U.S. patent application number 11/088004 was filed with the patent office on 2005-08-04 for methods of screening for b cell activity modulators.
This patent application is currently assigned to Board of Trustees of the Leland Stanford Junior University. Invention is credited to Glynne, Richard, Goodnow, Chris, Mack, David.
Application Number | 20050169841 11/088004 |
Document ID | / |
Family ID | 26867440 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050169841 |
Kind Code |
A1 |
Glynne, Richard ; et
al. |
August 4, 2005 |
Methods of screening for B cell activity modulators
Abstract
The invention provides for the identification of all genes,
whether known or novel, which are differentially expressed within
and among B cells, making possible the characterization of their
temporal regulation and function in the B cell response and/or in B
cell mediated disorders. Expression profiles, nucleic acids and
proteins are provided for differing states of B cells, including
resting, nave, activated, tolerant and immunosuppressed B cells.
The present invention makes possible the identification and
characterization of targets useful in prognosis, diagnosis,
monitoring, rational drug design, and/or therapeutic intervention
of immune system disorders.
Inventors: |
Glynne, Richard; (Palo Alto,
CA) ; Goodnow, Chris; (Ainslie, AU) ; Mack,
David; (Menlo Park, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Board of Trustees of the Leland
Stanford Junior University
Palo Alto
CA
Affymetrix, Inc.
Santa Clara
CA
|
Family ID: |
26867440 |
Appl. No.: |
11/088004 |
Filed: |
March 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11088004 |
Mar 22, 2005 |
|
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|
09747760 |
Dec 21, 2000 |
|
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|
60171796 |
Dec 22, 1999 |
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Current U.S.
Class: |
424/9.2 ;
435/6.16 |
Current CPC
Class: |
C40B 40/00 20130101;
G01N 2500/10 20130101; G01N 33/5052 20130101 |
Class at
Publication: |
424/009.2 ;
435/006 |
International
Class: |
A61K 049/00; C12Q
001/68 |
Claims
1-18. (canceled)
19. An array of probes, comprising a support bearing a plurality of
nucleic acid probes complementary to a plurality of mRNAs fewer
than 1000 in number, wherein the plurality of mRNA probes includes
an mRNA expressed by a gene selected from the group consisting of
Egr-1, Egr-2, Nur77, c-myc, MIP-1a, MIP-1b, BL34, gfi-1, NAB2,
neurogranin, SLAP, A1, E2-20K, SATB1, Cctq, kappa V, pcp-4, TGIF,
CD83, ApoE, Aeg-2, CD72, cyclin D2, 1ck, MEF-2C, bmk, IgD, Evi-2,
vimentin, CD36, c-fes, c-fos, TRAP, hIP30, Ly6E.1, LRG-21, Fos B,
gadd153, mafK, Ah-R, C/EBP beta, EZF, TIS7, TIS11, TIS11b, LSIRF,
MKP1, PAC-1, PEP, MacMARCKS, SNK, Stra13, kir/gem, EB12, IL1-R2,
MyD116, RP105, uPAR, 4F2, hRab30, Id3, BKLF, LKLF, EFP, bcl-3,
caspase 2, GILZ, hIFI-204, hRhoH, TRAF5, LT-beta, IFNg-R11, gadd45,
CDC47, NAG, scd2, kappa 0 ig, iap38, G7e, B29, and SCD2.
20. The array of claim 19, wherein the probes are cDNA
sequences.
21. The array of claim 19, comprising a plurality of sets of
probes, each set of probes complementary to subsequences from a
mRNA.
Description
TECHNICAL FIELD
[0001] The invention relates to the identification of expression
profiles and the nucleic acids involved in B cell activation,
immunosuppression and immunological tolerance and to the use of
such expression profiles and nucleic acids in methods for
identifying candidate agents which modulate B cell activity.
BACKGROUND OF THE INVENTION
[0002] B lymphocytes (also referred to as B cells) mature within
the bone marrow and leave the marrow expressing a unique
antigen-binding membrane receptor. The B-cell receptor is a
membrane-bound immunoglobulin glycoprotein. When a B cell
encounters the antigen for which its membrane-bound antibody is
specific, the cell begins to divide very rapidly; its progeny
differentiate into memory B cells and effector cells called plasma
cells. Memory B cells have a longer lifespan and continue to
express membrane-bound antibody with the same specificity as the
original parent cell. Plasma cells do not produce membrane-bound
antibody but instead produce the antibody in a form that can be
secreted.
[0003] Immunologic tolerance is a specific state of
non-responsiveness to an antigen. Immunologic tolerance generally
involves more than the absence of an immune response; this state is
an adaptive response of the immune system, one meeting the criteria
of antigen specificity and memory that are the hallmarks of any
immune response. Tolerance develops more easily in fetal and
neonatal animals than in adults, suggesting that immature T and B
cells are more susceptible to the induction of tolerance. Moreover,
studies have suggested that T cells and B cells differ in their
susceptibility to tolerance induction. Induction of tolerance,
generally, can be by clonal deletion or clonal anergy. In clonal
deletion, immature lymphocytes are eliminated during maturation. In
clonal anergy, mature lymphocytes present in the peripheral
lymphoid organs become functionally inactivated.
[0004] Treatment of transplant and autoimmune patients often
includes suppression of lymphocyte activation by tacrolimus (FK506)
or cyclosporin, both inhibitors of calcineurin. Borel, et al.,
(1976) Agents Actions 6: 468-75; Kino et al., (1987) J. Antibiot.
(Tokyo) 40, 1256-65; Ho, et al., (1996) Clin. Immunol.
Immunopathol. 80, S40-5; and Ruhlmann and Nordheim, (1997)
Immunobiology 198, 192-206. While effective during therapy, these
compounds do not allow (re-)establishment of immunological
tolerance to the offending autoantigen and instead can inhibit
tolerance induction. Prud'homme and Vanier, (1993) Clin. Immunol.
Immunopathol. 66: 185-92. Accordingly, the development of drugs
that could induce tolerance would be desirable.
[0005] Therefore, it is an object of this invention to identify the
expression profiles which are unique to B-cell tolerance,
activation and immunosuppression. It is further an object to use
the expression profiles in assays to identify agents which can be
used in the modulation of B cell activity including B cell
tolerance, activation, immunosuppression, mitosis, apoptosis,
differentiation and migration. It is further an object to use the
expression profiles as diagnostics to identify B cells which are
abnormal. It is further an object to provide assays to identify
agents for the treatment of B cell related disorders.
SUMMARY OF THE INVENTION
[0006] In one aspect of the invention, the identification of all
genes, whether known or novel, which are differentially expressed
within and among B cells are provided, making possible the
characterization of their temporal regulation and function in the B
cell response and/or in B cell mediated disorders. Thus, expression
profiles, nucleic acids and proteins are provided for differing
states of B cells, including resting, nave, activated, tolerant and
immunosuppressed B cells. Thus, the present invention makes
possible the identification and characterization of targets useful
in prognosis, diagnosis, monitoring, rational drug design, and/or
therapeutic intervention of immune system disorders.
[0007] The invention provides methods of screening drug candidates.
Such methods entail providing a cell that expresses an expression
profile gene selected from the group Egr-1, Egr-2, Nur77, c-myc,
MIP-1a, MIP-1b, BL34, gfi-1, NAB2, neurogranin, SLAP, A1, E2-20K,
SATB1, Cctq, kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2, CD72, cyclin
D2, 1ck, MEF-2C, bmk, IgD, Evi-2, vimentin, CD36, c-fes, c-fos,
TRAP, hIP30, Ly6E.1, LRG-21, Fos B, gadd153, mafK, Ah-R, C/EBP
beta, EZF, TIS7, TIS11, TIS11b, LSIRF, MKP1, PAC-1, PEP, MacMARCKS,
SNK, Stra13, kir/gem, EB12, IL1-R2, MyD116, RP105, uPAR, 4F2,
hRab30, Id3, BKLF, LKLF, EFP, bcl-3, caspase 2, GILZ, hIFI-204,
hRhoH, TRAF5, LT-beta, IFNg-RII, gadd45, CDC47, NAG, scd2, kappa 0
ig, iap38, G7e, B29, and SCD2. A drug candidate is added to the
cell. The effect of the drug candidate on the expression of the
expression profile gene is then determined.
[0008] In some methods the level of expression in the absence of
the drug candidate to the level of expression in the presence of
the drug candidate is compared. In other methods, the cell
expresses an expression profile gene set of at least one expression
profile gene, and the effect of the drug candidate on the
expression of the set is determined. In some such methods, the
profile gene set comprises a tolerance set comprising carb anh II,
IgD, CD72, SATB1, ApoE, CD83, cyclin D2, Cctq, MEF-2C, TGIF, Aeg-2,
Egr-1, 1ck, Egr-2, E2-20K, pcp-4, kappa V, neurogranin, NAB2, gfi-1
hIP-30, TRAP, bmk, CD36, Evi-2, vimetin, Ly6E.1, and c-fes. In some
such methods, the expression of hIP-30, TRAP, bmk, CD36, Evi-2, and
c-fes are decreased and the expression of carb anh 11, CD72, SATB1,
ApoE, CD83, cyclin D2, Cctq, MEF-2C, TGIF, Aeg-2, Egr-1, 1ck,
Egr-2, E2-20K, pcp-4, kappa V, neurogranin, NAB2, gfi-1 are
increased as a result of the introduction of the drug
candidate.
[0009] In some methods, the set comprises a stimulation set
comprising Egr-1, Egr-2, NAB2, mafK, LRG-21, c-fos, c-myc, Stra13,
AhR, gadd153, C/EBP beta, TIS11b, TIS11, gfi-1, EZF, Nur77, LSIRF,
SNK, PAC-1, kir/gem, MacMARCKS, PEP, MKP1, hRab30, MIP-1b, MIP-1a,
EB12, BL34, IL1-R2, TIS7, MyD116, A1, uPAR, RP105, Evi-2 4F2, CD72,
Id3, BKLF, LKLF, EFP, Stat1, bcl-3, hRhoH, TRAF5, SLAP, LT-beta,
IFNg-R11, GILZ, Caspase 2, gadd45, CDC47, NAG, scd2, kappa 0 ig,
B29, iap38, G7e, and hIFI-204. In some such methods, the expression
of Id3, BKLF, LKLF, EFP, Stat1, bcl-3, hRhoH, TRAF5, SLAP, LT-beta,
IFNg-R11, GILZ. Caspase 2, gadd45, CDC47, NAG, scd2, kappa 0 ig,
B29, iap38, G7e, and hIFI-204 are decreased and the expression of
Egr-1, Egr-2, NAB2, mafK, LRG-21, c-fos, c-myc, Stra13, AhR,
gadd153, C/EBP beta, TIS11b, TIS 11, gfi-1, EZF, Nur77, LSIRF, SNK,
PAC-1, kir/gem, MacMARCKS, PEP, MKP1, hRab30, MIP-1b, MIP-1a, EB12,
BL34, IL1-R2, TIS7, MyD116, Al, uPAR, RP 105, Evi-2 4F2, CD72 are
increased as a result of the introduction of the drug
candidate.
[0010] In some methods, the set comprises an immunosuppression set
comprising hIFI-204, hRhoH, caspase 2, B29, SLAP, NAG, iap38,
gadd45, BKLF, G7e, Id3, scd2, GILZ, Stat1, kappa 0 ig, LT-beta,
LKLF, IFNg-R11, mCDC47, EFP, TRAF5, and bcl-3. In some such
methods, the immunosuppressive set further comprises c-fos,
gadd153, EZF, C/EBP beta, Stra13, NAB2, mafK, and LRG-21. In some
such methods the expression of c-fos, gadd153, EZF, C/EBP beta,
Stra13, NAB2, mafK and LRG-21 are increased as a result of the
introduction of the drug candidate. In some methods the expression
of hIFI-204, hRhoH, caspase 2, B29, SLAP, NAG, iap38, gadd45, BKLF,
G7e, Id3, scd2, GILZ, Stat1, kappa 0 ig, LT-beta, LKLF, IFNg-RII,
mCDC47, EFP, TRAF5, and bcl-3 are decreased and the expression of
LSIRF, kir/gem, MKP1, hRab30, AhR, c-myc, Il1-R2, TIS11b, Evi-2,
A1, EB12, MyD116, MacMARCKS, MIP-1b, MIP-1a, PEP, CD72 are
increased as a result of the introduction of the drug
candidate.
[0011] The invention further provides methods of screening for a
bioactive agent capable of binding to a B lymphocyte modulator
protein (BLMP). The BLMP and a candidate bioactive agent are
combined. The binding of the candidate agent to the BLMP is then
determined. In some such methods, the BLMP is selected from the
group consisting of Egr-1, Egr-2, Nur77, c-myc, MIP-1a, MIP-1b,
BL34, gfi-1, NAB2, neurogranin, SLAP, A1, E2-20K, SATB1, Cctq,
kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2, CD72, cyclin D2, 1ck,
MEF-2C, bmk, IgD, Evi-2, vimentin, CD36, c-fes, c-fos, TRAP, hIP30,
Ly6E.1, LRG-21, Fos B, gadd153, mafK, Ah-R, C/EBP beta, EZF, TIS7,
TIS11, TIS 11b, LSIRF, MKP1, PAC-1, PEP, MacMARCKS, SNK, Stra13,
kir/gem, EB12, IL1-R2, MyD116, RP105, uPAR, 4F2, hRab30, Id3, BKLF,
LKLF, EFP, bcl-3, caspase 2, GILZ, hIFI-204, hRhoH, TRAF5, LT-beta,
IFNg-R11, gadd45, CDC47, NAG, scd2, kappa 0 ig, iap38, G7e, B29,
and SCD2.
[0012] The invention further provides methods for screening for a
bioactive agent capable of modulating the activity of a B
lymphocyte modulator protein (BLMP). The BLMP and a candidate
bioactive agent are combined. The effect of the candidate agent on
the bioactivity of the BLMP is then determined.
[0013] In some such methods the BLMP is selected from the group
consisting of Egr-1, Egr-2, Nur77, c-myc, MIP-1a, MIP-1b, BL34,
gfi-1, NAB2, neurogranin, SLAP, A1, E2-20K, SATB1, Cctq, kappa V,
pcp-4, TGIF, CD83, ApoE, Aeg-2, CD72, cyclin D2, 1ck, MF-2C, bmk,
IgD, Evi-2, vimentin, CD36, c-fes, c-fos, TRAP, hIP30, Ly6E.1,
LRG-21, Fos B, gadd153, mafK, Ah-R, C/EBP beta, EZF, TIS7, TIS11,
TIS11b, LSIRF, MKP1, PAC-1, PEP, MacMARCKS, SNK, Stra13, kir/gem,
EB12, IL1-R2, MyD116, RP105, uPAR, 4F2, hRab30, Id3, BKLF, LKLF,
EFP, bcl-3, caspase 2, GILZ, hIFI-204, hRhoH, TRAF5, LT-beta,
IFNg-RU, gadd45, CDC47, NAG, scd2, kappa 0 ig, iap38, G7e, B29, and
SCD2.
[0014] The invention further provides a method of evaluating the
effect of an immunosuppressive drug. In such methods, the drug is
administered to a patient; b) a cell sample is removed from the
patient; and c) the expression profile of the cell sample is
determined. Some such methods further comprise comparing the
expression profile to an expression profile of a healthy
individual. In some such methods the expression profile includes at
least one gene selected from the group consisting of Egr-1, Egr-2,
Nur77, c-myc, MIP-1a, MIP-1b, BL34, gfi-1, NAB2, neurogranin, SLAP,
A1, E2-20K, SATB1, Cctq, kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2,
CD72, cyclin D2, 1ck, MEF-2C, bmk, IgD, Evi-2, vimentin, CD36,
c-fes, c-fos, TRAP, hIP30, Ly6E.1, LRG-21, Fos B, gadd153, mafK,
Ah-R, C/EBP beta, EZF, TIS7, TIS11, TIS11b, LSIRF, MKP1, PAC-1,
PEP, MacMARCKS, SNK, Stra13, kir/gem, EB12, IL1-R2, MyD116, RP105,
uPAR, 4F2, hRab30, Id3, BKLF, LKLF, EFP, bcl-3, caspase 2, GILZ,
hIFI-204, hRhoH, TRAF5, LT-beta, IFNg-R11, gadd45, CDC47, NAG,
scd2, kappa 0 ig, iap38, G7e, B29, and SCD2.
[0015] The invention further provides an array of probes. The array
comprises a support bearing a plurality of nucleic acid probes
complementary to a plurality of mRNAs fewer than 1000 in number,
wherein the plurality of mRNA probes includes an mRNA expressed by
a gene selected from the group consisting of Egr-1, Egr-2, Nur77,
c-myc, MIP-1a, MIP-1b, BL34, gfi-1, NAB2, neurogranin, SLAP, A1,
E2-20K, SATB1, Cctq, kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2, CD72,
cyclin D2, 1ck, MEF-2C, bmk, IgD, Evi-2, vimentin, CD36, c-fes,
c-fos, TRAP, hIP30, Ly6E.1, LRG-21, Fos B, gadd153, mafK, Ah-R,
C/EBP beta, EZF, TIS7, TIS11, TIS11b, LSIRF, MKP1, PAC-1, PEP,
MacMARCKS, SNK, Stra13, kir/gem, EB12, IL1-R2, MyD116, RP105, uPAR,
4F2, hRab30, Id3, BKLF, LKLF, EFP, bcl-3, caspase 2, GILZ,
hIFI-204, hRhoH, TRAF5, LT-beta, IFNg-R11, gadd45, CDC47, NAG,
scd2, kappa 0 ig, iap38, G7e, B29, and SCD2. Some such arrays
comprise a plurality of sets of probes wherein each set of probes
iscomplementary to subsequences from a mRNA. In some arrays the
probes are cDNA sequences.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. Gene expression changes in B lymphocytes responding
to foreign antigen. A. Genes with increased mRNA levels after 1 hr
stimulation. 37 genes that showed significantly (p<0.00018)
increased expression (see methods) and showed a median fold change
of >1.75 were sorted by putative function. (CD72 is also shown
but only increased 1.5 fold. BL34 is represented twice on the
arrays, both sets of data are shown.) Each line represents one
experiment. The left end of the line shows hybridization intensity
in resting B cells mock stimulated in medium alone for one hour,
the right end of the line shows intensity in B cells stimulated for
one hour through the antigen receptor. Of the seven experiments
shown, 3 experiments were with Ig.sup.HEL transgenic B cells
stimulated with medium alone or with HEL, and 4 experiments were
with non-transgenic B cells stimulated with medium alone or with
anti-mu. Analysis of variance showed that the basal profiles and
responses to stimulation for Ig.sup.HEL and non-transgenic B cells
were essentially identical and the results have been presented
together for clarity. Spiking known concentrations of bacterial
transcripts allows an approximate calibration of 5 intensity
units/copy/cell assuming 300,000 transcripts per cell. B. Genes
with decreased mRNA levels after 1 hr stimulation. Hybridization
intensities are represented as for FIG. 1A. (GILZ is represented
twice on the arrays, both sets of data are shown.). C. 1 and 6 hr
timepoints of transcripts increased at 1 hr. Results are from 2
experiments showing HEL stimulation of Ig.sup.HEL transgenic B
cells. Each experiment is represented by a line. The left end of
the line is the intensity of the transcript in B cells mock
stimulated for 1 hr, the middle of the line is the intensity after
1 hr stimulation with HEL, the end of the line is the intensity
after 6 hr stimulation with HEL. Genes are shown in order of
exaggerated, sustained and transient increases relative to mock and
1 hr stimulated samples. D. 1 and 6 hr timepoints of transcripts
decreased at 1 hr. Results are from 2 experiments with HEL
stimulation of Ig transgenic B cells and are represented as in FIG.
1C.
[0017] FIG. 2. Gene expression changes in B lymphocytes responding
to self antigen. A. Genes upregulated in tolerant cells compared to
nave cells. The left end of each line represents hybridization
level in nave Ig.sup.HEL cells, the right end of the line
represents hybridization level in tolerant sHEL/Ig.sup.HEL cells.
Data points that are joined are from separate cell populations from
genetically distinct animals--each line represents samples prepared
in parallel on the same day. Five sets of data were derived from
negatively depleted B cell preparations and two sets from
FACS-sorted cells. One set of preparations included two tolerant
cell samples and one nave cell sample. This set is represented as 2
lines joining the nave cell hybridization intensity to each of the
tolerant cell intensities. B. Genes downregulated in tolerant cells
compared to nave cells. Data is represented as in FIG. 2A.
[0018] FIG. 3. Gene expression changes in B lymphocytes responding
to foreign antigen in the presence of FK506 or PD98059. A. FK506
sensitivity of the 1 hr upregulated genes defined in FIG. 1. B
cells were stimulated in the presence or absence of FK506, or were
mock stimulated. Data are shown from 5 experiments and genes are
shown in increasing order of median FK506 sensitivity. Each line
represents one experiment. The left end of the line is
hybridization intensity in resting B cells, the middle of the line
is intensity in B cells stimulated for one hour through the antigen
receptor and the right end of the line is intensity in B cells
stimulated for one hour in the presence of FK506. Of the five
experiments shown, 3 experiments were with IgHEL transgenic B cells
stimulated with medium, HEL or HEL/FK506, and 2 experiments were
with non-transgenic B cells stimulated with medium, anti-mu or
anti-mu/FK506. B. FK506 sensitivity of the 1 hr down-regulated
genes. Data is represented as for FIG. 3A. C. Correlation between
sensitivity to FK506 and sensitivity to EGTA for antigen-induced
transcripts. For the 37 induced genes defined in FIG. 1A, the
relative induction in the presence of EGTA was calculated as
average (antigen/EGTA-mock)/(antigen-mock), in two experiments with
IgHEL transgenic cells stimulated with HEL. For the same
transcripts, relative induction in the presence of FK506 was
calculated as median of (antigen/FK506-mock)/(antigen-mock) over 5
experiments. D. Upper two panels: upregulation of Egr-1 by anti-mu
stimulation of non-transgenic B cells is inhibited by PD98059 with
an IC50 of .about.5 .mu.M. Regulation of other 1 hour response
genes is less sensitive to PD98059. Lower panel: 3 transcripts
upregulated by both foreign and self antigen are sensitive to
PD98059. Left most four columns for each gene represent data from
non-transgenic B cells stimulated with anti-mu, right most 3
columns represent data from Ig.sup.HEL transgenic B cells
stimulated with HEL.
