by reference for all
purposes). Usually, purification procedures will include an
affinity chromatography step in which an ACT-4 polypeptide or a
binding fragment thereof is used as the immobilized reagent.
ACT-4-constant regions can be conveniently immobilized by binding
of the constant region moiety to protein A or G. ACT-4 ligands can
also be purified using anti-idiotypic antibodies to ACT-4 receptors
as the affinity reagent.
[0103] To determine the amino acid sequence or to obtain
polypeptide fragments of the receptor, the receptor may be digested
with trypsin. Peptide fragments may be separated by reversed-phase
high performance liquid chromatography (HPLC) and analyzed by
gas-phase sequencing. Other sequencing methods known in the art may
also be used. The sequence data can be used to design degenerate
probes for isolation of cDNA or genomic clones encoding ACT-4
ligands.
[0104] Alternatively, cDNA clones encoding ACT-4 ligands can be
obtained by expression cloning. In this approach, a cDNA library is
prepared from cells expressing an ACT-4 ligand (identified as
discussed, supra). The library is expressed in appropriate cells
(e.g., COS-7), and clones bearing the ACT-4 ligand are identified
by screening with labelled ACT-4 or binding fragment thereof,
optionally fused to a constant domain of an immunoglobulin heavy
chain.
[0105] The ACT-4 ligands or their binding domains can be used to
affinity purify respective ACT-4 receptors. ACT-4 ligands and
binding fragments thereof are also useful as agonists or
antagonists of ACT-4 ligand binding, and can be used in the
therapeutic methods discussed in Section VII, infra. For
membrane-bound ACT-4 ligands, binding fragments will comprise part
of the extracellular domain of an ACT-4 receptor. ACT-4 ligands and
fragments thereof are also useful in screening assays for
identifying agonists and antagonists of ACT-4 and/or its ligand.
ACT-4 ligands can be fused to other protein, such as toxins and
immunoglobulin constant domains, as discussed, supra, for ACT-4
receptors.
[0106] VI. Screening for Agonists and Antagonists ACT-4 receptor
and ACT-4 ligand fragments, analogs thereof, antibodies and
anti-idiotypic antibodies thereto, as well as other chemical or
biological agents are screened for their ability to block or
enhance binding of an ACT-4 ligand to its receptor. In addition,
they are tested for their ability to stimulate or inhibit metabolic
processes, such as DNA synthesis or protein phosphorylation in
cells bearing either an ACT-4 receptor or an ACT-4 ligand anchored
to their surfaces.
[0107] In some methods, the compound under test is screened for its
ability to block or enhance binding of a purified binding fragment
of an ACT-4 receptor (or fusion protein thereof) to a purified
binding fragment of an ACT-4 ligand (or fusion protein thereof). In
such experiments, either the receptor or ligand fragment is usually
immobilized to a solid support. The test compound then competes
with an ACT-4 ligand or receptor fragment (whichever is not
attached to the support) for binding to the support. Usually,
either the test compound or the competing ligand or receptor is
labelled.
[0108] In other methods, either or both of the ACT-4 receptor and
ligand, or binding fragments of these molecules, are expressed on a
cell surface. For example, ACT-4-h-1 antigen is expressed from
recombinant DNA in e.g., COS-7 cells (see Example 6). In these
methods, the existence of agonism or antagonism is determined from
the degree of binding between an ACT-4 receptor and its ligand that
occurs in the presence of the test compound. Alternatively,
activity of the test compound is assayed by measurement of
.sup.3H-thymidine incorporation into DNA or .sup.32p incorporation
into proteins in cells bearing an ACT-4 receptor and/or cells
bearing an ACT-4 ligand.
[0109] Compounds that block ACT-4-induced DNA synthesis or protein
phosphorylation are antagonists. Compounds that activate DNA
synthesis or phosphorylation via interaction with an ACT-4 receptor
or its ligand are agonists. Agonistic or antagonistic activity can
also be determined from other functional or physical endpoints of
leukocyte activation, or from clinically desirable or undesirable
outcomes, such as cytolytic activity, or extravasation of
leukocytes into tissues from blood vessels.
[0110] The ability of agents to agonize or antagonize T-cell
proliferation in vitro can be correlated with the ability to affect
the immune response in vivo. In vivo activity is typically assayed
using suitable animal models such as mice or rats. To assay the
effect of agents on allograft rejection, for example, potential
therapeutic agents can be administered to the animals at various
times before introduction of the allogeneic tissue; and the animals
can be monitored for graft rejection. Suitable methods for
performing the transplant and monitoring for graft rejection have
been described (see, e.g., Hislop et al., J. Thorac. Cardiovasc.
100:360-370 (1990)) (incorporated by reference for all
purposes).
VII. Therapeutic and Diagnostic Methods and Compositions
[0111] A. Diagnostic Methods
[0112] Diseases and conditions of the immune system associated with
an altered abundance, or functional mutation, of an ACT-4 receptor
or its mRNA, or an ACT-4 ligand or its mRNA may be diagnosed using
the probes and/or antibodies of the present invention. The
provision of antibodies against the ACT-4 receptor and nucleic acid
probes complementary to its mRNA allows activated CD4.sup.+ T-cells
to be distinguished from other leukocyte subtypes. The presence of
such cells is indicative of a MHC class II induced immune response
against, e.g., invading bacteria. Comparison of the numbers of
activated CD4.sup.+ cells and CD8.sup.+ cells may allow
differential diagnosis between bacterial and viral infections,
which predominantly induce these respective activated cell types.
