U.S. patent application number 13/299754 was filed with the patent office on 2012-06-07 for enhancing t cell activation using altered mhc-peptide ligands.
Invention is credited to Larry R. Pease.
Application Number | 20120141537 13/299754 |
Document ID | / |
Family ID | 46162460 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120141537 |
Kind Code |
A1 |
Pease; Larry R. |
June 7, 2012 |
ENHANCING T CELL ACTIVATION USING ALTERED MHC-PEPTIDE LIGANDS
Abstract
Materials and Methods for identifying and using MHC molecule
variants for activating self-reactive T cells in a peptide-specific
manner, and their use to focus autoimmune cellular responses
against diseases such as cancers and persisting viral infections,
are described.
Inventors: |
Pease; Larry R.; (Rochester,
MN) |
Family ID: |
46162460 |
Appl. No.: |
13/299754 |
Filed: |
November 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61415227 |
Nov 18, 2010 |
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Current U.S.
Class: |
424/277.1 ;
424/184.1; 424/278.1; 424/281.1; 506/10; 530/350 |
Current CPC
Class: |
A61K 39/001197 20180801;
A61K 39/001151 20180801; A61K 35/17 20130101; A61P 37/04 20180101;
A61K 39/001192 20180801; A61K 39/0011 20130101; G01N 2333/70539
20130101; C12N 7/00 20130101; A61K 2039/53 20130101; A61K 39/001191
20180801; A61K 39/001194 20180801; C12N 2710/10043 20130101; A61K
39/001186 20180801; A61K 39/001106 20180801; G01N 33/56977
20130101; A61K 39/001162 20180801; A61K 39/001193 20180801; A61K
38/00 20130101; C07K 14/70539 20130101; A61K 39/00115 20180801;
A61P 31/00 20180101; A61K 39/0008 20130101; A61K 39/001153
20180801; A61K 39/001157 20180801; A61K 2039/572 20130101; A61K
39/001182 20180801; A61K 39/001188 20180801; A61K 2039/5158
20130101; G01N 33/505 20130101; A61K 39/00117 20180801; A61P 35/00
20180101; A61K 39/001156 20180801 |
Class at
Publication: |
424/277.1 ;
424/278.1; 424/184.1; 424/281.1; 530/350; 506/10 |
International
Class: |
A61K 35/00 20060101
A61K035/00; C07K 14/74 20060101 C07K014/74; A61P 31/00 20060101
A61P031/00; A61P 37/04 20060101 A61P037/04; A61P 35/00 20060101
A61P035/00; A61K 35/12 20060101 A61K035/12; C40B 30/06 20060101
C40B030/06 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] Funding for the work described herein was provided in part
by the National Institutes of Health, grant number A1028320. The
federal government has certain rights in the invention.
Claims
1. A method for treating a subject in need thereof, comprising
administering to the subject: (a) a cell expressing on its surface
a variant of a major histocompatibility complex (MHC) molecule, the
variant comprising one or more amino acid changes from wild-type in
the part of the MHC molecule that interacts with a T cell receptor,
wherein the cell has been identified as having the ability to
activate a T cell in the presence of an epitope from the subject;
and (b) the epitope or a polypeptide comprising the epitope,
wherein the subject has a pathological condition that is amenable
to therapy by a T cell immune response.
2. The method of claim 1, wherein the subject is a human.
3. The method of claim 1, wherein at least one of the one or more
amino acid changes is at a residue of the MHC molecule that, on the
surface of a cell expressing the MHC molecule, is accessible for
interaction with a TCR.
4. The method of claim 1, wherein the MHC molecule is an HLA-A0201
MHC class I molecule, and wherein at least one of the one or more
amino acid changes is at position 72, 76, 79, 154, 158, 162, or 166
of the molecule.
5. The method of claim 1, wherein the epitope is from a polypeptide
associated with the pathological condition.
6. The method of claim 1, wherein the pathological condition is
cancer and the epitope is from a polypeptide expressed by a cancer
cell, or wherein the pathological condition is caused by an
infectious microorganism and the epitope is from a polypeptide
expressed by a cell infected with the infectious microorganism.
7. The method of claim 1, wherein the epitope is from a survivin,
GP100, MelA, survivin-2B, livin/ML-IAP, Bcl-2, Mcl-1, BcI-X(L),
mucin-1, NY-ESO-1, telomerase, CEA, MART-1, HER-2/neu, bcr-abl,
PSA, PSCA, tyrosinase, p53, hTRT, leukocyte proteinase-3, hTRT, gpl
OO, MAGE antigens, GASC, JMJD2C, JARD2 (JMJ), JHDM3a, WT-1,CA 9, or
protein kinase polypeptide.
8. A composition comprising: (a) a cell expressing on its surface a
variant of an MHC molecule, the variant comprising one or more
amino acid changes from wild-type in the part of the MHC molecule
that interacts with a T cell receptor, wherein the cell has been
identified as having the ability to activate a T cell in the
presence of a particular peptide epitope; and (b) a
pharmaceutically acceptable carrier; and optionally, the peptide
epitope.