[0019] FIG. 4A. Summary table of biochemical pathways in tolerant
cells and nave cells exposed to foreign antigen in the presence or
absence of FK506 and PD98059. B. Potential mechanisms of tolerance,
immunity and immunosuppression suggested by the gene expression
analysis. Font size reflects mRNA or protein expression level
relative to mock stimulated cells (immunosuppression and activation
panels) or nave cells (tolerance panel). Tolerant cells have
decreased surface IgM (sIgM) but increased IgD (mRNA and protein):
sIg engagement by self-antigen causes decreased tyrosine
phosphorylation relative to activated cells. Proximal signaling
from sIg can be modulated in activated and tolerant cells by
recruitment of SHP1 by increased CD72. Activation of nave cells
causes a robust calcium flux that triggers NFkB, JNK and NFAT. All
these pathways are blocked by FK506 through inhibition of
calcineurin: calmodulin action can be regulated in nave and
immunosuppressed cells by neurogranin and in tolerant cells by
neurogranin and pcp-4. Egr family transcription is predicted to be
different under the 3 conditions: in activated cells both Egr-1 and
Egr-2 are upregulated preceding upregulation of NAB2; in
immunosuppressed cells, only Egr-1 is upregulated; and in tolerant
cells Egr-1 and Egr-2 are only weakly upregulated and can have
different effects on transcription in the presence of increased
NAB2. The balance between mitosis and apoptosis is likely to be in
part determined by upregulation of the proto-oncogenes c-myc and
LSIRF and the anti-apoptotic gene A1 in activated cells. These
changes are blocked by tolerance and partially blocked by FK506.
Downregulation of LKLF, which is sufficient to cause T cell
activation, is partially inhibited by FK506 and is blocked in
tolerance. Upregulation of surface activation markers CD69 and B7.2
is uninhibited by FK506 but is blocked in tolerance. The level of
B7.2 on B cells is critical in interaction with antigen specific T
cells.
DETAILED DESCRIPTION
[0020] 1. Definitions
[0021] The term patient includes mammals, such as humans, domestic
animals (e.g., dogs or cats), farm animals (cattle, horses, or
pigs), monkeys, rabbits, rats, mice, and other laboratory
animals.
[0022] The terms "nucleic acid" or "nucleic acid molecule" refer to
a deoxyribonucleotide or ribonucleotide polymer in either single-
or double-stranded form, and unless otherwise limited, can
encompass known analogs of natural nucleotides that can function in
a similar manner as naturally occurring nucleotides.
[0023] A polynucleotide probe is a single stranded nucleic acid
capable of binding to a target nucleic acid of complementary
sequence through one or more types of chemical bonds, usually
through complementary base pairing, usually through hydrogen bond
formation. A polynucleotide probe can include natural (i.e., A, G,
C, or T) or modified bases (e.g., 7-deazaguanosine, inosine).
Therefore, polynucleotide probes can 5-10,000,10-5,000,10-500,
10-50, 10-25, 10-20, 15-25, and 15-20 bases long. Probes are
typically about 10-50 bases long, and are often 15-20 bases. In its
simplest embodiment, the array includes test probes (also referred
to as polynucleotide probes) more than 5 bases long, preferably
more than 10 bases long, and some more than 40 bases long. The
probes can also be less than 50 bases long. In some cases, these
polynucleotide probes can range from about 5 to about 45 or 5 to
about 50 nucleotides long, or from about 10 to about 40 nucleotides
long, or from about 15 to about 40 nucleotides in length. The
probes can also be about 20 or 25 nucleotides in length.
[0024] In addition, the bases in a polynucleotide probe can be
joined by a linkage other than a phosphodiester bond, so long as it
does not interfere with hybridization. Thus, polynucleotide probes
can be peptide nucleic acids in which the constituent bases are
joined by peptide bonds rather than phosphodiester linkages. The
length of probes used as components of pools for hybridization to
distal segments of a target sequence often increases as the spacing
of the segments increased thereby allowing hybridization to be
conducted under greater stringency to increase discrimination
between matched and mismatched pools of probes.
[0025] Relatively short polynucleotide probes can be sufficient to
specifically hybridize to and distinguish target sequences.
Therefore, the polynucleotide probes can be less than 50
nucleotides in length, generally less than 46 nucleotides, more
generally less than 41 nucleotides, most generally less than 36
nucleotides, preferably less than 31 nucleotides, more preferably
less than 26 nucleotides, and most preferably less than 21
nucleotides in length. The probes can also be less than 16
nucleotides, less than 13 nucleotides in length, less than 9
nucleotides in length and less than 7 nucleotides in length.
[0026] Typically, arrays can have polynucleotides as short as 10
nucleotides or 15 nucleotides. In addition, 20 or 25 nucleotides
can be used to specifically detect and quantify nucleic acid
expression levels. Where ligation discrimination methods are used,
the polynucleotide arrays can contain shorter polynucleotides.
Arrays containing longer polynucleotides are also suitable. High
density arrays can comprise greater than about 100, 1000, 16,000,
65,000, 250,000 or even greater than about 1,000,000 different
polynucleotide probes.
[0027] The term "target nucleic acid" refers to a nucleic acid
(often derived from a biological sample), to which the
polynucleotide probe is designed to specifically hybridize. It is
either the presence or absence of the target nucleic acid that is
to be detected, or the amount of the target nucleic acid that is to
be quantified. The target nucleic acid has a sequence that is
complementary to the nucleic acid sequence of the corresponding
probe directed to the target. The term target nucleic acid can
refer to the specific subsequence of a larger nucleic acid to which
the probe is directed or to the overall sequence (e.g., gene or
mRNA) whose expression level it is desired to detect. The
difference in usage can be apparent from context.
[0028] "Subsequence" refers to a sequence of nucleic acids that
comprise a part of a longer sequence of nucleic acids.
[0029] "Gene" refers to a unit of inheritable genetic material
found in a chromosome, such as in a human chromosome. Each gene is
composed of a linear chain of deoxyribonucleotides which can be
referred to by the sequence of nucleotides forming the chain. Thus,
"sequence" is used to indicate both the ordered listing of the
nucleotides which form the chain, and the chain which has that
sequence of nucleotides. The term "sequence" is used in the same
way in referring to RNA chains, linear chains made of
ribonucleotides. The gene includes regulatory and control
sequences, sequences which can be transcribed into an RNA molecule,
and can contain sequences with unknown function. Some of the RNA
products (products of transcription from DNA) are messenger RNAs
(mRNAs) which initially include ribonucleotide sequences (or
sequence) which are translated into a polypeptide and
ribonucleotide sequences which are not translated. The sequences
which are not translated include control sequences, introns and
sequences with unknowns function. It can be recognized that small
differences in nucleotide sequence for the same gene can exist
between different persons, or between normal cells and cancerous
cells, without altering the identity of the gene.
[0030] "Gene expression pattern" means the set of genes of a
specific tissue or cell type that are transcribed or "expressed" to
form RNA molecules. Which genes are expressed in a specific cell
line or tissue can depend on factors such as tissue or cell type,
stage of development or the cell, tissue, or target organism and
whether the cells are normal or transformed cells, such as
cancerous cells. For example, a gene can be expressed at the
embryonic or fetal stage in the development of a specific target
organism and then become non-expressed as the target organism
matures. Alternatively, a gene can be expressed in liver tissue but
not in brain tissue of an adult human.
[0031] Specific hybridization refers to the binding, duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence
under stringent conditions when that sequence is present in a
complex mixture (e.g., total cellular) DNA or RNA. Stringent
conditions are conditions under which a probe can hybridize to its
target subsequence, but to no other sequences. Stringent conditions
are sequence-dependent and are different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence at a defined ionic strength and pH. The
T.sub.m is the temperature (under defined ionic strength, pH, and
nucleic acid concentration) at which 50% of the probes
complementary to the target sequence hybridize to the target
sequence at equilibrium. (As the target sequences are generally
present in excess, at T.sub.m, 50% of the probes are occupied at
equilibrium). Typically, stringent conditions include a salt
concentration of at least about 0.01 to 1.0 M Na ion concentration
(or other salts) at pH 7.0 to 8.3 and the temperature is at least
about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides).
Stringent conditions can also be achieved with the addition of
destabilizing agents such as formamide or tetraalkyl ammonium
salts. For example, conditions of 5.times.SSPE (750 mM NaCl, 50 mM
Na Phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30.degree.
C. are suitable for allele-specific probe hybridizations. (See
Sambrook et al., Molecular Cloning 1989).
[0032] Terms used to describe sequence relationships between two or
more nucleotide sequences or amino acid sequences include
"reference sequence," "selected from," "comparison window,"
"identical," "percentage of sequence identity," "substantially
identical," "complementary," and "substantially complementary." For
sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a
sequence comparison algorithm, test and reference sequences are
entered into a computer, subsequence coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. Default program parameters are used. Methods of
alignment of sequences for comparison are well-known in the art.
Optimal alignment of sequences for comparison can be conducted,
e.g., by the local homology algorithm of Smith & Waterman, Adv.
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. Nat'l. Acad.
Sci. USA 85: 2444 (1988), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Current Protocols in Molecular Biology (Ausubel et al.,
eds 1995 supplement)).
[0033] One example of a useful algorithm is PILEUP. PILEUP uses a
simplification of the progressive alignment method of Feng &
Doolittle, J. Mol. Evol. 35: 351-360 (1987). The method used is
similar to the method described by Higgins & Generally,
stringent conditions are selected to be about 5.degree. C. lower
than the thermal melting point (T.sub.m) for the specific sequence
at a defined ionic strength and pH. The T.sub.m is the temperature
(under defined ionic strength, pH, and nucleic acid concentration)
at which 50% of the probes complementary to the target sequence
hybridize to the target sequence at equilibrium. (As the target
sequences are generally present in excess, at T.sub.m, 50% of the
probes are occupied at equilibrium). Typically, stringent
conditions include a salt concentration of at least about 0.01 to
1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and
the temperature is at least about 30.degree. C. for short probes
(e.g., 10 to 50 nucleotides). Stringent conditions can also be
achieved with the addition of destabilizing agents such as
formamide or tetraalkyl ammonium salts. For example, conditions of
5.times.SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4)
and a temperature of 25-30.degree. C. are suitable for
allele-specific probe hybridizations. (See Sambrook et al.,
Molecular Cloning 1989).
[0034] Terms used to describe sequence relationships between two or
more nucleotide sequences or amino acid sequences include
"reference sequence," "selected from," "comparison window,"
"identical," "percentage of sequence identity," "substantially
identical," "complementary," and "substantially complementary."
[0035] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters are used. Methods of
alignment of sequences for comparison are well-known in the art.
Optimal alignment of sequences for comparison can be conducted,
e.g., by the local homology algorithm of Smith & Waterman, Adv.
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. Nat'l. Acad.
Sci. USA 85: 2444 (1988), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Current Protocols in Molecular Biology (Ausubel et al.,
eds 1995 supplement)).
[0036] One example of a useful algorithm is PILEUP. PILEUP uses a
simplification of the progressive alignment method of Feng &
Doolittle, J. Mol. Evol. 35: 351-360 (1987). The method used is
similar to the method described by Higgins & Sharp, CABIOS 5:
151-153 (1989). Using PILEUP, a reference sequence is compared to
other test sequences to determine the percent sequence identity
relationship using the following parameters: default gap weight
(3.00), default gap length weight (0.10), and weighted end gaps.
PILEUP can be obtained from the GCG sequence analysis software
package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:
387-395 (1984).
[0037] Another example of algorithms that are suitable for
determining percent sequence identity and sequence similarity are
the BLAST and the BLAST 2.0 algorithm, which are described in
Altschul et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et
al., Nucleic Acids Res. 25: 3389-3402 (1977)). Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). The BLASTN program (for nucleotide
sequences) uses as defaults a word length (W) of 11, alignments (B)
of 50, expectation (E) of 10, M=5, N-4, and a comparison of both
strands. The BLASTP program (for amino acid sequences) uses as
defaults a word length (W) of 3, and expectation (E) of 10, and the
BLOSUM62 scoring matrix (see Henikoff& Henikoff, Proc. Natl.
Acad. Sci. USA 89: 10915 (1989)).
[0038] The term antibody is used to include intact antibodies and
binding fragments thereof Typically, fragments compete with the
intact antibody from which they were derived and with other
antibodies for specific binding to an antigen. The term antibody
includes polyclonal antibodies, monoclonal antibodies, chimeric
antibodies and humanized antibodies, produced by immunization, from
hybridomas, or recombinantly.
[0039] The term molecule is used broadly to mean an organic or
inorganic chemical such as a drug; a peptide, including a variant
or modified peptide or peptide-like substance such as a
peptidomimetic or peptoid; or a protein such as an antibody or a
growth factor receptor or a fragment thereof, such as an F.sub.v,
F.sub.c or F.sub.ab fragment of an antibody, which contains a
binding domain. A molecule can be nonnaturally occurring, produced
as a result of in vitro methods, or can be naturally occurring,
such as a protein or fragment thereof expressed from a cDNA
library.
[0040] The term specific binding (and equivalent phrases) refers to
the ability of a binding moiety (e.g., a receptor, antibody, ligand
or antiligand) to bind preferentially to a particular target
molecule (e.g., ligand or antigen) in the presence of a
heterogeneous population of proteins and other biologics (i.e.,
without significant binding to other components present in a test
sample). Typically, specific binding between two entities, such as
a ligand and a receptor, means a binding affinity of at least about
10.sup.6 M.sup.-1, and preferably at least about 10.sup.7,
10.sup.8, 10.sup.9, or 10.sup.10 M.sup.-1. In some embodiments
specific (or selective) binding is assayed (and specific binding
molecules identified) according to the method of U.S. Pat. No.
5,622,699; this reference and all references cited therein are
incorporated herein by reference. Typically a specific or selective
reaction according to this assay is at least about twice background
signal or noise and more typically at least about 5 or at least
about 100 times background, or more.
[0041] When the binding moiety is an antibody, a variety of
immunoassay formats can be used to select antibodies that are
specifically immunoreactive with a particular protein. For example,
solid-phase ELISA immunoassays are routinely used to select
monoclonal antibodies specifically immunoreactive with an antigen.
See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold
Spring Harbor Publications, New York, for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity (this reference and references cited
therein are incorporated herein by reference).
[0042] The term "autoimmune disease" refers to a spontaneous or
induced malfunction of the immune system of mammals in which the
immune system fails to distinguish between foreign immunogenic
substances within the mammal and/or autologous ("self") substances
and, as a result, treats autologous ("self") tissues and substances
as if they were foreign and mounts an immune response against them.
Autoimmune disease is characterized by production of either
antibodies that react with self tissue, and/or the activation of
immune effector T cells that are autoreactive to endogenous self
antigens. Three main immunopathologic mechanisms act to mediate
autoimmune diseases: 1) autoantibodies are directed against
functional cellular receptors or other cell surface molecules, and
either stimulate or inhibit specialized cellular function with or
without destruction of cells or tissues; 2)
autoantigen--autoantibody immune complexes form in intercellular
fluids or in the general circulation and ultimately mediate tissue
damage; and 3) lymphocytes produce tissue lesions by release of
cytokines or by attracting other destructive inflammatory cell
types to the lesions. These inflammatory cells in turn lead to
production of lipid mediators and cytokines with associated
inflammatory disease.
[0043] The term "inflammation" refers to both acute responses
(i.e., responses in which the inflammatory processes are active)
and chronic responses (i.e., responses marked by slow progression
and formation of new connective tissue). Acute and chronic
inflammation may be distinguished by the cell types involved. Acute
inflammation often involves polymorphonuclear neutrophils; whereas
chronic inflammation is normally characterized by a
lymphohistiocytic and/or granulomatous response. Inflammation
includes reactions of both the specific and non-specific defense
systems. A specific defense system reaction is a specific immune
system reaction response to an antigen (possibly including an
autoantigen). A non-specific defense system reaction is an
inflammatory response mediated by leukocytes incapable of
immunological memory. Such cells include granulocytes, macrophages,
neutrophils and eosinophils. Examples of specific types of
inflammation are diffuse inflammation, focal inflammation, croupous
inflammation, interstitial inflammation, obliterative inflammation,
parenchymatous inflammation, reactive inflammation, specific
inflammation, toxic inflammation and traumatic inflammation.
[0044] The term "immune-mediated" refers to a process that is
either autoimmune or inflammatory in nature.
[0045] The term "perfect match probe" refers to a probe that has a
sequence that is perfectly complementary to a particular target
sequence. The test probe is typically perfectly complementary to a
portion (subsequence) of the target sequence. The perfect match
(PM) probe can be a "test probe," a "normalization control" probe,
an expression level control probe and the like. A perfect match
control or perfect match probe is, however, distinguished from a
"mismatch control" or "mismatch probe."
[0046] The term "mismatch control" or "mismatch probe" refer to
probes whose sequence is deliberately selected not to be perfectly
complementary to a particular target sequence. For each mismatch
(MM) control in a high-density array there typically exists a
corresponding perfect match (PM) probe that is perfectly
complementary to the same particular target sequence. The mismatch
can comprise one or more bases. While the mismatch(s) can be
located anywhere in the mismatch probe, terminal mismatches are
less desirable as terminal mismatch is less likely to prevent
hybridization of the target sequence.
[0047] The term "probe set" comprises at least a plurality of genes
perfectly matched with a known target sequence.
[0048] The terms "background" or "background signal intensity"
refer to hybridization signals resulting from non-specific binding,
or other interactions, between the labeled target nucleic acids and
components of the polynucleotide array (e.g. the polynucleotide
probes, control probes, or the array substrate). Background signals
can also be produced by intrinsic fluorescence of the array
components themselves. A single background signal can be calculated
for the entire array, or a different background signal can be
calculated for each region of the array. In some embodiments,
background is calculated as the average hybridization signal
intensity for the lowest 1% to 10% of the probes in the array, or
region of the array. In expression monitoring arrays (i.e., where
probes are preselected to hybridize to specific nucleic acids
(genes), a different background signal can be calculated for each
target nucleic acid. Where a different background signal is
calculated for each target gene, the background signal is
calculated for the lowest 1% to 10% of the probes for each gene.
Where the probes to a particular gene hybridize well and thus
appear to be specifically binding to a target sequence, they should
not be used in a background signal calculation. Alternatively,
background can be calculated as the average hybridization signal
intensity produced by hybridization to probes that are not
complementary to any sequence found in the sample (e.g., probes
directed to nucleic acids of the opposite sense or to genes not
found in the sample such as bacterial genes where the sample is of
mammalian origin). Background can also be calculated as the average
signal intensity produced by regions of the array that lack any
probes at all.
[0049] The term "quantifying" when used in the context of
quantifying nucleic acid abundance or concentrations (e.g.,
transcription levels of a gene) can refer to absolute or to
relative quantification. Absolute quantification can be
accomplished by inclusion of known concentration(s) of one or more
target nucleic acids (e.g. control nucleic acids such as BioB or
with known amounts the target nucleic acids themselves) and
referencing the hybridization intensity of unknowns with the known
target nucleic acids (e.g., through generation of a standard
curve). Alternatively, relative quantification can be accomplished
by comparison of hybridization signals between two or more genes,
or between two or more treatments to quantify the changes in
hybridization intensity and, by implication, transcription
level.
[0050] The term "cluster" or "clustering" refers to clustering
algorithms, such as principal components analysis and variable
clustering analysis. These algorithms serve to "cluster" cells into
groups. The purpose of clustering is to place the isolates into
groups or clusters suggested by the data, not defined a priori,
such that isolates in a given cluster tend to be similar and
isolates in different clusters tend to be dissimilar. Methods of
clustering are described in Tamayo et al., Proc. Natl. Acad. Sci
U.S.A. (1999) 96: 2907-2912.
[0051] 2. Gene Expression Profiles
[0052] The present invention provides novel methods for screening
for compositions which modulate B cell activity. The expression
levels of genes are determined for different cellular states of B
cells to provide expression profiles. A B cell expression profile
of a particular B cell state can be a "fingerprint" of the state;
while two states can have any particular gene similarly expressed,
the evaluation of a number of genes simultaneously allows the
generation of a gene expression profile that is unique to the state
of the cell. By comparing expression profiles of B cells in nave,
activated, immunosuppressed, tolerant or resting states,
information regarding which genes are important (including both up-
and down-regulation of genes) in each of these states is obtained.
This information can then be used in a number of ways. For example,
the evaluation of a particular treatment regime can be evaluated:
does an immunosuppressive drug act like an immunosuppressive drug
in this particular patient. Similarly, diagnosis can be done or
confirmed: does this patient have the gene expression profile of
immunosuppressed B cells. Furthermore, these gene expression
profiles can be used in drug candidate screening to find drugs that
mimic a particular expression profile; for example, screening can
be done for drugs that induce B cell tolerance as evidenced by a
tolerant expression profile. Accordingly, genes are identified and
described which are differentially expressed within and among B
cells in different states, from which the expression profiles are
generated as further described herein. For example, determinations
of differentially expressed nucleic acids are provided herein for B
cells which are resting, activated, immunosuppressed, nave, and
tolerant.
[0053] "Differential expression," or grammatical equivalents as
used herein, refers to both qualitative as well as quantitative
differences in the genes' temporal and/or cellular expression
patterns within and among B cells. Thus, a differentially expressed
gene can qualitatively have its expression altered, including an
activation or inactivation in, for example, tolerant versus
immunosuppressed cells, rested, nave or activated cells, or in a
healthy B cell response versus an abnormal B cell response. Genes
can be turned on or turned off in a particular state, relative to
another state. Any comparison of two or more states can be made.
Such a qualitatively regulated gene will exhibit an expression
pattern within a state or cell type which can be detectable by
standard techniques in one such state or cell type, but can be not
detectable in both. Alternatively, the determination can be
quantitative in that expression is increased or decreased; that is,
the expression of the gene is either upregulated, resulting in an
increased amount of transcript, or downregulated, resulting in a
decreased amount of transcript. The degree to which expression
differs need only be large enough to quantify using standard
characterization techniques, for example, by using Affymetrix
GeneChip.TM. expression arrays (Lockhart, Nature Biotechnology,
(1996) 14: 1675-1680; this reference and all references cited
therein are incorporated by reference). Other methods include, but
are not limited to, quantitative reverse transcriptase PCR,
Northern analysis and RNase protection. Preferably the change or
modulation in expression (i.e., upregulation or downregulation) is
at least about 5%, more preferably at least about 10%, more
preferably, at least about 20%, more preferably, at least about
30%, or more preferably by at least about 50%, or at least about
75%, and more preferably at least about 90%.