The presence of activated CD4.sup.+ cells is also indicative of
undesirable diseases and conditions of the immune system, such as
allograft rejection, graft versus host disease, autoimmune
diseases, allergies and inflammation. The efficacy of therapeutic
agents in treating such diseases and conditions can be
monitored.
[0113] Diagnosis can be accomplished by removing a cellular sample
(e.g., blood sample, lymph node biopsy or tissue) from a patient.
The sample is then subjected to analysis for determining: (1) the
amount of expressed ACT-4 receptor or ligand in individual cells of
the sample (e.g., by immunohistochemical staining of fixed cells
with an antibody or FACS.TM. analysis), (2) the amount of ACT-4
receptor or ligand mRNA in individual cells (by in situ
hybridization with a labelled complementary polynucleotide probe),
(3) the amount of ACT-4 receptor or ligand mRNA in the cellular
sample by RNA extraction followed by hybridization to a labeled
complementary polynucleotide probe (e.g., by Northern blotting, dot
blotting, solution hybridization or quantitative PCR), or (4) the
amount of ACT-4 receptor or ligand in the cellular sample (e.g., by
cell disruption followed by immunoassay or Western blotting of the
resultant cell extract).
[0114] Diagnosis can also be achieved by in vivo administration of
a diagnostic reagent (e.g., a labelled anti-ACT-4 receptor antibody
for diagnosis of activated CD4.sup.+ T-cells) and detection by in
vivo imaging. The concentration of diagnostic agent administered
should be sufficient that the binding to those cells having the
target antigen is detectable compared to the background signal.
Further, it is desirable that the diagnostic reagent can be rapidly
cleared from the circulatory system in order to give the best
target-to-background signal ratio. The diagnostic reagent can be
labelled with a radioisotope for camera imaging, or a paramagnetic
isotope for magnetic resonance or electron spin resonance
imaging.
[0115] A change (typically an increase) in the level of protein or
mRNA of an ACT-4 receptor or ligand in a cellular sample from an
individual, which is outside the range of clinically established
normal levels, may indicate the presence of an undesirable immune
reaction in the individual from whom the sample was obtained,
and/or indicate a predisposition of the individual for developing
(or progressing through) such a reaction. Protein or mRNA levels
may be employed as a differentiation marker to identify and type
cells of certain lineages (i.e., activated CD4.sup.+ cells for the
ACT-4 receptor) and developmental origins. Such cell-type specific
detection may be used for histopathological diagnosis of undesired
immune responses.
[0116] B. Diagnostic Kits
[0117] In another aspect of the invention, diagnostic kits are
provided for the diagnostic methods described supra. The kits
comprise container(s) enclosing the diagnostic reagents, such as
labelled antibodies against ACT-4 receptors, and reagents and/or
apparatus for detecting the label. Other components routinely found
in such kits may also be included together with instructions for
performing the test.
[0118] C. Pharmaceutical Compositions
[0119] The pharmaceutical compositions used for prophylactic or
therapeutic treatment comprise an active therapeutic agent, for
example, an ACT-4 receptor, ligand, fragments thereof, and
antibodies and idiotypic antibodies thereto, and a variety of other
components. The preferred form depends on the intended mode of
administration and therapeutic application. The compositions may
also include, depending on the formulation desired,
pharmaceutically-acceptable, non-toxic carriers or diluents, which
are defined as vehicles commonly used to formulate pharmaceutical
compositions for animal or human administration. The diluent is
selected so as not to affect the biological activity of the
combination. Examples of such diluents are distilled water,
physiological saline, Ringer's solutions, dextrose solution, and
Hank's solution. In addition, the pharmaceutical composition or
formulation may also include other carriers, adjuvants, or
nontoxic, nontherapeutic, nonimmunogenic stabilizers and the
like.
[0120] D. Therapeutic Methods
[0121] The therapeutic methods employ the therapeutic agents
discussed above for treatment of various diseases in humans or
animals, particularly vertebrate mammals. The therapeutic agents
include ACT-4 receptors, binding fragments thereof, ACT-4 ligands,
binding fragments thereof, anti-ACT-4 receptor and ligand
antibodies and anti-idiotypic antibodies thereto, binding fragments
of these antibodies, humanized versions of these antibodies,
immunotoxins, and other agents discussed, supra. Some therapeutic
agents function by blocking or otherwise antagonizing the action of
an ACT-4 receptor with its ligand. Other therapeutic agents
function by killing cells bearing a polypeptide against which the
agent is targeted. For example, anti-ACT-4 receptor antibodies with
effector functions or which are conjugated to toxins, radioisotopes
or drugs are capable of selectively killing activated CD4.sup.+
T-cells. Selective elimination of such cells is particularly
advantageous because an undesirable immune response can be reduced
or eliminated, while preserving a residual immune capacity in the
form of inactivated CD4.sup.+ cells and CD8.sup.+ cells to combat
invading microorganisms to which a patient may subsequently be
exposed. Other therapeutic agents function as agonists of the
interaction between the ACT-4 receptor and ligand.
