U.S. patent application number 14/336944 was filed with the patent office on 2015-03-12 for methods and systems for detecting mhc class i binding peptides.
The applicant listed for this patent is Beckman Coulter, Inc.. Invention is credited to Sylvain Monseaux, Felix A. Montero-Julian.
Application Number | 20150072886 14/336944 |
Document ID | / |
Family ID | 32068790 |
Filed Date | 2015-03-12 |
United States Patent
Application |
20150072886 |
Kind Code |
A1 |
Montero-Julian; Felix A. ;
et al. |
March 12, 2015 |
Methods and Systems for Detecting MHC Class I Binding Peptides
Abstract
The present invention is based on the discovery that MHC heavy
chain monomers immobilized to a solid surface are still capable of
forming detectable conformational epitopes and being detected by
conformation-dependent antibodies. Methods for detecting peptide
binding to HLA monomers, and methods for measuring the relative
degree of binding between two MHC-binding peptides as well as a
method of measurement for the rate of dissociation of peptides from
MHC complexes are provided. The present invention also provides
systems and kits useful for conducting the methods of the present
invention.
Inventors: |
Montero-Julian; Felix A.;
(Marseille, FR) ; Monseaux; Sylvain; (Marseille,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Beckman Coulter, Inc. |
Brea |
CA |
US |
|
|
Family ID: |
32068790 |
Appl. No.: |
14/336944 |
Filed: |
July 21, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13029902 |
Feb 17, 2011 |
8815528 |
|
|
14336944 |
|
|
|
|
10269473 |
Oct 11, 2002 |
|
|
|
13029902 |
|
|
|
|
Current U.S.
Class: |
506/9 ; 435/7.9;
436/501; 506/18 |
Current CPC
Class: |
B01J 2219/00454
20130101; B01J 2219/0074 20130101; G01N 33/54306 20130101; B01J
2219/00725 20130101; B01J 19/0046 20130101; B01J 2219/00605
20130101; B01J 2219/005 20130101; G01N 2333/70539 20130101; B01J
2219/00637 20130101; B01J 2219/00315 20130101; G01N 2458/00
20130101; G01N 33/6854 20130101; B01J 2219/00621 20130101; B01J
2219/0061 20130101; B01J 2219/0063 20130101; G01N 33/56977
20130101 |
Class at
Publication: |
506/9 ; 506/18;
436/501; 435/7.9 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Claims
1. A system comprising a solid surface, wherein the surface has
attached thereto one or more MHC monomer or modified MHC monomer
wherein the monomer denatures under denaturing conditions and
reconstitutes to form a ternary complex containing a suitable
MHC-binding peptide under reconstituting conditions.
2. The system of claim 1, wherein the solid surface is a bead.
3. The system of claim 1, wherein the solid surface is in a
microtiter plate.
4. The system of claim 1, wherein the solid surface is suitable for
screening in a high throughput system.
5. The system of claim 1, wherein attachment of the monomer to the
solid surface is reversible.
6. The system of claim 1, wherein attachment of the monomer to the
solid surface is cleavable.
7. The system of claim 1, wherein the solid surface is coated with
a first binding ligand and the C-terminal end of the monomer is
provided with a second binding ligand, wherein the first ligand
binds specifically with the second ligand.
8. The system of claim 7, wherein the first binding ligand is
selected from avidin, streptavidin, neutravidin, StrepTactin and
monomeric avidin and the second binding ligand is biotin.
9. The system of claim 8, wherein the second binding ligand is
attached to the monomer via a C-terminal end.
10. The system of claim 1, wherein the denaturing conditions
comprise a pH of about 2 to about 4.
11. The system of claim 1, wherein the system further comprises an
anti-MHC antibody that binds specifically to a conformational
epitope that is present in the reconstituted monomer and absent in
the denatured monomer.
12. The system of claim 1, wherein the reconstituting conditions
include a pH of from about 7 to about 8.5.
13. The system of claim 1, wherein the system further comprises
beta-2 microglobulin.
14. The system of claim 1, wherein the monomer is HLA class I.
15. The system of claim 14 further comprising an anti-MHC class I
monoclonal antibody, wherein the monoclonal antibody specifically
binds to a reconstituted monomer and does not bind to a denatured
monomer.
16. The system of claim 15, wherein the monomer is HLA class I and
the system further comprises beta-2 microglobulin and a suitable
HLA-binding peptide of from about 8 to about 12 amino acids;
wherein a reconstituted monomer binds to the beta-2 microglobulin
and the suitable peptide under reconstituting conditions.
17. The system of claim 15, wherein the monoclonal antibody is
produced by hybridoma B9.12.1
18. The system of claim 1 or 16, wherein the solid surface coated
with the monomers is in a dried form.
19. A kit comprising the system of claim 1, 16 or 17.
20. The kit of claim 19 further comprising an instruction.
21. The kit of claim 20 further comprising a control peptide to
which the MHC monomer binds in a reconstituted form.
22. A method for determining binding between a MHC monomer or
modified MHC monomer and a putative MHC-binding peptide therefor,
said method comprising: incubating under reconstituting conditions
a solid surface having attached thereto a plurality of MHC monomers
or modified MHC monomers in the presence and absence of the
putative MHC-binding peptide, wherein the monomers have been
denatured and reconstitute to form a ternary complex containing a
suitable MHC-binding peptide under reconstituting conditions, and
determining binding to the MHC monomers after contact therewith of
a monoclonal antibody that binds to the ternary complex but does
not bind to dissociated components of the complex, which binding of
the antibody indicates binding of the monomers with the putative
MHC-binding peptide.
23. The method of claim 22, wherein the denaturing conditions
include a pH in the range from about 2 to about 4.
24. The method of claim 22, further comprising separately
incubating the monomers with a standard MHC-binding peptide for the
monomers under the reconstituting conditions in the presence of the
monoclonal antibody, and wherein the determining includes comparing
binding of the antibody caused by the standard peptide to the
binding of the antibody caused by the putative MHC-binding
peptide.
25. The method of claim 24, wherein the monomers are HLA class I,
the monoclonal antibody is an anti-MHC-class I antibody, and the
reconstituting conditions include the presence of sufficient beta-2
microglobulin for reconstitution of the monomers.
26. The method of claim 25, wherein the monomers are HLA subclass
A, B or C.
27. The method of claim 22, wherein the monoclonal antibody is
provided with a detectable label and the determining includes
detecting the detectable label.
28. The method of claim 27, wherein the detectable label is a
secondary antibody that specifically binds to the monoclonal
antibody.
29. The method of claim 28, wherein the detectable label is
fluorescent.
30. The method of claim 22, wherein the solid surface is the wells
of a microtiter plate or beads and the determining includes reading
fluorescence with a fluorimeter.
31. The method of claim 30, wherein the detecting further comprises
detecting the fluorescence using high throughput scanning.
32. The method of claim 22, wherein the solid surface is coated
with avidin and the monomers are biotinylated to attach to the
solid surface.
33. The method of claim 22, wherein attachment of the monomers to
the solid surface is reversible.
34. The method of claim 22, wherein attachment of the monomers to
the solid surface is cleavable.
35. A method for determining the degree of binding affinity of an
MHC monomer or modified MHC monomer for a putative MHC-binding
peptide therefor, said method comprising: incubating at least one
denatured MHC monomer or modified MHC monomer attached to a solid
surface with the putative MHC-binding peptide and a monoclonal
antibody that specifically binds to a conformational epitope in a
ternary complex containing a corresponding reconstituted MHC
monomer and does not bind to any dissociated component of the
ternary complex, wherein the incubation is under reconstituting
conditions; and comparing binding of the monoclonal antibody to a
ternary complex that contains the putative MHC-binding peptide with
binding of the monoclonal antibody to a corresponding ternary
complex containing the monomer and a known MHC-binding peptide,
wherein a difference in the bindings indicates the relative degree
of binding affinity of the reconstituted monomer for the putative
MHC-binding peptide.
36. The method of claim 35, wherein the reconstituting conditions
include a temperature in the range from about -18.degree. C. to
about 37.degree. C.
37. The method of claim 35, wherein the reconstituting conditions
include a temperature in the range from about 4.degree. C. to about
8.degree. C.
38. The method of claim 35, wherein the reconstituting conditions
include a pH in the range from about 7 to about 8.5.
39. The method of claim 35, wherein the reconstituting conditions
include the presence of a suitable reconstitution buffer.
40. The method of claim 39, wherein the suitable reconstitution
buffer comprises polyoxyethylene(20) sorbitan monolaurate.
41. The method of claim 35, wherein the incubating is for a period
of from about 12 hours to about 48 hours.
42. The method of claim 35, wherein the antibody is provided with a
detectable label and wherein the comparison of binding comprises
detecting a difference in the respective signals produced by the
detectable label resulting from binding of the antibody
thereto.
43. The method of claim 42, wherein the antibody is labeled with a
fluorescent label and the comparison of binding comprises detecting
a difference in the respective fluorescence resulting from binding
of the antibody thereto.
44. The method of claim 43, wherein the solid surface is the wells
of a microtiter plate or beads and the detecting includes reading
the fluorescence with a fluorimeter.
45. The method of claim 44, wherein the detecting further comprises
detecting the fluorescence using a high throughput scanning.
46. The method of claim 35 wherein the fluorescent label is
fluorescein isothiocyanate (FITC).
47. The method of claim 35, wherein the monomers are HLA class I
and further bind with beta-2 microglobulin in reconstituting
conditions and the monoclonal antibody is an anti-MHC-class I
monoclonal antibody.
48. The method of claim 47, wherein the monomers are selected from
HLA-A, HLA-B, and HLA C.
49. The method of claim 47, wherein the monomers are chimeric.
50. The method of claim 35, wherein the monoclonal antibody is
produced by hybridoma B9.12.1.