[0054] Any one, two, three, four, five, or ten or more genes can be
evaluated. These genes include, but are not limited to, Egr-1,
Egr-2, Nur77, c-myc, MIP-1a, MIP-1b, BL34, gfi-1, NAB2,
neurogranin, SLAP, A1, E2-20K, SATB1, Cctq, kappa V, pcp4, TGIF,
CD83, ApoE, Aeg-2, CD72, cyclin D2, 1ck, MEF-2C, bmk, IgD, Evi-2,
vimentin, CD36, c-fes, c-fos, TRAP, hIP30, Ly6E.1, LRG-21, Fos B,
gadd153, mafK, Ah-R, C/EBP beta, EZF, TIS7, TIS11, TIS11b, LSIRF,
MKP1, PAC-1, PEP, MacMARCKS, SNK, Stra13, kir/gem, EB12, IL1-R2,
MyD116, RP105, uPAR, 4F2, hRab30, Id3, BKLF, LKLF, EFP, bcl-3,
caspase 2, GILZ, hIFI-204, hRhoH, TRAF5, LT-beta, IFNg-R11, gadd45,
CDC47, NAG, scd2, kappa 0 ig, iap38, G7e, B29, and SCD2 (the
accession numbers for these genes can be found in Table 1).
Generally, oligonucleotide sequences used in the evaluation of
these genes are derived from their 3' untranslated regions.
[0055] Differentially expressed genes can represent "expression
profile genes", which includes "target genes". "Expression profile
gene," as used herein, refers to a differentially expressed gene
whose expression pattern can be used in methods for identifying
compounds useful in the modulation of B cell states or activity, or
the treatment of disorders, or alternatively, the gene can be used
as part of a prognostic or diagnostic evaluation of immune
disorders. For example, the effect of the compound on the
expression profile gene normally displayed in connection with a
particular state, such as tolerance, for example, can be used to
evaluate the efficacy of the compound to modulate that state, or
preferably, to induce or maintain that state. Such assays are
further described below. Alternatively, the gene can be used as a
diagnostic or in the treatment of immune disorders as also further
described below. In some instances, only a fragment of an
expression profile gene is used, as further described below.
[0056] "Expression profile," as used herein, refers to the pattern
of gene expression generated from two up to all of the expression
profile genes which exist for a given state. As outlined above, an
expression profile is in a sense a "fingerprint" or "blueprint" of
a particular cellular state; while two or more states have genes
that are similarly expressed, the total expression profile of the
state will be unique to that state. The gene expression profile
obtained for a given B cell state can be useful for a variety of
applications, including diagnosis of a particular disease or
condition and evaluation of various treatment regimes. In addition,
comparisons between the expression profiles of different B cell
states can be similarly informative. An expression profile can
include genes which do not appreciably change between two states,
so long as at least two genes which are differentially expressed
are represented. The gene expression profile can also include at
least one target gene, as defined below. Alternatively, the profile
can include all of the genes which represent one or more states.
Specific expression profiles are described below.
[0057] Gene expression profiles can be defined in several ways. For
example, a gene expression profile can be the relative transcript
level of any number of particular set of genes. Alternatively, a
gene expression profile can be defined by comparing the level of
expression of a variety of genes in one state to the level of
expression of the same genes in another state. For example, genes
can be either upregulated, downregulated, or remain substantially
at the same level in both states.
[0058] The expression profile for an activated B cell compared to a
nave resting B cell following lymphocyte activation for one hour is
shown in FIG. 1. Lymphocyte activation as used herein refers to the
antigen induced progression of B cells from the G0 phase to the G1
phase of the cell cycle. FIG. 1A shows the following upregulated
genes after lymphocyte activation for 1 hour: Egr-1, Egr-2, NAB2,
mafK, LRG-21, Fos B, c-fos, c-myc, Stra3, AhR, gadd153, C/EBP beta,
TIS11, TIS11b, gfi-1, EZF, Nur77, LSIRF, SNK, PAC-1, kir/gem,
MacMARCKS, PEP, MKP1, hRab30, MIP-1a, MIP-1b, EBI2, BL34, IL1-R2,
TIS7, MyD116, A1, uPAR, RP105, Evi-2, 4F2 and CD72; these genes are
referred to herein as upregulated early activation B cell
expression profile genes. FIG. 1B shows the following genes that
are downregulated after lymphocyte activation for one hour: Id3,
BKLF, LKLF, EFP, Stat1, bcl-3, hRhoH, TRAF5, SLAP, LT-beta,
IFNg-RII, GILZ, Caspase 2, gadd45, mCDC47, NAG, scd2, kappa 0 Ig,
B29, iap38, G7e, and hIFI-204; these genes are referred to herein
as downregulated early activation B cell expression profile
genes.
[0059] Also provided herein are gene expression profiles for
tolerant B cells compared to nave B cells after activation by self
or foreign antigen. Tolerance is generally defined as a state of
altered responsiveness to a particular antigen that prevents
development of either a cellular- or antibody-based immune response
to that antigen. FIG. 2A shows genes that are upregulated in
tolerant cells compared to nave cells after activation by
self-antigen: IgD, carb anh 11, CD72, SATB1, ApoE, CD83, cyclin D2,
Cctq, MEF-2C, TGIF, Aeg-2, Egr-1, 1ck, Egr-2, E2-20K, pcp-4, kappa
V, neurogranin, NAB2 and gfi-1. FIG. 2B shows the following genes
that are downregulated in tolerant cells compared to nave cells
after activation by self-antigen: Ly6E.1, vimentin, hIP-30, TRAP,
bmk, CD36, Evi-2, and c-fes.
[0060] Also provided herein are gene expression profiles for B cell
activation inhibited by immunosuppressive agents, as generally
outlined below. Examples of immunosuppressive drugs which inhibit B
cell activation include FK506 (see, e.g., Wicker, L. S. et al., Eur
J. Immunol (1990) 20: 2277-83) or cyclosporin A (see, e.g., Clin
Immunol Immunopathol (1996) 80(3 Pt 2): S40-5). As used herein,
immunosuppression and tolerance include the suppression of B
lymphocyte activation. Agents which modulate immunosuppression are
referred to herein as immunosuppressants, immunosuppressant
modulators, or immunosuppressive agents. The expression profile for
immunosuppressed B cells compared to activated and resting B cells
is shown in FIG. 3. FIG. 3A and FIG. 3B show the upregulated and
downregulated early activation B cell expression profile genes
where each line individually shows one gene in the resting state,
activated state and immunosuppressed state by reading the line left
to right respectively. Thus, a gene sensitive to immunosuppression
is represented by a peak for upregulated genes (FIG. 3A) and
valleys for downregulated genes (see FIG. 3B). FIG. 3A shows the
immunosuppressive sensitivity of the following upregulated early
activation B cell expression profile genes in order of sensitivity,
where the right side of FIG. 3A shows the most sensitive genes.
"Sensitive" in this context means that gene expression is
downregulated as compared to the active state. Sensitive
upregulated early activation B cell expression profile genes
include: LSIRF, kir/gem, MKP1, hRab30, AhR, c-myc, IL1-R2, TIS11b,
Evi-2, A1, EB12, MyD116, MacMARCKS, MIP-1b, Egr-2, MIP-1a, PEP and
CD72. Upregulated early activation B cell expression profile genes
that are less than 30% inhibited by immunosuppressive agents
include: c-fos, gadd153, EZF, C/EBP beta, Stra13, mafK, LRG-21,
BL34, SNK, uPAR, TIS7, PAC-1, Fos B, TIS11, gfi-1, Egr-1, 4F2,
RP10S and Nur77.
[0061] FIG. 3B shows the immunosuppressive sensitivity of the
down-regulated early activation B cell expression profile genes in
order of sensitivity, where the right side of FIG. 3B shows the
most sensitive genes. Sensitive downregulated early activation B
cell expression profile genes include: LKLF, IFNg-R11, CDC47, EFP,
TRAF5 and bcl-3. Downregulated early activation B cell expression
profile genes that are less than 30% inhibited by immunosuppressive
agents include: hIFI-204, hRhoH, caspase 2, B29, SLAP, NAG iap38,
gadd45, BKLF, G7e, Id3, scd2, GILZ, Stat1, kappa 0 ig, and
LT-beta.
[0062] A gene expression profile can include a combination of at
least two of Egr-1, Nur77, c-myc, MIP-1a, MIP-1b, BL34, gfi-1,
NAB2, neurogranin, and SLAP. A1 can also be included in the
expression profile of tolerant cells as shown in FIG. 4. Another
target gene for tolerance is B7.2, which upregulation is inhibited
in tolerance but not in immunosuppression. A preferred target gene
for tolerance or tolerance modulation is NAB2 which is upregulated
in tolerant B cells compared to resting or nave cells. Moreover,
target genes for tolerance include: CD72, neurogranin, pcp4, Egr-1,
Egr-2, NAB2, myc, LSIRF, A1, and LKLF which are downregulated in
tolerance; these changes appear to be unique to the tolerance
phenotype and are not seen in response to an activating signal.
Agents which modulate or induce a state of tolerance are referred
to herein as tolerants. In a preferred expression profile, the
total expression profile is recreated by at least one small
molecule (e.g., FK506 or cyclosporin A) or other pharmacological
intervention. In one embodiment, at least one of or all of Egr-1,
Egr-2, c-myc and c-fos are suppressed while NAB2 is
upregulated.
[0063] 3. Target and Pathway Genes
[0064] In addition to expression profile genes, the present
invention also provides target genes. "Target gene," as used
herein, refers to a differentially expressed expression profile
gene whose expression is unique for a particular state, such that
the presence or absence of the transcript of a target gene(s) can
indicate the state the cell is in. A target gene can be completely
unique to a particular state; the presence or absence of the gene
is only seen in a particular cell state, or alternatively, cells in
all other states express the gene but it is not seen in the first
state. Thus for example NAB2 is not expressed in nave B cells but
is expressed in all other states. Alternatively, target genes can
be identified as relevant to a comparison of two states, that is,
the state is compared to another particular state or standard to
determine the uniqueness of the target gene. Target genes can be
used in the diagnostic, prognostic, and compound identification
methods described herein.
[0065] It should be understood that a target gene for a first state
can be an expression profile gene for a second state. The presence
or absence of a particular target gene in one state can be
diagnostic of the state; the same gene in a different state can be
an expression profile gene.
[0066] Further, pathway genes are provided herein. "Pathway genes"
are defined by the ability of their gene products to interact with
expression profile genes. Pathway genes can also exhibit target
gene and/or expression profile gene characteristics and can be
included as modulators of expression profile genes as further
described below.
[0067] The present invention includes the products of such
expression profile, target, and pathway genes, as well as
antibodies to such gene products. Furthermore, the engineering and
use of cell- and animal-based models of B cell states to which such
profiles, genes and gene products can contribute, are also
described.
[0068] 4. Sample Preparation
[0069] To measure the transcription level (and thereby the
expression level) of a gene or genes, a nucleic acid sample
comprising mRNA transcript(s) of the gene or genes, or nucleic
acids derived from the mRNA transcript(s) is provided. A nucleic
acid derived from an mRNA transcript refers to a nucleic acid for
whose synthesis the mRNA transcript or a subsequence thereof has
ultimately served as a template. Thus, a cDNA reverse transcribed
from an mRNA, an RNA transcribed from that cDNA, a DNA amplified
from the cDNA, an RNA transcribed from the amplified DNA, are all
derived from the mRNA transcript and detection of such derived
products is indicative of the presence and/or abundance of the
original transcript in a sample. Thus, suitable samples include
mRNA transcripts of the gene or genes, cDNA reverse transcribed
from the mRNA, cRNA transcribed from the cDNA, DNA amplified from
the genes, RNA transcribed from amplified DNA, and the like.
[0070] In some methods, a nucleic acid sample is the total mRNA
isolated from a biological sample. The term "biological sample", as
used herein, refers to a sample obtained from an organism or from
components (e.g., cells) or an organism. The sample can be of any
biological tissue or fluid. Frequently the sample is from a
patient. Such samples include sputum, blood, blood cells (e.g.,
white cells), tissue or fine needle biopsy samples, urine,
peritoneal fluid, and fleural fluid, or cells therefrom. Biological
samples can also include sections of tissues such as frozen
sections taken for histological purposes. Often two samples are
provided for purposes of comparison. The samples can be, for
example, from different cell or tissue types, from different
species, from different individuals in the same species or from the
same original sample subjected to two different treatments (e.g.,
drug-treated and control).
[0071] 5. Method
[0072] (A) Generation of cDNAs
[0073] For example, methods of isolation and purification of
nucleic acids are described in detail in WO 97/10365, WO 97/27317,
Chapter 3 of Laboratory Techniques in Biochemistry and Molecular
Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and
Nucleic Acid Preparation, P. Tijssen, ed. Elsevier, N.Y. (1993) and
Chapter 3 of Laboratory Techniques in Biochemistry and Molecular
Biology: Hybridization With Nucleic Acid Probes, Part 1. Theory and
Nucleic Acid Preparation, P. Tijssen, ed. Elsevier, N.Y.
(1993)).
[0074] The total nucleic acid can be isolated from a given sample
using, for example, an acid quanidinium-phenol-choloroform
extraction method and poly A.sup.+ mRNA is isolated by oligo dT
column chromatography or by using (dT)n magnetic beads (see, e.g.,
Sambrook et al., Molecular Cloning: A Laboratory Manual (2.sup.nd
ed.), Vols 1-3, Cold Spring Harbor Laboratory, (1989), or Current
Protocols in Molecular Biology, F. Ausubel et al., ed., Breene
Publishing and Wiley-Interscience, N.Y. (1987)).
[0075] The sample mRNA can be reverse transcribed with a reverse
transcriptase and a primer consisting of oligo dT and a sequence
encoding the phage T7 promoter to provide single stranded DNA
template. The second DNA strand is polymerized using a DNA
polymerase. Methods of in vitro polymerization are well known (see,
e.g., Sambrook, supra) and this particular method is described in
detail by Van Gelder, et al., Proc. Natl. Acad. Sci. U.S.A 87:
1663-1667 (1990) report that in vitro amplification according to
this method preserves the relative frequencies of the various RNA
transcripts. Eberwine et al., Proc. Natl. Acad. Sci. U.S.A 89:
3010-3014 provide a further protocol that uses two round of
amplification via in vitro transcription thereby permitting
expression monitoring. Eberwine et al. describe another method of
amplification in Methods (1996) 10(3): 283-8. Another method of
amplification is described in Dixon et al., Nucleic Acids Res
(1998) 26(19): 4426-31. A still further method of amplification is
the amplification method described in Dulac et al., Cell (1995) 83:
195-206. An alternative method of amplification is described in
U.S. Ser. No. 60/126,796 filed on Mar. 30, 1999, which is herein
incorporated by reference.
[0076] After amplification, the nucleic acids are typically cleaved
into smaller fragments. Cleavage can be achieved by DNaseI
digestion, restriction enzyme digestion, or sonication. Nucleic
acids are typically labeled. Label can be introduced during
amplification either by linkage to one of the primers or by one of
the nucleotides being incorporated. Alternatively, labeling can be
effected after amplification and cleavage by end-labeling.
Detectable labels suitable for use in the present invention include
any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means;
see WO 97/10365.
[0077] In general, nucleic acid probes comprising the expression
profile genes, including differentially expressed genes and target
genes, can be attached to a solid support, generally in an array
format, to allow for gene expression monitoring. "Gene" in this
context includes full length genes and fragments thereof, and can
comprise either the coding strand or its complement, and can be a
portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA
including mRNA and rRNA.
[0078] In some cases, the differentially expressed nucleic acid can
be a fragment, or expressed sequence tag (EST). Once a
differentially expressed nucleic acid which is not a full length
gene is identified, it can be cloned and, if necessary, its
constituent parts recombined to form an entire full length or
mature differentially expressed nucleic acid. Using methods
described herein and known in the art, it can be used to identify
the full length clone. Wherein the full length nucleic acid has a
signal peptide and/or transmembrane region(s), it can be modified
to exclude one or more of these regions so as to encode a peptide
in its mature soluble form. Once isolated from its natural source,
e.g., contained within a plasmid or other vector or excised
therefrom as a linear nucleic acid segment, the recombinant
differentially expressed nucleic acid can be further-used as a
probe to identify and isolate other differentially expressed
nucleic acid acids. It can also be used as a "precursor" nucleic
acid to make modified or variant differentially expressed nucleic
acid acids and proteins. Where two or more nucleic acids overlap,
the overlapping portion(s) of one of the overlapping nucleic acids
can be omitted and the nucleic acids combined for example by
ligation to form a longer linear differentially expressed nucleic
acid so as to, for example, encode the full length or mature
peptide. The same applies to the amino acid sequences of
differentially expressed polypeptides in that they can be combined
so as to form one contiguous peptide.
[0079] It should be noted that the nucleic acid probes used herein
need not be identical to the wild-type genes listed above. Nucleic
acids having sequence identity with differentially expressed
nucleic acids preferably have about 65% or 75%, more preferably
greater than about 80%, even more preferably greater than about 85%
and most preferably greater than 90% sequence identity. In some
embodiments the sequence identity will be as high as about 93 to 95
or 98%. Sequence identity will be determined using standard
techniques known in the art, including, but not limited to, the
local sequence identity algorithm of Smith & Waterman, Adv.
Appl. Math. 2: 482 (1981), by the sequence identity 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 (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit
sequence program described by Devereux et al., Nucl. Acid Res. 12:
387-395 (1984), preferably using the default settings, or by
inspection.
[0080] The PCR method of amplification is described in 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 et al., 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). Nucleic acids in a target sample are usually labeled
in the course of amplification by inclusion of one or more labeled
nucleotides in the amplification mix. Labels can also be attached
to amplification products after amplification e.g., by
end-labeling. The amplification product can be RNA or DNA depending
on the enzyme and substrates used in the amplification
reaction.
[0081] Other suitable amplification methods include the ligase
chain reaction (LCR) (see Wu and Wallace, Genomics 4, 560 (1989),
Landegren et al., Science 241, 1077 (1988), transcription
amplification (Kwoh et al., Proc. Natl. Acad. Sci. U.S.A 86, 1173
(1989)), and self-sustained sequence replication (Guatelli et al.,
Proc. Nat. Acad. Sci. U.S.A 87, 1874 (1990)) and nucleic acid based
sequence amplification (NASBA). The latter two amplification
methods involve isothermal reactions based on isothermal
transcription, which produce both single stranded RNA (ssRNA) and
double stranded DNA (dsDNA) as the amplification products in a
ratio of about 30 or 100 to 1, respectively.
[0082] A variety of labels can be incorporated into target nucleic
acids in the course of amplification or after amplification.
Suitable labels include fluorescein or biotin, the latter being
detected by staining with phycoerythrin-streptavidin after
hybridization. In some methods, hybridization of target nucleic
acids is compared with control nucleic acids. Optionally, such
hybridizations can be performed simultaneously using different
labels are used for target and control samples. Control and target
samples can be diluted, if desired, prior to hybridization to
equalize fluorescence intensities.
[0083] 6. Supports
[0084] Supports can be made of a variety of materials, such as
glass, silica, plastic, nylon or nitrocellulose. Supports are
preferably rigid and have a planar surface. Supports typically have
from 1-10,000,000 discrete spatially addressable regions, or cells.
Supports having 10-1,000,000 or 100-100,000 or 1000-100,000 cells
are common. The density of cells is typically at least 1000,
10,000, 100,000 or 1,000,000 cells within a square centimeter.
Typically a single probe per cell. In some supports, all cells are
occupied by pooled mixtures of probes. In other supports, some
cells are occupied by pooled mixtures of probes, and other cells
are occupied, at least to the degree of purity obtainable by
synthesis methods, by a single type of polynucleotide. The
strategies for probe design described in the present application
can be combined with other strategies, such as those described by
WO 95/11995, EP 717,113 and WO 97/29212 in the same array.
[0085] The location and sequence of each different polynucleotide
probe in the array is generally known. Moreover, the large number
of different probes can occupy a relatively small area providing a
high density array having a probe density of generally greater than
about 60, more generally greater than about 100, and most generally
greater than about 600, often greater than about 1000, more often
greater than about 5,000, most often greater than about 10,000,
preferably greater than about 40,000 more preferably greater than
about 100,000, and most preferably greater than about 400,000
different polynucleotide probes per cm.sup.2. The small surface
area of the array (often less than about 10 cm.sup.2, preferably
less than about 5 cm.sup.2 more preferably less than about 2
cm.sup.2, and most preferably less than about 1.6 cm.sup.2) permits
the use of small sample volumes and extremely uniform hybridization
conditions.
[0086] 7. Synthesis of Probe Arrays
[0087] Arrays of probes can be synthesized in a step-by-step manner
on a support or can be attached in presynthesized form. A preferred
method of synthesis is VLSIPS.TM. (see Fodor et al, 1991, Fodor et
al., 1993, Nature 364, 555-556; McGall et al, U.S. Ser. No.
08/445,332; U.S. Pat. No. 5,143,854; EP 476,014), which entails the
use of light to direct the synthesis of polynucleotide probes in
high-density, miniaturized arrays. Algorithms for design of masks
to reduce the number of synthesis cycles are described by Hubbel et
al, U.S. Pat. No. 5,571,639 and U.S. Pat. No. 5,593,839. Arrays can
also be synthesized in a combinatorial fashion by delivering
monomers to cells of a support by mechanically constrained
flowpaths. See Winkler et al, EP 624,059. Arrays can also be
synthesized by spotting monomers reagents on to a support using an
ink jet printer. See id.; Pease et al., EP 728,520.
[0088] After hybridization of control and target samples to an
array containing one or more probe sets as described above and
optional washing to remove unbound and nonspecifically bound probe,
the hybridization intensity for the respective samples is
determined for each probe in the array. For fluorescent labels,
hybridization intensity can be determined by, for example, a
scanning confocal microscope in photon counting mode. Appropriate
scanning devices are described by e.g., Trulson et al., U.S. Pat.
No. 5,578,832; Stern et al., U.S. Pat. No. 5,631,734 and are
available from Affymetrix, Inc., under the GeneChip.TM. label. Some
types of label provide a signal that can be amplified by enzymatic
methods (see Broude, et al, Proc. Natl. Acad. Sci. U.S.A. 91,
3072-3076 (1994))
[0089] 8. Design of Arrays
[0090] (A) Customized and Generic Arrays
[0091] The design of arrays for expression monitoring is generally
described, for example, WO 97/27317 and WO 97/10365 (these
references are herein incorporated by reference). There are two
principal categories of arrays. One type of array detects the
presence and/or levels of particular mRNA sequences that are known
in advance. In these arrays, polynucleotide probes can be selected
to hybridize to particular preselected subsequences of mRNA gene
sequence. Such expression monitoring arrays can include a plurality
of probes for each mRNA to be detected. For analysis of mRNA
nucleic acids, the probes are designed to be complementary to the
region of the mRNA that is incorporated into the nucleic acids
(i.e., the 3' end). The array can also include one or more control
probes.