[0122] 1. Dosages and Methods of Administration
[0123] In therapeutic applications, a pharmaceutical composition
(e.g., comprising an anti-ACT-4 receptor antibody) is administered,
in vivo or ex vivo, to a patient already suffering from an
undesirable immune response (e.g., transplant rejection), in an
amount sufficient to cure, partially arrest, or detectably slow the
progression of the condition, and its complications. An amount
adequate to accomplish this is defined as a "therapeutically
effective dose" or "efficacious dose." Amounts effective for this
use will depend upon the severity of the condition, the general
state of the patient, and the route of administration, and
combination with other immunosuppressive drugs, if any, but
generally range from about 10 ng to about 1 g of active agent per
dose, with single dosage units of from 10 mg to 100 mg per patient
being commonly used. Pharmaceutical compositions can be
administered systemically by intravenous infusion, or locally by
injection. The latter is particularly useful for localized
undesired immune response such as host versus graft rejection. For
a brief review of methods for drug delivery, see Langer, Science
249:1527-1533 (1990) (incorporated by reference for all
purposes).
[0124] In prophylactic applications, pharmaceutical compositions
are administered to a patients at risk of, but not already
suffering an undesired immune reaction (e.g., a patient about to
undergo transplant surgery). The amount of antibody to be
administered is a "prophylactically effective dose," the precise
amounts of which will depend upon the patient's state of health and
general level of immunity, but generally range from 10 ng to 1 g
per dose, especially 10 mg to 100 mg per patient.
[0125] Because the therapeutic agents of the invention are likely
to be more selective and generally less toxic than conventional
immunomodulating agents, they will be less likely to cause the side
effects frequently observed with the conventional agents. Moreover,
because some of the therapeutic agents are human protein sequences
(e.g., binding fragments of an ACT-4 receptor or ligand or
humanized antibodies), they are less likely to cause immunological
responses such as those observed with murine anti-CD3 antibodies.
The therapeutic agents of the present invention can also be
combined with traditional therapeutics, and can be used to lower
the dose of such agents to levels below those associated with side
effects. For example, other immunosuppressive agents such as
antibodies to the .alpha.3 domain, T cell antigens (e.g., OKT4 and
OKT3), antithymocyte globulin, as well as chemotherapeutic agents
such as cyclosporine, glucocorticoids, azathioprine, prednisone can
be used in conjunction with the therapeutic agents of the present
invention.
[0126] For destruction of a specific population of target cells, it
can be advantageous to conjugate the therapeutic agents of the
present invention to another molecule. For example, the agents can
be joined to liposomes containing particular immunosuppressive
agents, to a specific monoclonal antibody or to a cytotoxin or
other modulator of cellular activity, whereby binding of the
conjugate to a target cell population will result in alteration of
that population. A number of protein toxins have been discussed
supra. Chemotherapeutic agents include, for example, doxorubicin,
daunorubicin, methotrexate, cytotoxin, and anti-sense RNA.
Antibiotics can also be used. In addition, radioisotopes such as
yttrium-90, phosphorus-32, lead-212, iodine-131, or palladium-109
can be used. The emitted radiation destroys the targeted cells.
[0127] 2. Diseases and Conditions Amenable to Treatment
[0128] The pharmaceutical compositions discussed above are suitable
for treating several diseases and conditions of the immune
system.
[0129] a. Transplant Rejection
[0130] Over recent years there has been a considerable improvement
in the efficiency of surgical techniques for transplanting tissues
and organs such as skin, kidney, liver, heart, lung, pancreas and
bone marrow. Perhaps the principal outstanding problem is the lack
of satisfactory agents for inducing immunotolerance in the
recipient to the transplanted allograft or organ. When allogeneic
cells or organs are transplanted into a host (i.e., the donor and
donee are different individual from the same species), the host
immune system is likely to mount an immune response to foreign
antigens in the transplant (host-versus-graft disease) leading to
destruction of the transplanted tissue. CD8.sup.+ cells, CD4.sup.+
cells and monocytes are all involved in the rejection of transplant
tissues. The therapeutic agents of the present invention are useful
to block alloantigen-induced immune responses in the donee (e.g.,
blockage or elimination of allogen-activation of CD4.sup.+ T-cells
by anti-ACT-4 receptor antibodies) thereby preventing such cells
from participating in the destruction of the transplanted tissue or
organ.
[0131] b. Graft Versus Host Disease
[0132] A related use for the therapeutic agents of the present
invention is in modulating the immune response involved in "graft
versus host" disease (GVHD). GVHD is a potentially fatal disease
that occurs when immunologically competent cells are transferred to
an allogeneic recipient. In this situation, the donor's
immunocompetent cells may attack tissues in the recipient. Tissues
of the skin, gut epithelia and liver are frequent targets and may
be destroyed during the course of GVHD. The disease presents an
especially severe problem when immune tissue is being transplanted,
such as in bone marrow transplantation; but less severe GVHD has
also been reported in other cases as well, including heart and
liver transplants. The therapeutic agents of the present invention
are used to block activation of, or eliminate, the donor T-cells
(particularly activated CD4.sup.+ T-cells, for therapeutic agents
targeted against the ACT-4 receptor), thereby inhibiting their
ability to lyse target cells in the host.
[0133] c. Autoimmune diseases
[0134] A further situation in which immune suppression is desirable
is in treatment of autoimmune diseases such as insulin-dependent
diabetes mellitus, multiple sclerosis, stiff man syndrome,
rheumatoid arthritis, myasthenia gravis and lupus erythematosus. In
these disease, the body develops a cellular and/or humoral immune
response against one of its own antigens leading to destruction of
that antigen, and potentially crippling and/or fatal consequences.