51. The method of claim 42, wherein the difference in the binding
is compared after incubating the ternary complex containing the
putative MHC binding peptide and the known peptide under conditions
comprising a dissolution-testing temperature for a time sufficient
to indicate the relative dissolution rate of the putative
MHC-binding peptide.
52. The method of claim 51, wherein the dissolution-testing
temperature is in the range from about 4.degree. C. to about
37.degree. C.
53. The method of claim 52, wherein the time is from about 2 hours
to about 48 hours.
54. The method of claim 28, wherein the detectable label is
peroxidase.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to the field of
immunoassays, especially using immunoassays to detect and measure
binding of peptides to MHC alleles.
BACKGROUND OF THE INVENTION
[0002] The Class I histocompatibility ternary complex consists of
three parts associated by noncovalent bonds. A transmembrane
protein, called the MHC heavy chain is mostly exposed at the cell
surface. The cell surface domains of the MHC heavy chain contain
two segments of alpha helix that form two ridges with a groove
between them. A short peptide binds noncovalently ("fits") into
this groove between the two alpha helices, and a molecule of beta-2
microglobulin binds noncovalently along side the outer two domains
of the MHC monomer, forming a ternary complex. Peptides that bind
noncovalently to one MHC subtype heavy chain usually will not bind
to another subtype. However, all bind with the same type of beta-2
microglobulin. MHC molecules are synthesized and displayed by most
of the cells of the body.
[0003] In humans, MHC molecules are referred to as HLA molecules.
Humans primarily synthesize three different sub-types of MHC class
I molecules designated HLA-A, HLA-B and HLA-C, differing only in
the heavy chains.
[0004] The MHC works coordinately with a specialized type of T cell
(the cytotoxic T cell) to rid the body of "nonself" or foreign
viral proteins. The antigen receptor on T-cells recognizes an
epitope that is a mosaic of the bound peptide and portions of the
alpha helices of the making up the groove flanking it. Following
generation of peptide fragments by cleavage of a foreign protein,
the presentation of peptide fragments by the MHC molecule allows
for MHC-restricted cytotoxic T cells to survey cells for the
expression of "nonself" or foreign viral proteins. A functional
T-cell will exhibit a cytotoxic immune response upon recognition of
an MHC molecule containing bound antigenic peptide for which the
T-cell is specific.
[0005] In the performance of these functions in humans, HLA-A, B,
and C heavy chains interact with a multitude of peptides of about 8
to about 10, possibly about 8 to about 11, or about 8 to about 12
amino acids in length. Only certain peptides bind into the binding
pocket in the heavy chain of each HLA sub-type as the monomer
folds, although certain subtypes cross-react. By 1995, complete
coding region sequences had been determined for each of 43 HLA-A,
89 HLA-B and 11 HLA-C alleles (P. Parham et al., Immunology Review
143:141-180, 1995).
[0006] Class II histocompatibility molecules consist of two
transmembrane polypeptides that interact to form a groove at their
outer end which, like the groove in class I molecules,
non-covalently associates with an antigenic peptide. However, the
antigenic peptides bound to class II molecules are derived from
antigens that the cell has taken in from its surroundings. In
addition, peptides that hind to class II histocompatibility
molecules are 15 to about 25 or to about 30 amino acids in length.
Only cells, such as macrophages, dendritic cells and B lymphocytes,
that specialize in taking up antigen from extracellular fluids,
express class II molecules.
[0007] It has long been thought that discovery of which antigen
fragments will be recognized by class I MHC-restricted T-cells can
lead to development of effective vaccines against cancer and viral
infections. A number of approaches have been developed wherein
algorithms are used to predict the amino acid sequence of HLA A, B,
or C-binding peptides and several are available on the internet.
For example, U.S. Pat. No. 6,037,135 describes a matrix-based
algorithm that ranks peptides for likelihood of binding to any
given HLA-A allele. Similarly, most prediction methods are limited
to a set of alleles. Consequently, the predicted peptides may not
bind to MHC monomers from a whole population of patients and thus
may not be globally effective.
[0008] Another approach to identifying MHC-binding peptides uses a
competition-based binding assay. All competition assays yield a
comparison of binding affinities of different peptides. However,
such assays do not yield an absolute dissociation constant since
the result is dependent on the reference peptide used.
[0009] Still another approach used for determining MHC-binding
peptides is the classical reconstitution assay, e.g. using "T2"
cells, in which cells expressing an appropriate MHC allele are
"stripped" of a native binding peptide by incubating at pH 2-3 for
a short period of time. Then, to determine the binding affinity of
a putative MHC-binding peptide for the same MHC allele, the
stripped MHC monomer is combined in solution with the putative
MHC-binding peptide, beta2-microglobulin and a
conformation-dependent monoclonal antibody. The difference in
fluorescence intensity determined between cells incubated with and
without the test binding peptide after labeling, for example,
either directly with the labeled monoclonal antibody or a
fluorescence-labeled secondary antibody, can be used to determine
binding of the test peptide. However, soluble MHC monomers stripped
at low pH aggregate immediately, making their use in high
through-put assays difficult and impractical.
[0010] There are currently a series of in vitro assays for cell
mediated immunity which use cells from the donor. The assays
include situations where the cells are from the donor, however,
many assays provide a source of antigen presenting cells from other
sources, e.g., B cell lines. These in vitro assays include the
cytotoxic T lymphocyte assay; lymphoproliferative assays, e.g.,
tritiated thymidine incorporation; the protein kinase assays, the
ion transport assay and the lymphocyte migration inhibition
function assay (Hickling, J. K. et al, J. Virol., 61: 3463 (1987);
Hengel, H. et al, J. Immunol., 139: 4196 (1987); Thorley-Lawson, D.
A. et al, Proc. Natl. Acad. Sci. USA, 84: 5384 (1987); Kadival, G.
J. et al, J. Immunol., 139: 2447 (1987); Samuelson, L. E. et al, J.
Immunol., 139: 2708 (1987); Cason, J. et al, J. Immunol. Meth.,
102: 109 (1987); and Tsein, R. J. et al, Nature, 293: 68 (1982)).
These assays are disadvantageous in that they may lack true
specificity for cell mediated immunity activity, they require
antigen processing and presentation by an APC of the same MHC type,
they are slow (sometimes lasting several days), and some are
subjective and/or require the use of radioisotopes.
[0011] Yet another approach to identifying MHC class I-binding
peptides utilizes formation of MHC tetramers, which are complexes
of four MHC monomers with streptavidin, a molecule having
tetrameric binding sites for biotin, to which is bound a
fluorochrome, e.g., phycoerythrin. For class I monomers, soluble
subunits of .beta.2-microglobulin, the peptide fragment containing
a putative T-cell epitope, and of a MHC heavy chain corresponding
to the predicted MHC subtype of the peptide fragment of interest,
are obtained by expression of the polypeptides in host cells. Each
of the four monomers contained in the MHC tetramer is produced as a
monomer by refolding these soluble subunits under conditions that
favor assembly of the soluble units into reconstituted monomers,
each containing a beta2-microglobulin, a peptide fragment, and the
corresponding MHC heavy chain. An MHC tetramer is constructed from
the monomers by biotinylation of the monomers and subsequent
contact of the biotinylated reconstituted monomers with
fluorochrome-labeled streptavidin. When contacted with a diverse
population of T cells, such as is contained in a sample of the
peripheral blood lymphocytes (PBLs) of a subject, those tetramers
containing reconstituted monomers that are recognized by a T cell
in the sample will bind to the matched T cell. Contents of the
reaction is analyzed using fluorescence flow cytometry, to
determine, quantify and/or isolate those T-cells having a MHC
tetramer bound thereto (See U.S. Pat. No. 5,635,363).
[0012] At least one other test is required to determine whether a
test peptide recognized by a T-cell by the MHC tetramer assay will
activate the T-cell to generate an immune response, a so-called
"functional test". The enzyme-linked immunospot (ELISpot) assay has
been adapted for the detection of individual cells secreting
specific cytokines or other effector molecules by attachment of a
monoclonal antibody specific for a cytokine or effector molecule on
a microplate. Cells stimulated by an antigen are contacted with the
immobilized antibody. After washing away cells and any unbound
substances, a tagged polyclonalantibody or more often, a monoclonal
antibody, specific for the same cytokine or other effector molecule
is added to the wells. Following a wash, a colorant that binds to
the tagged antibody is added such that a blue-black colored
precipitate (or spot) forms at the sites of cytokine localization.
The spots can be counted manually or with automated ELISpot reader
system to quantitated the response. A final confirmation of T-cell
activation by the test peptide may require in vivo testing, for
example in a mouse model. Thus, the route to final confirmation of
the efficacy of a MHC-binding peptide is expensive and time
consuming.
[0013] Thus, there is still a need in the art for new and better
systems and methods for preliminary screening assays identifying
putative MHC class I-binding peptides and for measuring peptide
binding to MHC class I alleles, such as HLA-A, B or C, especially
an in vitro assay in solid phase format. There is also a need in
the art to develop methods to determine the MHC-binding affinity of
MHC-binding peptides and for a measurement for the dissociation
rate of a bound peptide from the MHC molecule.
SUMMARY OF THE INVENTION
[0014] The present invention is based on the discovery that MHC
class I monomers when immobilized to a solid surface are still
capable of reconstituting to incorporate from solution an
MHC-binding peptide and form a ternary complex.
[0015] Accordingly, in one embodiment the invention provides a
system comprising a solid surface, wherein the surface has attached
thereto one or more MHC monomer or modified MHC monomer, wherein
the monomer denatures in a denaturing condition and reconstitutes
to form a ternary complex containing a suitable MHC-binding peptide
in the binding pocket under reconstituting conditions. In another
embodiment a kit comprising the invention system is also
provided.