[0092] Generic arrays can include all possible nucleotides of a
given length; that is, polynucleotides having sequences
corresponding to every permutation of a sequence. Thus since the
polynucleotide probes of this invention preferably include up to 4
bases (A, G, C, T) or (A, G, C, U) or derivatives of these bases,
an array having all possible nucleotides of length X contains
substantially 4.sup.X different nucleic acids (e.g., 16 different
nucleic acids for a 2 mer, 64 different nucleic acids for a 3 mer,
65536 different nucleic acids for an 8 mer). Some small number of
sequences can be absent from a pool of all possible nucleotides of
a particular length due to synthesis problems, and inadvertent
cleavage). An array comprising all possible nucleotides of length X
refers to an array having substantially all possible nucleotides of
length X. All possible nucleotides of length X includes more than
90%, typically more than 95%, preferably more than 98%, more
preferably more than 99%, and most preferably more than 99.9% of
the possible number of different nucleotides. Generic arrays are
particularly useful for comparative hybridization analysis between
two mRNA populations or nucleic acids derived therefrom.
[0093] (B) Variations
[0094] (1) Constant Regions
[0095] In both customized and generic array, probes can comprise
additional constant regions fused with the variable regions that
mediate hybridization to target nucleic acid. In some arrays,
constant regions are double stranded thereby providing a site at
which hybridized target can ligate to immobilized probes. A
constant domain is a nucleotide subsequence that is common to
substantially all of the polynucleotide probes. Constant domains
are typically located at the terminus of the polynucleotide probe
closest to the substrate (i.e., attached to the linker/anchor
molecule). The constant regions can comprise virtually any
sequence. Some constant regions comprise a sequence or subsequence
complementary to the sense or antisense strand of a restriction
site (a nucleic acid sequence recognized by a restriction
enzyme).
[0096] Constant regions can be synthesized de novo on the array or
prepared in a separate procedure and then coupled intact to the
array. Since the constant domain can be synthesized separately and
then the intact constant subsequences coupled to the high density
array, the constant domain can be virtually any length. Some
constant domains range from 3 nucleotides to about 500 nucleotides
in length, more typically from about 3 nucleotides in length to
about 100 nucleotides in length, most typically from 3 nucleotides
in length to about 50 nucleotides in length. Constant domains can
also range from 3 nucleotides to about 45 nucleotides in length, or
from 3 nucleotides in length to about 25 nucleotides in length or
from 3 to about 15 or even 10 nucleotides in length. Constant
domains can also range from about 5 nucleotides to about 15
nucleotides in length.
[0097] (2) Control Probes
[0098] Either customized or generic probe arrays can contain
control probes in addition to the probes described above.
[0099] (a) Normalization Controls
[0100] Normalization controls are typically perfectly complementary
to one or more labeled reference polynucleotides that are added to
the nucleic acid sample. The signals obtained from the
normalization controls after hybridization provide a control for
variations in hybridization conditions, label intensity, reading
and analyzing efficiency and other factors that can cause the
signal of a perfect hybridization to vary between arrays. Signals
(e.g., fluorescence intensity) read from all other probes in the
array can be divided by the signal (e.g., fluorescence intensity)
from the control probes thereby normalizing the measurements.
[0101] Virtually any probe can serve as a normalization control.
However, hybridization efficiency can vary with base composition
and probe length. Normalization probes can be selected to reflect
the average length of the other probes present in the array,
however, they can also be selected to cover a range of lengths. The
normalization control(s) can also be selected to reflect the
(average) base composition of the other probes in the array.
However one or a fewer normalization probes can be used and they
can be selected such that they hybridize well (i.e., no secondary
structure) and do not match any target-specific probes.
[0102] Normalization probes can be localized at any position in the
array or at multiple positions throughout the array to control for
spatial variation in hybridization efficiently. The normalization
controls can be located at the corners or edges of the array as
well as in the middle of the array.
[0103] (b) Expression Level Controls
[0104] Expression level controls can be probes that hybridize
specifically with constitutively expressed genes in the biological
sample. Expression level controls can be designed to control for
the overall health and metabolic activity of a cell. Examination of
the covariance of an expression level control with the expression
level of the target nucleic acid can indicate whether measured
changes or variations in expression level of a gene is due to
changes in transcription rate of that gene or to general variations
in health of the cell. Thus, for example, when a cell is in poor
health or lacking a critical metabolite the expression levels of
both an active target gene and a constitutively expressed gene are
expected to decrease. The converse can also be true. Thus where the
expression levels of both an expression level control and the
target gene appear to both decrease or to both increase, the change
can be attributed to changes in the metabolic activity of the cell
as a whole, not to differential expression of the target gene in
question. Conversely, where the expression levels of the target
gene and the expression level control do not covary, the variation
in the expression level of the target gene can be attributed to
differences in regulation of that gene and not to overall
variations in the metabolic activity of the cell.
[0105] Virtually any constitutively expressed gene can provide a
suitable target for expression level controls. Typically expression
level control probes can have sequences complementary to
subsequences of constitutively expressed genes including, but not
limited to the .beta.-actin gene, the transferrin receptor gene,
the GAPDH gene, and the like.
[0106] (c) Mismatch Controls
[0107] Mismatch controls can also be provided for the probes to the
target genes, for expression level controls or for normalization
controls. Mismatch controls are typically employed in customized
arrays containing probes matched to known mRNA species. For
example, some such arrays contain a mismatch probe corresponding to
each match probe. The mismatch probe is the same as its
corresponding match probe except for at least one position of
mismatch. A mismatched base is a base selected so that it is not
complementary to the corresponding base in the target sequence to
which the probe can otherwise specifically hybridize. One or more
mismatches are selected such that under appropriate hybridization
conditions (e.g. stringent conditions) the test or control probe
can be expected to hybridize with its target sequence, but the
mismatch probe cannot hybridize (or can hybridize to a
significantly lesser extent). Mismatch probes can contain a central
mismatch. Thus, for example, where a probe is a 20 mer, a
corresponding mismatch probe can have the identical sequence except
for a single base mismatch (e.g., substituting a G, a C or a T for
an A) at any of positions 6 through 14 (the central mismatch).
[0108] In generic (e.g., random, arbitrary, or haphazard) arrays,
since the target nucleic acid(s) are unknown perfect match and
mismatch probes cannot be a priori determined, designed, or
selected. In this instance, the probes can be provided as pairs
where each pair of probes differ in one or more preselected
nucleotides. Thus, while it is not known a priori which of the
probes in the pair is the perfect match, it is known that when one
probe specifically hybridizes to a particular target sequence, the
other probe of the pair can act as a mismatch control for that
target sequence. The perfect match and mismatch probes need not be
provided as pairs, but can be provided as larger collections (e.g.,
3, 4, 5, or more) of probes that differ from each other in
particular preselected nucleotides.
[0109] In both customized and generic arrays mismatch probes can
provide a control for non-specific binding or cross-hybridization
to a nucleic acid in the sample other than the target to which the
probe is complementary. Mismatch probes thus can indicate whether a
hybridization is specific or not. For example, if the complementary
target is present the perfect match probes can be consistently
brighter than the mismatch probes. In addition, if all central
mismatches are present, the mismatch probes can be used to detect a
mutation. Finally, the difference in intensity between the perfect
match and the mismatch probe (I(PM)-I(MM) can provide a good
measure of the concentration of the hybridized material.
[0110] (d) Sample Preparation, Amplification, and Quantitation
Controls
[0111] Arrays can also include sample preparation/amplification
control probes. These can be probes that are complementary to
subsequences of control genes selected because they do not normally
occur in the nucleic acids of the particular biological sample
being assayed. Suitable sample preparation/amplification control
probes can include, for example, probes to bacterial genes (e.g.,
Bio B) where the sample in question is a biological sample from a
eukaryote.
[0112] The RNA sample can then be spiked with a known amount of the
nucleic acid to which the sample preparation/amplification control
probe is directed before processing. Quantification of the
hybridization of the sample preparation/amplification control probe
can then provide a measure of alteration in the abundance of the
nucleic acids caused by processing steps (e.g., PCR, reverse
transcription, or in vitro transcription).
[0113] Quantitation controls can be similar. Typically they can be
combined with the sample nucleic acid(s) in known amounts prior to
hybridization. They are useful to provide a quantitation reference
and permit determination of a standard curve for quantifying
hybridization amounts (concentrations).
[0114] 9. Methods of Detection
[0115] In one method of detection, mRNA or nucleic acid derived
therefrom, typically in denatured form, are applied to an array.
The component strands of the nucleic acids hybridize to
complementary probes, which are identified by detecting label.
Optionally, the hybridization signal of matched probes can be
compared with that of corresponding mismatched or other control
probes. Binding of mismatched probe serves as a measure of
background and can be subtracted from binding of matched probes. A
significant difference in binding between a perfectly matched
probes and a mismatched probes signifies that the nucleic acid to
which the matched probes are complementary is present. Binding to
the perfectly matched probes is typically at least 1.2, 1.5, 2, 5
or 10 or 20 times higher than binding to the mismatched probes.
[0116] In a variation of the above method, nucleic acids are not
labeled but are detected by template-directed extension of a probe
hybridized to a nucleic acid strand with the nucleic acid strand
serving as a template. The probe is extended with a labeled
nucleotide, and the position of the label indicates, which probes
in the array have been extended. By performing multiple rounds of
extension using different bases bearing different labels, it is
possible to determine the identity of additional bases in the tag
than are determined through complementarity with the probe to which
the tag is hybridized. The use of target-dependent extension of
probes is described by U.S. Pat. No. 5,547,839.
[0117] In a further variation, probes can be extended with inosine.
The inosine strand can be labeled. The addition of degenerate
bases, such as inosine (it can pair with all other bases), can
increase duplex stability between the polynucleotide probe and the
denatured single stranded DNA nucleic acids. The addition of 1-6
inosines onto the end of the probes can increase the signal
intensity in both hybridization and ligation reactions on a generic
ligation array. This can allow for ligations at higher
temperatures. The use of degenerate bases is described in WO
97/27317.
[0118] Ligation reactions can offer improved discriminate between
fully complementary hybrids and those that differ by one or more
base pairs, particularly in cases where the mismatch is near the 5'
terminus of the polynucleotide probes. Use of a ligation reaction
in signal detection increases the stability of the hybrid duplex,
improves hybridization specificity (particularly for shorter
polynucleotide probes (e.g., 5 to 12-mers), and optionally,
provides additional sequence information. Ligation reactions used
in signal detection are described in WO 97/27317. Optionally,
ligation reactions can be used in conjunction with
template-directed extension of probes, either by inosine or other
bases.
[0119] 10. Analysis of Hybridization Patterns
[0120] The position of label is detected for each probe in the
array using a reader, such as described by U.S. Pat. No. 5,143,854,
WO 90/15070, and Trulson et al., supra. For customized arrays, the
hybridization pattern can then be analyzed to determine the
presence and/or relative amounts or absolute amounts of known mRNA
species in samples being analyzed as described in e.g., WO
97/10365. Comparison of the expression patterns of two samples is
useful for identifying mRNAs and their corresponding genes that are
differentially expressed between the two samples.
[0121] The quantitative monitoring of expression levels for large
numbers of genes can prove valuable in elucidating gene function,
exploring the causes and mechanisms of disease, and for the
discovery of potential therapeutic and diagnostic targets.
Expression monitoring can be used to monitor the expression
(transcription) levels of nucleic acids whose expression is altered
in a disease state. For example, a cancer can be characterized by
the overexpression of a particular marker such as the HER2
(c-erbB-2/neu) protooncogene in the case of breast cancer.
[0122] Expression monitoring can be used to monitor expression of
various genes in response to defined stimuli, such as a drug. This
is especially useful in drug research if the end point description
is a complex one, not simply asking if one particular gene is
overexpressed or underexpressed. Therefore, where a disease state
or the mode of action of a drug is not well characterized, the
expression monitoring can allow rapid determination of the
particularly relevant genes.
[0123] In generic arrays, the hybridization pattern is also a
measure of the presence and abundance of relative mRNAs in a
sample, although it is not immediately known, which probes
correspond to which mRNAs in the sample.
[0124] However the lack of knowledge regarding the particular genes
does not prevent identification of useful therapeutics. For
example, if the hybridization pattern on a particular generic array
for a healthy cell is known and significantly different from the
pattern for a diseased cell, then libraries of compounds can be
screened for those that cause the pattern for a diseased cell to
become like that for the healthy cell. This provides a detailed
measure of the cellular response to a drug.
[0125] Generic arrays can also provide a powerful tool for gene
discovery and for elucidating mechanisms underlying complex
cellular responses to various stimuli. For example, generic arrays
can be used for expression fingerprinting. Suppose it is found that
the mRNA from a certain cell type displays a distinct overall
hybridization pattern that is different under different conditions
(e.g., when harboring mutations in particular genes, in a disease
state). Then this pattern of expression (an expression
fingerprint), if reproducible and clearly differentiable in the
different cases can be used as a very detailed diagnostic. It is
not required that the pattern be fully interpretable, but just that
it is specific for a particular cell state (and preferably of
diagnostic and/or prognostic relevance).
[0126] Both customized and generic arrays can be used in drug
safety studies. For example, if one is making a new antibiotic,
then it should not significantly affect the expression profile for
mammalian cells. The hybridization pattern can be used as a
detailed measure of the effect of a drug on cells, for example, as
a toxicological screen.
[0127] The sequence information provided by the hybridization
pattern of a generic array can be used to identify genes encoding
mRNAs hybridized to an array. Such methods can be performed using
DNA nucleic acids of the invention as the target nucleic acids
described in WO 97/27317. DNA nucleic acids can be denatured and
then hybridized to the complementary regions of the probes, using
standard conditions described in WO 97/27317. The hybridization
pattern indicates which probes are complementary to nucleic acid
strands in the sample. Comparison of the hybridization pattern of
two samples indicates which probes hybridize to nucleic acid
strands that derive from mRNAs that are differentially expressed
between the two samples. These probes are of particular interest,
because they contain complementary sequence to mRNA species subject
to differential expression. The sequence of such probes is known
and can be compared with sequences in databases to determine the
identity of the full-length mRNAs subject to differential
expression provided that such mRNAs have previously been sequenced.
Alternatively, the sequences of probes can be used to design
hybridization probes or primers for cloning the differentially
expressed mRNAs. The differentially expressed mRNAs are typically
cloned from the sample in which the mRNA of interest was expressed
at the highest level. In some methods, database comparisons or
cloning is facilitated by provision of additional sequence
information beyond that inferable from probe sequence by template
dependent extension as described above.
[0128] 11. Screening for B Cell Activity Modulators
[0129] (A) Candidate Bioactive Agents
[0130] Having identified a number of suitable expression profiles,
the information is used in a wide variety of ways. In a preferred
method, the expression profiles can be used in conjunction with
high throughput screening techniques, to allow monitoring for
expression profile genes after treatment with a candidate agent,
Zlokarnik, et al., Science 279, 84-8 (1998), Heid et al., Genome
Res. (1996) 6: 986. In a preferred method, the candidate agents are
added to cells.
[0131] The term "candidate bioactive agent" or "drug candidate" or
grammatical equivalents as used herein describes any molecule,
e.g., protein, oligopeptide, small organic molecule,
polysaccharide, polynucleotide, to be tested for bioactive agents
that are capable of directly or indirectly altering the activity of
a B cell. In preferred methods, the bioactive agents modulate the
expression profiles, or expression profile nucleic acids or
proteins provided herein. In a particularly preferred method, the
candidate agents induce an immunosuppressive tolerant response, or
maintain such a response as indicated, for example, by the effect
of the agent on the expression profile, nucleic acids, proteins or
B cell activity as further described below. Generally a plurality
of assay mixtures are run in parallel with different agent
concentrations to obtain a differential response to the various
concentrations. Typically, one of these concentrations serves as a
negative control, i.e., at zero concentration or below the level of
detection.
[0132] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 100 and less than
about 2,500 daltons. Candidate agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Particularly preferred are peptides.
[0133] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents can be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0134] In some preferred embodiment, the candidate bioactive agents
are proteins. By "protein" herein is meant at least two covalently
attached amino acids, which includes proteins, polypeptides,
oligopeptides and peptides. The protein can be made up of naturally
occurring amino acids and peptide bonds, or synthetic
peptidomimetic structures. Thus "amino acid", or "peptide residue",
as used herein means both naturally occurring and synthetic amino
acids. For example, homo-phenylalanine, citrulline and noreleucine
are considered amino acids for the purposes of the invention.
"Amino acid" also includes imino acid residues such as proline and
hydroxyproline. The side chains can be in either the (R) or the (S)
configuration. In some preferred embodiment, the amino acids are in
the (S) or L-configuration. If non-naturally occurring side chains
are used, non-amino acid substituents can be used, for example to
prevent or retard in vivo degradations.
[0135] In a preferred method, the candidate bioactive agents are
naturally occurring proteins or fragments of naturally occurring
proteins. Thus, for example, cellular extracts containing proteins,
or random or directed digests of proteinaceous cellular extracts,
can be used. In this way libraries of procaryotic and eucaryotic
proteins can be made for screening in the methods of the invention.
The libraries can be bacterial, fungal, viral, and mammalian
proteins, with the latter being preferred, and human proteins.
[0136] In some methods, the candidate bioactive agents are peptides
of from about 5 to about 30 amino acids, with from about 5 to about
20 amino acids being preferred, and from about 7 to about 15 being
particularly preferred. The peptides can be digests of naturally
occurring proteins as is outlined above, random peptides, or
"biased" random peptides. By "randomized" or grammatical
equivalents herein is meant that each nucleic acid and peptide
consists of essentially random nucleotides and amino acids,
respectively. Since generally these random peptides (or nucleic
acids, discussed below) are chemically synthesized, they can
incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins
or nucleic acids, to allow the formation of all or most of the
possible combinations over the length of the sequence, thus forming
a library of randomized candidate bioactive proteinaceous
agents.
[0137] In some methods, the library can be fully randomized, with
no sequence preferences or constants at any position. In other
methods, the library can be biased. Some positions within the
sequence are either held constant, or are selected from a limited
number of possibilities. For example, in some methods, the
nucleotides or amino acid residues are randomized within a defined
class, for example, of hydrophobic amino acids, hydrophilic
residues, sterically biased (either small or large) residues,
towards the creation of nucleic acid binding domains, the creation
of cysteines, for cross-linking, prolines for SH-3 domains,
serines, threonines, tyrosines or histidines for phosphorylation
sites, or to purines. In other methods, the candidate bioactive
agents are nucleic acids, as defined above.
[0138] As described above generally for proteins, nucleic acid
candidate bioactive agents can be naturally occurring nucleic
acids, random nucleic acids, or "biased" random nucleic acids. For
example, digests of procaryotic or eucaryotic genomes can be used
as is outlined above for proteins.
[0139] In some methods, the candidate bioactive agents are organic
chemical moieties.
[0140] (B) Drug Screening Methods
[0141] Several different drug screening methods can be accomplished
to identify drugs or bioactive agents that modulate B cell
activity. One such method is the screening of candidate agents that
can induce a particular expression profile, thus preferably
generating the associated phenotype. Candidate agents that can
mimic or produce an expression profile similar to an
immunosuppressive expression profile as shown herein is expected to
result in the immunosuppressive phenotype. Similarly, candidate
agents that can mimic or produce an expression profile similar to a
tolerant expression profile as shown herein is expected to result
in the tolerant phenotype. Thus, in some methods, candidate agents
can be determined that mimic an expression profile or change one
profile to another.
[0142] In other methods, after having identified the differentially
expressed genes important in any one state, candidate agent
screening can be run to alter the expression of individual genes.
For example, particularly in the case of target genes whose
presence or absence is unique between two states, screening for
modulators of the target gene expression can be done.
[0143] In other methods, screening can be done to alter the
biological function of the expression product of the differentially
expressed gene. Again, having identified the importance of a gene
in a particular state, screening for agents that bind and/or
modulate the biological activity of the gene product can be
performed as outlined below.
[0144] Thus, screening of candidate agents that modulate B cell
activity either at the level of gene expression or protein level
can be accomplished.
[0145] In some methods, a candidate agent can be administered to B
cells in any state, that thus has an associated B cell activity
expression profile. By "administration" or "contacting" herein is
meant that the candidate agent is added to the cells in such a
manner as to allow the agent to act upon the cell, whether by
uptake and intracellular action, or by action at the cell surface.
In some embodiments, nucleic acid encoding a proteinaceous
candidate agent (i.e., a peptide) can be put into a viral construct
such as a retroviral construct and added to the cell, such that
expression of the peptide agent is accomplished; see PCT
US97/01019, hereby expressly incorporated by reference.
[0146] Once the candidate agent has been administered to the cells,
the cells can be washed if desired and are allowed to incubate
under preferably physiological conditions for some period of time.
The cells are then harvested and a new gene expression profile is
generated, as outlined herein.
[0147] For example, activated B cells can be screened for agents
that produce a tolerant phenotype. A change in at least one gene of
the expression profile indicates that the agent has an effect on B
cell activity. In a preferred method, an immunosuppressive tolerant
profile is induced or maintained, before, during, and/or after
stimulation with antigen. By defining such a signature for
immunological tolerance, screens for new drugs that mimic the
tolerance phenotype can be devised. With this approach, the drug
target need not be known and need not be represented in the
original expression screening platform, nor does the level of
transcript for the target protein need to change. In some methods,
the agent induces or maintains a profile which indicates a
selective block immune response while still permitting tolerance to
be actively (re)established. For example, in one such embodiment,
the agent suppresses at least one of Egr-1, Egr-2, c-myc and c-fos
while sparing upregulation of NAB2.
[0148] In some preferred methods, screens can be done on individual
genes and gene products. After having identified a particular
differentially expressed gene as important in a particular state,
screening of modulators of either the expression of the gene or the
gene product itself can be completed. The gene products of
differentially expressed genes are sometimes referred to herein as
"B lymphocyte modulator proteins" or BLMPs.
[0149] Thus, in some preferred methods, screening for modulators of
expression of specific genes can be completed. This will be done as
outlined above, but in general the expression of only one or a few
genes are evaluated. In some methods, screens are designed to first
find candidate agents that can bind to differentially expressed
proteins, and then these agents can be used in other assays that
evaluate the ability of the candidate agent to modulate
differentially expressed activity. There are a number of different
assays which can be completed, such as binding assays and activity
assays.
[0150] In a preferred method, binding assays are performed. In
general, purified or isolated gene product is used; that is, the
gene products of one or more differentially expressed nucleic acids
are made. Using the nucleic acids of the present invention which
encode a differentially expressed protein in a B cell state, a
variety of expression vectors can be made. The expression vectors
can be either self-replicating extrachromosomal vectors or vectors
which integrate into a host genome. Generally, these expression
vectors include transcriptional and translational regulatory
nucleic acid operably linked to the nucleic acid encoding a
differentially expressed protein. The term "control sequences"
refers to DNA sequences necessary for the expression of an operably
linked coding sequence in a particular host organism. The control
sequences that are suitable for prokaryotes, for example, include a
promoter, optionally an operator sequence, and a ribosome binding
site. Eukaryotic cells are known to utilize promoters,
polyadenylation signals, and enhancers.