9. The composition of claim 8, wherein at least one of the one or
more amino acid changes is at a residue of the MHC molecule that is
accessible for interaction with a TCR.
10. The composition of claim 8, wherein the MHC molecule is an
HLA-A0201 MHC class I molecule, and wherein at least one of the one
or more amino acid changes is at position 72, 76, 79, 154, 158,
162, or 166 of the molecule.
11. The composition of claim 8, wherein the peptide epitope is from
a polypeptide expressed by a cancer cell or by a cell infected with
an infectious microorganism.
12. The composition of claim 8, wherein the peptide epitope is from
a survivin, GP100, MelA, survivin-2B, livin/ML-IAP, Bcl-2, Mcl-1,
BcI-X(L), mucin-1, NY-ESO-1, telomerase, CEA, MART-1, HER-2/neu,
bcr-abl, PSA, PSCA, tyrosinase, p53, hTRT, leukocyte proteinase-3,
hTRT, gpl OO, MAGE antigens, GASC, JMJD2C, JARD2 (JMJ), JHDM3a,
WT-1,CA 9, or protein kinase polypeptide.
13. The composition of claim 8, wherein the pharmaceutically
acceptable carrier is selected from the group consisting of water,
saline solution, binding agents, fillers, lubricants,
disintegrates, and wetting agents.
14. The composition of claim 8, further comprising an adjuvant
selected from the group consisting of Freund's adjuvant, aluminum
hydroxide, lysolecithin, pluronic polyols, polyanions, peptides,
oil emulsions, keyhole limpet hemocyanin, dinitrophenol, cytokines,
and bacterial products.
15. A method comprising contacting a cell in a subject with a virus
particle comprising a nucleic acid that encodes a variant of an MHC
molecule, the variant comprising one or more amino acid changes
from wild-type in a portion of the MHC molecule that interacts with
a T cell receptor.
16. The method of claim 15, wherein the subject is a human.
17. The method of claim 15, wherein the virus is an adenovirus.
18. The method of claim 15, wherein the MHC molecule is a class I
MHC molecule.
19. A variant of an MHC molecule, comprising one or more amino acid
changes from wild-type in the part of the MHC molecule that
interacts with a T cell receptor, wherein the MHC molecule, in the
presence of a particular epitope, has been identified as having the
ability to activate a T cell.
20. A method of selecting a MHC molecule variant for activation of
an immune response, the method comprising: providing a panel of
cells, each cell of the panel expressing on its surface a variant
of an MHC molecule, the variant comprising one or more amino acid
changes from wild-type in the part of the MHC molecule that
interacts with a T cell receptor; testing the ability of each cell
of the panel to activate a T cell in the presence of a selected
peptide epitope; and selecting the MHC molecule expressed by a cell
of the panel that activates a T cell in the presence of the peptide
epitope.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority from U.S.
Provisional Application Ser. No. 61/415,227, filed on Nov. 18,
2010.
TECHNICAL FIELD
[0003] This document relates to materials and methods for
activating self-reactive T cells in a peptide-specific manner, to
focus autoimmune T cellular responses against, for example, cancers
and persisting virus infections.
BACKGROUND
[0004] During development of the repertoire of the normal T cell
population, affinity/avidity thresholds for cellular activation are
set, defined by the ability of the expressed T cell receptors to
bind to major histocompatibility complex (MHC) molecules that
present peptide epitopes derived from the body's own proteins
(Jenkins et al. (2010) Annu. Rev. Immunol. 28:275-294). This means
that T cells bearing antigen-specific T cell receptors (TCR)
capable of binding self peptides presented by self MHC molecules
are not present functionally in the immune repertoire. T cells with
receptors just below this affinity/avidity threshold are presumably
present, but they are believed to be functionally blind to self
antigen.
[0005] A T cell response can be visualized as having two phases
with respect to cellular activation. The first is the transition of
naive T cells to activated T cells. This happens when T cells first
encounter non-self antigens presented by MHC molecules capable of
binding their receptors with affinity/avidity above the set
activation threshold. Once these cells are activated, they undergo
a series of cell divisions, acquire a primed state characterized by
the development of effector function capabilities, and enter the
blood in search of cells expressing the inciting antigen presented
in the context of self MHC. Upon engaging the inciting antigen
presented in the context of self MHC in the peripheral tissues, the
primed T cells release cytokines and granule proteins, inducing
cell death and controlling the replication and infectivity of
pathogens. The threshold for reactivation of primed cells is
thought to be lower than the threshold for generating primed cells
from naive cells.
SUMMARY
[0006] The normal immune system contains T cells (e.g., CD4.sup.+
and CD8.sup.+ T cells) bearing antigen-specific TCR that are
composed of two chains (mostly a and (3 chains), and that are not
normally reactive to self This document is related in part to the
development of methods to prime self-reactive T cells in a peptide
specific manner, and the discovery that the primed self-reactive T
cells can execute their effector functions in peripheral tissues
with specificity. Thus, activated cells of this kind can be
incorporated into therapeutic schemes to focus autoimmune cellular
responses against, for example, cancers and persisting pathogenic
(e.g., viral) infections.