[0016] In yet another embodiment, the invention provides methods
for determining binding between a MHC monomer or modified MHC
monomer and a putative MHC-binding peptide therefor. In this method
for assaying binding of a putative MHC-binding peptide, a solid
surface having attached thereto a plurality of previously denatured
MHC monomers or modified MHC monomers is incubated under
reconstituting conditions in the presence and absence of the
putative MHC-binding peptide such that the monomers reconstitute to
form a ternary complex containing a suitable MHC-binding peptide
under the reconstituting conditions. Binding to the ternary complex
of a monoclonal antibody that does not bind to dissociated
components of the complex indicates binding between the putative
MHC-binding peptide and the monomers.
[0017] In still another embodiment, the invention provides methods
for determining the degree of binding affinity of an MHC monomer or
modified MHC monomer for a putative MHC-binding peptide therefor.
In this embodiment, at least one denatured MHC monomer or modified
MHC monomer attached to a solid surface is incubated under
reconstituting conditions with the putative MHC-binding peptide and
a monoclonal antibody that specifically binds to a conformational
epitope of a corresponding reconstituted MHC monomer that is not
present in the denatured monomer. Binding of the monoclonal
antibody to a monomer that binds to the putative MHC-binding
peptide is compared with binding of the monoclonal antibody to a
corresponding monomer having a known MHC-binding peptide bound
thereto. The difference in the binding indicates the relative
degree of binding affinity of the reconstituted monomer for the
putative MHC-binding peptide.
[0018] In still another embodiment, the invention provides methods
for determining the stability at 37.degree. C. of an MHC monomer or
modified MHC monomer for a putative MHC-binding peptide therefor.
In this embodiment, at least one denatured MHC monomer or modified
MHC monomer attached to a solid surface is incubated under
reconstituting conditions with the putative MHC-binding peptide and
a monoclonal antibody that specifically binds to a conformational
epitope of a corresponding reconstituted MHC monomer that is not
present in the denatured monomer. After the reconstituted ternary
complex with the monoclonal antibody is incubated at different
temperatures and different times. The difference in the signal
obtained at different temperatures and different times, indicates
the relative stability of the reconstituted monomer for the
putative MHC-binding peptide.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 is a schematic representation of the immunoassay.
[0020] FIG. 2 is a graph showing calibration of the anti-HLA-class
I-FITC mAb for fluorometric assay.
[0021] FIG. 3 is a graph showing a decrease in binding of
anti-HLA-class I-FITC mAb to a reconstituted HLA heavy chain
monomer Mart1 26-35 with increasing temperature as determined by
fluorescence of bound antibody.
[0022] FIG. 4 is a graph showing the binding of an anti-HLA-class
I-FITC monoclonal antibody to human and mouse alleles as determined
by fluorescence of bound antibody.
[0023] FIG. 5 is a graph showing renaturation in various buffer
solutions of the MHC heavy chain monomers attached to a plate as
detected by an anti-HLA-class I-FITC mAb.
[0024] FIG. 6 is a graph showing antibody binding to monomer at
concentrations of anti-HLA-class I mAb of 1 to 2 .mu.g/ml for
various HLA heavy chain monomer concentrations to determine the
optimal concentration of the anti-HLA-class I antibody for use with
a microtiter plate assay.
[0025] FIGS. 7A and 7B are graphs showing the dose response curve
obtained with two different HLA heavy chain monomers FIG. 7A shows
the results with HLA-A*0201/Mart1 2635L (Linear regression
equation: y=1555.5x+39.787; R2=0.9889. FIG. 7B shows the results
with HLA heavy chain monomer HLA-A*0201/HIVpol (Linear regression
equation: y=1487.1X+13.927, R2=0.9982)
[0026] FIG. 8 is a graph showing the specificity of the
anti-HLA-class I antibody for various HLA-A and HLA-B alleles.
[0027] FIG. 9 is a schematic drawing showing formation of a
human-mouse chimeric MHC modified monomer according to the
invention.
[0028] FIGS. 10A-D show graphs of the dissociation curves for
renatured peptides (HBV core peptide; 26-35L; 26-35; 27-35,
respectively).
[0029] FIGS. 10E-H show graphs of the off rates for peptides HBV
core; 26-35L; 26-35; and 27-35, respectively.
[0030] FIG. 10I shows the effect of temperature on monomer
dissociation.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The present invention relates in general to immunoassays
directed to detection and measurement of the binding affinity of
MHC heavy chain monomers, especially MHC heavy chain monomers
immobilized on a surface, for putative MHC-binding peptides. It is
the discovery of the present invention that MHC heavy chain
monomers and modified MHC monomers immobilized to a solid surface
are still capable of refolding so as to bind from solution
beta2-microglobulin and a MHC-binding peptide that has the
requisite binding. Moreover, it is the discovery of the present
invention that such binding can be detected in an immunoassay
format, such as one utilizing a conformation-dependent monoclonal
antibody that specifically binds to a ternary complex containing
such refolded or reconstituted MHC monomers but does not bind to
dissociated components of the ternary complex.
[0032] As used herein, the terms "MHC monomer" and "HLA monomer"
refer to a class I MHC heavy chain that maintains the ability to
assemble into a ternary complex with an appropriate MHC-binding or
HLA-binding peptide and beta-2 microglobulin under renaturing
conditions. The terms "MHC monomer" and HLA monomer" are also used
to refer to the denatured form of the monomer that results from
subjecting the ternary complex to denaturing conditions, causing
the monomer to unfold and dissociate from a MHC-binding peptide and
from beta-2 microglobulin.
[0033] As used herein, the terms "modified MHC monomer" and
"modified HLA monomer" refer to class I monomers as described
above, but which have been engineered to introduce modifications as
described below. These terms also encompass functional fragments of
the MHC monomer that maintain the ability to assemble into a
ternary complex with an appropriate MHC-binding or HLA-binding
peptide and beta-2 microglobulin under renaturing conditions and to
dissociate under denaturing conditions. For example, a functional
fragment can comprise only the .alpha..sub.1, .alpha..sub.2,
.alpha..sub.3, domains, or only .alpha..sub.1, .alpha..sub.2
domains, of the class I heavy chain, i.e., the cell surface
domains, that participate in formation of the ternary complex. In
another embodiment, modified MHC monomers can be class I heavy
chain molecules, or functional fragments thereof, contained in a
fusion protein or "single chain" molecule and may further include
an amino acid sequence functioning as a linker between cell surface
domains of the monomer, a detectable marker or as a ligand to
attach the molecule to a solid support that is coated with a second
ligand with which the ligand in the fusion protein reacts. Moreover
the terms "modified MHC monomer" and "modified HLA monomer" are
intended to encompass chimera containing domains of class I heavy
chain molecules from more than one species or from more than one
class I subclass. FIG. 9 herein illustrates preparation of a
chimera by substitution of a mouse H-2Kb domain for one of the
three alpha domains in a human HLA-A2 fragment. Such a molecule is
conveniently expressed as a single chain with optional amino acid
linkers between subunits or as a fusion protein as is known in the
art.
Preparation of Monomers
[0034] The Class I MHC in humans is located on chromosome 6 and has
three loci, HLA-, HLA-B, and HLA-C. The first two loci have a large
number of alleles encoding alloantigens. These are found to consist
of a 44 Kd heavy chain subunit and a 12 Kd
.beta.sub.2-microglobulin subunit which is common to all antigenic
specificities. For example, soluble HLA-A2 can be purified after
papain digestion of plasma membranes from the homozygous human
lymphoblastoid cell line J-Y as described by Turner, M. J. et al.,
J. Biol. Chem. (1977) 252:7555-7567. Papain cleaves the 44 Kd heavy
chain close to the transmembrane region, yielding a molecule
comprised of .alpha..sub.1, .alpha..sub.2, .alpha..sub.3 domains
and beta-2 microglobulin.
[0035] The MHC monomers can be isolated from appropriate cells or
can be recombinantly produced, for example as described by Paul et
al, Fundamental Immunology, 2d Ed., W. E. Paul, ed., Ravens Press
N.Y. 1989, Chapters 16-18) and readily modified, as described
below.
[0036] The term "isolated" as applied to MHC monomers herein refers
to an MHC glycoprotein heavy chain of MHC class I, which is in
other than its native state, for example, not associated with the
cell membrane of a cell that normally expresses MHC. This term
embraces a full length subunit chain, as well as a functional
fragment of the MHC monomer. A functional fragment is one
comprising an antigen binding site and sequences necessary for
recognition by the appropriate T cell receptor. It typically
comprises at least about 60-80%, typically 90-95% of the sequence
of the full-length chain. As described herein, the "isolated" MHC
subunit component may be recombinantly produced or solubilized from
the appropriate cell source.
[0037] It is well known that native forms of "mature" MHC
glycoprotein monomers will vary somewhat in length because of
deletions, substitutions, and insertions or additions of one or
more amino acids in the sequences. Thus, MHC monomers are subject
to substantial natural modification, yet are still capable of
retaining their functions. Modified protein chains can also be
readily designed and manufactured utilizing various recombinant DNA
techniques well known to those skilled in the art and described in
detail, below. For example, the chains can vary from the naturally
occurring sequence at the primary structure level by amino acid
substitutions, additions, deletions, and the like. These
modifications can be used in a number of combinations to produce
the final modified protein chain.
[0038] In general, modifications of the genes encoding the MHC
monomer may be readily accomplished by a variety of well-known
techniques, such as site-directed mutagenesis. The effect of any
particular modification can be evaluated by routine screening in a
suitable assay for the desired characteristic. For instance, a
change in the immunological character of the subunit can be
detected by competitive immunoassay with an appropriate antibody.
The effect of a modification on the ability of the monomer to
activate T cells can be tested using standard in vitro cellular
assays or the methods described in the example section, below.
Modifications of other properties such as redox or thermal
stability, hydrophobicity, susceptibility to proteolysis, or the
tendency to aggregate are all assayed according to standard
techniques.