[0151] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice. The transcriptional and
translational regulatory nucleic acid will generally be appropriate
to the host cell used to express a differentially expressed
protein; for example, transcriptional and translational regulatory
nucleic acid sequences from Bacillus are preferably used to express
a differentially expressed protein in Bacillus. Numerous types of
appropriate expression vectors, and suitable regulatory sequences
are known in the art for a variety of host cells.
[0152] In general, the transcriptional and translational regulatory
sequences can include, but are not limited to, promoter sequences,
ribosomal binding sites, transcriptional start and stop sequences,
translational start and stop sequences, and enhancer or activator
sequences. In a preferred method, the regulatory sequences include
a promoter and transcriptional start and stop sequences.
[0153] Promoter sequences encode either constitutive or inducible
promoters. The promoters can be either naturally occurring
promoters or hybrid promoters. Hybrid promoters, which combine
elements of more than one promoter, are also known in the art, and
are useful in the present invention.
[0154] In addition, the expression vector can comprise additional
elements. For example, the expression vector can have two
replication systems, thus allowing it to be maintained in two
organisms, for example in mammalian or insect cells for expression
and in a procaryotic host for cloning and amplification.
Furthermore, for integrating expression vectors, the expression
vector contains at least one sequence homologous to the host cell
genome, and preferably two homologous sequences which flank the
expression construct. The integrating vector can be directed to a
specific locus in the host cell by selecting the appropriate
homologous sequence for inclusion in the vector. Constructs for
integrating vectors are well known in the art. Preferred methods to
effect homologous recombination are described in PCT US93/03868 and
PCT US98/05223, hereby incorporated by reference.
[0155] In some methods, the expression vector contains a selectable
marker gene to allow the selection of transformed host cells.
Selection genes are well known in the art and will vary with the
host cell used.
[0156] A preferred expression vector system is a retroviral vector
system such as is generally described in PCT/US97/01019 and
PCT/US97/01048, both of which are hereby expressly incorporated by
reference.
[0157] The differentially expressed proteins of the present
invention are produced by culturing a host cell transformed with an
expression vector containing nucleic acid encoding a differentially
expressed protein, under the appropriate conditions to induce or
cause expression of a differentially expressed protein. The
conditions appropriate for differentially expressed protein
expression will vary with the choice of the expression vector and
the host cell, and will be easily ascertained by one skilled in the
art through routine experimentation. For example, the use of
constitutive promoters in the expression vector will require
optimizing the growth and proliferation of the host cell, while the
use of an inducible promoter requires the appropriate growth
conditions for induction. In some methods, the timing of the
harvest is important. For example, the baculoviral systems used in
insect cell expression are lytic viruses, and thus harvest time
selection can be crucial for product yield.
[0158] Appropriate host cells include yeast, bacteria,
archebacteria, fungi, and insect and animal cells, including
mammalian cells. Of particular interest are Drosophila melangaster
cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus
subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO,
COS, and HeLa cells. In some preferred methods, B cells are host
cells as provided herein, which for example, include
non-recombinant cell lines, such as primary cell lines. In
addition, purified primary B cells derived from either transgenic
or non-transgenic strains can also be used. The B cells can be in a
particular state, or be induced to be in a particular state. The
host cell can alternatively be a B cell known to have a B cell
disorder.
[0159] In a preferred method, the differentially expressed proteins
are expressed in mammalian cells. Mammalian expression systems can
include retroviral systems. A mammalian promoter is any DNA
sequence capable of binding mammalian RNA polymerase and initiating
the downstream (3') transcription of a coding sequence for
differentially expressed protein into mRNA. A promoter will have a
transcription initiating region, which is usually placed proximal
to the 5' end of the coding sequence, and a TATA box, using a
located 25-30 base pairs upstream of the transcription initiation
site. The TATA box is thought to direct RNA polymerase II to begin
RNA synthesis at the correct site. A mammalian promoter will also
contain an upstream promoter element (enhancer element), typically
located within 100 to 200 base pairs upstream of the TATA box. An
upstream promoter element determines the rate at which
transcription is initiated and can act in either orientation. Of
particular use as mammalian promoters are the promoters from
mammalian viral genes, since the viral genes are often highly
expressed and have a broad host range. Examples include the SV40
early promoter, mouse mammary tumor virus LTR promoter, adenovirus
major late promoter, herpes simplex virus promoter, and the CMV
promoter.
[0160] Typically, transcription termination and polyadenylation
sequences recognized by mammalian cells are regulatory regions
located 3' to the translation stop codon and thus, together with
the promoter elements, flank the coding sequence. The 3' terminus
of the mature mRNA is formed by site-specific post-translational
cleavage and polyadenylation. Examples of transcription terminator
and polyadenlytion signals include those derived form SV40.
[0161] The methods of introducing nucleic acid into mammalian
hosts, as well as other hosts, is well known in the art, and will
vary with the host cell used. Techniques include dextran-mediated
transfection, calcium phosphate precipitation, polybrene mediated
transfection, protoplast fusion, electroporation, viral infection,
encapsulation of the polynucleotide(s) in liposomes, and direct
microinjection of the DNA into nuclei.
[0162] In some methods, differentially expressed proteins are
expressed in bacterial systems which are well known in the art.
[0163] In other methods, differentially expressed proteins can be
produced in insect cells. Expression vectors for the transformation
of insect cells, and in particular, baculovirus-based expression
vectors, are well known in the art.
[0164] In some methods, differentially expressed proteins are
produced in yeast cells. Yeast expression systems are well known in
the art, and include expression vectors for Saccharomyces
cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha,
Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P.
pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
[0165] A differentially expressed protein can also be made as a
fusion protein, using techniques well known in the art. For
example, for the creation of monoclonal antibodies, if the desired
epitope is small, the differentially expressed protein can be fused
to a carrier protein to form an immunogen. Alternatively, a
differentially expressed protein can be made as a fusion protein to
increase expression. For example, when a differentially expressed
protein is a differentially expressed peptide, the nucleic acid
encoding the peptide can be linked to other nucleic acid for
expression purposes. Similarly, differentially expressed proteins
of the invention an be linked to protein labels, such as green
fluorescent protein (GFP), red fluorescent protein (RFP), yellow
fluorescent protein (YFP), and blue fluorescent protein (BFP).
[0166] Preferably, the proteins are recombinant. A "recombinant
protein" is a protein made using recombinant techniques, i.e.,
through the expression of a recombinant nucleic acid as depicted
above. A recombinant protein is distinguished from naturally
occurring protein by at least one or more characteristics. For
example, the protein can be isolated or purified away from some or
all of the proteins and compounds with which it is normally
associated in its wild type host, and thus can be substantially
pure. For example, an isolated protein is unaccompanied by at least
some of the material with which it is normally associated in its
natural state, preferably constituting at least about 0.5%, more
preferably at least about 5% by weight of the total protein in a
given sample. A substantially pure protein comprises at least about
75% by weight of the total protein, with at least about 80% being
preferred, and at least about 90% being particularly preferred. The
definition includes the production of a differentially expressed
protein from one organism in a different organism or host cell.
Alternatively, the protein can be made at a significantly higher
concentration than is normally seen, through the use of a inducible
promoter or high expression promoter, such that the protein is made
at increased concentration levels. Alternatively, the protein can
be in a form not normally found in nature, as in the addition of an
epitope tag or amino acid substitutions, insertions and deletions,
as discussed below.
[0167] In some preferred methods, when the differentially expressed
protein is to be used to generate antibodies, the protein must
share at least one epitope or determinant with the full length
transcription product of the differentially expressed nucleic acids
shown herein. By "epitope" or "determinant" herein is meant a
portion of a protein which will generate and/or bind an antibody.
Thus, in most instances, antibodies made to a smaller protein
should be able to bind to the full length protein. In a preferred
embodiment, the epitope is unique; that is, antibodies generated to
a unique epitope show little or no cross-reactivity.
[0168] In some preferred methods, the antibodies provided herein
can be capable of reducing or eliminating the biological function
of a differentially expressed protein, as is described below. The
addition of antibodies (either polyclonal or preferably monoclonal)
to the protein (or cells containing the differentially expressed
protein) can reduce or eliminate the protein's activity. Generally,
at least a 25% decrease in activity is preferred, with at least
about 50% being particularly preferred and about a 95-100% decrease
being especially preferred.
[0169] In addition, the proteins can be variant proteins,
comprising one more amino acid substitutions, insertions and
deletions.
[0170] In a preferred method, a differentially expressed protein is
purified or isolated after expression. Differentially expressed
proteins can be isolated or purified in a variety of ways. Standard
purification methods include electrophoretic, molecular,
immunological and chromatographic techniques, including ion
exchange, hydrophobic, affinity, and reverse-phase HPLC
chromatography, and chromatofocusing. For example, a differentially
expressed protein can be purified using a standard
anti-differentially expressed protein antibody column.
Ultrafiltration and diafiltration techniques, in conjunction with
protein concentration, are also useful. For general guidance in
suitable purification techniques, see Scopes, R., Protein
Purification, Springer-Verlag, NY (1982). The degree of
purification necessary will vary depending on the use of the
differentially expressed protein. In some instances no purification
will be necessary.
[0171] Once the gene product of the differentially expressed gene
is made, binding assays can be done. These methods comprise
combining a differentially expressed protein and a candidate
bioactive agent, and determining the binding of the candidate agent
to the differentially expressed protein. Preferred methods utilize
a human differentially expressed protein, although other mammalian
proteins can also be used, including rodents (mice, rats, hamsters,
guinea pigs), farm animals (cows, sheep, pigs, horses) and
primates. These latter methods can be preferred for the development
of animal models of human disease. In some methods, variant or
derivative differentially expressed proteins can be used, including
deletion differentially expressed proteins as outlined above.
[0172] The assays herein utilize differentially expressed proteins
as defined herein. In some assays, portions of differentially
expressed proteins can be utilized. In other assays, portions
having differentially expressed activity can be used. In addition,
the assays described herein can utilize either isolated
differentially expressed proteins or cells comprising the
differentially expressed proteins. In some methods, the
differentially expressed protein or the candidate agent is
non-diffusably bound to an insoluble support having isolated sample
receiving areas (e.g., a microtiter plate or an array). The
insoluble supports can be made of any composition to which the
compositions can be bound, is readily separated from soluble
material, and is otherwise compatible with the overall method of
screening. The surface of such supports can be solid or porous and
of any convenient shape. Examples of suitable insoluble supports
include microtiter plates, arrays, membranes and beads. These are
typically made of glass, plastic (e.g., polystyrene),
polysaccharides, nylon or nitrocellulose, and teflon.TM..
Microtiter plates and arrays are especially convenient because a
large number of assays can be carried out simultaneously, using
small amounts of reagents and samples. In some cases magnetic beads
and the like are included. The particular manner of binding of the
composition is not crucial so long as it is compatible with the
reagents and overall methods of the invention, maintains the
activity of the composition and is nondiffusable. Preferred methods
of binding include the use of antibodies (which do not sterically
block either the ligand binding site or activation sequence when
the protein is bound to the support), direct binding to "sticky" or
ionic supports, chemical crosslinking, the synthesis of the protein
or agent on the surface. Following binding of the protein or agent,
excess unbound material is removed by washing. The sample receiving
areas can then be blocked through incubation with bovine serum
albumin (BSA), casein or other innocuous protein or other moiety.
Also included in this invention are screening assays wherein solid
supports are not used.
[0173] In other methods, the differentially expressed protein is
bound to the support, and a candidate bioactive agent is added to
the assay. Alternatively, the candidate agent is bound to the
support and the differentially expressed protein is added. Novel
binding agents include specific antibodies, non-natural binding
agents identified in screens of chemical libraries, and peptide
analogs. Of particular interest are screening assays for agents
that have a low toxicity for human cells. A wide variety of assays
can be used for this purpose, including labeled in vitro
protein-protein binding assays, electrophoretic mobility shift
assays, immunoassays for protein binding, functional assays (such
as phosphorylation assays) and the like.
[0174] The determination of the binding of the candidate bioactive
agent to a differentially expressed protein can be done in a number
of ways. In some methods, the candidate bioactive agent is labeled,
and binding determined directly. For example, this can be done by
attaching all or a portion of a differentially expressed protein to
a solid support, adding a labeled candidate agent (for example a
fluorescent label), washing off excess reagent, and determining
whether the label is present on the solid support. Various blocking
and washing steps can be utilized.
[0175] By "labeled" herein is meant that the compound is either
directly or indirectly labeled with a label which provides a
detectable signal, e.g., radioisotope, fluorescers, enzyme,
antibodies, particles such as magnetic particles, chemiluminescers,
or specific binding molecules. Specific binding molecules include
pairs, such as biotin and streptavidin, digoxin and antidigoxin.
For the specific binding members, the complementary member would
normally be labeled with a molecule which provides for detection,
in accordance with known procedures, as outlined above. The label
can directly or indirectly provide a detectable signal.
[0176] In some methods, only one of the components is labeled. For
example, the proteins (or proteinaceous candidate agents) can be
labeled at tyrosine positions using .sup.125I, or with
fluorophores. Alternatively, more than one component can be labeled
with different labels; using .sup.125I for the proteins, for
example, and a fluorophor for the candidate agents.
[0177] In other methods, the binding of the candidate bioactive
agent is determined through the use of competitive binding assays.
In this method, the competitor is a binding moiety known to bind to
the target molecule such as an antibody, peptide, binding partner,
or ligand. Under certain circumstances, there can be competitive
binding as between the bioactive agent and the binding moiety, with
the binding moiety displacing the bioactive agent. This assay can
be used to determine candidate agents which interfere with binding
between differentially expressed proteins and the competitor.
[0178] In some methods, the candidate bioactive agent is labeled.
Either the candidate bioactive agent, or the competitor, or both,
is added first to the protein for a time sufficient to allow
binding, if present. Incubations can be performed at any
temperature which facilitates optimal activity, typically between 4
and 40.degree. C. Incubation periods are selected for optimum
activity, but can also be optimized to facilitate rapid high
through put screening. Typically between 0.1 and 1 hour will be
sufficient. Excess reagent is generally removed or washed away. The
second component is then added, and the presence or absence of the
labeled component is followed, to indicate binding.
[0179] In other methods, the competitor is added first, followed by
the candidate bioactive agent. Displacement of the competitor is an
indication that the candidate bioactive agent is binding to the
differentially expressed protein and thus is capable of binding to,
and potentially modulating, the activity of the differentially
expressed protein. In this method, either component can be labeled.
For example, if the competitor is labeled, the presence of label in
the wash solution indicates displacement by the agent.
Alternatively, if the candidate bioactive agent is labeled, the
presence of the label on the support indicates displacement.
[0180] In other methods, the candidate bioactive agent is added
first, with incubation and washing, followed by the competitor. The
absence of binding by the competitor can indicate that the
bioactive agent is bound to the differentially expressed protein
with a higher affinity. Thus, if the candidate bioactive agent is
labeled, the presence of the label on the support, coupled with a
lack of competitor binding, can indicate that the candidate agent
is capable of binding to the differentially expressed protein.
[0181] Competitive binding methods can also be run as differential
screens. These methods can comprise a differentially expressed
protein and a competitor in a first sample. A second sample
comprises a candidate bioactive agent, a differentially expressed
protein and a competitor. The binding of the competitor is
determined for both samples, and a change, or difference in binding
between the two samples indicates the presence of an agent capable
of binding to the differentially expressed protein and potentially
modulating its activity. If the binding of the competitor is
different in the second sample relative to the first sample, the
agent is capable of binding to the differentially expressed
protein.
[0182] Other methods utilize differential screening to identify
drug candidates that bind to the native differentially expressed
protein, but cannot bind to modified differentially expressed
proteins. The structure of the differentially expressed protein can
be modeled, and used in rational drug design to synthesize agents
that interact with that site. Drug candidates that affect
differentially expressed bioactivity are also identified by
screening drugs for the ability to either enhance or reduce the
activity of the protein.
[0183] In some methods, screening for agents that modulate the
activity of differentially expressed proteins are performed. In
general, this will be done on the basis of the known biological
activity of the differentially expressed protein. In these methods,
a candidate bioactive agent is added to a sample of the
differentially expressed protein, as above, and an alteration in
the biological activity of the protein is determined. "Modulating
the activity" includes an increase in activity, a decrease in
activity, or a change in the type or kind of activity present.
Thus, in these methods, the candidate agent should both bind to
differentially expressed (although this may not be necessary), and
alter its biological or biochemical activity as defined herein. The
methods include both in vitro screening methods, as are generally
outlined above, and in vivo screening of cells for alterations in
the presence, distribution, activity or amount of the
differentially expressed protein.
[0184] Some methods comprise combining a differentially expressed
sample and a candidate bioactive agent, then evaluating the effect
on B cell activity. By "differentially expressed activity" or
grammatical equivalents herein is meant one of B cell biological
activities, including, but not limited to, its ability to affect
suppression, tolerance and activation. One activity herein is the
capability to bind to a target gene, or modulate an expression
profile. Preferably, expression profiles are induced or maintained
and/or the desired B cell state is induced or maintained.
[0185] In other methods, the activity of the differentially
expressed protein is increased; in other methods, the activity of
the differentially expressed protein is decreased. Thus, bioactive
agents that are antagonists are preferred in some methods, and
bioactive agents that are agonists can be preferred in other
methods.
[0186] The invention provides methods for screening for bioactive
agents capable of modulating the activity of a differentially
expressed protein. These methods comprise adding a candidate
bioactive agent, as defined above, to a cell comprising
differentially expressed proteins. Preferred cell types include
almost any cell. The cells contain a recombinant nucleic acid that
encodes a differentially expressed protein. In a preferred method,
a library of candidate agents are tested on a plurality of cells.
The effect of the candidate agent on B cell activity is then
evaluated.
[0187] Positive controls and negative controls can be used in the
assays. Preferably all control and test samples are performed in at
least triplicate to obtain statistically significant results.
Incubation of all samples is for a time sufficient for the binding
of the agent to the protein. Following incubation, all samples are
washed free of non-specifically bound material and the amount of
bound, generally labeled agent determined. For example, where a
radiolabel is employed, the samples can be counted in a
scintillation counter to determine the amount of bound
compound.
[0188] A variety of other reagents can be included in the screening
assays. These include reagents like salts, neutral proteins (e.g.,
albumin and detergents) which can be used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Reagents that otherwise improve the efficiency of the
assay, (such as protease inhibitors, nuclease inhibitors,
anti-microbial agents) can also be used. The mixture of components
can be added in any order that provides for the requisite
binding.
[0189] The components provided herein for the assays provided
herein can also be combined to form kits. The kits can be based on
the use of the protein and/or the nucleic acid encoding the
differentially expressed proteins. Assays regarding the use of
nucleic acids are further described below.
[0190] (C) Animal Models
[0191] In a preferred method, nucleic acids which encode
differentially expressed proteins or their modified forms can also
be used to generate either transgenic animals, including "knock-in"
and "knock out" animals which, in turn, are useful in the
development and screening of therapeutically useful reagents. A
non-human transgenic animal (e.g., a mouse or rat) is an animal
having cells that contain a transgene, which transgene is
introduced into the animal or an ancestor of the animal at a
prenatal, e.g., an embryonic stage. A transgene is a DNA which is
integrated into the genome of a cell from which a transgenic animal
develops, and can include both the addition of all or part of a
gene or the deletion of all or part of a gene. In some methods,
cDNA encoding a differentially expressed protein can be used to
clone genomic DNA encoding a differentially expressed protein in
accordance with established techniques and the genomic sequences
used to generate transgenic animals that contain cells which either
express (or overexpress) or suppress the desired DNA. Methods for
generating transgenic animals, particularly animals such as mice or
rats, have become conventional in the art and are described, for
example, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically,
particular cells would be targeted for a differentially expressed
protein transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding a
differentially expressed protein introduced into the germ line of
the animal at an embryonic stage can be used to examine the effect
of increased expression of the desired nucleic acid. Such animals
can be used as tester animals for reagents thought to confer
protection from, for example, pathological conditions associated
with its overexpression. In accordance with this facet of the
invention, an animal is treated with the reagent and a reduced
incidence of the pathological condition, compared to untreated
animals bearing the transgene, would indicate a potential
therapeutic intervention for the pathological condition. Similarly,
non-human homologues of a differentially expressed protein can be
used to construct a transgenic animal comprising a differentially
expressed protein "knock out" animal which has a defective or
altered gene encoding a differentially expressed protein as a
result of homologous recombination between the endogenous gene
encoding a differentially expressed protein and altered genomic DNA
encoding a differentially expressed protein introduced into an
embryonic cell of the animal. For example, cDNA encoding a
differentially expressed protein can be used to clone genomic DNA
encoding a differentially expressed protein in accordance with
established techniques. A portion of the genomic DNA encoding a
differentially expressed protein can be deleted or replaced with
another gene, such as a gene encoding a selectable marker which can
be used to monitor integration. Typically, several kilobases of
unaltered flanking DNA (both at the 5' and 3' ends) are included in
the vector (see, e.g., Thomas and Capecchi, Cell (1987) 51: 503 for
a description of homologous recombination vectors). The vector is
introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has
homologously recombined with the endogenous DNA are selected (see,
e.g., Li et al., Cell (1992) 69: 915). The selected cells are then
injected into a blastocyst of an animal (e.g., a mouse or rat) to
form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas
and Embryonic Stem Cells: A Practical Approach, E. J. Robertson,
ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then
be implanted into a suitable pseudopregnant female foster animal
and the embryo brought to term to create a "knock out" animal.
Progeny harboring the homologously recombined DNA in their germ
cells can be identified by standard techniques and used to breed
animals in which all cells of the animal contain the homologously
recombined DNA. Knockout animals can be characterized for instance,
for their ability to defend against certain pathological conditions
and for their development of pathological conditions due to absence
of a differentially expressed protein polypeptide.
[0192] Animal models for B cell related disorders, or having a
particular state of B cell activity can include, for example,
genetic models. For example, such animal models can include the
nonobese diabetic (NOD) mouse (see, e.g., McDuffie, M., Curr Opin
Immunol. (1998) 10(6): 704-9; Tochino, Y., Crit Rev Immunol (1987)
8(1): 49-81), and experimental autoimmune encephalomyelitis (RAE)
(see, e.g., Wong, F. S., Immunol Rev (1999) 169: 93-104). See also
Schwartz, R. S. and Datta, S. K., Autoimmunity and Autoimmune
Diseases, Ch. 31, in Fundamental Immunology, Paul, W. E. (ed.)
(Raven Press 1989). Other models can include studies involving
transplant rejection.