[0007] In one aspect, this document features a method for treating
a subject in need thereof. The method can include administering to
the subject (a) a cell expressing on its surface a variant of a MHC
molecule, the variant having one or more amino acid changes from
wild-type in the part of the MHC molecule that interacts with a T
cell receptor, where the cell has been identified as having the
ability to activate a T cell in the presence of an epitope from the
subject, and (b) the epitope or a polypeptide comprising the
epitope, wherein the subject has a pathological condition that is
amenable to therapy by a T cell immune response. The subject can be
a human. At least one of the one or more amino acid changes can be
at a residue of the MHC molecule that, on the surface of a cell
expressing the MHC molecule, is accessible for interaction with a
TCR. The MHC molecule can be an HLA-A0201 MHC class I molecule,
where at least one of the one or more amino acid changes is at
position 72, 76, 79, 154, 158, 162, or 166 of the molecule. The
epitope can be from a polypeptide associated with the pathological
condition. For example, the pathological condition can be cancer
and the epitope can be from a polypeptide expressed by a cancer
cell, or the pathological condition can be caused by an infectious
microorganism and the epitope can be from a polypeptide expressed
by a cell infected with the infectious microorganism. In some
embodiments, the epitope can be from a survivin, GP100, MelA,
survivin-2B, livin/ML-IAP, Bcl-2, Mcl-1, BcI-X(L), mucin-1,
NY-ESO-1, telomerase, CEA, MART-1, HER-2/neu, bcr-abl, PSA, PSCA,
tyrosinase, p53, hTRT, leukocyte proteinase-3, hTRT, gpl OO, MAGE
antigens, GASC, JMJD2C, JARD2 (JMJ), JHDM3a, WT-1,CA 9, or protein
kinase polypeptide.
[0008] In another aspect, this document features a composition that
includes (a) a cell expressing on its surface a variant of an MHC
molecule, the variant having one or more amino acid changes from
wild-type in the part of the MHC molecule that interacts with a T
cell receptor, wherein the cell has been identified as having the
ability to activate a T cell in the presence of a particular
peptide epitope; and (b) a pharmaceutically acceptable carrier; and
optionally, the peptide epitope. At least one of the one or more
amino acid changes can be at a residue of the MHC molecule that is
accessible for interaction with a TCR. The MHC molecule can be an
HLA-A0201 MHC class I molecule, wherein at least one of the one or
more amino acid changes is at position 72, 76, 79, 154, 158, 162,
or 166 of the molecule. The peptide epitope can be from a
polypeptide expressed by a cancer cell or by a cell infected with
an infectious microorganism. For example, the peptide epitope can
be from a survivin, GP100, MelA, survivin-2B, livin/ML-IAP, Bcl-2,
Mcl-1, BcI-X(L), mucin-1, NY-ESO-1, telomerase, CEA, MART-1,
HER-2/neu, bcr-abl, PSA, PSCA, tyrosinase, p53, hTRT, leukocyte
proteinase-3, hTRT, gpl OO, MAGE antigens, GASC, JMJD2C, JARD2
(JMJ), JHDM3a, WT-1,CA 9, or protein kinase polypeptide. The
pharmaceutically acceptable carrier can be selected from the group
consisting of water, saline solution, binding agents, fillers,
lubricants, disintegrates, and wetting agents. The composition can
further comprise an adjuvant selected from the group consisting of
Freund's adjuvant, aluminum hydroxide, lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanin, dinitrophenol, cytokines, and bacterial products.
[0009] In another aspect, this document features a method that
includes contacting a cell in a subject with a virus particle
containing a nucleic acid that encodes a variant of an MHC
molecule, the variant having one or more amino acid changes from
wild-type in a portion of the MHC molecule that interacts with a T
cell receptor. The subject can be a human. The virus can be an
adenovirus. The MHC molecule can be a class I MHC molecule.
[0010] In yet another aspect, this document features a variant of
an MHC molecule, where the variant has one or more amino acid
changes from wild-type in the part of the MHC molecule that
interacts with a T cell receptor, and where the MHC molecule, in
the presence of a particular peptide epitope, has been identified
as having the ability to activate a T cell.
[0011] This document also features a method of selecting a MHC
molecule variant for activation of an immune response. The method
can include: providing a panel of cells, each cell of the panel
expressing on its surface a variant of an MHC molecule, the variant
having one or more amino acid changes from wild-type in the part of
the MHC molecule that interacts with a T cell receptor; testing the
ability of each cell of the panel to activate a T cell in the
presence of a selected peptide epitope; and selecting the MHC
molecule expressed by a cell of the panel that activates a T cell
in the presence of the peptide epitope.
[0012] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0013] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0014] FIG. 1A is a depiction of the structure of a TCR:MHC:Peptide
complex (2C TcR:H-2 Kb:pdEV8; Garcia et al. (1997) Proc. Nat. Acad.