[0039] This invention provides amino acid sequence modification of
MHC monomers prepared with various objectives in mind, including
increasing the affinity of the subunit for antigenic peptides
and/or T cell receptors, facilitating the stability, purification
and preparation of the subunits. The monomers may also be modified
to modify plasma half life, improve therapeutic efficacy, or to
lessen the severity or occurrence of side effects during
therapeutic use of complexes of the present invention. The amino
acid sequence modifications of the subunits are usually
predetermined variants not found in nature or naturally occurring
alleles. The variants typically exhibit the same biological
activity (for example, MHC-peptide binding) as the naturally
occurring analogue.
[0040] Insertional modifications of the present invention are those
in which one or more amino acid residues are introduced into a
predetermined site in the MHC monomer and which displace the
preexisting residues. For instance, insertional modifications can
be fusions of heterologous proteins or polypeptides to the amino or
carboxyl terminus of the subunits.
[0041] Other modifications, include fusions of the monomer with a
heterologous signal sequence and fusions of the monomer to
polypeptides having enhanced plasma half life (ordinarily>about
20 hours) such as immunoglobulin chains or fragments thereof as is
known in the art.
[0042] Substitutional modifications are those in which at least one
residue has been removed and a different residue inserted in its
place. Nonnatural amino acid (i.e., amino acids not normally found
in native proteins), as well as isosteric analogs (amino acid or
otherwise) are also suitable for use in this invention.
[0043] Substantial changes in function or immunological identity
are made by selecting substituting residues that differ in their
effect on maintaining the structure of the polypeqptide backbone
(e.g., as a sheet or helical conformation), the charge or
hydrophobicity of the molecule at the target site, or the bulk of
the side chain. The substitutions which in general are expected to
produce the greatest changes in function will be those in which (a)
a hydrophilic residue, e.g., serine or threonine, is substituted
for (or by) a hydrophobic residue, e.g. leucine, isoleucine,
phenylalanine, valine or alanine; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, e.g., lysine, arginine, or histidine,
is substituted for (or by) an electronegative residue, e.g.,
glutamine or aspartine; or (d) a residue having a bulky side chain,
e.g., phenylalanine, is substituted for (or by) one not having a
side chain, e.g., glycine.
[0044] Substitutional modifications of the monomers also include
those where functionally homologous (having at least about 70%
homology) domains of other proteins are substituted by routine
methods for one or more of the MHC subunit domains. Particularly
preferred proteins for this purpose are domains from other species,
such as murine species as illustrated in FIG. 9 herein.
[0045] Another class of modifications are deletional modifications.
Deletions are characterized by the removal of one or more amino
acid residues from the MHC monomer sequence. Typically, the
transmembrane and cytoplasmic domains are deleted. Deletions of
cysteine or other labile residues also may be desirable, for
example in increasing the oxidative stability of the MHC complex.
Deletion or substitutions of potential proteolysis sites, e.g.,
ArgArg, is accomplished by deleting one of the basic residues or
substituting one by glutaminyl or histidyl residues.
[0046] A preferred class of substitutional or deletional
modifications comprises those involving the transmembrane region of
the subunit. Transmembrane regions of MHC monomers are highly
hydrophobic or lipophilic domains that are the proper size to span
the lipid bilayer of the cellular membrane. They are believed to
anchor the MHC molecule in the cell membrane. Inactivation of the
transmembrane domain, typically by deletion or substitution of
transmembrane domain hydroxylation residues, will facilitate
recovery and formulation by reducing its cellular or membrane lipid
affinity and improving its aqueous solubility. Alternatively, the
transmembrane and cytoplasmic domains can be deleted to avoid the
introduction of potentially immunogenic epitopes. Inactivation of
the membrane binding function is accomplished by deletion of
sufficient residues to produce a substantially hydrophilic
hydropathy profile at this site or by substitution with
heterologous residues which accomplish the same result.
[0047] A principal advantage of the transmembrane-inactivated MHC
monomer is that it may be secreted into the culture medium of
recombinant hosts. This variant is soluble in body fluids such as
blood and does not have an appreciable affinity for cell membrane
lipids, thus considerably simplifying its recovery from recombinant
cell culture. Typically, modified MHC monomers of this invention
will not have a functional transmembrane domain and preferably will
not have a functional cytoplasmic sequence. Such modified MHC
monomers will consist essentially of the effective portion of the
extracellular domain of the MHC monomer. In some circumstances, the
monomer comprises sequences from the transmembrane region (up to
about 10 amino acids), so long as solubility is not significantly
affected.
[0048] For example, the transmembrane domain may be substituted by
any amino acid sequence, e.g., a random or predetermined sequence
of about 5 to 50 serine, threonine, lysine, arginine, glutamine,
aspartic acid and like hydrophilic residues, which altogether
exhibit a hydrophilic hydropathy profile. Like the deletional
(truncated) monomer, these monomers are secreted into the culture
medium of recombinant hosts.
[0049] Glycosylation variants are included within the scope of this
invention. They include variants completely lacking in
glycosylation (unglycosylated) and variants having at least one
less glycosylated site than the native form (deglycosylated) as
well as variants in which the glycosylation has been changed.
Included are deglycosylated and unglycosylated amino acid sequence
variants, deglycosylated and unglycosylated subunits having the
native, unmodified amino acid sequence. For example, substitutional
or deletional mutagenesis is employed to eliminate the N- or
O-linked glycosylation sites of the subunit, e.g., the asparagine
residue is deleted or substituted for by another basic residue such
as lysine or histidine. Alternatively, flanking residues making up
the glycosylation site are substituted or deleted, even though the
asparagine residues remain unchanged, in order to prevent
glycosylation by eliminating the glycosylation recognition site.
Additionally, unglycosylated MHC monomers which have the amino acid
sequence of the native monomers are produced in recombinant
prokaryotic cell culture because prokaryotes are incapable of
introducing glycosylation into polypeptides.
[0050] Glycosylation variants are conveniently produced by
selecting appropriate host cells or by in vitro methods. Yeast, for
example, introduce glycosylation which varies significantly from
that of mammalian systems. Similarly, mammalian cells having a
different species (e.g., hamster, murine, insect, porcine, bovine
or ovine) or tissue origin (e.g., lung, liver, lymphoid,
mesenchymal or epidermal) than the MHC source are routinely
screened for the ability to introduce variant glycosylation as
characterized for example by elevated levels of mannose or variant
ratios of mannose, fucose, sialic acid, and other sugars typically
found in mammalian glycoproteins. In vitro processing of the
subunit typically is accomplished by enzymatic hydrolysis, e.g.,
neuraminidase digestion.
[0051] MHC glycoproteins suitable for use in the present invention
have been isolated from a multiplicity of cells using a variety of
techniques including solubilization by treatment with papain, by
treatment with 3M KCl, and by treatment with detergent. For
example, detergent extraction of Class I protein followed by
affinity purification can be used. Detergent can then be removed by
dialysis or selective binding beads. The molecules can be obtained
by isolation from any MHC I bearing cell, for example from an
individual suffering from a targeted cancer or viral disease.
[0052] Isolation of individual heavy chain from the isolated MHC
glycoproteins is easily achieved using standard techniques known to
those skilled in the art. For example, the heavy chain can be
separated using SDS/PAGE and electroelution of the heavy chain from
the gel (see, e.g., Dornmair et al., supra and Hunkapiller, et al.,
Methods in Enzymol. 91:227-236 (1983). Separate subunits from MHC I
molecules are also isolated using SDS/PAGE followed by
electroelution as described in Gorga et al. J. Biol. Chem.
262:16087-16094 (1987) and Dornmair et al. Cold Spring Harbor Symp.
Quant. Biol. 54:409-416 (1989) Those of skill will recognize that a
number of other standard methods of separating molecules can be
used, such as ion exchange chromatography, size exclusion
chromatography or affinity chromatography.
[0053] Alternatively, the amino acid sequences of a number of Class
I proteins are known, and the genes have been cloned, therefore,
the heavy chain monomers can be expressed using recombinant
methods. These techniques allow a number of modifications of the
MHC monomers as described above. For instance, recombinant
techniques provide methods for carboxy terminal truncation which
deletes the hydrophobic transmembrane domain. The carboxy termini
can also be arbitrarily chosen to facilitate the conjugation of
ligands or labels, for example, by introducing cysteine and/or
lysine residues into the molecule. The synthetic gene will
typically include restriction sites to aid insertion into
expression vectors and manipulation of the gene sequence. The genes
encoding the appropriate monomers are then inserted into expression
vectors, expressed in an appropriate host, such as E. coli, yeast,
insect, or other suitable cells, and the recombinant proteins are
obtained.
[0054] As the availability of the gene permits ready manipulation
of the sequence, a second generation of construction includes
chimeric constructs, as illustrated in FIG. 9. The .alpha..sub.1,
.alpha..sub.2, .alpha..sub.3, domains of the class I heavy chain
are linked typically by the .alpha..sub.3 domain of Class I with
beta-2 microglobulin and coexpressed to stabilize the complex. The
transmembrane and intracellular domains of the Class I gene can
optionally also be included.
[0055] Construction of expression vectors and recombinant
production from the appropriate DNA sequences are performed by
methods known in the art. Standard techniques are used for DNA and
RNA isolation, amplification, and cloning. Generally enzymatic
reactions involving DNA ligase, DNA polymerase, restriction
endonucleases, and the like, are performed according to the
manufacturer's specifications. These techniques and various other
techniques are generally performed according to Sambrook et al.,
Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989. The procedures therein
are believed to be well known in the art.
[0056] Expression can be in procaryotic or eucaryotic systems.