Activated CD4.sup.+ T-cells are believed to play a major role in
many autoimmune diseases. Autoimmune diseases are treated by
administering one of the therapeutic agents of the invention,
particularly agents targeted against an ACT-4 receptor. Optionally,
the autoantigen, or a fragment thereof, against which the
autoimmune disease is targeted can be administered shortly before,
concurrently with, or shortly after the immunosuppressive agent. In
this manner, tolerance can be induced to the autoantigen under
cover of the suppressive treatment, thereby obviating the need for
continued immunosuppression. See, e.g., Cobbold et al., WO 90/15152
(1990).
[0135] d. Inflammation
[0136] Inflammation represents the consequence of capillary
dilation with accumulation of fluid and migration of phagocytic
leukocytes, such as granulocytes and monocytes. Inflammation is
important in defending a host against a variety of infections but
can also have undesirable consequences in inflammatory disorders,
such as anaphylactic shock, arthritis and gout. Activated T-cells
have an important modulatory role in inflammation, releasing
interferon .gamma. and colony stimulating factors that in turn
activate phagocytic leukocytes. The activated phagocytic leukocytes
are induced to express a number of specific cells surface molecules
termed homing receptors, which serve to attach the phagocytes to
target endothelial cells. Inflammatory responses can be reduced or
eliminated by treatment with the therapeutic agents of the present
invention. For example, therapeutic agents targeted against the
ACT-4 receptor function by blocking activation of, or eliminating
activated, CD4.sup.+ cells, thereby preventing these cells from
releasing molecules required for activation of phagocytic cell
types.
[0137] e. Infectious Agents
[0138] The invention also provides methods of augmenting the
efficacy of vaccines in preventing or treating diseases and
conditions resulting from infectious agents. Therapeutic agents
having the capacity to activate CD4.sup.+ T-cells (e.g., certain
monoclonal antibodies against a ACT-4-h-1 receptor polypeptide) are
administered shortly before, concurrently with, or shortly after
the vaccine containing a selected antigen. The therapeutic agent
serves to augment the immune response against the selected antigen.
These methods may be particularly advantageous in patients
suffering from immune deficiency diseases.
[0139] The following examples are offered to illustrate, but not to
limit, the invention.
EXAMPLES
Example 1
A Monoclonal Antibody Against ACT-4-h-1
[0140] Mice were immunized with PHA-transformed T-lymphoblasts.
Splenocytes from immunized mice were fused with SP2/O myeloma cells
and hybridomas secreting antibodies specific for the T-cell clone
were selected. The hybridomas were cloned by limiting dilution. A
monoclonal antibody, designated L106, produced by one of the
resulting hybridoma, was selected for further characterization. The
L106 antibody was found to have an IgG1 isotype. A hybridoma
producing the antibody, designated HBL106 has been deposited at the
American Type Culture Collection at______, on______, and assigned
ATCC Accession No.______.
Example 2
Cellular Distribution of Polypeptide Recognized by L106
Antibody
[0141] Samples containing the antibody L106 were made available to
certain participants at the Fourth International Workshop and
Conference on Human Leucocyte Differentiation Antigens (Vienna
1989) for the purpose of identifying tissue and cell types which
bind to the L106 antibody. The data from the workshop are presented
in Leukocyte Typing IV (ed. W, Knapp, Oxford U. Press, 1989)
(incorporated by reference for all purposes) and an accompanying
computer data base available from Walter R. Gilks, MRC
Biostatistics Unit, Cambridge University, England. This reference
reports the L106 antibody binds a polypeptide of about 50 kDa. This
polypeptide was reported to be present on HUT-102 cells (a
transformed T-cell line), PHA-activated peripheral blood
lymphocytes, an EBV-transformed B-lymphoid cell line, and HTLV-II
transformed T-cell line, PMA-activated tonsil cells, ConA- or
PHA-activated PBLs, and PMA-activated monocytes. The polypeptide
was reported to be substantially absent on inter alia resting
basophils, endothelial cells, fibroblasts, interferon
.gamma.-activated monocytes, peripheral non-T-cells, peripheral
granulocytes, peripheral monocytes, peripheral mononuclear cells,
peripheral T cells, and peripheral red blood cells.
[0142] The present inventors have obtained data indicating that the
50 kDa polypeptide (hereinafter "ACT-4-h-1 receptor") is
preferentially expressed on the CD4.sup.+ subspecies of activated
T-cells. In one series of experiments, cell-specific ACT-4-h-1
expression was analyzed on unfractionated PBLs by a two-color
staining method. PBL were activated with PHA for about two days
(using the culture conditions described in Example 3), and analyzed
for cell-surface expression of ACT-4-h-1 on different cellular
subtypes by staining with two differently-labelled antibodies (FITC
and PE labels). Labels were detected by FACS.TM. analysis
essentially as described by Picker et al., J. Immunol.
150:1105-1121 (1993) (incorporated by reference for all purposes).
One antibody, L106, was specific for ACT-4-h-1, the other antibody
was specific for a particular leukocyte subtype. FIG. 1 shows three
charts in which L106 staining is shown on the Y-axis of each chart,
and anti-CD4, anti-CD8 and anti-CD19 staining as the X-axes of the
respective charts. For the chart stained with anti-CD4, many cells
appear as double positives (i.e., express both CD4 and ACT-4-h-1).