[0193] Animal models exhibiting B cell related disorder-like
symptoms can be engineered by utilizing, for example,
differentially expressed sequences in conjunction with techniques
for producing transgenic animals that are well known to those of
skill in the art. For example, gene sequences can be introduced
into, and overexpressed in, the genome of the animal of interest,
or, if endogenous target gene sequences are present, they can
either be overexpressed or, alternatively, can be disrupted in
order to underexpress or inactivate target gene expression.
[0194] In order to overexpress a target gene sequence, the coding
portion of the target gene sequence can be ligated to a regulatory
sequence which is capable of driving gene expression in the animal
and cell type of interest. Such regulatory regions will be well
known to those of skill in the art, and can be utilized in the
absence of undue experimentation.
[0195] For underexpression of an endogenous target gene sequence,
such a sequence can be isolated and engineered such that when
reintroduced into the genome of the animal of interest, the
endogenous target gene alleles will be inactivated. Preferably, the
engineered target gene sequence is introduced via gene targeting
such that the endogenous target sequence is disrupted upon
integration of the engineered target sequence into the animal's
genome.
[0196] Animals of any species, including, but not limited to, mice,
rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human
primates, e.g., baboons, monkeys, and chimpanzees can be used to
generate animal models of B cell related disorders or being a
perpetually desired state of the B cell.
[0197] (D) Nucleic Acid Based Therapeutics
[0198] Nucleic acids encoding differentially expressed
polypeptides, antagonists or agonists can also be used in gene
therapy. Broadly speaking, a gene therapy vector is an exogenous
polynucleotide which produces a medically useful phenotypic effect
upon the mammalian cell(s) into which it is transferred. A vector
can or can not have an origin of replication. For example, it is
useful to include an origin of replication in a vector for
propagation of the vector prior to administration to a patient.
However, the origin of replication can often be removed before
administration if the vector is designed to integrate into host
chromosomal DNA or bind to host mRNA or DNA. Vectors used in gene
therapy can be viral or nonviral. Viral vectors are usually
introduced into a patient as components of a virus. Nonviral
vectors, typically dsDNA, can be transferred as naked DNA or
associated with a transfer-enhancing vehicle, such as a
receptor-recognition protein, lipoamine, or cationic lipid.
[0199] (1) Viral-Based Methods
[0200] Viral vectors, such as retroviruses, adenoviruses,
adenoassociated viruses and herpes viruses, are often made up of
two components, a modified viral genome and a coat structure
surrounding it (see generally Smith et al., Ann. Rev. Microbiol.
(1995) 49, 807-838; this reference and all references cited therein
are incorporated herein by reference), although sometimes viral
vectors are introduced in naked form or coated with proteins other
than viral proteins. Most current vectors have coat structures
similar to a wildtype virus. This structure packages and protects
the viral nucleic acid and provides the means to bind and enter
target cells. However, the viral nucleic acid in a vector designed
for gene therapy is changed in many ways. The goals of these
changes are to disable growth of the virus in target cells while
maintaining its ability to grow in vector form in available
packaging or helper cells, to provide space within the viral genome
for insertion of exogenous DNA sequences, and to incorporate new
sequences that encode and enable appropriate expression of the gene
of interest. Thus, vector nucleic acids generally comprise two
components: essential cis-acting viral sequences for replication
and packaging in a helper line and the transcription unit for the
exogenous gene. Other viral functions are expressed in trans in a
specific packaging or helper cell line.
[0201] (a) Retroviruses
[0202] Retroviruses comprise a large class of enveloped viruses
that contain single-stranded RNA as the viral genome. During the
normal viral life cycle, viral RNA is reverse-transcribed to yield
double-stranded DNA that integrates into the host genome and is
expressed over extended periods. As a result, infected cells shed
virus continuously without apparent harm to the host cell. The
viral genome is small (approximately 10 kb), and its prototypical
organization is extremely simple, comprising three genes encoding
gag, the group specific antigens or core proteins; pol, the reverse
transcriptase; and env, the viral envelope protein. The termini of
the RNA genome are called long terminal repeats (LTRs) and include
promoter and enhancer activities and sequences involved in
integration. The genome also includes a sequence required for
packaging viral RNA and splice acceptor and donor sites for
generation of the separate envelope mRNA. Most retroviruses can
integrate only into replicating cells, although human
immunodeficiency virus (HIV) appears to be an exception. This
property restricts the use of retroviruses as vectors for gene
therapy.
[0203] Retrovirus vectors are relatively simple, containing the 5'
and 3' LTRs, a packaging sequence, and a transcription unit
composed of the gene or genes of interest, which is typically an
expression cassette. To grow such a vector, one must provide the
missing viral functions in trans using a so-called packaging cell
line. Such a cell is engineered to contain integrated copies of
gag, pol, and env but to lack a packaging signal so that no helper
virus sequences become encapsidated. Additional features added to
or removed from the vector and packaging cell line reflect attempts
to render the vectors more efficacious or reduce the possibility of
contamination by helper virus.
[0204] The main advantage of retroviral vectors is that they
integrate and are therefore potentially capable of long-term
expression. They can be grown in relatively large amounts, but care
is needed to ensure the absence of helper virus.
[0205] (b) Adenoviruses
[0206] Adenoviruses comprise a large class of nonenveloped viruses
containing linear double-stranded DNA. The normal life cycle of the
virus does not require dividing cells and involves productive
infection in permissive cells during which large amounts of virus
accumulate. The productive infection cycle takes about 32-36 hours
in cell culture and comprises two phases, the early phase, prior to
viral DNA synthesis, and the late phase, during which structural
proteins and viral DNA are synthesized and assembled into virions.
In general, adenovirus infections are associated with mild disease
in humans.
[0207] Adenovirus vectors are somewhat larger and more complex than
retrovirus or AAV vectors, partly because only a small fraction of
the viral genome is removed from most current vectors. If
additional genes are removed, they are provided in trans to produce
the vector, which so far has proved difficult. Instead, two general
types of adenovirus-based vectors have been studied, E3-deletion
and E1-deletion vectors. Some viruses in laboratory stocks of
wildtype lack the E3 region and can grow in the absence of helper.
This ability does not mean that the E3 gene products are not
necessary in the wild, only that replication in cultured cells does
not require them. Deletion of the E3 region allows insertion of
exogenous DNA sequences to yield vectors capable of productive
infection and the transient synthesis of relatively large amounts
of encoded protein.
[0208] Deletion of the E1 region disables the adenovirus, but such
vectors can still be grown because there exists an established
human cell line (called "293") that contains the E1 region of Ad5
and that constitutively expresses the E1 proteins. Most recent gene
therapy applications involving adenovirus have utilized E1
replacement vectors grown in 293 cells.
[0209] The main advantages of adenovirus vectors are that they are
capable of efficient episomal gene transfer in a wide range of
cells and tissues and that they are easy to grow in large amounts.
The main disadvantage is that the host response to the virus
appears to limit the duration of expression and the ability to
repeat dosing, at least with high doses of first-generation
vectors.
[0210] (c) Adeno-Associated Virus (AAV)
[0211] AAV is a small, simple, nonautonomous virus containing
linear single-stranded DNA. See Muzycka, Current Topics Microbiol.
Immunol. (1992) 158, 97-129; this reference and all references
cited therein are incorporated herein by reference. The virus
requires co-infection with adenovirus or certain other viruses in
order to replicate. AAV is widespread in the human population, as
evidenced by antibodies to the virus, but it is not associated with
any known disease. AAV genome organization is straightforward,
comprising only two genes: rep and cap. The termini of the genome
comprises terminal repeats (ITR) sequences of about 145
nucleotides.
[0212] AAV-based vectors typically contain only the ITR sequences
flanking the transcription unit of interest. The length of the
vector DNA cannot greatly exceed the viral genome length of 4680
nucleotides. Currently, growth of AAV vectors is cumbersome and
involves introducing into the host cell not only the vector itself
but also a plasmid encoding rep and cap to provide helper
functions. The helper plasmid lacks ITRs and consequently cannot
replicate and package. In addition, helper virus such as adenovirus
is often required. The potential advantage of AAV vectors is that
they appear capable of long-term expression in nondividing cells,
possibly, though not necessarily, because the viral DNA integrates.
The vectors are structurally simple, and they can therefore provoke
less of a host-cell response than adenovirus. A major limitation at
present is that AAV vectors are extremely difficult to grow in
large amounts.
[0213] (2) Non-Viral Gene Transfer Methods
[0214] Nonviral nucleic acid vectors used in gene therapy include
plasmids, RNAs, antisense oligonucleotides (e.g., methylphosphonate
or phosphorothiolate), polyamide nucleic acids, and yeast
artificial chromosomes (YACs). Such vectors typically include an
expression cassette for expressing a protein or RNA. The promoter
in such an expression cassette can be constitutive, cell
type-specific, stage-specific, and/or modulatable (e.g., by
hormones such as glucocorticoids; MMTV promoter). Transcription can
be increased by inserting an enhancer sequence into the vector.
Enhancers are cis-acting sequences of between 10 to 300 bp that
increase transcription by a promoter. Enhancers can effectively
increase transcription when either 5' or 3' to the transcription
unit. They are also effective if located within an intron or within
the coding sequence itself. Typically, viral enhancers are used,
including SV40 enhancers, cytomegalovirus enhancers, polyoma
enhancers, and adenovirus enhancers. Enhancer sequences from
mammalian systems are also commonly used, such as the mouse
immunoglobulin heavy chain enhancer.
[0215] Gene therapy vectors of all kinds can also include a
selectable marker gene. Examples of suitable markers include, the
dihydrofolate reductase gene (DHFR), the thymidine kinase gene
(TK), or prokaryotic genes conferring drug resistance, gpt
(xanthine-guanine phosphoribosyltransferase, which can be selected
for with mycophenolic acid; neo (neomycin phosphotransferase),
which can be selected for with G418, hygromycin, or puromycin; and
DHFR (dihydrofolate reductase), which can be selected for with
methotrexate (Mulligan & Berg, Proc. Natl. Acad. Sci. U.S.A.
(1981) 78, 2072; Southern & Berg, J. Mol. Appl. Genet. (1982)
1, 327).
[0216] Before integration, the vector has to cross many barriers
which can result in only a very minor fraction of the DNA ever
being expressed. Limitations to high level gene expression include:
loss of vector due to nucleases present in blood and tissues;
inefficient entry of DNA into a cell; inefficient entry of DNA into
the nucleus of the cell and preference of DNA for other
compartments; lack of DNA stability in the nucleus (factor limiting
nuclear stability can differ from those affecting other cellular
and extracellular compartments), efficiency of integration into the
chromosome; and site of integration.
[0217] These potential losses of efficiency can be addressed by
including additional sequences in a nonviral vector besides the
expression cassette from which the product effecting therapy is to
be expressed. The additional sequences can have roles in conferring
stability both outside and within a cell, mediating entry into a
cell, mediating entry into the nucleus of a cell and mediating
integration within nuclear DNA. For example, aptamer-like DNA
structures, or other protein binding sites can be used to mediate
binding of a vector to cell surface receptors or to serum proteins
that bind to a receptor thereby increasing the efficiency of DNA
transfer into the cell.
[0218] Other DNA sequences can directly or indirectly result in
avoidance of certain compartments and preference for other
compartments, from which escape or entry into the nucleus is more
efficient. Other DNA sites and structures directly or indirectly
bind to receptors in the nuclear membrane or to other proteins that
go into the nucleus, thereby facilitating nuclear uptake of a
vector. Other DNA sequences directly or indirectly affect the
efficiency of integration. For integration by homologous
recombination, important factors are the degree and length of
homology to chromosomal sequences, as well as the frequency of such
sequences in the genome (e.g., alu repeats). The specific sequence
mediating homologous recombination is also important, since
integration occurs much more easily in transcriptionally active
DNA. Methods and materials for constructing homologous targeting
constructs are described by e.g., Mansour et al., Nature (1988)
336: 348; Bradley et al., Bio/Technology (1992) 10: 534.
[0219] For nonhomologous, illegitimate and site-specific
recombination, recombination is mediated by specific sites on the
therapy vector which interact with cell encoded recombination
proteins (e.g., cre/10.times. and flp/frt systems). For example
Baubonis & Sauer, Nuc. Acids Res. (1993) 21, 2025-2029 report
that a vector including a loxP site becomes integrated at a loxP
site in chromosomal DNA in the presence of cre enzyme.
[0220] Nonviral vectors encoding products useful in gene therapy
can be introduced into an animal by means such as lipofection,
biolistics, virosomes, liposomes, immunoliposomes, polycation:
nucleic acid conjugates, naked DNA, artificial virions,
agent-enhanced uptake of DNA, ex vivo transduction. Lipofection is
described in e.g. U.S. Pat. Nos. 5,049,386, 4,946,787; and
4,897,355) and lipofection reagents are sold commercially (e.g.,
Transfectam.TM. and Lipofectin.TM.). Cationic and neutral lipids
that are suitable for efficient receptor-recognition lipofection of
polynucleotides include those of Felgner, WO 91/17424, WO
91/16024.
[0221] Unlike existing viral-based gene therapy vectors which can
only incorporate a relatively small non-viral polynucleotide
sequence into the viral genome because of size limitations for
packaging virion particles, naked DNA or lipofection complexes can
be used to transfer large (e.g., 50-5,000 kb) exogenous
polynucleotides into cells. This property of nonviral vectors is
particularly advantageous since many genes which can be delivered
by therapy span over 100 kilobases (e.g., amyloid precursor protein
(APP) gene, Huntington's chorea gene) and large homologous
targeting constructs or transgenes can be required for efficient
integration. Optionally, such large genes can be delivered to
target cells as two or more fragments and reconstructed by
homologous recombination within a cell (see WO 92/03917).
[0222] (3) Applications of Gene Therapy
[0223] Gene therapy vectors can be delivered in vivo by
administration to an individual patient, typically by systemic
administration (e.g., intravenous, intraperitoneal, intramuscular,
subdermal, or intracranial infusion) or topical application.
Alternatively, vectors can be delivered to cells ex vivo, such as
cells explanted from an individual patient (e.g., lymphocytes, bone
marrow aspirates, tissue biopsy) or universal donor hematopoietic
stem cells, followed by reimplantation of the cells into a patient,
usually after selection for cells which have incorporated the
vector.
[0224] 12. Diagnostic Methods
[0225] In addition to assays, the creation of animal models, and
nucleic acid based therepeutics, identification of important
differentially expressed genes allows the use of these genes in
diagnosis (e.g., diagnosis of cell states and abnormal B cell
conditions). Disorders based on mutant or variant differentially
expressed genes can be determined. The invention also provides
methods for identifying cells containing variant differentially
expressed genes comprising determining all or part of the sequence
of at least one endogeneous differentially expressed genes in a
cell. As will be appreciated by those in the art, this can be done
using any number of sequencing techniques. The invention also
provides methods of identifying the differentially expressed
genotype of an individual comprising determining all or part of the
sequence of at least one differentially expressed gene of the
individual. This is generally done in at least one tissue of the
individual, and can include the evaluation of a number of tissues
or different samples of the same tissue. The method can include
comparing the sequence of the sequenced differentially expressed
gene to a known differentially expressed gene, i.e., a wild-type
gene.
[0226] The sequence of all or part of the differentially expressed
gene can then be compared to the sequence of a known differentially
expressed gene to determine if any differences exist. This can be
done using any number of known sequence identity programs, such as
Bestfit, and others outlined herein. In some preferred methods, the
presence of a difference in the sequence between the differentially
expressed gene of the patient and the known differentially
expressed gene is indicative of a disease state or a propensity for
a disease state, as outlined herein.
[0227] Similarly, diagnosis of B cell states can be done using the
methods of the invention. By evaluating the gene expression profile
of B cells from a patient, the B cell state can be determined. This
is particularly useful to verify the action of a drug, for example
an immunosuppressive drug. Other methods comprise administering the
drug to a patient and removing a cell sample, particularly of B
cells, from the patient. The gene expression profile of the cell is
then evaluated, as outlined herein, for example by comparing it to
the expression profile from an equivalent sample from a healthy
individual. In this manner, both the efficacy (i.e., whether the
correct expression profile is being generated from the drug) and
the dose (is the dosage correct to result in the correct expression
profile) can be verified.
[0228] The present discovery relating to the role of differentially
expressed in B cells thus provides methods for inducing or
maintaining differing B cell states. In a preferred method, the
differentially expressed proteins, and particularly differentially
expressed fragments, are useful in the study or treatment of
conditions which are mediated by B cell activity, i.e., to
diagnose, treat or prevent B cell-mediated disorders. Thus, "B cell
mediated disorders" or "disease states" can include conditions
involving, for example, arthritis, diabetes, or multiple
sclerosis.
[0229] Methods of modulating B cell activity in cells or organisms
are provided. Some methods comprise administering to a cell an
anti-differentially expressed antibody or other agent identified
herein or by the methods provided herein, that reduces or
eliminates the biological activity of the endogeneous
differentially expressed protein. Alternatively, the methods
comprise administering to a cell or organism a recombinant nucleic
acid encoding a differentially expressed protein or modulator
including anti-sense nucleic acids. As will be appreciated by those
in the art, this can be accomplished in any number of ways. In some
preferred methods, the activity of differentially expressed is
increased by increasing the amount of differentially expressed in
the cell, for example by overexpressing the endogeneous
differentially expressed or by administering a differentially
expressed gene, using known gene therapy techniques, for example.
In a preferred method, the gene therapy techniques include the
incorporation of the exogenous gene using enhanced homologous
recombination (EHR), for example as described in PCT/US93/03868,
hereby incorporated by reference in its entirety.
[0230] In some methods, the invention provides methods for
diagnosing an B cell activity related condition in an individual.
The methods comprise measuring the activity of differentially
expressed protein in a tissue from the individual or patient, which
can include a measurement of the amount or specific activity of the
protein. This activity is compared to the activity of
differentially expressed from either a unaffected second individual
or from an unaffected tissue from the first individual. When these
activities are different, the first individual can be at risk for
an B cell activity mediated disorder.
[0231] Furthermore, nucleotide sequences encoding a differentially
expressed protein can also be used to construct hybridization
probes for mapping the gene which encodes that differentially
expressed protein and for the genetic analysis of individuals with
genetic disorders. The nucleotide sequences provided herein can be
mapped to a chromosome and specific regions of a chromosome using
known techniques, such as in situ hybridization, linkage analysis
against known chromosomal markers, and hybridization screening with
libraries.
[0232] 13. Antibodies
[0233] In some methods, the differentially expressed proteins of
the present invention can be used to generate polyclonal and
monoclonal antibodies to differentially expressed proteins, which
are useful as described herein. A number of immunogens are used to
produce antibodies that specifically bind differentially expressed
polypeptides. Full-length differentially expressed polypeptides are
suitable immunogens. Typically, the immunogen of interest is a
peptide of at least about 3 amino acids, more typically the peptide
is at least 5 amino acids in length, preferably, the fragment is at
least 10 amino acids in length and more preferably the fragment is
at least 15 amino acids in length. The peptides can be coupled to a
carrier protein (e.g., as a fusion protein), or are recombinantly
expressed in an immunization vector. Antigenic determinants on
peptides to which antibodies bind are typically 3 to 10 amino acids
in length. Naturally occurring polypeptides are also used either in
pure or impure form. Recombinant polypeptides are expressed in
eukaryotic or prokaryotic cells and purified using standard
techniques. The polypeptide, or a synthetic version thereof, is
then injected into an animal capable of producing antibodies.
Either monoclonal or polyclonal antibodies can be generated for
subsequent use in immunoassays to measure the presence and quantity
of the polypeptide.
[0234] These antibodies find use in a number of applications. For
example, the differentially expressed antibodies can be coupled to
standard affinity chromatography columns and used to purify
differentially expressed proteins as further described below.
[0235] The antibodies can also be used as blocking polypeptides, as
outlined above, since they will specifically bind to the
differentially expressed protein.
[0236] The anti-differentially expressed protein antibodies can
comprise polyclonal antibodies. Methods for producing polyclonal
antibodies are known to those of skill in the art. In brief, an
immunogen, preferably a purified polypeptide, a polypeptide coupled
to an appropriate carrier (e.g., GST and keyhole limpet
hemocyanin), or a polypeptide incorporated into an immunization
vector such as a recombinant vaccinia virus (see, U.S. Pat. No.
4,722,848) is mixed with an adjuvant and animals are immunized with
the mixture. The animal's immune response to the immunogen
preparation is monitored by taking test bleeds and determining the
titer of reactivity to the polypeptide of interest. When
appropriately high titers of antibody to the immunogen are
obtained, blood is collected from the animal and antisera are
prepared. Further fractionation of the antisera to enrich for
antibodies reactive to the polypeptide is performed where desired.
See, e.g., Coligan (1991) CURRENT PROTOCOLS IN IMMUNOLOGY
Wiley/Greene, NY; and Harlow and Lane (1989) ANTIBODIES: A
LABORATORY MANUAL Cold Spring Harbor Press, NY.
[0237] Antibodies, including binding fragments and single chain
recombinant versions thereof, against predetermined fragments of
differentially expressed proteins are raised by immunizing animals,
e.g., with conjugates of the fragments with carrier proteins as
described above.
[0238] The anti-differentially expressed protein antibodies can,
alternatively, be monoclonal antibodies. The monoclonal antibodies
are prepared from cells secreting the desired antibody. These
antibodies are screened for binding to normal or modified
polypeptides, or screened for agonistic or antagonistic activity,
e.g., activity mediated through the differentially expressed
proteins. In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, and humans. Description of techniques for preparing such
monoclonal antibodies are found in, e.g., Stites et al. (eds.)
BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange Medical Publications,
Los Altos, Calif., and references cited therein; Harlow and Lane,
Supra; Goding (1986) Monoclonal Antibodies: Principles and Practice
(2d ed.) Academic Press, New York, N.Y.; and Kohler and Milstein
(1975) Nature 256: 495-497.
[0239] The immunizing agent will typically include the
differentially expressed protein polypeptide or a fusion protein
thereof Generally, either peripheral blood lymphocytes ("PBLs") are
used if cells of human origin are desired, or spleen cells or lymph
node cells are used if non-human mammalian sources are desired. The
lymphocytes are then fused with an immortalized cell line using a
suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell (Goding, Monoclonal Antibodies: Principles and
Practice Academic Press, (1986) pp. 59-103). Immortalized cell
lines are usually transformed mammalian cells, particularly myeloma
cells of rodent, bovine and human origin. Usually, rat or mouse
myeloma cell lines are employed. The hybridoma cells can be
cultured in a suitable culture medium that preferably contains one
or more substances that inhibit the growth or survival of the
unfused, immortalized cells. For example, if the parental cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which substances prevent the growth of HGPRT-deficient
cells.
[0240] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Rockville, Md. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63).