Suitable eucaryotic systems include yeast, plant and insect
systems, such as the Drosophila expression vectors under an
inducible promoter. Procaryotes most frequently are represented by
various strains of E. coli. However, other microbial strains may
also be used, such as bacilli, for example Bacillus subtilis,
various species of Pseudomonas, or other bacterial strains. In such
procaryotic systems, plasmid vectors which contain replication
sites and control sequences derived from a species compatible with
the host are used. For example, E. coli is typically transformed
using derivatives of pBR322, a plasmid derived from an E. coli
species by Bolivar et al., Gene (1977) 2:95. Commonly used
procaryotic control sequences, which are defined herein to include
promoters for transcription initiation, optionally with an
operator, along with ribosome binding site sequences, including
such commonly used promoters as the .beta.-lactamase
(penicillinase) and lactose (lac) promoter systems (Change et al.,
Nature (1977) 198:1056) and the tryptophan (trp) promoter system
(Goeddel et al., Nucleic Acids Res. (1980) 8:4057) and the
lambda-derived P.sub.L promoter and N-gene ribosome binding site
(Shimatake et al., Nature (1981) 292:128). Any available promoter
system compatible with procaryotes can be used.
[0057] The expression systems useful in the eucaryotic hosts
comprise promoters derived from appropriate eucaryotic genes. A
class of promoters useful in yeast, for example, include promoters
for synthesis of glycolytic enzymes, including those for
3-phosphoglycerate kinase (Hitzeman, et al., J. Biol. Chem. (1980)
255:2073). Other promoters include, for example, those from the
enolase gene (Holland, M. J., et al. J. Biol. Chem. (1981)
256:1385) or the Leu2 gene obtained from YEp13 (Broach, J., et al.,
Gene (1978) 8:121). A Drosophila expression system under an
inducible promoter (Invitrogen, San Diego, Calif.) can also be
used.
[0058] Suitable mammalian promoters include the early and late
promoters from SV40 (Fiers, et al., Nature (1978) 273:113) or other
viral promoters such as those derived from polyoma, adenovirus II,
bovine papilloma virus or avian sarcoma viruses. Suitable viral and
mammalian enhancers are cited above.
[0059] The expression system is constructed from the foregoing
control elements operably linked to the MHC sequences using
standard methods, employing standard ligation and restriction
techniques which are well understood in the art. Isolated plasmids,
DNA sequences, or synthesized oligonucleotides are cleaved,
tailored, and religated in the form desired.
[0060] Site-specific DNA cleavage is performed by treatment with
the suitable restriction enzyme (or enzymes) under conditions which
are generally understood in the art, and the particulars of which
are specified by the manufacturer of these commercially available
restriction enzymes. In general, about 1 .mu.g of plasmid or DNA
sequence is cleaved by one unit of enzyme in about 20 .mu.l of
buffer solution; an excess of restriction enzyme may be used to
insure complete digestion of the DNA substrate. After each
incubation, protein is removed by extraction with
phenol/chloroform, and may be followed by ether extraction, and the
nucleic acid recovered from aqueous fractions by precipitation with
ethanol followed by running over a Sephadex G-50 spin column. If
desired, size separation of the cleaved fragments may be
performed.
[0061] Restriction cleaved fragments may be blunt ended by treating
with the large fragment of E. coli DNA polymerase I (Klenow) in the
presence of the four deoxynucleotide triphosphates (dNTPs) After
treatment with Klenow, the mixture is extracted with
phenol/chloroform and ethanol precipitated followed by running over
a Sephadex G-50 spin column.
[0062] Synthetic oligonucleotides are prepared using commercially
available automated oligonucleotide synthesizers. In the proteins
of the invention, however, a synthetic gene is conveniently
employed. The gene design can include restriction sites which
permit easy manipulation of the gene to replace coding sequence
portions with these encoding analogs.
[0063] Correct ligations for plasmid construction can be confirmed
by first transforming E. coli strain MM294 obtained. from E. coli
Genetic Stock Center, CGSC #6135, or other suitable host, with the
ligation mixture. Successful transformants can be selected by
ampicillin, tetracycline or other antibiotic resistance or by using
other markers depending on the mode of plasmid construction, as is
understood in the art. Plasmid from the transformants are then
prepared, optionally following chloramphenicol amplification. The
isolated DNA is analyzed by restriction and/or sequenced by the
dideoxy method of Sanger, F., et al., Proc. Natl. Acad. Sci. USA
(1977) 74:5463 as further described by Messing, et al., Nucleic
Acids Res. (1981) 9:309, or by the method of Maxam, et al., Methods
in Enzymology (1980) 65:499.
[0064] The constructed vector is then transformed into a suitable
host for production of the protein. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride, as
described by Cohen, S. N., Proc. Natl. Acad. Sci. USA (1972)
69:2110, or the RbCl method described in Maniatis, et al.,
Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor
Press, p. 254 is used for procaryotes or other cells which contain
substantial cell wall barriers. For mammalian cells without such
cell walls, the calcium phosphate precipitation method of Graham
and van der Eb, Virology (1978) 52:546 or electroporation is
preferred. Transformations into yeast are carried out according to
the method of Van Solingen, P., et al., J. Bacter. (1977) 130:946
and Hsiao, C. L., et al., Proc. Natl. Acad. Sci. USA (1979)
76:3829.
[0065] The transformed cells are then cultured under conditions
favoring expression of the MHC sequence and the recombinantly
produced protein recovered from the culture.
MHC-Binding Peptides
[0066] It is believed that the presentation of antigen by the MHC
glycoprotein on the surface of antigen-presenting cells (APCs)
occurs subsequent to the hydrolysis of antigenic proteins into
smaller peptide units. The location of these smaller segments
within the antigenic protein can be determined empirically. These
MHC-binding peptides are thought to be about 8 to about 10,
possibly about 8 to about 11, or about 8 to about 12 residues in
length, and contain both the agretope (recognized by the MHC
molecule) and the epitope (recognized by T cell receptor on the T
cell). The epitope is a contiguous or noncontiguous sequence of
about 5-6 amino acids that is recognized by the antigen-specific T
cell receptor. The agretope is a continuous or noncontiguous
sequence that is responsible for binding of the peptide with the
MHC glycoproteins. The invention provides systems, kits, and assays
for evaluating putative MHC-binding peptides to determine whether
such fragments can be incorporated into a ternary complex with an
MHC monomer or modified MHC monomer.
[0067] Thus, the invention provides systems, kits and screening
methods to be used in screening of candidate peptides for use in
diagnostic assays, vaccines, and other treatment modalities.
Putative MHC-binding peptides for use in the invention methods can
be made using any method known in the art, the most convenient
being peptide synthesis for fragments of 8 to 12 amino acids in
length.
[0068] Accordingly, in one embodiment the invention provides a
system comprising a solid surface having attached thereto one or
more MHC monomer or modified MHC monomer wherein the monomer
denatures in a denaturing condition and reconstitutes to form a
functional binding pocket containing a suitable MHC-binding peptide
under reconstituting conditions. For example, a plurality of the
monomers can be bound to a single surface. The surface of the
system can be any known or later discovered solid surface
including, without any limitation, any solid, polymer, membrane,
synthetic surface, and the like. For example, the solid surface of
the invention system can be a microtiter plate, such as the wells
of a microtiter plate, or a bead, such as an agarose A bead, an
agarose G bead, and the like. In one aspect, the solid surface of
the invention system is suitable for use in a high throughput
scanning system, e.g., the surface is compatible with the high
throughput system or allows a system to work with the entities
associated with the surface in a high throughput manner, such as
fluorescence determined flow cytometry.
[0069] Recently, a short peptide sequence (streptagII) has been
identified that demonstrates binding affinity
(Kd.about.1.times.10-6M) for the biotin-binding site of a mutated
streptavidin molecule, called StrepTactin. The molecule d-biotin,
which binds with higher affinity to strepTactin
(Kd.about.1.times.10-13 M) effectively competes with the StrepTagII
for the binding site. (Knabel, M., Franz, T. J., Schiemann, M.,
Wulf, A., Villmow, B., Schmidt, B., Bernhard, H., Wagner, H.,
Busch, D. H. (2002) Reversible MHC multimer staining for functional
isolation of T-cell populations and effective adoptive transfer.
Nature Medicine Vol. 8 No. 6, June 2002. pp: 631-637). Attachment
of the MHC monomers to the solid surface can be accomplished by any
method known in the art. For example, the solid surface can be
coated with a first binding ligand, such as avidin, and the monomer
is then provided with a second binding ligand, such as biotin,
wherein the first ligand binds specifically with the second ligand.
The second binding ligand may optionally be attached to the
monomers via a C-terminal end. Attachment of the one or more
monomers to the solid surface is optionally reversible or
cleavable. For example, a cleavable binding complex is commercially
available from Amersham Bioscience Bioscience (Orsay France) such
as Factor Xa, PreScission Protease and thrombin. All of these
proteases can be used with the GST affinity tag from proteins
expressed using pGEX-T vectors
[0070] The invention system comprising a solid support with
attached MHC monomers is preferably stored in a renatured state, by
causing formation of a ternary complex with the MHC monomer
containing a MHC binding peptide of 8 to 12, or about 9 to 11 amino
acids in the binding pocket and a beta-2 microglobulin molecule
bound thereto, as described herein.
[0071] Formation of the ternary complex containing a MHC heavy
chainor modified MHC attached to a solid support is referred to
herein as "renaturation" and is accomplished under renaturing
conditions as is know in the art and described herein. For example,
renaturing conditions typically include the presence of a suitable
MHC binding peptide for the monomer, the presence of beta-2
microglobulin, and a suitable refolding buffer having a pH of from
about 7 to about 8.5. Suitable refolding buffers are illustrated in
the Examples herein and are known in the art.
[0072] In further preparation for storage, the solid support with
bound MHC monomer(s) can be dried while in a renatured state, for
example by exposure to a buffer containing sugars. In preparation
for use of the solid support of the invention system to test
putative MHC-binding peptides, the solid support and attached MHC
monomers in ternary complex are exposed to denaturing conditions to
cause dissociation and unfolding of the monomers. For example,
denaturing conditions can comprise exposure of the solid support
and bound monomers to a pH of about 2 to about 4 for sufficient
time to cause dissociation of the ternary complexes without damage
to the monomers.