For the chart stained with anti-CD8, far fewer cells appear as
double positives. For the chart stained with anti-CD19 (a B-cell
marker), double-positive cells are substantially absent.
[0143] In another series of experiments expression of ACT-4-h-1 was
analyzed by single-color staining on isolated cell types. Cells
were stained with fluorescently labelled L106 antibody and the
label was detected by FACS.TM. analysis. See Engleman et al., J.
Immunol. 127:2124-2129 (1981) (incorporated by reference for all
purposes). In some experiments, cells were activated by PHA
stimulation for about two days (again using the culture conditions
described in Example 3). The results from this experiment, together
with those from the two-color staining experiment described supra,
are summarized in Table 1. Table 1 shows that about 80% of
activated CD4.sup.+ cells expressed ACT-4-h-1 with a mean channel
fluorescence of >20, irrespective whether the CD4.sup.+ cells
are isolated (one-color staining) or in unfractionated PBLs
(two-color staining). The level of expression of ACT-4-h-1 on
activated CD8.sup.+ cells is much lower than on activated CD4.sup.+
T-cells in the two-color staining experiment, and very much lower
in the one-color staining. Thus, the extent of expression on
activated CD8.sup.+ cells appears to depend on whether the C8.sup.+
cells are fractionated from other PBLs before activation. In
unfractionated CD8.sup.+ cells (two-color staining), about 10% of
cells express ACT-4-h-1, with a mean channel fluorescence of about
4. In the fractionated cells, only about 4% of cells express
ACT-4-h-1 with a mean channel fluorescence of about 2. These data
suggest that ACT-4-h-1 is expressed only on a small subtype of
activated CD8.sup.+ cells and that this subtype is somewhat more
prevalent when the CD8.sup.+ cells are activated in the presence of
other PBLs.
[0144] Table 1 also indicates that ACT-4-h-1 was substantially
absent on all resting leukocyte subtypes tested (i.e., CD4.sup.+
T-cells, CD8.sup.+ T-cells, CD19.sup.+ B-cells, CD14.sup.+
monocytes, granulocytes and platelets), and was also substantially
absent on activated B-cells and monocytes. ACT-4-h-1 was also found
to be substantially absent on most tumor cell lines tested.
However, Molt3, Raji and NC37 cell lines did show a low level of
expression. TABLE-US-00001 TABLE 1 CELL SPECIFICITY OF ACT-4-h-1
EXPRESSION Expression of ACT-4-h-1 % Cells MCF.sup.1 Two Color
Staining CD4.sup.+ T-Cells (resting) <2 <2 CD4.sup.+ T-Cells
(activated).sup.2 80 25 CD8.sup.+ T-Cells (resting) <2 <2
CD8.sup.+ T-Cells (activated) 10 4 CD19.sup.+ B-Cells (resting)
<2 <2 CD19.sup.+ B-Cells (activated) <2 <2 CD14.sup.+
Monocytes (resting) <2 <2 CD14.sup.+ Monocytes (activated)
<2 <2 One Color Staining PBLs (resting) <2 3 PBLs
(activated) 50 27 CD4.sup.+ (resting) <2 <2 CD4.sup.+
(activated) 80 22 CD8.sup.+ (resting) <2 <2 CD8.sup.+
(activated) 4 2 Granulocytes <2 <2 Platelets <2 <2
Tumor Lines Molt-4, CEM, Hut 78, H9, Jurkat <2 <2 HPB-ALL,
Sezary, T-AU <2 <2 Molt-3 20 3 B-LCL, Arent, RML, JY, KHY,
PGf <2 <2 MSAB, CESS, 9037, 9062 <2 <2 Dandi, Ramos,
Namalwa <2 <2 Raji, NC37 30 4 U937, THP-1, HL-60 <2 <2
Kgla, K562, HEL <2 <2 .sup.1MCF = Mean Channel Fluorescence.
.sup.2Cells indicated as "activated" had been stimulated with PHA
for about three days.
Example 3
Time Course of ACT-4-h-1 Expression Responsive to CD4.sup.+ T-cell
Activation
[0145] CD4.sup.+ T-cells were tested for expression of ACT-4-h-1
receptors in response to various activating stimuli. CD4.sup.+
T-cells were purified from peripheral blood mononuclear cells by
solid-phase immunoadsorption ("panning"). 5.times.10.sup.4
CD4.sup.+ T-cells were cultured with an activating agent in
microtiter wells containing RPMI medium supplemented with 10% human
serum. Three different activating agents were used: (1)
5.times.10.sup.4 irradiated (3000 rads) monocytes, (2) PHA (1
.mu.g/ml) and (3) tetanus toxoid (5 .mu.g/ml). .sup.3H-thymidine
was added to the cultures 12-16 h before harvest. After harvest,
cells were tested for the expression of cell surface antigens by
incubation with various labelled antibodies (L106, anti-CD4 and
anti-CD8), as described by Engleman et al., J. Immunol.
127:2124-2129 (1981).