[0241] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against differentially expressed protein. Preferably, the
binding specificity of monoclonal antibodies produced by the
hybridoma cells is determined by immunoprecipitation or by an in
vitro binding assay, such as radioimmunoassay (RIA) or
enzyme-linked immunosorbent assay (ELISA). Such techniques and
assays are known in the art. The binding affinity of the monoclonal
antibody can, for example, be determined by the Scatchard analysis
of Munson and Pollard, Anal. Biochem. 107: 220 (1980).
[0242] After the desired hybridoma cells are identified, the clones
can be subcloned by limiting dilution procedures and grown by
standard methods (Goding, supra). Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells can be
grown in vivo as ascites in a mammal.
[0243] The monoclonal antibodies secreted by the subclones can be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0244] Other suitable techniques involve selection of libraries of
recombinant antibodies in phage or similar vectors. See, Huse et
al. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature
341: 544-546.
[0245] Also, recombinant immunoglobulins may be produced. See, U.S.
Pat. No. 4,816,567 (Cabilly); and Queen et al. (1989) Proc. Nat'l
Acad. Sci. USA 86: 10029-10033.
[0246] Briefly, nucleic acids encoding light and heavy chain
variable regions, optionally linked to constant regions, are
inserted into expression vectors. The light and heavy chains can be
cloned in the same or different expression vectors. The DNA
segments encoding antibody chains are operably linked to control
sequences in the expression vector(s) that ensure the expression of
antibody chains. Such control sequences include a signal sequence,
a promoter, an enhancer, and a transcription termination sequence.
Expression vectors are typically replicable in the host organisms
either as episomes or as an integral part of the host
chromosome.
[0247] E. coli is one procaryotic host particularly for expressing
antibodies of the present invention. Other microbial hosts suitable
for use include bacilli, such as Bacillus subtilus, and other
enterobacteriaceae, such as Salmonella, Serratia, and various
Pseudomonas species. In these prokaryotic hosts, one can also make
expression vectors, which typically contain expression control
sequences compatible with the host cell (e.g., an origin of
replication) and regulatory sequences such as a lactose promoter
system, a tryptophan (trp) promoter system, a beta-lactamase
promoter system, or a promoter system from phage lambda.
[0248] 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.
[0249] Mammalian tissue cell culture can also be used to express
and produce the antibodies of the present invention (See Winnacker,
From Genes to Clones (VCH Publishers, N.Y., 1987). Eukaryotic cells
are preferred, because a number of suitable host cell lines capable
of secreting intact antibodies have been developed. Preferred
suitable host cells for expressing nucleic acids encoding the
immunoglobulins of the invention include: monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293) (Graham et al, 1977, J. Gen. Virol. 36: 59); baby
hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster
ovary-cells-DHFR(CHO, Urlaub and Chasin, 1980, Proc. Natl. Acad.
Sci. U.S.A. 77: 4216); mouse sertoli cells (TM4, Mather, 1980,
Biol. Reprod. 23: 243-251); monkey kidney cells (CV1 ATCC CCL 70);
african green monkey kidney cells (VERO-76, ATCC CRL 1587); human
cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells
(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL
1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep
G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); and,
TR1 cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383: 4446);
baculovirus cells.
[0250] The vectors containing the polynucleotide sequences of
interest (e.g., the heavy and light chain encoding sequences and
expression control sequences) can be transferred into the host
cell. Calcium chloride transfection is commonly utilized for
prokaryotic cells, whereas calcium phosphate treatment or
electroporation can be used for other cellular hosts. (See
generally Sambrook et al., Molecular Cloning A Laboratory Manual
(Cold Spring Harbor Press, 2nd ed., 1989) (incorporated by
reference in its entirety for all purposes). When heavy and light
chains are cloned on separate expression vectors, the vectors are
co-transfected to obtain expression and assembly of intact
immunoglobulins. After introduction of recombinant DNA, cell lines
expressing immunoglobulin products are cell selected. Cell lines
capable of stable expression are preferred (i.e., undiminished
levels of expression after fifty passages of the cell line).
[0251] Once expressed, the whole antibodies, their dimers,
individual light and heavy chains, or other immunoglobulin forms of
the present invention can be purified according to standard
procedures of the art, including ammonium sulfate precipitation,
affinity columns, column chromatography, gel electrophoresis and
the like (See generally Scopes, Protein Purification
(Springer-Verlag, N.Y., 1982). Substantially pure immunoglobulins
of at least about 90 to 95% homogeneity are preferred, and 98 to
99% or more homogeneity most preferred.
[0252] Frequently, the polypeptides and antibodies will be labeled
by joining, either covalently or non-covalently, a substance which
provides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Thus, an antibody used
for detecting an analyte can be directly labeled with a detectable
moiety, or may be indirectly labeled by, for example, binding to
the antibody a secondary antibody that is, itself directly or
indirectly labeled.
[0253] The antibodies of this invention are also used for affinity
chromatography in isolating differentially expressed proteins.
Columns are prepared, e.g. with the antibodies linked to a solid
support, e.g., particles, such as agarose, Sephadex, or the like,
where a cell lysate is passed through the column, washed, and
treated with increasing concentrations of a mild denaturant,
whereby purified differentially expressed polypeptides are
released.
[0254] 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. Such B cells can
be obtained from a human immunized with the desired antigen,
fragments, longer polypeptides containing the antigen or fragments
or anti-idiotypic antibodies. Optionally, such B cells are obtained
from an individual who has not been exposed to the antigen. B cell
can also be obtained from transgenic non-human animals expressing
human immunoglobulin sequences. The transgenic non-human animals
can be immunized with an antigen or collection of antigens. The
animals can also be unimmunized. B cell mRNA sequences encoding
human antibodies are used to generate cDNA using reverse
transcriptase. The V region encoding segments of the cDNA sequences
are then cloned into a DNA vector that directs expression of the
antibody V regions. Typically the V region sequences are
specifically amplified by PCR prior to cloning. Also typically, the
V region sequences are cloned into a site within the DNA vector
that is constructed so that the V region is expressed as a fusion
protein. Examples of such fusion proteins include m13 coliphage
gene 3 and gene 8 fusion proteins. The collection of cloned V
region sequences is then used to generate an expression library of
antibody V regions. To generate an expression library, the DNA
vector comprising the cloned V region sequences is used to
transform eukaryotic or prokaryotic host cells. In addition to V
regions, the vector can optionally encode all or part of a viral
genome, and can comprise viral packaging sequences. In some cases
the vector does not comprise an entire virus genome, and the vector
is then used together with a helper virus or helper virus DNA
sequences. The expressed antibody V regions are found in, or on the
surface of, transformed cells or virus particles from the
transformed cells. This expression library, comprising the cells or
virus particles, is then used to identify V region sequences that
encode antibodies, or antibody fragments reactive with
predetermined antigens. To identify these V region sequences, the
expression library is screened or selected for reactivity of the
expressed V regions with the predetermined antigens. The cells or
virus particles comprising the cloned V region sequences, and
having the expressed V regions, are screened or selected by a
method that identifies or enriches for cells or virus particles
that have V regions reactive (e.g., binding association or
catalytic activity) with a predetermined antigen. For example,
radioactive or fluorescent labeled antigen that then binds to
expressed V regions can be detected and used to identify or sort
cells or virus particles. Antigen bound to a solid matrix or bead
can also be used to select cells or virus particles having reactive
V regions on the surface. The V region sequences thus identified
from the expression library can then be used to direct expression,
in a transformed host cell, of an antibody or fragment thereof,
having reactivity with the predetermined antigen.
[0255] 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, U.S. Pat. Nos.
5,871,907, 5,858,657, 5,837,242, 5,733,743 and 5,565,332 (each of
which is incorporated by reference in its entirety for all
purposes). In these methods, libraries of phage are produced in
which members (display packages) display different antibodies on
their outer surfaces. Antibodies are usually displayed as Fv or Fab
fragments. Phage displaying antibodies with a desired specificity
can be selected by affinity enrichment to the antigen or fragment
thereof Phage display combined with immunized transgenic non-human
animals expressing human immunoglobulin genes can be used to obtain
antigen specific antibodies even when the immune response to the
antigen is weak.
[0256] In a variation of the phage-display method, human antibodies
having the binding specificity of a selected murine antibody can be
produced. See, for example, Winter, WO 92/20791. In this method,
either the heavy or light chain variable region of the selected
murine antibody is used as a starting material. If, for example, a
light chain variable region is selected as the starting material, a
phage library is constructed in which members display the same
light chain variable region (i.e., the murine starting material)
and a different heavy chain variable region. The heavy chain
variable regions are obtained from a library of rearranged human
heavy chain variable regions. A phage showing strong specific
binding (e.g., at least 10.sup.8 and preferably at least 10.sup.9
M.sup.-1) can then be selected. The human heavy chain variable
region from this phage then serves as a starting material for
constructing a further phage library. In this library, each phage
displays the same heavy chain variable region (i.e., the region
identified from the first display library) and a different light
chain variable region. The light chain variable regions are
obtained from a library of rearranged human variable light chain
regions. Again, phage showing strong specific binding for the
selected are selected. Artificial antibodies that are similar to
human antibodies can be obtained from phage display libraries that
incorporate random or synthetic sequences, for example, in CDR
regions.
[0257] In another embodiment of the invention, fragments of
antibodies against differentially expressed protein or protein
analogs are provided. Typically, these fragments exhibit specific
binding to the differentially expressed protein receptor similar to
that of a complete immunoglobulin. Antibody fragments include
separate heavy chains, light chains F.sub.ab, F.sub.ab'F.sub.(ab')2
and F.sub.v. Fragments are produced by recombinant DNA techniques,
or by enzymic or chemical separation of intact immunoglobulins.
[0258] The antibodies can be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the F.sub.c region so
as to prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0259] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art.
[0260] 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 U.S.
Pat. No. 5,585,089 (Queen et al.). Humanized forms of non-human
(e.g., murine) antibodies are immunoglobulins, immunoglobulin
chains or fragments thereof (such as F.sub.v, F.sub.ab, F.sub.ab',
F.sub.ab2 or other antigen-binding subsequences of antibodies)
which contain minimal sequence derived from non-human
immunoglobulin. Humanized antibodies include human immunoglobulins
(recipient antibody) in which residues from a complementary
determining region (CDR) of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and
capacity. In some instances, F.sub.v framework residues of the
human immunoglobulin are replaced by corresponding non-human
residues. Humanized antibodies can also comprise residues which are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an F region, typically
that of a human immunoglobulin (Jones et al., Nature (1986) 321:
522-525; Riechmann et al., Nature (1988) 332: 323-329; and Presta,
Curr. Op. Struct. Biol. (1992) 2: 593-596).
[0261] Chimeric and humanized antibodies have the same or similar
binding specificity and affinity as a mouse or other nonhuman
antibody that provides the starting material for construction of a
chimeric or humanized antibody. Chimeric antibodies are antibodies
whose light and heavy chain genes have been constructed, typically
by genetic engineering, from immunoglobulin gene segments belonging
to different species. For example, the variable (V) segments of the
genes from a mouse monoclonal antibody may be joined to human
constant (C) segments, such as IgG.sub.1 and IgG.sub.4. Human
isotype IgG is preferred. A typical chimeric antibody is thus a
hybrid protein consisting of the V or antigen-binding domain from a
mouse antibody and the C or effector domain from a human
antibody.
[0262] Humanized antibodies have variable region framework residues
substantially from a human antibody (termed an acceptor antibody)
and complementarity determining regions substantially from a
mouse-antibody (referred to as the donor immunoglobulin). See,
Queen et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86: 10029-10033
and WO 90/07861, U.S. Pat. No. 5,693,762, U.S. Pat. No. 5,693,761,
U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,530,101 and Winter, U.S.
Pat. No. 5,225,539 (incorporated by reference in their entirety for
all purposes). The constant region(s), if present, are also
substantially or entirely 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.
[0263] For example, when an amino acid differs between a murine
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: (1) noncovalently binds antigen directly, (2) is adjacent to
a CDR region, (3) otherwise interacts with a CDR region (e.g. is
within about 6 A of a CDR region), or (4) participates in the VL-VH
interface.
[0264] 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 mouse donor antibody or
from the equivalent positions of more typical human
immunoglobulins. Other candidates for substitution are acceptor
human framework amino acids that are unusual for a human
immunoglobulin at that position. The variable region frameworks of
humanized immunoglobulins usually show at least 85% sequence
identity to a human variable region framework sequence or consensus
of such sequences.
[0265] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
discussed above (Hoogenboom and Winter, J. Mol. Biol. (1991) 227:
381; Marks et al., J. Mol. Biol. (1991) 222: 581). The techniques
of Cole et al. and Boemer et al. are also available for the
preparation of human monoclonal antibodies (Cole et al., Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and
Boemer et al., J. Immunol. (1991) 147(1): 86-95). Similarly, human
antibodies can be made by introducing of human immunoglobulin loci
into transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which
closely resembles that seen in humans in all respects, including
gene rearrangement, assembly, and antibody repertoire. This
approach is described, for example, in U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; see also
Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al.,
Nature (1994)368: 856-859; Morrison, Nature (1994)368: 812-13;
Fishwild et al., Nature Biotechnology (1996) 14: 845-51; Neuberger,
Nature Biotechnology (1996) 14: 826; Lonberg and Huszar, Intern.
Rev. Immunol. (1995) 13: 65-93.
[0266] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for the differentially expressed protein, the
other one is for any other antigen, and preferably for a
cell-surface protein or receptor or receptor subunit.
[0267] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities Milstein and Cuello, Nature (1983) 305:
537-539). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker et al., EMBO J. (1991) 10: 3655-3659.
[0268] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology
(1986) 121: 210.
[0269] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for treatment of HIV infection (WO
91/00360; WO 92/200373; EP 03089). It is contemplated that the
antibodies can be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins can be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0270] The anti-differentially expressed protein antibodies of the
invention have various utilities. For example, anti-differentially
expressed protein antibodies can be used in diagnostic assays for a
differentially expressed protein, e.g., detecting its expression in
specific cells, tissues, or serum. Various diagnostic assay
techniques can be used, such as competitive binding assays, direct
or indirect sandwich assays and immunoprecipitation assays
conducted in either heterogeneous or homogeneous phases (Zola,
Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc.
(1987) pp. 147-158). The antibodies used in the diagnostic assays
can be labeled with a detectable moiety. The detectable moiety
should be capable of producing, either directly or indirectly, a
detectable signal. For example, the detectable moiety can be a
radioisotope, such as 3H, .sup.14C, .sup.32P, .sup.35S, or
.sup.125I, a fluorescent or chemiluminescent compound, such as
fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme,
such as alkaline phosphatase, beta-galactosidase or horseradish
peroxidase. Any method known in the art for conjugating the
antibody to the detectable moiety can be employed, including those
methods described by Hunter et al., Nature (1962) 144: 945; David
et al., Biochemistry (1974) 13: 1014; Pain et al., J. Immunol.
Meth. (1981) 40: 219; and Nygren, J. Histochem. and Cytochem.
(1982) 30: 407.
[0271] Anti-differentially expressed protein antibodies also are
useful for the affinity purification of differentially expressed
protein from recombinant cell culture or natural sources. In this
process, the antibodies against differentially expressed protein
are immobilized on a suitable support, such a Sephadex resin or
filter paper, using methods well known in the art. The immobilized
antibody then is contacted with a sample containing the
differentially expressed protein to be purified, and thereafter the
support is washed with a suitable solvent that will remove
substantially all the material in the sample except the
differentially expressed protein, which is bound to the immobilized
antibody. Finally, the support is washed with another suitable
solvent that will release the differentially expressed protein from
the antibody.
[0272] 14. Pharmaceutical Compositions and Methods of
Administration
[0273] The anti-differentially expressed protein antibodies can
also be used in treatment. In some methods, the genes encoding the
antibodies are provided, such that the antibodies bind to and
modulate the differentially expressed protein within the cell. In
other methods, a therapeutically effective amount of a
differentially expressed protein, agonist or antagonist is
administered to a patient. A "therapeutically effective amount",
"pharmacologically acceptable dose", "pharmacologically acceptable
amount" means that a sufficient amount of an immunosuppressive
agent or combination of agents is present to achieve a desired
result, e.g., preventing, delaying, inhibiting or reversing a
symptom of a disease or disorder or the progression of disease or
disorder when administered in an appropriate regime.
[0274] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present invention (see, e.g. Remington's
Pharmaceutical Sciences, 17.sup.th ed., 1989). The pharmaceutical
compositions of the present invention generally comprise a
differentially expressed protein, agonist or antagonist in a form
suitable for administration to a patient. The pharmaceutical
compositions are generally formulated as sterile, substantially
isotonic and in full compliance with all Good Manufacturing
Practice (GMP) regulations of the U.S. Food and Drug
Administration.
[0275] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the packaged
nucleic acid suspended in diluents, such as water, saline or PEG
400; (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as liquids, solids,
granules or gelatin; (c) suspensions in an appropriate liquid; and
(d) suitable emulsions. Tablet forms can include one or more of
lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato starch, microcrystalline cellulose, gelatin,
colloidal silicon dioxide, talc, magnesium stearate, stearic acid,
and other excipients, colorants, fillers, binders, diluents,
buffering agents, moistening agents, preservatives, flavoring
agents, dyes, disintegrating agents, and pharmaceutically
compatible carriers. Lozenge forms can comprise the active
ingredient in a flavor, usually sucrose and acacia or tragacanth,
as well as pastilles comprising the active ingredient in an inert
base, such as gelatin and glycerin or sucrose and acacia emulsions,
gels, and the like containing, in addition to the active
ingredient, carriers known in the art.
[0276] In some preferred methods, the pharmaceutical compositions
are in a water soluble form, such as being present as
pharmaceutically acceptable salts, which is meant to include both
acid and base addition salts. "Pharmaceutically acceptable acid
addition salt" refers to those salts that retain the biological
effectiveness of the free bases and that are not biologically or
otherwise undesirable, formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid and the like, and organic acids such as acetic
acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid and the like. "Pharmaceutically acceptable base
addition salts" include those derived from inorganic bases such as
sodium, potassium, lithium, ammonium, calcium, magnesium, iron,
zinc, copper, manganese, aluminum salts and the like. Particularly
preferred are the ammonium, potassium, sodium, calcium, and
magnesium salts. Salts derived from pharmaceutically acceptable
organic non-toxic bases include salts of primary, secondary, and
tertiary amines, substituted amines including naturally occurring
substituted amines, cyclic amines and basic ion exchange resins,
such as isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, and ethanolamine.
[0277] The nucleic acids, alone or in combination with other
suitable components, can be made into aerosol formulations (i.e.,
they can be "nebulized") to be administered via inhalation. Aerosol
formulations can be placed into pressurized acceptable propellants,
such as dichlorodifluoromethane, propane, nitrogen, and the
like.
[0278] Suitable formulations for rectal administration include, for
example, suppositories, which consist of the packaged nucleic acid
with a suppository base. Suitable suppository bases include natural
or synthetic triglycerides or paraffin hydrocarbons. In addition,
it is also possible to use gelatin rectal capsules which consist of
a combination of the packaged nucleic acid with a base, including,
for example, liquid triglycerides, polyethylene glycols, and
paraffin hydrocarbons.
[0279] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. In the practice
of this invention, compositions can be administered, for example,
by intravenous infusion, orally, topically, intraperitoneally,
intravesically or intrathecally. Parenteral administration and
intravenous administration are the preferred methods of
administration. Formulations for injection can be presented in unit
dosage form, e.g., in ampules or in multidose containers, with an
added preservative. The compositions are formulated as sterile,
substantially isotonic and in full compliance with all Good
Manufacturing Practice (GMP) regulations of the U.S. Food and Drug
Administration.
[0280] Injection solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described. Cells transduced by the packaged nucleic acid as
described above in the context of ex vivo therapy can also be
administered intravenously or parenterally as described above.
[0281] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
therapeutic response in the patient over time. The dose will be
determined by the efficacy of the particular vector employed and
the condition of the patient, as well as the body weight or surface
area of the patient to be treated. The size of the dose also will
be determined by the existence, nature, and extent of any adverse
side-effects that accompany the administration of a particular
vector, or transduced cell type in a particular patient.
[0282] In determining the effective amount of the vector to be
administered in the treatment or prophylaxis of conditions
resulting from expression of the differentially expressed proteins
of the invention, the physician evaluates circulating plasma levels
of the vector, vector toxicities, progression of the disease, and
the production of anti-vector antibodies. In general, the dose
equivalent of a naked nucleic acid from a vector is from about 1
.mu.g to 100 .mu.g for a typical 70 kilogram patient, and doses of
vectors which include a retroviral particle are calculated to yield
an equivalent amount of therapeutic nucleic acid.
[0283] For administration, inhibitors and transduced cells of the
present invention can be administered at a rate determined by the
LD.sub.50 of the inhibitor, vector, or transduced cell type, and
the side-effects of the inhibitor, vector or cell type at various
concentrations, as applied to the mass and overall health of the
patient. Administration can be accomplished via single or divided
doses.
[0284] Transduced cells are prepared for reinfusion according to
established methods (see Abrahamsen et al., J. Clin. Apheresis 6:
48-53 (1991); Carter et al., J. Clin. Arpheresis 4: 113-117 (1998);
Aebersold et al., J. Immunol. Meth. 112: 1-7 (1998); Muul et al.,
J. Immunol. Methods 101: 171-181 (1987); and Carter et al.,
Transfusion 27: 362-365 (1987)). After a period of about 24 weeks
in culture, the cells should number between 1.times.10.sup.8 and
1.times.10.sup.12. In this regard, the growth characteristics of
cells vary from patient to patient and from cell type to cell type.
About 72 hours prior to reinfusion of the transduced cells, an
aliquot is taken for analysis of phenotype, and percentage of cells
expressing the therapeutic agent.
[0285] 15. Kits
[0286] The differentially expressed protein, agonist or antagonist
of the present invention or their homologs are useful tools for
examining expression and regulation of Slo family potassium
channels. Reagents that specifically hybridize to nucleic acids
encoding differentially expressed proteins of the invention
(including probes and primers of the differentially expressed
proteins), and reagents that specifically bind to the
differentially expressed proteins, e.g., antibodies, are used to
examine expression and regulation.
[0287] Nucleic acid assays for the presence of differentially
expressed proteins in a sample include numerous techniques are
known to those skilled in the art, such as Southern analysis,
northern analysis, dot blots, RNase protection, S1 analysis,
amplification techniques such as PCR and LCR, high density
oligonucleotide array analysis, and in situ hybridization. In in
situ hybridization, for example, the target nucleic acid is
liberated from its cellular surroundings in such as to be available
for hybridization within the cell while preserving the cellular
morphology for subsequent interpretation and analysis. The
following articles provide an overview of the art of in situ
hybridization: Singer et al., Biotechniques 4: 230-250 (1986);
Haase et al., Methods in Virology, vol. VII, pp. 189-226 (1984);
and Nucleic Acid Hybridization: A Practical Approach (Hames et al.,
eds. 1987). In addition, a differentially expressed protein can be
detected with the various immunoassay techniques described above.