Sci. USA 94:13838-13843; and Garcia et al. (1998) Science
279:1166-1172) derived from x-ray crystallographic data. FIG. 1B is
a depiction of the structure of an Ab (antibody):MHC:Peptide
complex (25-D1.16:H-2 Kb:pdEV8; Mareeva et al. (2008) J. Biol.
Chem. 283:29053-29059) derived from x-ray crystallographic data.
FIG. 1C is a depiction of the structure of an MHC heavy chain
(derived from x-ray crystallographic data), showing the Kbm3
mutations (D77S and K89A).
[0015] FIG. 2 is a graph plotting percent lysis in an OT-1 T cell
killing assay. OT-1 T cells activated with the Kb-SIINFEKL (SEQ ID
NO:1) pMHC-peptide complex lysed EL4 (Kb) targets presenting
SIINFEKL (SEQ ID NO:1), while OT-1 T cells activated with the Kb-Q7
(SEQ ID NO:2) pMHC-peptide complex did not. OT1 spleen cells were
cultured with 10 .mu.g/ml peptide in culture for 5 days prior to
use in a standard .sup.51Cr release cytotoxicity assay against
target cells (EL4 or EL4 pulsed with the SIINFEKL (SEQ ID NO:1)
peptide or the Q7 (SEQ ID NO:2) peptide.
[0016] FIG. 3 is a graph plotting percent lysis in an OT-1 T cell
killing assay. OT-1 cells activated with the Kb-SIINFEKL (SEQ ID
NO:1) pMHC-peptide complex lysed EL4 (Kb) target cells presenting
SIINFEKL (SEQ ID NO:1) or Q7 (SEQ ID NO:2) peptide, but not EL4
cells not pulsed with peptide. OT-1 T cells were activated and
assayed as in FIG. 2.
[0017] FIG. 4 is a graph plotting lack of diabetes occurrence in
RIP-OVA mice challenged with OT-1 and Q7 peptide (SEQ ID NO:2)
pulsed antigen presenting cells (APCs).
[0018] FIG. 5 is a graph plotting growth of lymphoma tumor grafts
in wild type (B6) mice or in mice having an amino acid substitution
in the peptide binding domain of the H-2 K (bm3 and bm8) or D
(bm14) class I antigen presenting molecules. The lymphoma cells
grew out in the wild type B6 hosts genetically matched with the
tumor, but were rejected in the mutant mice.
[0019] FIG. 6 is a diagram showing comparable molecular
interactions defined for mouse 2C TcR CDR2a region with H-2 Kb
.alpha.2 helix (Garcia (1998), supra) and for human A6 TcR
CDR2.alpha. with the HLA-A0201 .alpha.2 of heavy chain (Garboczi et
al. (1996) Nature 384:134-141).
DETAILED DESCRIPTION
[0020] This document provides materials and methods for priming
self-reactive T cells in a peptide specific manner, such that the
cells can execute their effector functions in peripheral tissues
(cancers and pathogen infected cells) with specificity. Activated
cells of this kind can be incorporated into therapeutic schemes to
focus autoimmune cellular responses against cancers and persisting
virus infections, for example.
[0021] MHC genes contain polymorphisms, and vary greatly from
individual to individual. There are two general classes of MHC
molecules. Class I MHC (pMHC) molecules are found on almost all
cells and present peptides to cytotoxic T lymphocytes (CTL). Class
II MHC molecules are found mainly on antigen-presenting immune
cells (APCs), which ingest polypeptide antigens (in, for example,
microbes) and digest them into peptide fragments. The MHC-II
molecules then present the peptide fragments to helper T cells,
which, after activation, provide generally required helper activity
for responses of other cells of the immune system (e.g., CTL or
antibody-producing B cells).
[0022] The interaction between the peptide bound in the binding
cleft of the heavy chain of MHC class I (pMHC) and the
complementary determining regions (CDR) of the T cell receptor
(TCR) determines the potential for T cell activation during the
afferent and efferent stages of cellular immunity. The affinity
that exists between TCR and MHC-peptide complex regulates T cell
fate during development, initial activation, and during execution
of effector functions. The crystal structure of the TCR/MHC complex
indicates that the highly variable CDR3 regions of the V.alpha. and
V.beta. domains determine the energetics of TCR/MHC interactions,
with the predominate contacts occurring between the TCR CDR3
regions and peptide bound by the MHC molecule.
[0023] This document provides isolated MHC polypeptides that
contain one or more (e.g., one, two, three, four, five, more than
five, or any range between one and five) substitutions, additions,
or deletions. As used herein, a "polypeptide" is any chain of amino
acid residues, regardless of post-translational modification (e.g.,
phosphorylation or glycosylation). An "isolated" polypeptide is a
polypeptide that (1) is not associated with proteins found in
nature, (2) is free of other proteins from the same source (e.g.,
free of human proteins), (3) is expressed by a cell from a
different species, or (4) does not occur in nature. An isolated
polypeptide can be, for example, encoded by DNA or RNA, including
synthetic DNA or RNA, or some combination thereof.