[0073] Optionally, the invention system may further comprise a
monoclonal antibody, described in greater detail below, that binds
specifically to a conformational epitope that is present in the
ternary complex and absent in the dissociated components of such a
complex. For example, the conformational epitope may be formed in
the reconstituted MHC monomers or modified MHC monomers used in the
system and absent in the denatured monomers. The invention system
may further contain a supply of beta-2 microglobulin.
[0074] The MHC monomer used in the invention systems and methods
can be any MHC monomer or modified MHC monomer, i.e., class I heavy
chain, capable of binding a peptide in the range of 8 to 11 amino
acids, for example 8 to 10 amino acids under renaturing conditions.
The MHC monomer can be encoded by any partial or full-length
modified or unmodified MHC gene sequence from any species or
subtype, or a combination thereof, including without limitation
human and murine species, and chimera thereof. Preferred MHC
encoding gene sequences are those encoding any HLA allele genotype
and any variation or polymorphism thereof. For example, the MHC
monomer utilized in the invention systems and methods can be any
partial or full-length HLA heavy chain that binds an HLA-binding
peptide under renaturing conditions, i.e., any subtype or allele of
HLA-A, HLA-B, or HLA-C,
[0075] For example, in one embodiment, the MHC monomer is modified
by truncation to include only the .alpha..sub.1, .alpha..sub.2 and
the .alpha..sub.3 domains of an HLA heavy chain. In still another
embodiment, the MHC monomer can be a chimeric, such as a fusion
protein, containing these MHC domains and an anchor domain, wherein
the MHC domain binds to a MHC-binding a peptide, as described
herein, while the anchor domain is suitable for immobilizing the
MHC monomer to a surface. The anchor domain can be a polypeptide
fused with the HLA domain to form a fusion protein or can be any
entity coupled to the HLA domain through any suitable means known
in the art, e.g., biotinylated MHC monomer.
[0076] The MHC monomer can be attached to the solid surface by any
suitable means known in the art. For example, the MHC monomer can
be immobilized to a surface either directly or indirectly, e.g.,
via an anchoring or connecting entity. In one embodiment, the solid
surface of the invention system is coated with a first ligand
entity, which binds to or interacts with a second ligand connected
to or within the MHC monomer, e.g., via covalent or noncovalent
bond. In another embodiment, the surface is coated with avidin or
its derivatives, e.g., streptavidin, and the MHC monomer contains
biotin or its derivatives as its anchor domain. Attachment of the
MHC monomer to the solid surface, in one embodiment of the
invention, is reversible or cleavable.
[0077] The MHC monomer coated or immobilized to a solid surface can
be denatured, e.g., stripped or dissociated in a denaturing
condition, and then renatured, e.g., refolded from a denatured form
under a non-denaturing or renaturing condition so as to bind an
appropriate MHC-binding peptide. In one embodiment, the surface
coated with the MHC monomer provided by the present invention can
be dried and stored for use at a later time. Preferably, the
storage is at 4 degrees C.
[0078] In addition to the surface coated with the MHC monomer the
system of the invention can further include a monoclonal antibody
and a peptide. The peptide can be any peptide that binds to the HLA
heavy chain monomers, e.g., MHC-binding peptides. In one
embodiment, the peptide has high affinity to the MHC monomer, e.g.,
HBc high affinity peptide.
[0079] The monoclonal antibody used in the invention systems and
methods can be any monoclonal antibody that specifically binds to a
conformational epitope present only in a ternary complex of an MHC
monomer and not present in dissociated components of the ternary
complex. For example, the conformational epitope can be present in
beta-2 microglobulin when incorporated into the ternary complex.
Alternatively, the monoclonal antibody can recognize a
conformational epitope present in the MHC monomer or modified MHC
monomer being used in a particular invention system or method. The
monoclonal antibody may be species-matched to the MHC monomers, for
example, when the solid support has attached HLA class I monomers,
the monoclonal antibody is a murine, human or humanized anti-MHC
class I monoclonal antibody. However, when the modified MHC monomer
is a chimera containing domains from more than one species, the
anti-MHC monoclonal antibody can be selected to bind to a
conformational epitope present in only one of the domains. For
example, as illustrated in FIG. 9, a ternary complex containing
modified MHC monomer that is a chimera containing alpha-1 and alpha
2 domains of HLA-A2 heavy chain and a murine alpha-3 domain of H-2
Kb can be detected by a murine monoclonal antibody that binds to a
conformational domain in the murine alpha-3 domain.
[0080] When the MHC monomer is an HLA monomer, the monoclonal
antibody can be any anti-MHC class I monoclonal antibody that
recognizes any subclass of HLA monomer in a ternary complex, i.e.,
HLA-A, HLA-B or HLA-C. A preferred anti-MHC-class I monoclonal
antibody for use in the invention systems and methods is a mouse
IgG2a conformational dependent anti-HLA monoclonal antibody
produced by hybridoma B9.12.1, which as been deposited under the
provisions of the Budapest Treaty on the International Recognition
of the Deposit of Microorganisms for the Purpose of Patent
Procedure and the Regulations thereunder (Budapest Treaty) at
Collection Nationale de Cultures de Microorganismes (CNCM),
Institut Pasteur 25, Rue du Docteur Roux, F75724 Paris Cedex 15
France, under registration number CNCM 1-2941. This assures
maintenance of viable cultures for 30 years from the date of
deposit. The organisms will be made publicly available by CNCM
under the terms of the Budapest Treaty and assures permanent and
unrestricted availability of the progeny of the culture to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn.122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn.1.14
with particular reference to 886 OG 638).
[0081] In one embodiment, the monoclonal antibody used in the
invention systems and methods is provided with a detectable label,
i.e., a label that produces a detectable signal as is known in the
art. Labels may be conjugated to the antibody using any of a
variety of procedures known in the art. Alternatively, the antibody
can be produced to include a label, such as a radioactive amino
acid. Labels suitable for use in the invention systems, kits and
methods include, but are not limited to, radioisotopes,
fluorochromes, enzymes, biotin and electron dense molecules.
Binding of the monoclonal antibody indicates formation of a ternary
complex by binding of a MHC-binding peptide to the monomer and can
be easily detected and/or quantified by detecting the signals
produced by the signal entity after washing away unbound antibody
and other components of the system. A detectable label presently
preferred is a fluorescent label, e.g., FITC. The binding of
fluorescently labeled antibodies on the solid support can be
readily detected using a fluorimeter or by fluorescence determined
flow cytometry.
[0082] The invention system can be provided either as part of
another system or as a kit. For example, microtiter plates coated
with the MHC monomers or modified monomers, e.g., in dried form,
can be provided in a kit, which can optionally additionally
include, in separate vials or containers, an anti-MHC monoclonal
antibody or an anti-beta-2 microglobulin antibody, as described
herein, and a control peptide that binds specifically to the
monomers attached to the solid support. In one embodiment, the kit
includes an instruction explaining the procedures for using the
system to conduct immunoassays, e.g., the methods provided by the
present invention. The kit can optionally also include any or all
of the following: denaturing or refolding buffers, controls for the
MHC monomers, the peptide, or the monoclonal antibody.
[0083] In yet another embodiment, the invention provides methods
for determining binding between a MHC monomer or modified MHC
monomer and a putative MHC-binding peptide to be tested for binding
to the monomer(s). In this method for assaying binding of a
putative MHC-binding peptide, a solid surface having attached
thereto a plurality of MHC monomers or modified MHC monomers is
incubated in the presence and absence of the putative MHC-binding
peptide. Preferably the solid surface is one belonging to an
invention system or kit and is prepared as described herein. If the
MHC monomers attached to the solid support at the start of the
assay procedure are in a reconstituted form, the MHC monomers are
prepared for the assay by exposure to denaturing conditions as
described herein, for example by exposure to a pH in the range from
about 2 to about 4, or exposure overnight to a temperature higher
than about 37.degree. C. After denaturation, unbound MHC-binding
peptides are washed away.
[0084] For the assay, the solid support with attached denatured MHC
monomers or modified MHC monomers is incubated with a putative
MHC-binding peptide under reconstituting conditions for a suitable
period of time to allow for formation of ternary complexes. The
reconstituting conditions will include the presence of a sufficient
amount of beta-2 microglobulin (or beta 2 microglobulin modified to
increase binding or stabilize ternary complex formation) to
saturate the MHC monomers. For example, it is contemplated that the
beta 2-microglobulin may be modified by attachment thereto of a
stabilizing molecule, such as a leucine zipper, or the like, to
stabilize ternary complex formation. Incubation with the putative
MHC-binding peptide and beta-2 microglobulin will typically be
required from about 12 hours or overnight to about 48 hours to
allow for complex formation. The reconstituting conditions may also
include a temperature in the range from about minus 18.degree. C.
to about 37.degree. C., for example about 4.degree. C. to about
8.degree. C.
[0085] After the reconstituting incubation, binding to the MHC
monomers of the putative MHC-binding peptide is determined by
contacting the MHC monomers on the solid support with a monoclonal
antibody that binds to a conformational epitope present only in
ternary complex, for example a conformational epitope present in
the refolded MHC monomer of the ternary complex and not present in
a denatured MHC monomer. Binding of the antibody with the ternary
complex attached to the solid support indicates that the putative
MHC-binding peptide is an MHC-binding peptide specific for the MHC
monomers or modified MHC monomers used in the assay. For purposes
of comparison of the binding of the putative MHC-binding peptide to
that of a standard MHC-binding peptide, a parallel assay (e.g.,
under the same reconstituting conditions, same monomer, and in the
presence of the same monoclonal antibody) may be conducted using
the monomers. Binding of the monoclonal antibody in the parallel
assay to the ternary complex containing the standard MHC-binding
peptide can be compared to binding of the monoclonal antibody to
the ternary complex in the test assay to aid in determining the
binding efficiency of the putative MHC-binding peptide, using
computational methods known in the art.