The test sample is typically compared to both a positive control
(e.g., a sample expressing recombinant differentially expressed
protein) and a negative control.
[0288] The present invention also provides for kits for screening B
cell activity modulators. Such kits can be prepared from readily
available materials and reagents. For example, such kits can
comprise any one or more of the following materials: the
differentially expressed proteins, agonists, or antagonists of the
present invention, reaction tubes, and instructions for testing the
activities of differentially expressed genes. A wide variety of
kits and components can be prepared according to the present
invention, depending upon the intended user of the kit and the
particular needs of the user. For example, the kit can be tailored
for in vitro or in vivo assays for measuring the activity of a
differentially expressed proteins or B cell activity modulators of
the present invention.
[0289] The invention further provides kits comprising probe arrays
as described above. Optional additional components of the kit
include, for example, other restriction enzymes,
reverse-transcriptase or polymerase, the substrate nucleoside
triphosphates, means used to label (for example, an avidin-enzyme
conjugate and enzyme substrate and chromogen if the label is
biotin), and the appropriate buffers for reverse transcription,
PCR, or hybridization reactions.
[0290] Usually, the kits of the present invention also contain
instructions for carrying out the methods.
EXAMPLES
[0291] Methods
[0292] B Cell Purification and Stimulation
[0293] Splenic B cells from non-transgenic, Ig.sup.HEL or
sHEL/Ig.sup.HEL transgenic mice were purified at room temperature
in 1% bovine calf serum in RPMI. The spleen cells were stained with
CD4, CD8 and Mac-1 FITC conjugated antibodies (Caltag) and depleted
of T cells and macrophages with sheep anti-FITC magnetic beads
(Perseptive Biosystems). The remaining cells were 85-95% B220
positive and were either lysed immediately (nave and tolerant cell
preps) or stimulated in RPMI with 1% serum at 37.degree. C. at
2-3.times.10.sup.6 cells/ml. For stimulation experiments, HEL
(Sigma) was used at 500 ng/ml, goat anti-mu (Jackson Labs) at 10
.mu.g/ml, FK506 at 10 ng/ml, PD98059 (NEB) at 20 .mu.M unless
stated otherwise, ionomycin at 1 .mu.M and EGTA at 3 mM. Cells were
preincubated for 45 minutes with PD98059, 15 minutes with FK506 and
2 minutes with EGTA before addition of HEL or anti-mu. Mock
stimulations were performed by addition of carrier alone for
stimuli or inhibitors. At the end of the incubation, the cells were
pelleted by centrifugation, resuspended in a minimal volume of
medium (about 50 .mu.l) by pipetting and lysed in 0.5-1 ml Trizol
(Gibco BRL).
[0294] Nave and tolerant B cells were also purified by FACS. Spleen
cells from Ig.sup.HEL and sHEL/Ig.sup.HEL mice were stained for
B220 and CD21 and sorted for B220 positive, CD21.sup.medium cells.
Marginal zone cells (CD21 high) were excluded from the gate. FACS
allowed us to control for the fact that B cells in sHEL/Ig.sup.HEL
mice are generally present at lower numbers than Ig mice and the
marginal zone B cell subset is absent. Thus, expression changes
between anergic and nave cells determined from samples purified by
negative depletion that are also seen in cells purified by FACS are
unlikely to be due to systematic differences in the amount of
marginal zone B cells or non-B cells in the two samples.
[0295] RNA Purification, cDNA Synthesis In Vitro Transcription
(IVT) and Array Hybridization
[0296] Trizol lysates were phenol-extracted and precipitated with
isopropanol. Poly A+ RNA was purified with Oligotex (Qiagen). CDNA
was synthesised with a SuperscriptII cDNA synthesis kit (Gibco BRL)
using a T7:(dT)24 oligo to prime the first strand and purified by
phenol extraction and ethanol precipitation. The cDNA was used as a
template for in vitro transcription (IVT) using the Megascript
system (Ambion) with the inclusion of biotinylated CTP and UTP. The
IVT product was separated from unincorporated nucleotides using a
RNeasy column (Qiagen) and was fragmented with 150 mM magnesium
chloride at 95.degree. C. Fragmented cRNA was hybridised in a
volume of 200 .mu.l 1 M sodium chloride, 10 mM Tris pH 7.4, 0.005%
Triton X100 including 1 mg/ml BSA, 0.1 mg/ml herring sperm DNA and
bacterial transcripts spiked at known concentrations. Hybridization
was for 12-16 hours at 40.degree. C. with rotation. The arrays were
incubated at 50.degree. C. for 1 hr then washed to 0.5.times.SSPE,
room temperature. Biotinylated hybridised cRNA was developed by
staining with strepavidin-PE and the chips were scanned with a
Molecular Dynamics scanner (see also Lipshutz, R. J. et al., Nat.
Genet (1999) 21: 20-4; this reference and references cited therein
are herein incorporated by reference).
[0297] Statistical Analysis and Querying
[0298] Each gene on the arrays was tiled as a collection of
approximately 20 probe pairs. Each probe pair contains a 25mer
oligo that is exactly complementary to the transcript (the perfect
match oligo) and a second oligo that contains a single base
mismatch at the middle base. The perfect match--mismatch
intensities (PM-MM) were calculated for each probe pair for each
gene in each experiment. For each probe pair, the PM-MM values were
compared by t test between the two conditions (e.g. resting vs
stimulated, matched t test; nave vs anergic, unmatched t test).
Positive t values associated with a probability of less than 0.1
were scored as increased, negative t values with a probability of
less than 0.1 were scored as decreased. The number of increased and
decreased probe pairs associated with each gene was determined and
called npos and nneg respectively (of the approximately 20 probe
pairs tiled for each gene). The distribution of npos and nneg is
binomial where p=0.1 and n=number of probe pairs for that gene. The
probability of scoring npos or more of the total number of probe
pairs was determined. The same analysis was done for decreased
genes using nneg. A probability value was chosen so that the
probability of one or more false positive in any query was less
than 5% after correcting for the number of genes queried. For
example, 280 genes had a median fold change after 1 hour activation
of 1.75 or greater. The adjusted 5% probability for 280 trials is
0.00018, and 59 of the 280 changes were significant at this level.
We also analysed our data using an analysis of variance approach
where the variation in PM-MM values for each gene was partitioned
into that due to error (within group), probe pair, or the
experimental factor (e.g. resting vs stimulated). The results were
broadly the same as for the t test strategy described above.
However, the t test strategy is more robust to rare probes that had
very high and anomalous signals.
[0299] To analyse the 6 hr timepoint (2 experiments) and the sorted
nave and tolerant B cells we used the following query. For a given
transcript, each probe pair was scored as increased in sample A
relative to sample B if (PM-MM).sub.A-(PM-MM).sub.B>30 and
(PM-MM).sub.A>1.3.times.(PM-M- M).sub.B and decreased by the
reverse. A transcript was scored as increased in each pairwise
comparison if the number of increased probe pairs was 3 or greater,
the ratio of increased to decreased probe pairs was greater than 3
and the ratio of average difference intensities was greater than
1.8. Decreased genes were determined by the reverse of this
algorithm. Based on comparisons of all genes between closely
matched samples the false positive rate of this query was
empirically determined to be approximately 1 in 18 transcripts in
any pairwise comparison. Consistent changes across the 2
experiments have a false positive rate of approximately 1 in
300.
[0300] Measurement of Gene Expression
[0301] An intensity for each gene was calculated based on a trimmed
mean of the PM-MM values. Values less than 5 were considered
indistinguishable and were set to 5. The resulting average
difference intensities were used to represent expression levels in
the figures and to calculate fold changes.
[0302] Results
[0303] To identify molecular events distinguishing activation and
tolerance in peripheral lymphocytes, gene expression profiles were
analyzed in lymphocytes undergoing these opposing processes that
were in all other respects as closely matched as possible.
Homogeneous populations of B lymphocytes specific for a well
defined antigen, hen egg lysozyme (HEL), were obtained from the
spleen of mice transgenic for a B cell antigen receptor against HEL
(Ig.sup.HEL mice). Resting B cells from Ig.sup.HEL mice are
antigenically nave and in G0 of cell cycle. Acute stimulation with
foreign HEL triggers their activation and entry into G1, promoting
clonal proliferation and antibody secretion provided that T cells
or bacterial lipopolysaccharides are present as costimuli. In
parallel, homogeneous populations of self-tolerant (anergic) B
cells were obtained from the spleen of double transgenic mice which
carry the same Ig.sup.HEL receptor transgene but also express HEL
as a self antigen (sHEL:Ig.sup.HEL mice). Despite expressing the
same HEL-specific receptors and being matched for stage of
development, the tolerant cells are unable to make a proliferative
or antibody response to HEL. Instead, repeated stimulation of their
receptors by self HEL causes them to make responses that actively
reinforce tolerance, such as altered migration, Fas-dependent and
Fas-independent apoptosis, and inhibition of plasma cell
differentiation. Peripheral tolerant B cells in the sHEL:Ig.sup.HEL
mice first encountered antigen during development in the bone
marrow and have a life span of about 2 weeks. Despite the fact that
these cells have been exposed to self-antigen for differing lengths
of time, they appear homogenous with respect to the aspects of
tolerance listed above, and as measured by continuous calcium
oscillations and uniformly low expression of the activation markers
B7-2 and CD69.
[0304] 6,500 genes for changes in expression during activation or
tolerance using a set of Affymetrix GeneChip.TM. expression arrays
(reviewed in Lipshutz et al.). Hybridization intensity levels
correspond only approximately to absolute expression levels as the
protocol relies on an amplification scheme that can not be strictly
quantitative for all transcripts. However, relative expression
levels of the same transcripts across different samples are
conserved and absolute levels are accurate within +/- 2 fold.
[0305] A relatively small set of response genes was associated with
the initial phase of B lymphocyte activation, one hour after
foreign antigen stimulation ex vivo (FIG. 1). Of the 6,500 genes
screened in seven independent replicate experiments, mRNAs for only
thirty seven were significantly increased and twenty two were
decreased (FIG. 1, p<0.00018). A large fraction of the increased
and decreased transcripts encode transcriptional regulators. While
a small number of these 59 transcripts have been previously
identified as early response genes in B cells, validating the data
obtained here, most were not previously known to participate in B
cell responses. Many of these genes encode proteins with
established roles in mitotic and anti-apoptotic responses by
lymphocytes or other cell types. For example, LSIRF is necessary
for mitogenesis as B cells from mice deficient in this gene are
unable to proliferate in response to anti-IgM. Furthermore,
expression of A1, a bcl-2 homologue, can be sufficient to prevent
apoptosis after antigen receptor engagement on B cells. Conversely,
down-regulation of LKLF can be obligatory for B cell activation as
T cells deficient in LKLF have a spontaneously activated cell
surface phenotype. Finally, c-myc, c-fos, and FosB are associated
with mitogenesis through their status as oncogenes and Egr-1 and
Egr-2 have been specifically implicated in B cell mitogenesis.
[0306] The pattern of gene expression was much more extensively
altered after six hours of antigen stimulation. While mRNAs for
many of the 1 hr induced genes had decreased by this time (e.g.,
Egr-1, PAC-1, c-fos, FosB in FIG. 1C), others in the early response
set showed sustained or exaggerated responses at 6 hours (e.g., A1
and MIP-1a/b in FIG. 1C, LKLF and GILZ in FIG. 1D). Many of the
additional gene expression changes are consistent with movement to
the G1 phase of the cell cycle, including upregulation of CDK4 and
cyclin D2.
[0307] The same set of 6,500 genes was screened for expression
changes in anergic B cells undergoing peripheral tolerance
responses to HEL antigen in vivo. Expression was compared between
five tolerant B cell preparations from sHEL:Ig.sup.HEL mice and
four nave B cell preparations from Ig.sup.HEL mice, where paired
samples were purified by negative selection with magnetic beads. A
further two preparations each of tolerant and nave B cells were
purified by positive selection on a fluoresence activated cell
sorter (FACS). Using an algorithm that requires consistency between
both purification methods, expression of only twenty genes was
significantly increased and eight genes decreased in tolerant cells
(FIG. 2, p<0.00034). One of these changes can be due to
contaminating erythroid cells (carbonic anhydrase II) because this
mRNA species was much less abundant in FACS-sorted B cells.
[0308] To determine the extent of overlap between the responses to
the same antigen when presented as self or foreign the data was
compared using both stringent (p value corrected for number of
trials) and non-stringent (uncorrected) queries. Of the 19 genes
upregulated by self-antigen (excluding carbonic anhydrase II), 7
were also upregulated by foreign antigen after 1 hour
(p<0.00018) or 6 hours (2 of 2 experiments): NAB2 and
neurogranin were comparably upregulated by both forms of antigen;
Egr-1, Egr-2, Gfi-1, cyclin D2 and Cctq were upregulated to a
greater extent by foreign antigen than by self-antigen. Of the
remaining 12 transcripts upregulated in tolerant cells, there was
weaker evidence for upregulation of 4 genes after 6 hours exposure
to foreign antigen (SATB1, CD83, TGIF and CD72, 1 of 2
experiments). For 8 of the 19 transcripts upregulated by
self-antigen, there was no evidence for upregulation by foreign
antigen. Seven of 8 transcripts downregulated by self-antigen were
downregulated by 6 hours exposure to foreign antigen in 2 of 2
experiments (4 transcripts) or 1 of 2 experiments (3 transcripts).
In summary, most, but not all, of the transcript changes induced by
self-antigen were also regulated by foreign antigen, though to
differing degrees.
[0309] Only 16 of more than 500 transcript changes caused by
foreign antigen were also regulated by self-antigen (p<0.05,
fold change>1.8, at least 1 of 2 experiments with sorted cells):
nearly all of the response to foreign antigen is blocked in
tolerant cells. The response to foreign antigen is measured after
in vitro stimulation whereas the response to self-antigen occurs in
vivo. This was necessary because of technical limitations on the
length of time required to isolate and purify activated cells after
stimulation in vivo relative to the time of activation. However, an
analysis of transcript changes caused by in vitro incubation in the
absence of antigen is not consistent with this causing a partial
activation response: in fact, some of the genes that were
upregulated by antigen are downregulated by in vitro incubation and
vice vers. Therefore, the differences that described between
exposure to self and foreign antigen can reflect biological
differences between tolerance and immunity rather than an
"adjuvant" effect of in vitro incubation.
[0310] FK506, a commonly used immunosuppressant drug, can block B
cell activation and can be a phenocopy of tolerance. B cells were
stimulated as for FIG. 1 but in the presence of 10 ng/ml FK506.
This concentration was chosen as it is within the range maintained
in the blood of kidney and liver transplant patients receiving
FK506 (also called Tacrolimus and Prograf, information on dosing
from http://www.fujisawa.com/info/medinfo/- mnpginst.htm). Of the
59 genes defined previously as increasing or decreasing 1 hr after
B lymphocyte activation, only one third of these were efficiently
suppressed by this dose of FK506 (FIG. 3). Some early response
genes (for example, gadd153) were superinduced in the presence of
drug. By this analysis, the suppressive effects of FK506 on
lymphocyte activation are much more limited than the suppression
achieved by peripheral tolerance. The response genes blocked by the
drug include genes that are triggered by self-antigen, such as
Egr-2 and CD72, which can contribute to the active maintenance of
tolerance. Cells stimulated in the presence of FK506 do not
activate NFAT, NFkB nor JNK though signaling through Erk is intact.
Self-antigen causes apparent activation of more signaling pathways,
as signaling through both Erk and NFAT is intact, but the response
to antigen measured by transcript profiling is much more repressed
than is achieved by FK506.
[0311] Nave cells were stimulated in the presence of EGTA. This
reagent had essentially the same effect on the transcript profile
as FK506 (FIG. 3C), confirming that FK506 affected transcript
levels through a calcium/calcineurin-dependent pathway. A notable
exception to this was MyD116. Antigen induced upregulation of this
transcript was repressed by FK506 (n=5) but not by EGTA (n=2),
which can be indicative of secondary effects of the drug other than
calcineurin inhibition. However, there were no FK506-induced
changes in transcripts other than those altered in the antigen
activation response.
[0312] The effects of tolerance and pharmacological reagents on B
cell activation can be used to assign transcriptional events
downstream of particular signaling pathways (FIG. 4A).
Transcriptional events that are suppressed by FK506 and EGTA after
antigen stimulation of nave cells can be firmly assigned to be
downsteam of the calcium/calcineurin pathway. These can be further
subdivided on the basis of their expression levels in tolerant
cells. In these cells, self-antigen evokes chronic low calcium
oscillations that are sufficient to induce nuclear translocation of
NFAT but not to activate NFkB nor JNK, though all three pathways
are dependent on calcium/calcineurin. Thus, FK506-sensitive
upregulation of genes not altered in tolerant cells is suggestive
of signaling through NFkB or JNK, whereas FK506-sensitive genes
that are also upregulated in tolerance would be expected to be
downstream of NFAT (for example, Egr-2 and CD72). Upregulation of
A1 is FK506 sensitive but blocked in tolerance.
[0313] In addition to NFAT, the ERK pathway is also activated in
foreign or self antigen-stimulated cells. To determine downstream
transcriptional effects of ERK, the effect of MEK, an ERK kinase,
on B cell activation was determined. The MEK inhibitor PD98059 was
titrated in B cell activation experiments and gene expression was
monitored using one of the four arrays in the set (approximately
1600 genes). Upregulation of Egr-1 was totally inhibited by 20
.mu.M PD98059 and was 50% inhibited at 5-10 .mu.M, consistent with
the potency of PD98059 against recombinant MEK (FIG. 3D).
Regulation of other early response genes was less sensitive than
Egr-1. Induction of three transcripts (Egr-1, NAB2 and Gfi-1) that
are upregulated by both self and foreign antigen was sensitive to
PD98059 (FIG. 3D) but insensitive to FK506 (FIG. 3A). Continuous
activation of the ERK pathway by self-antigen can have
transcriptional consequences which are distinct from those
downstream of NFAT (FIG. 4A).
[0314] The strategy followed here, statistically comparing the
expression of large numbers of genes in replicate cell samples that
were closely matched to eliminate secondary effects, provides the
first molecular picture of how self-tolerance prevents lymphocyte
mitogenesis (FIG. 4B). Given the continuous signaling through the
ERK and NFAT pathways in response to self antigen, it is remarkable
how few of the mitogenic response genes are triggered. The loss of
a mitotic response to antigen in tolerant cells is explained by the
failure to upregulate LSIRF, a B cell myeloma protooncogene that is
an essential transcription factor for B cell mitogenic responses,
and failure to upregulate A1, an anti-apoptotic protein that is
sufficient and apparently necessary to block apoptotic responses to
antigen in B cells. The block to induction of the B cell lymphoma
protooncogene, c-myc, is also likely to contribute since increased
expression of c-myc is sufficient to promote B cell blastogenesis
in transgenic mice. By comparison, it is surprising how little of
the early mitogenic response is suppressed by FK506 given its
ability to block foreign-antigen stimulated NFAT, NFkB and JNK.
Inhibition of A1 by FK506 can alone be sufficient to explain the
anti-mitogenic effects of FK506, since activation in the presence
of FK506 is associated with increased B cell death.
[0315] The small number of foreign-response genes that are still
triggered in the tolerance response can be inhibited or subverted
from pro-mitogenic roles by other gene products in the tolerant
cells. The Egr-1 and Egr-2 transcription factors have been
specifically implicated in B cell mitogenesis, but are induced at
lower levels in tolerant cells than after activation (FIG. 2B), a
quantitative difference also true for Egr-1 protein. Their
mitogenic activity in tolerant cells is likely to be repressed by
relatively high expression of NAB2, an Egr family inhibitor, and it
Egr/NAB2 heterodimers can activate tolerance-specific genes.
Inhibition by FK506 of Egr-2, and other shared activation/tolerance
response genes such as CD72, shows that this immunosuppressive drug
can also interfere with components of the active self-tolerance
response. This effect can further limit its efficacy in
establishing or restoring tolerance in autoimmunity and
transplantation.
[0316] Many of the genes associated with the tolerance response can
be predicted to have negative regulatory functions for maintaining
the tolerant state. The function of these genes in this context is
unknown but clues can be found in previous work. The largest change
is an increase in mRNA from Aeg-2 (also called CRISP-3), a gene
known to be controlled by Oct-2 in B cells which encodes a secreted
protein of unknown function. Two others, neurogranin and pcp-4,
encode related gene products that have been implicated in
regulation of calcium signaling through calmodulin and they can
have a role in regulation of the downstream effects caused by low
level calcium spiking in tolerant cells (FIG. 4B). Two cell surface
proteins upregulated in tolerant cells regulate proximal signaling
pathways necessary for the maintenance of tolerance. IgD is the
primary receptor isotype expressed on tolerant B cells, through
which repeated binding of self antigen can trigger calcium
oscillations. Proximal signaling can be decreased relative to nave
cells by increased levels of CD72, which has been shown to recruit
the inhibitory tyrosine phosphatase, SHP-1, and diminish BCR
signaling (FIG. 4B). Increased IgD and CD72 in tolerant cells have
been confirmed at the protein level.
[0317] The molecular definition of lymphocyte activation,
tolerance, and FK506-immunosuppression established here can provide
a guide to search for more efficient immunosuppressive drugs. In
particular, the unique transcript signature associated with
self-reactive cells can be used as a surrogate marker for
tolerance, the phenotypes of which are not easily assayed in a high
throughput way. Recent advances in high throughput screening
techniques allow monitoring of gene expression after treatment of
cells with a chemical library of potential drug leads. By defining
a molecular signature for peripheral tolerance, screens for new
drugs that better mimic the tolerance phenotype can be screened.
With this approach, the drug target need not be known and need not
be represented in the original expression screening platform, nor
does the level of transcript for the target protein itself need to
change. To develop drugs that better emulate the active process of
peripheral tolerance, the desired small molecule would suppress
members of the activation-only early response gene subset defined
here, while leaving unaffected the subset of early response genes
that also participate in tolerance. A drug with this profile could
likely to block immunity but not tolerance, which can be key to
(re)establishing immunological unresponsiveness in autoimmunity,
allergy, or tranplantation.
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[0350] Although the foregoing invention has been described in
detail for purposes of clarity of understanding, it will be obvious
that certain modifications can be practiced within the scope of the
appended claims. All publications and patent documents cited above
are hereby incorporated by reference in their entirety for all
purposes to the same extent as if each were so individually
denoted.
* * * * *
References