[0024] The polypeptides provided herein can contain an amino acid
tag. A "tag" is generally a short amino acid sequence that provides
a ready means of detection or purification through interactions
with an antibody against the tag or through other compounds or
molecules that recognize the tag. For example, tags such as c-myc,
hemagglutinin, polyhistidine, or FLAG.RTM. can be used to aid
purification and detection of a polypeptide. As an example, a
polypeptide with a polyhistidine tag can be purified based on the
affinity of histidine residues for nickel ions (e.g., on a Ni-NTA
column), and can be detected in western blots by an antibody
against polyhistidine (e.g., the Penta-His antibody; Qiagen,
Valencia, Calif.). Tags can be inserted anywhere within the
polypeptide sequence, although insertion at the amino- or
carboxy-terminus is particularly useful.
[0025] The quality of the TCR/MHC interaction can be changed in
both positive and negative directions by altering the peptides at
their interface with the CDR3 loops of the TCR. The data presented
herein show that changes in the structure of the MHC heavy chain
can increase pMHC binding to the TCR, enhancing T cell activation.
Rosetta Protein Modeling Suite, a computer modeling approach, was
used to design more efficient pMHC to stimulate T cells in a
peptide-dependent manner. The naturally occurring mouse pMHC
molecule H-2 Kbm3 mutant was of particular interest, due to a
single point mutation existing at position 77 of the heavy chain
that increases TCR/pMHC affinity.
[0026] This document also provides methods for generating a library
of modified MHC (e.g., pMHC) molecules, and methods for using the
library in selection of a particular modified MHC that can
potentiate an immune response against a particular peptide. For
example, an MHC heavy chain can be modified to contain one or more
(e.g., one, two, three, four, five, or more than five) amino acid
substitutions, deletions, or additions. These modifications can be
located, for example, at amino acid residues that are involved in
interactions with TCR. In some cases, one or more modifications can
be made to the two .alpha.-helices (between which lies the peptide
binding cleft) of MHC molecules. Such amino acid residues would
generally be on the "upper" surface of these .alpha.-helices that,
on the surface of a cell expressing the MHC molecule, faces
outwards from the cell and thus is most accessible for interaction
with a TCR of a T cell in the vicinity of the cell expressing the
MHC molecule. The modifications can be such that the
affinity/avidity of the MHC-peptide complex for the TCR is altered
(e.g., increased or decreased, compared to a wild type MHC heavy
chain). Exemplary modifications to the mouse pMHC heavy chain are
described in the Examples herein. These changes can be extrapolated
to human MHC molecules.
[0027] In some embodiments, a modified MHC molecule can contain one
or more conservative substitutions. Amino acid substitutions can be
made, in some cases, by selecting substitutions that do not differ
significantly in their effect on maintaining (a) the structure of
the peptide backbone in the area of the substitution, (b) the
charge or hydrophobicity of the molecule at the target site, or (c)
the bulk of the side chain. For example, naturally occurring
residues can be divided into groups based on side-chain properties:
(1) hydrophobic amino acids (norleucine, methionine, alanine,
valine, leucine, and isoleucine); (2) neutral hydrophilic amino
acids (cysteine, serine, and threonine); (3) acidic amino acids
(aspartic acid and glutamic acid); (4) basic amino acids
(asparagine, glutamine, histidine, lysine, and arginine); (5) amino
acids that influence chain orientation (glycine and proline); and
(6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine).
Substitutions made within these groups can be considered
conservative substitutions. Examples of useful substitutions
include, without limitation, substitution of valine for alanine,
lysine for arginine, glutamine for asparagine, glutamic acid for
aspartic acid, serine for cysteine, asparagine for glutamine,
aspartic acid for glutamic acid, proline for glycine, arginine for
histidine, leucine for isoleucine, isoleucine for leucine, arginine
for lysine, leucine for methionine, leucine for phenyalanine,
glycine for proline, threonine for serine, serine for threonine,
tyrosine for tryptophan, phenylalanine for tyrosine, and/or leucine
for valine.
[0028] In some embodiments, a modified MHC chain (e.g., a pMHC
heavy chain) can include one or more non-conservative
substitutions. Non-conservative substitutions typically entail
exchanging a member of one of the classes described above for a
member of another class.