[0146] FIG. 2 shows the appearance of ACT-4-h-1 in response to
alloantigen activation. Before activation, no expression was
observed. The percentage of cells expressing the ACT-4-h-1 receptor
increases with time, peaking at about 30% after about seven days of
alloantigen activation. The results also show that essentially all
cells expressing ACT-4-h-1 also expressed the CD4 receptor and that
essentially no such cells expressed the CD8 receptor. FIG. 3
presents similar data for the appearance of ACT-4-h-1 in response
to tetanus toxoid activation. Again, the percentage of cells
expressing ACT-4-h-1 peaked at about seven days. However, at this
time a higher percentage of cells (about 60%) expressed the
receptor. FIG. 4 presents similar data for the appearance of
ACT-4-h-1 on CD4.sup.+ T-cells in response to PHA activation. In
this situation, the percentage of CD4.sup.+ T-cells expressing the
receptor peaks at about 65% after three days of activation.
[0147] It is concluded that ACT-4-h-1 is a CD4.sup.+ T-cell
activation antigen that is expressed in response to diverse
activating stimuli.
Example 4
Cloning ACT-4-h-1 cDNA
[0148] The cDNA clone for the ACT-4-h-1 receptor was isolated using
a slightly modified COS cell expression system, first developed by
Aruffo & Seed, supra. RNA was isolated from 72-hour PHA
activated human peripheral blood lymphocytes. Total RNA was
extracted with TRI-reagent (Molecular Research Center), and
poly(A)+RNA was isolated by oligo dT-magnetic bead purification
(Promega). cDNA was synthesized by the method of Gubler &
Hoffman, Gene 25:263-369 (1982) using superscript reverse
transcriptase (Gibco/BRL) and an oligo dT primer. The blunted cDNA
was ligated to non-self-complementary BstXl adaptors and passed
over a sephacryl S-400 spin column to remove unligated adaptors and
small fragments (<300 base pairs). The linkered cDNA was then
ligated into a BstXl cut eukaryotic expression vector, pcDNA-IRL,
an ampicillin resistant version of pcDNA-I(Invitrogen). The
precipitated and washed products of the ligation reaction were
electroporated into E. coli strain WM1100(BioRad). Plating and
counting of an aliquot of the transformed bacteria revealed a total
count of 2 million independent clones in the unamplified library.
Average insert size was determined to be 1.2 kb. The bulk of the
library was amplified in liquid culture, 250 ml standard LB media.
Plasmid was recovered by alkaline lysis and purified over an
ion-exchange column (Qiagen).
[0149] Sub-confluent COS-7 cells were transfected with the purified
plasmid DNA by electroporation. Cells were plated on 100 mm dishes
and allowed to grow for 48 hours. Cells were recovered from the
plates with PBS-EDTA solution, incubated with monoclonal antibody
L106, and were panned according to standard procedures. A second
round panning revealed enrichment as numerous COS cells adsorbed to
the plates. Episomal DNA was recovered from the immunoselected
cells by the Hirt method, and electroporated into bacteria for
amplification.
[0150] Bacteria transformed with plasmid from the second round Hirt
preparation were diluted into small pools of about 100 colonies.
The pools were amplified and their DNA purified and tested for the
ability to confer expression of the L106 antigen on COS-7 cells by
immunofluorescence. Phycoerythrin-conjugated L106 antibody was used
to stain COS-7 cell monolayers and the cells were then examined by
manual immunofluorescence microscopy. Miniprep DNA from four out of
eight pools was positive when tested for expression. The pool with
the best expression, pool E, was divided into smaller pools of
.sup..about.12 colonies. Three out of eight sub-pools were
positive, and sub-pool E1 was plated to allow for the analysis of
single colonies. Clone E1-27 was found to confer high level
expression of ACT-4-h-1 receptor on the surface of transfected COS
cells.
Example 5
cDNA Sequence Analysis
[0151] The insert from the clone designated E1-27 was subcloned
into pBluescript and sequenced by the dideoxy chain termination
method, using the T7 polymerase autoread sequencing kit (Pharmacia)
on an ALF sequencer (Pharmacia). Restriction mapping revealed
several convenient sites for subcloning. Five subclones were
generated in pBluescript and were sequenced on both strands with
M13 forward and universal primers.
[0152] The cDNA and deduced amino acid sequences of ACT-4-h-1 are
shown in FIG. 5. The ACT-4-h-1 cDNA sequence of 1,137 base pairs
contains a 14-bp 5' untranslated region and a 209-bp 3'
untranslated region. An AATAAA polyadenylation signal is present at
position 1,041 followed by an 80-bp poly A tail starting at
position 1,057. The longest open reading frame begins with the
first ATG at position 15 and ends with a TGA at position 846. The
predicted amino acid sequence is that of a typical type 1 integral
membrane protein. Hydrophobicity analysis reveals a putative signal
sequence following the initiating ATG, with a short stretch of
basic residues followed by a longer stretch of hydrophobic
residues. A predicted signal peptide cleavage site is present at
residue 22 or 24 (the latter being the more likely by the criteria
of von Heijne, Nucleic Acids Res. 14, 4683-4690 (1986))
(incorporated by reference for all purposes), leaving a mature
protein of 253 amino acid residues (or 255 amino acids, if cleavage
occurs at the less probable site). Hydrophobicity analysis also
reveals a single large stretch of 27 hydrophobic residues predicted
to be the transmembrane domain, which predicts an extracellular
domain of 189 (or 191) amino acids and an intracellular domain of
37 amino acids. The extracellular domain is cysteine rich, where 18
cysteines are found within a stretch of 135 amino acids. The
predicted molecular mass (Mr) for the mature protein is 27,400, and
there are two potential N-glycosylation sites at amino acid
residues 146 and 160.