[0086] In still another embodiment, the invention provides methods
for determining the degree of binding affinity of an MHC monomer or
modified MHC monomer for a putative MHC-binding peptide. In this
embodiment, at least one denatured MHC monomer or modified MHC
monomer attached to a solid surface is incubated under
reconstituting conditions with the putative MHC-binding peptide and
a monoclonal antibody that specifically binds to a conformational
epitope created by formation of a ternary complex containing a
corresponding reconstituted MHC monomer that is not present in any
of the dissociated components of the complex. For reconstitution, a
suitable amount of beta-2 microglobulin for complex formation of
the total amount of monomer in the assay must also be present
Binding of the monoclonal antibody to the ternary complex so
formed, is compared with binding of the monoclonal antibody to a
corresponding ternary complex containing the same MHC monomer or
modified MHC monomer and a known MHC-binding peptide. The
difference in the binding indicates the relative degree of binding
affinity of the reconstituted MHC monomer or modified MHC monomer
for the putative MHC-binding peptide. For the determination of the
binding affinity of a peptide the test is done in multiples using
different peptide concentrations in each parallel test. In practice
of the invention methods, the MHC monomers may belong to any
species for which determination of appropriate class I binding
peptides is desired, including, without limitation, murine and
human or a chimera containing monomer subunits from a combination
of species or subtypes.
[0087] Various readily available means can be used to determine the
specific binding of the monoclonal antibody to the ternary complex
containing the reconstituted MHC monomer. For example, the binding
can be detected by directly labeling the monoclonal antibody with a
detectable label, i.e., one that produces a detectable signal, and
detecting the signal or via a secondary antibody which is
detectably labeled and recognizes the monoclonal antibody that
binds to the ternary complex containing the MHC monomer used in the
assay. Suitable detectable labels that can be used for this purpose
are well known in the art and include labels selected from the
group consisting of radioisotopes, fluorochromes, enzymes, biotin,
electron dense molecules, and the like. Fluorochromes or
fluorescent labels are currently preferred since binding can
readily be detected by subjecting the solid support to a
fluorimeter. For example, when the solid support is a plate, such
as a 96 well microtiter plate, or beads, such as agarose A or
agarose G beads, the assay can take advantage of high through-put
florescence scanning using any of the methods known in the art.
[0088] The following examples are intended to illustrate but not to
limit the invention in any manner, shape, or form, either
explicitly or implicitly. While they are typical of those that
might be used, other procedures, methodologies, or techniques known
to those skilled in the art may alternatively be used.
Example 1
Detection of Correctly Folded HLA Heavy Chain Monomers
[0089] This experiment demonstrates that MHC monomers when attached
to a solid support can be reconstituted so as to form a ternary
complex and be recognized and specifically bound by a
conformation-dependent anti-MHC monoclonal antibody. In other
words, MHC monomers bound to a solid support will correctly fold to
bind MHC-binding peptides. Table 1 below summarizes the major steps
for detecting the correctly folded HLA monomers upon peptide
binding. (See also FIG. 1.)
TABLE-US-00001 TABLE 1 Step 1 Step 2 Step 3 Incubation of HLA After
washing, Washing and read out heavy chain coated incubation with in
the fluorimeter plates with low pH different concentrations
solution. of peptide and a Washing constant concentration of beta-2
microglobulin and anti-HLA-class I- FITC mAb. Incubation time:
overnight or 24 h
Example 2
Calibration of Anti-HLA-FTIC Antibody
[0090] In this example BSA-Biotin-Avidin coated 96-well microtiter
plates were prepared used for a fluorimetric assay. HLA-A2m monomer
in ternary complex with binding peptide Mart-1 26-35L was incubated
at various concentrations with an anti-HLA-ABC-FITC or
anti-HLA-FITC monoclonal antibody at concentrations of 0, 0.25,
0.5, 1, 2, and 4 .mu.g/ml. Specifically, for each antibody
concentration, the HLA monomer was added at concentrations of 0,
0.0078, 0.0156, 0.03125, 0.0625, 0.125, 0.25, and 0.5 .mu.g/ml.
[0091] In this experiment the HLA heavy chain and the anti-HLA-FITC
antibody were incubated together for 40 min at room temperature
under shaking. The total fluorescence was read before washing the
plates to remove unbound antibody. Then, plates were washed three
times to remove any unbound antibody, and the fluorescence of the
bound monomers was read.
[0092] As shown in FIG. 2, saturation occurred when the antibody
concentration reached 0.25 and 0.5 .mu.g/ml. However, the
fluorescence signal increased when the antibody was added at 1, 2
and 4 .mu.g/ml. This observation indicates that the antibody binds
two MHC monomers when added at 0.5 and 0.25 .mu.g/ml. In contrast,
upon incubation at 1, 2 or 4 .mu.g/ml, the antibody binds only one
HLA monomer. This explains the signal increase, e.g., 300
Fluorescence units (FU) with 0.5 .mu.g/ml of antibody and 0.5
.mu.g/ml of HLA heavy chain as compared with 600 FU with 1 .mu.g/ml
of antibody and 0.5 .mu.g/ml of HLA heavy chain. Another
observation was that between the concentrations of 2 and 4 .mu.g/ml
of antibody the signal remains constant. It was determined,
therefore, that 2 .mu.g/ml of anti-HLA-FITC mAb was an appropriate
saturation concentration to use for the assay.
Example 3
Specificity of Anti-HLA-FITC Monoclonal Antibody
A. Conformational Specificity
[0093] Experiments were designed to determine if the signal
produced from anti-HLA-class I-FITC antibody differs as a function
of the degree of dissociation of a stressed HLA monomer. The
particular HLA monomer used for the experiments was HLA heavy chain
monomer HLA-A*0201 containing binding peptide Mart1 27-35 in
ternary complex.
[0094] Different solutions containing the ternary complex at a
concentration of 10 .mu.g/ml were prepared and incubated overnight
at the temperatures of 37.degree. C., 30.degree. C., 25.degree. C.,
or 4-8.degree. C. Antibody binding experiments as described above
in Example 2 were carried out using 2 .mu.g/ml of anti-HLA-FITC
conjugate to detectably label the HLA monomers remaining in ternary
complex attached to the solid support. A solution containing a
ternary complex of HLA monomer and Mart1 27-35 at a concentration
of 640 .mu.g/ml were incubated at -18.degree. C. as a control. A
solution, from a sample stored at -18.degree. C. at the
concentration of 640 .mu.g/ml, containing a ternary complex of
HLA-monomer and Mart-1 27-35 was diluted at the same concentration
than the other samples and included as control.
[0095] As shown in FIG. 3, it was found that incubation of the
ternary complex bound to the solid support at highest temperature
gave the weakest fluorescent signal, indicating that the ternary
complex of HLA heavy chain monomer gradually dissociated as the
temperature was increased. At one point, the anti-HLA-FITC
conjugate could no longer recognize the HLA monomer because of the
degree of dissociation of the ternary complex dissociation and the
fluorescence signal diminished accordingly, indicating that the
anti-HLA-FTIC conjugate specifically recognizes correctly folded
reconstituted HLA monomers, but not denatured monomers.
B. Heavy Chain Monomer Specificity
[0096] As shown in Table 2, different HLA heavy chain monomers for
human alleles, A2, A3, A11, B7, B8, as well as one mouse allele Kd
were incubated with the anti-HLA-Class I-FITC antibody.
TABLE-US-00002 TABLE 2 Human alleles Mouse allele HLA-A*0201/Mart1
2635L H-2Kd/Flu HLA-A*0301/EBV HLA-A*1101/EBV HLA-B*0702/gp41
HLA-B*0801/Nef
[0097] The antibody binding experiments were carried out as
described above. The concentration of the anti-HLA-class I-FITC
antibody used was 2 .mu.g/ml.
[0098] As shown in FIG. 4, all the reconstituted HLA-A and -B
monomers were detectable with the anti-HLA-class I monoclonal
antibody. As expected, no signal was detected when a ternary
complex containing the mouse allele (H-2Kd/Flu) was attached to the
plate, confirming the specificity of the antibody to human HLA.
Variations of signal between different alleles were likely due to
concentration precision and storage conditions of the HLA monomers,
e.g., freeze, thaw, etc.
Coating of MHC Heavy Chain Monomers to Plates, Plate Storage and
Reconstitution
[0099] Biotinylated MHC monomers in a ternary complex with Mart1
27-35 peptide at the concentration of 5 .mu.g/ml were attached to
avidin coated plates. After saturating the plates with a
sugar-containing buffer overnight at 4.degree. C. to 8.degree. C.,
the plates were dried overnight at 30.degree. C. and 19% humidity.
After the plates were dried under these conditions, it was found
that the HLA monomers were dissociated from the ternary complex.
Therefore, it was not necessary to strip the MHC-binding peptide
from the monomers with low pH in preparation for use of the plates
in the binding assay.
[0100] For the antibody binding assay, 10 nM to 100 .mu.M of HBc
high affinity peptide (the affinity can be calculated as
1.8.times.10.sup.-7M) were incubated with 10 .mu.g/ml of .beta.2
microglobulin and the monomer-coated plates were incubated with one
of the three different buffers containing ingredients as described
below: [0101] Buffer 1: Tris, Arginine, EDTA, GSH, GSSG and BSA
[0102] Buffer 2: Tris, NaCl, EDTA, NaN.sub.3, BSA and 0.05% TWEEN
20.RTM. detergent [0103] Buffer 3: Tris, NaCl, EDTA, NaN.sub.3, BSA
and 0.05% NONIDET.RTM. P40 detergent.