[0029] Methods for making modified polypeptides are known in the
art. By way of example and not limitation, a polypeptide can be
obtained by extraction from a natural source (e.g., from isolated
cells, tissues or bodily fluids), by chemical synthesis (e.g., by
solid-phase synthesis or other methods well known in the art,
including synthesis with an ABI peptide synthesizer; Applied
Biosystems, Foster City, Calif.), or by expression of a recombinant
nucleic acid encoding the polypeptide. Thus, in addition to
modified MHC polypeptides, this document provides isolated nucleic
acids encoding modified MHC polypeptides as described herein. The
term "isolated" as used herein with reference to nucleic acid
refers to a naturally-occurring nucleic acid that is not
immediately contiguous with both of the sequences with which it is
immediately contiguous (one on the 5' end and one on the 3' end) in
the naturally-occurring genome of the organism from which it is
derived. An isolated nucleic acid can be, e.g., a recombinant DNA
molecule of any length, provided one of the nucleic acid sequences
normally found immediately flanking that recombinant DNA molecule
in a naturally-occurring genome is removed or absent. Thus, an
isolated nucleic acid can be, without limitation, a recombinant DNA
that exists as a separate molecule (e.g., a cDNA or a genomic DNA
fragment produced by PCR or restriction endonuclease treatment)
independent of other sequences as well as recombinant DNA that is
incorporated into a vector, an autonomously replicating plasmid, a
virus (e.g., a retrovirus, adenovirus, or herpes virus), or into
the genomic DNA of a prokaryote or eukaryote. In addition, an
isolated nucleic acid can include a recombinant DNA molecule that
is part of a hybrid or fusion nucleic acid sequence.
[0030] The term "isolated" as used herein with reference to nucleic
acid also includes any non-naturally-occurring nucleic acid since
non-naturally-occurring nucleic acid sequences are not found in
nature and do not have immediately contiguous sequences in a
naturally-occurring genome. For example, non-naturally-occurring
nucleic acid such as an engineered nucleic acid is considered to be
isolated nucleic acid. Engineered nucleic acids can be made using
common molecular cloning or chemical nucleic acid synthesis
techniques. Isolated non-naturally-occurring nucleic acid can be
independent of other sequences, or incorporated into a vector, an
autonomously replicating plasmid, a virus (e.g., a retrovirus,
adenovirus, or herpes virus), or the genomic DNA of a prokaryote or
eukaryote. In addition, a non-naturally-occurring nucleic acid can
include a nucleic acid molecule that is part of a hybrid or fusion
nucleic acid sequence. A nucleic acid existing among hundreds to
millions of other nucleic acids within, for example, cDNA libraries
or genomic libraries, or gel slices containing a genomic DNA
restriction digest, is not to be considered an isolated nucleic
acid.
[0031] As used herein, the term "nucleic acid" refers to both RNA
and DNA, including mRNA, cDNA, genomic DNA, synthetic (e.g.,
chemically synthesized) DNA, and nucleic acid analogs. The nucleic
acid can be double-stranded or single-stranded, and where
single-stranded, can be the sense strand or the antisense strand.
In addition, nucleic acid can be circular or linear. Nucleic acid
analogs can be modified at the base moiety, sugar moiety, or
phosphate backbone to improve, for example, stability,
hybridization, or solubility of a nucleic acid. Modifications at
the base moiety include deoxyuridine for deoxythymidine, and
5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for
deoxycytidine. Modifications of the sugar moiety can include
modification of the 2' hydroxyl of the ribose sugar to form
2'-O-methyl or 2'-O-allyl sugars. The deoxyribose phosphate
backbone can be modified to produce morpholino nucleic acids, in
which each base moiety is linked to a six-membered, morpholino
ring, or peptide nucleic acids, in which the deoxyphosphate
backbone is replaced by a pseudopeptide backbone and the four bases
are retained. See, for example, Summerton and Weller (1997)
Antisense Nucleic Acid Drug Dev. 7:187-195; and Hyrup et al. (1996)
Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate
backbone can be replaced with, for example, a phosphorothioate or
phosphorodithioate backbone, a phosphoroamidite, or an alkyl
phosphotriester backbone.
[0032] Isolated nucleic acids also can be chemically synthesized,
either as a single nucleic acid molecule (e.g., using automated DNA
synthesis in the 3' to 5' direction using phosphoramidite
technology) or as a series of oligonucleotides. For example, one or
more pairs of long oligonucleotides (e.g., >100 nucleotides) can
be synthesized that contain the desired sequence, with each pair
containing a short segment of complementarity (e.g., about 15
nucleotides) such that a duplex is formed when the oligonucleotide
pair is annealed. DNA polymerase can be used to extend the
oligonucleotides, resulting in a single, double-stranded nucleic
acid molecule per oligonucleotide pair, which then can be ligated
into a vector.
[0033] Isolated nucleic acids also can be obtained by mutagenesis.
For example, a nucleic acid sequence encoding a MHC heavy chain
polypeptide can be mutated using standard techniques such as, for
example, oligonucleotide-directed mutagenesis and/or site-directed
mutagenesis through PCR. See, Short Protocols in Molecular Biology,
Chapter 8, Green Publishing Associates and John Wiley & Sons,
Edited by Ausubel et al., 1992.
[0034] This document also provides vectors containing a nucleic
acid provided herein. As used herein, a "vector" is a replicon,
such as a plasmid, phage, or cosmid, into which another DNA segment
can be inserted so as to bring about the replication of the
inserted segment. A vector can be an expression vector. An
"expression vector" is a vector that includes one or more
expression control sequences, and an "expression control sequence"
is a DNA sequence that controls and regulates the transcription
and/or translation of another DNA sequence.