[0153] Comparison of the amino acid sequence of ACT-4-h-1 with
known sequences in the swiss-prot database using the BLAZE program
reveals a sequence similarity with members of the nerve growth
factor receptor superfamily. Amino acid sequences are at least 20%
identical for NGF-R, TNF-R, CD40, 41-BB, and fas/APO-1,. and 62%
for OX-40, allowing for gaps and deletions. Alignments of the
various proteins reveal the conservation of multiple cysteine rich
motifs. Three of these motifs are present in ACT-4-h-1 and OX-40,
compared with four such motifs in NGF-R and CD40.
[0154] Comparison of the nucleotide sequence of ACT-4-h-1 with
known sequences in the Genbank and EMBL databases using the
programs BLAST and FASTDB revealed a high degree of sequence
similarity with only one member of the nerve growth factor receptor
family, OX-40. Allowing for gaps and insertions, the sequence
identity is 66%. Comparison of the ACT-4-h-1 and OX-40 nucleotide
sequences reveals that both contain a 14-bp 5' untranslated region,
and both contain approximately 80-bp poly A tails. In ACT-4-h-1,
however, there is a slight lengthening of the 3' untranslated
region from 187-bp to 209-bp, and there is a lengthening of the
coding region from 816-bp to 834-bp, a difference of 18-bp or 6
amino acid insertions. Aligning the two amino acid sequences
reveals that four of the amino acid insertions occur prior to the
signal sequence cleavage site. Thus, the mature ACT-4-h-1 receptor
protein contains one more amino acid residue than OX-40 (i.e., 253
vs. 252 amino acids). Remarkably, the ACT-4-h-1 nucleotide sequence
is much more GC rich, than the OX-40 sequence (70% v. 55%)
indicating that the two sequences will not hybridize under
stringent conditions.
Example 6
Production of Stable ACT-4-h-1 Transfectants
[0155] An XbaI-HindIII fragment was excised from the construct
described in Example 4, and inserted into XbaI/HindIII-digested
pcDNA-I-neo (Invitrogen) to generate an expression vector termed
ACT-4-h-1-neo (FIG. 6). This vector was linearized with Sf1 and
electroporated into three eukaryotic cell lines. These cell lines
were SP2/O (a mouse myeloma derived from the Balb/c strain), Jurkat
(a transformed human T-cell line) and COS-7 (an adherent monkey
cell line). After a 48-h recovery period, transformed cells were
selected in 1 mg/ml G418 (Gibco). After three weeks of selection,
neo-resistant cell lines were incubated with a saturating
concentration of L106 antibody, washed and overlayered onto 100 mm
petri dishes coated with goat anti-mouse IgG to select for cells
expressing ACT-4-h-1. After washing off unbound cells, adherent
cells were recovered and expanded in tissue culture. Cell lines
were subject to two further rounds of panning and expression. The
resulting cell lines were shown by direct immunofluorescence
staining to express abundant STAN-4-h-1 (FIG. 7).
Example 7
Production of an ACT-4-h-1-Immunoglobulin Fusion Protein
[0156] A soluble fusion protein has been constructed in which the
extracellular domain of ACT-4-h-1 is linked via its C-terminal to
the N-terminal of the constant domain of a human immunoglobulin.
The vector encoding ACT-4-h-1 described in Example 4 was cleaved
with SmaI and NotI to excise all ACT-4-h-1 sequences downstream of
the SmaI site including the transmembrane, cytoplasmic and 3'
untranslated regions. The remaining region encodes the soluble
extracellular portion of ACT-4-h-1 (FIG. 8). The source of the
immunoglobulin constant region to be joined to the ACT-4-h-1
extracellular domain was a plasmid termed 5K-41BB-Eg1 (Proc. Natl.
Acad. Sci. (USA) 89: 10360-10364) (incorporated by reference for
all purposes). This plasmid contains a 1.3 kb BamHI/EagI genomic
fragment encoding the hinge, CH2 and terminal CH3 domains of human
Ig, isotype gamma 1. The fragment required modification for
insertion into the SmaI/NotI ends of the ACT-4-h-1 vector, while
preserving the peptide reading frame across the SmaI junction to be
formed by blunt-end ligation. The vector 5k-41BB-Eg1 was cut with
BamHI and the resulting 5' extensions were filled with Klenow
fragment. The vector was then cut with EagI releasing the 1.3 kb
fragment with blunt and NotI compatible ends. This fragment was
ligated with SmaI/NotI digested ACT-4-h-1 vector. The ligation mix
was electroporated into E. coli and multiple transformant clones
screened with PCR using ACT-4-h-1 and IgG1 nucleotide fragments as
primers.
[0157] Plasmids containing the ACT-4-h-1-IgG1 coding were
electroporated into COS cells. The cells were allowed to grow for
five days at which point their supernatants were harvested and
sterile filtered through a 0.2 micron membrane. The supernatants
were tested for expression of ACT-4-h-1-IgG1 by dot blotting.
Supernatants were blotted onto mitrocellulose and blocked with 5%
nonfat dry milk. Replica blots were probed with antibody L106 or
alkaline phosphatase-labelled goat anti-human immunoglobulin IgG
(American Qualex). Antibody L106 was detected with an alkaline
phosphatase labelled goat anti-mouse IgG. NBT/BCIP (Pierce) was
used as a colorimetric substrate. High producing positive clones
were sequenced at the junction site to confirm proper vector
construction. The resulting fusion gene is depicted in FIG. 9.