[0104] It was found that peptide binding and reconstitution of the
monomers occurred at 2 temperatures: 4.degree. C.-8.degree. C. and
room temperature.
[0105] Renaturation of the HLA monomers was tested after 24 hours
and 48 hours of incubation with 2 .mu.g/ml of anti-HLA-class I-FITC
conjugate. As shown in FIG. 5, the FITC signal increased as a
function of the peptide concentration. This result shows that the
HLA monomer renaturated by incorporation into a ternary complex and
that renaturation of MHC monomers can be effectively detected with
an anti-HLA-class I-FITC antibody. It was found that the best
renaturation buffer was the Buffer 2 containing TWEEN 20.RTM..
Interestingly no refolding was measured with Buffer 1.
[0106] Under the conditions tested here, the best temperature for
the antibody binding assay was 4.degree. C.-8.degree. C. and the
best incubation period to allow renaturation was 24 hours.
Material and Methods
A. Reagents.
[0107] Fine chemicals, unless otherwise stated, were from Merck
(Darmstadt, Germany) and CarloErba (Rodeno, Italy). Biotinylated
BSA as well as avidin was obtained from Immunotech (Marseille,
France). LUMITRAC-600 White 96-well microtiter plates were from
Greiner [PN: 655074 LUMITRAC 600; (Frickenhausen, Germany). SA-PE
as well as HLA-A*0301/EBV HLA heavy chain were from Immunomics
((San Diego, Calif.). Anti-HLA-class I monoclonal antibody
conjugated to FITC (clone: B9.12.1) was from Antibody Manufacturing
Service of Immunotech. Part Number: IM1838. This antibody is a
mouse IgG2a monoclonal antibody.
B. Preparation of Avidin Coated 96-Well Microtiter Plates.
[0108] Each well of white 96-well microtiter plates were coated
with 200 .mu.l of a 5 .mu.g/ml biotinylated BSA solution in PBS and
the plates were incubated for 16 hours at 4.degree. C. The plates
were washed and then 200 .mu.l/well of avidin solution at 5
.mu.g/ml was added. The plates were then incubated for another 16
hours at 4.degree. C. Subsequently the plates were washed and a
blocking, drying solution was added. The plates were incubated
again for another 16 hours. Afterwards, the solution was poured off
and the plates were slapped face down on paper towels. Then the
plates were placed in a special drying room for 24 hours.
Afterwards the plates were placed individually in a self-locking
bag until use.
C. Monomer Immunoassay Procedure.
[0109] The assay procedure was as follows. Each sample 200
.mu.l/well containing the HLA monomer in ternary complex at 0.25
.mu.g/ml and diluted in Tris 10 mM, NaCl 150 mM, EDTA 0.5 mM, NaN3
0.1%, BSA 0.2%, was loaded into wells of the avidin-coated plate
and incubated for 1 hour at room temperature on an orbital shaker
in the dark. The wells were then rinsed three times with an
automatic washer (SLT, Salzburg, Austria) using 300 .mu.l of a 9
g/l NaCl solution containing 0.05% TWEEN 80.RTM.. Subsequently 200
.mu.l/well of FITC-conjugated anti-HLA-class I antibody at 2
.mu.g/ml were added. The plates were incubated for 45 min at room
temperature on an orbital shaker in the dark, washed three times,
and 200 .mu.l/well of Tris 10 mM, NaCl 150 mM, EDTA 0.5 mM, NaN3
0.1%, BSA 0.2% were added. The FITC fluorescence was measured with
a Perkin Elmer LS-50B fluorimeter following these parameters:
[0110] Excitation=405 nm [0111] Emission=525 nm [0112] Emission
filter=515 nm [0113] Band pass (Exc,Emi)=5.15 nm [0114] 0.5
sec/well
[0115] The assay procedure is further summarized in Table 3
below.
TABLE-US-00003 TABLE 3 Step 1 Step 2 Step 3 Step 4 Mix IILA
Incubate 200 .mu.l/ Three washes Three washes heavy well of each
sample Add 200 .mu.l/well Add 200 .mu.l of chain and in the 96-well
of anti-HLA-class I buffer streptavidin streptavidin coated mAb at
2 .mu.g/ml Fluorescence PE white plates. Incubate 45 min at
determination Incubate 1 hour at room temperature in room
temperature in the dark under the dark under agitation
agitation
Calibration of the Anti-HLA-Class I-FITC Antibody
[0116] HLA-A*0201/Mar1 reconstituted monomers in various
concentrations was incubated with various concentrations of the
anti-HLA-class I-FITC mAb. As shown in FIG. 6, a plateau was
reached with concentrations of anti-HLA-class I mAb at 1 to 2
.mu.g/ml for all HLA heavy chain monomer concentrations.
[0117] A dose response curve at various concentration of
reconstituted monomers was plotted using using 2 .mu.g/ml of
anti-HLA-ABC mAb. As shown in FIGS. 7A and 7B, the signal remained
linear with increasing concentrations until 0.5 .mu.g/ml of
reconstituted HLA monomer was used. Concentrations of the
reconstituted HLA monomer higher than 0.5 .mu.g/ml provided signals
that were very close to a plateau. The data summarized in FIGS. 7A
and 7B demonstrate that for the best result, the assay conditions
should include 0.25 .mu.g/ml of reconstituted HLA monomer and 2
.mu.g/ml of anti-HLA-class I FITC mAb. These data also indicated
that the sensitivity of the assay is about 4 to 6 ng/ml of the
reconstituted HLA monomer.
Specificity
[0118] The specificity of the anti-HLA-class I-FITC antibody for
HLA monomer in ternary complex was tested against different human
alleles. A dose response curve was prepared as described above for
each of the the following HLA monomer/peptide ternary complexes.
[0119] HLA-A*0201/Mart1 2635L [0120] HLA-A*0301/EBV [0121]
HLA-B*0702/H1V [0122] HLA-B*0801/H1V [0123] HLA-A-*1101/EBV [0124]
H-2Db/HA1
[0125] As shown in FIG. 8, all the human alleles were recognized
very well by the same anti-HLA-class I-FITC antibody. No signal was
obtained when a human allele was replaced by a mouse allele,
indicating that the antibody used is specific for human class I
alleles and should be used only in assays involving human alleles.
A conformational anti-mouse H-2 antibody was found suitable for use
in the assays involving mouse HLA monomers.
Example 4
Measurement of the Peptide-MHC Off Rate
[0126] For effective CD8+T cell responses, class I MHC molecules
must bind many peptides of diverse sequence in sufficient abundance
for a long period of time. Many tumor cells appear to escape the
immune response because antigenic peptides do not bind well to
class I MHC molecules that present them. If a peptide does not bind
efficiently to the MHC molecule, circulating T cells will not
recognize the MHC ternary complex, and cells presenting them will
not be eliminated.
[0127] Typical half-lives of immunodominant peptides are greater
than 20 hours at 37.degree. C. (Stuber, et al., (1994) Eur. J.
immunol. 24, 765-768, and Pogue, et al., (1995) Proc. Natl. Acad.
Sci. US 92, 8166-8170). From this evidence, a test was developed to
use the invention solid phase assay to determine the stability of
various complexes at different temperatures, and thus calculate the
off rate of the peptides. This parameter is very valuable to know
when peptides are used in vaccination for the purpose of eliciting
an immune response.
[0128] Measurement of the peptide off rate: Monomer
HLA-A*0201/Mart-1 2635L was loaded in four different 96-well avidin
coated plates. The plate was incubated for two hours under shaking
at room temperature. After washing and stripping with citrate
phosphate buffer at pH 3.2 the monomer was reconstituted with high
affinity peptides HbV core, Mart-1 2635L, with intermediate
affinity peptide Mart-1 26-35 as well as the low affinity peptide
Mart-1 27-35. Free beta2 microglobulin as well as the
anti-HLA-ABC-FITC monoclonal antibody was added at the same time
with the peptide. The plates were incubated at 21.degree. C. under
shaking overnight. After that, the plates were washed and the level
of the fluorescence determined. After this Tris buffer containing
the BSA was added to each well and the plates were re-incubated at
different temperatures, one plate was incubated at 4.degree. C.,
one at 21.degree. C., one at 32.5.degree. C. and the last one at
37.degree. C., respectively. Some strips of each plate were washed
at different times--4 hours, 24 hours and 48 hours--and the
fluorescence at different times was determined.
[0129] B0 is the fluorescence determined at time zero. The time
zero corresponds to the moment when the plates were washed once the
monomer was reconstituted and the plates were placed at different
temperatures. B is the fluorescence obtained at each time. After
the Ln (Fluorescence Emission) as a function of the time was
plotted. Linear regression was calculated and the Half life was
calculated as T1/2=0.69/slope of the curves.
[0130] Results of these assays are shown in Table 4 below:
TABLE-US-00004 TABLE 4 T.sub.1/2 hours Peptide 4.degree. C.
21.degree. C. 32.5.degree. C. 37.degree. C. HBVcore 13800 493 101
21.5 2635L >1725 345 98.6 22.4 2635 186 20.3 2.5 1 2735 56.1 8.8
1.3 0.96
[0131] It was observed that high affinity peptides, such as HBV
core and Mart-1 27-35 had a very good stability at 37.degree. C.
and 32.5.degree. C. In contrast, peptide Mart-1 26-35 as well
peptide Mart-1 27-35 showed a very high off rate at 37.degree. C.
Differences were found also when complexes were incubated at
21.degree. C. These results indicate that the assay can be used to
determine the off rate of peptides from the MHC ternary complex
(see FIG. 10; FIGS. 10A-D show graphs of the dissociation curves
for renatured peptides. FIGS. 10E-H shows graphs of the off rates
for peptides. FIG. 10I shows the effect of temperature on monomer
dissociation).
[0132] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
* * * * *