[0035] In an expression vector provided herein, the nucleic acid
can be operably linked to one or more expression control sequences.
As used herein, "operably linked" means incorporated into a genetic
construct so that expression control sequences effectively control
expression of a coding sequence of interest. Examples of expression
control sequences include promoters, enhancers, and transcription
terminating regions. A coding sequence is "operably linked" and
"under the control" of expression control sequences in a cell when
RNA polymerase is able to transcribe the coding sequence into mRNA,
which then can be translated into the polypeptide encoded by the
coding sequence.
[0036] Suitable expression vectors include, without limitation,
plasmids and viral vectors derived from, for example,
bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses,
cytomegalovirus, retroviruses, poxviruses, adenoviruses, and
adeno-associated viruses. Numerous vectors and expression systems
are commercially available from such corporations as Novagen
(Madison, Wis.), Clontech Laboratories (Mountain View, Calif.),
Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies
(Carlsbad, Calif.).
[0037] An expression vector can include a tag sequence designed to
facilitate subsequent manipulation of the expressed nucleic acid
sequence (e.g., purification or localization). Tag sequences, such
as green fluorescent protein (GFP), glutathione S-transferase
(GST), polyhistidine, c-myc, hemagglutinin, or Flag.TM. tag (Kodak,
New Haven, Conn.) sequences typically are expressed as a fusion
with the encoded polypeptide. Such tags can be inserted anywhere
within the polypeptide including at either the carboxyl or amino
terminus.
[0038] This document also provides host cells containing a nucleic
acid molecule and/or nucleic acid vector provided herein. The term
"host cell" refers to prokaryotic cells and eukaryotic cells into
which a nucleic acid molecule or vector can be introduced. Any
method can be used to introduce nucleic acid into a cell. For
example, naked DNA can be delivered directly to cells in vivo as
described elsewhere (U.S. Pat. Nos. 5,580,859 and 5,589,466). In
addition, calcium phosphate precipitation, electroporation, heat
shock, lipofection, microinjection, and viral-mediated nucleic acid
transfer can be used introduce nucleic acid into cells. In some
cases, for example, a nucleic acid molecule (e.g., a cDNA) encoding
a modified MHC molecule, a particular peptide epitope, or a
polypeptide that includes a particular peptide epitope can be
incorporated into a viral vector (e.g., an adenoviral vector, an
adeno-associated virus vector, a herpes virus vector, a
cytomegalovirus vector, a retrovirus vector, or a poxvirus vector).
High titer virus can be prepared using standard methods, and the
virus can be used to infect host cells such as, without limitation,
cell lines in culture, or tumor cells in situ.
[0039] As described herein, a library or panel containing cells
(e.g., RMAS cells) expressing modified MHC molecules can be
generated. The cells in such a panel can be used in a screening
method to determine which, if any, member or members of the panel
contain a modified MHC molecule that can stimulate an immune
response to a particular peptide. For example, a method can include
providing a panel of cells, each of which expresses on its surface
a variant of an MHC molecule, where the variant has one or more
amino acid changes from wild-type in the part of the MHC molecule
that interacts with a T cell receptor; testing the ability of each
panel member to activate a T cell in the presence of a peptide
epitope of interest; and selecting the MHC molecule expressed by a
member of the panel that activates a T cell in the presence of the
peptide epitope. Self peptides in general can be from proteins
against which tolerance was established, but against which it is
desired to activate a response because they are highly expressed
in, for example, certain cancers. Thus, for example, a self peptide
antigen from a cancer cell can be used as a peptide of interest,
and a modified MHC molecule that stimulates an immune response
against the peptide can be selected for potential therapeutic use.
Examples of self peptides include, without limitation, peptides
that are contained within proteins such as survivin, GP100, MelA,
survivin-2B, livin/ML-IAP, Bcl-2, Mcl-1, BcI-X(L), mucin-1,
NY-ESO-1, telomerase, CEA, MART-1, HER-2/neu, bcr-abl, PSA, PSCA,
tyrosinase, p53, hTRT, leukocyte proteinase-3, hTRT, gpl OO, MAGE
antigens, GASC, JMJD2C, JARD2 (JMJ), JHDM3a, WT-1,CA 9, and protein
kinases. See, also, WO 2010/037395, which discloses suitable cancer
antigenic peptides.
[0040] In the methods provided herein, the modified MHC molecule
can be a Class I (pMHC) or Class II molecule. Testing can be
conducted in vivo or in vitro. In vitro testing can include, for
example, the use of T cells from a cloned T cell line, a polyclonal
T cell population, or T cells expressing recombinant TCR chains.
The T cells can be CD4.sup.+ or CD8.sup.+ T cells. In vivo methods
can include, e.g., administering to a subject (e.g., a human or a
non-human mammal) one or more modified MHC molecules, nucleic acids
encoding the one or more modified MHC molecule, or cells expressing
the one or more modified MHC molecule, and testing for activation
of T cell by the variant MHC-peptide complex by methods known in
the art. A non-human mammal can be, e.g., a transgenic non-human
mammal expressing a recombinant TCR (e.g., a human TCR) on, for
example, all of its T cells, all of its CD4.sup.+ T cells, or all
of its CD8.sup.+ T cells.