[0158] For the purposes of clarity and understanding, the invention
has been described in these examples and the above disclosure in
some detail. It will be apparent, however, that certain changes and
modifications may be practiced within the scope of the appended
claims. All publications and patent applications are hereby
incorporated by reference for all purposes to the same extent as if
each were individually denoted as being incorporated by reference.
Sequence CWU 1
1
2 1 1057 DNA Homo sapiens CDS (15)..(845) ACT-4-h-1 cDNA 1
cagcagagac gagg atg tgc gtg ggg gct cgg cgg ctg ggc cgc ggg ccg 50
Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro 1 5 10 tgt gcg gct
ctg ctc ctc ctg ggc ctg ggg ctg agc acc gtg acg ggg 98 Cys Ala Ala
Leu Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly 15 20 25 ctc
cac tgt gtc ggg gac acc tac ccc agc aac gac cgg tgc tgc cac 146 Leu
His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His 30 35
40 gag tgc agg cca ggc aac ggg atg gtg agc cgc tgc agc cgc tcc cag
194 Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln
45 50 55 60 aac acg gtg tgc cgt ccg tgc ggg ccg ggc ttc tac aac gac
gtg gtc 242 Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp
Val Val 65 70 75 agc tcc aag ccg tgc aag ccc tgc acg tgg tgt aac
ctc aga agt ggg 290 Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn
Leu Arg Ser Gly 80 85 90 agt gag cgg aag cag ctg tgc acg gcc aca
cag gac aca gtc tgc cgc 338 Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr
Gln Asp Thr Val Cys Arg 95 100 105 tgc cgg gcg ggc acc cag ccc ctg
gac agc tac aag cct gga gtt gac 386 Cys Arg Ala Gly Thr Gln Pro Leu
Asp Ser Tyr Lys Pro Gly Val Asp 110 115 120 tgt gcc ccc tgc cct cca
ggg cac ttc ttc cca ggc gac aac cag gcc 434 Cys Ala Pro Cys Pro Pro
Gly His Phe Ser Pro Gly Asp Asn Gln Ala 125 130 135 140 tgc aag ccc
tgg acc aac tgc acc ttg gct ggg aag cac acc ctg cag 482 Cys Lys Pro
Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln 145 150 155 ccg
gcc agc aat agc tcg gac gca atc tgt gag gac agg gac ccc cca 530 Pro
Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro 160 165
170 gcc acg cag ccc cag gag acc cag ggc ccc ccg gcc agg ccc atc act
578 Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr
175 180 185 gtc cag ccc act gaa gcc tgg ccc aga acc tca cag gga ccc
tcc acc 626 Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro
Ser Thr 190 195 200 cgg ccc gtg gag gtc ccc ggg ggc cgt gcg gtt gcc
gcc atc ctg ggc 674 Arg Pro Val Glu Val Pro Gly Gly Arg Ala Val Ala
Ala Ile Leu Gly 205 210 215 220 ctg ggc ctg gtg ctg ggg ctg ctg ggc
ccc ctg gcc atc ctg ctg gcc 722 Leu Gly Leu Val Leu Gly Leu Leu Gly
Pro Leu Ala Ile Leu Leu Ala 225 230 235 ctg tac ctg ctc cgg agg gac
cag agg ctg ccc ccc gat gcc cac aag 770 Leu Tyr Leu Leu Arg Arg Asp
Gln Arg Leu Pro Pro Asp Ala His Lys 240 245 250 ccc cct ggg gga ggc
agt ttc cgg acc ccc atc caa gag gag cag gcc 818 Pro Pro Gly Gly Gly
Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala 255 260 265 gac gcc cac
tcc acc ctg gcc aag atc tgacctgggc ccaccaaggt 865 Asp Ala His Ser
Thr Leu Ala Lys Ile 270 275 ggacgctggg ccccgccagg ctggagcccg
gagggtctgc tgggcgagca gggcaggtgc 925 aggccgcctg ccccgccacg
ctcctgggcc aactctgcac cgttctaggt gccgatggct 985 gcctccggct
ctctgcttac gtatgccatg catacctcct gccccgcggg accacaataa 1045
aaaccttggc ag 1057 2 277 PRT Homo sapiens deduced amino acid
sequence of ACT-4-h-1 2 Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly
Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr
Val Thr Gly Leu His Cys Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn
Asp Arg Cys Cys His Glu Cys Arg Pro 35 40 45 Gly Asn Gly Met Val
Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys 50 55 60 Arg Pro Cys
Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro 65 70 75 80 Cys
Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys 85 90
95 Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly
100 105 110 Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala
Pro Cys 115 120 125 Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala
Cys Lys Pro Trp 130 135 140 Thr Asn Cys Thr Leu Ala Gly Lys His Thr
Leu Gln Pro Ala Ser Asn 145 150 155 160 Ser Ser Asp Ala Ile Cys Glu
Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175 Gln Glu Thr Gln Gly
Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr 180 185 190 Glu Ala Trp
Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200 205 Val
Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val 210 215
220 Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu
225 230 235 240 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro
Pro Gly Gly 245 250 255 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln
Ala Asp Ala His Ser 260 265 270 Thr Leu Ala Lys Ile 275
* * * * *