[0041] In some embodiments, an epitope of interest can be from any
polypeptide against which an immune response is desired. For
example, an epitope can be from a polypeptide expressed by a cancer
cell, or by a cell infected with an infectious microorganism (e.g.,
a virus, bacteria, or protozoan). It is noted that an epitope (also
referred to herein as a peptide epitope) "from" a particular
polypeptide does not need to be physically isolated from that
polypeptide, but also can be chemically synthesized or made
recombinantly, for example, provided that the epitope has a
sequence contained within the polypeptide.
[0042] This document also provides methods for treating an
individual in need thereof (e.g., an individual in whom it is
desired to stimulate an immune response against a particular
peptide). The methods can include administering to the subject (a)
a cell expressing on its surface a variant of an MHC molecule,
where the variant has one or more amino acid changes from wild-type
in the part of the MHC molecule that interacts with a T cell
receptor, and where the cell has been identified as having the
ability to activate a T cell (e.g., a CD4.sup.+ T cell or a
CD8.sup.+ T cell) in the presence of a peptide epitope from the
subject; and (b) the peptide epitope or a polypeptide containing
the peptide epitope. The subject can have, or be likely to have, a
pathological condition (e.g., cancer or an infectious disease, such
as a viral, bacterial, or protozoan infection) that is amenable to
therapy by a T cell immune response. A subject that is likely to
have a pathological condition would be one having one or more
symptoms of the condition. Symptoms of cancer and infectious
diseases are well known in the art. The peptide epitope can be from
a polypeptide that is expressed by a cancer cell or a cell infected
with an infectious microorganism (e.g., a virus or an intracellular
bacteria or protozoans).
[0043] The methods provided herein can include administering to a
mammal (e.g., a human or a non-human mammal) an effective amount of
a modified MHC polypeptide, nucleic acid encoding the modified MHC
polypeptide, or cell expressing the modified MHC polypeptide or an
effective amount of a composition containing such a
polypeptide/nucleic acid/cell. In some cases, a method can include
administering a nucleic acid encoding a modified MHC polypeptide by
a virus-mediated transfer method (e.g., by direct injection into a
selected tissue of viral particles encoding the modified MHC
molecule).
[0044] As used herein, the term "effective amount" is an amount of
a molecule, cell, or composition that is sufficient to increase an
immune response against a peptide of interest. For example, in some
embodiments, an "effective amount" of a cell expressing a modified
MHC polypeptide can be an amount that is sufficient to increase T
cell activation in a peptide specific manner. The degree of T cell
activation can be determined by, for example, detecting or
measuring CTL activity or helper activity [e.g., the production of
cytokines such as interleukins (IL) (e.g., IL-2, IL-4, IL-5, IL-10,
IL-12, or IL-13), or interferons (IFN) (e.g., IFN-.alpha.,
IFN-.beta., or IFN-.gamma.)]. Activation also can be assessed by
flow cytometry (e.g., to look for particular antigen-specific
cells, and to monitor populations of cells, such as CD4.sup.+ vs.
CD8.sup.+ cells, or to look for CD2, CD27, CD28, CD45RA, CD45RO,
CD62L, and/or CCR7, which are surface markers unique to T cells in
various differentiation states). In some embodiments, T cell
activation can be evaluated using ELISPOT, by adding the antigenic
peptide optionally associated with a MHC monomer or MHC multimer or
adding antigenic polypeptide comprising antigenic peptide, followed
by measurement of IFN-gamma secretion from a population of cells or
from individual cells. T cell activation also can be measured with
quantaferon-like detection assays, e.g., using indirect detection,
such as by adding the antigenic peptide optionally associated with
a MHC monomer or MHC multimer or adding antigenic polypeptide
comprising antigenic peptide, followed by measurement of IFN-gamma
secretion from a population of cells or from individual cells.
[0045] In addition, this document provides compositions and methods
for their administration to a subject. For example, the modified
MHC polypeptides described herein, nucleic acids encoding the
modified MHC polypeptides, or cells expressing the modified MHC
polypeptides can be incorporated into compositions for
administration to a subject (e.g., a subject having cancer or a
viral or bacterial infection). In some embodiments, a composition
can contain a peptide epitope of interest, or a polypeptide
containing the peptide epitope.
[0046] Methods for formulating and subsequently administering
therapeutic compositions are well known to those in the art.
Dosages typically are dependent on the responsiveness of the
subject to the composition, with the course of treatment lasting
from a single treatment to several days or several months, or until
a suitable response is achieved. Persons of ordinary skill in the
art routinely determine optimum dosages, dosing methodologies and
repetition rates. Optimum dosages can vary depending on the
relative potency of a composition, and in some embodiments can be
estimated based on the EC.sub.50 found to be effective in in vitro
and/or in vivo animal models.
[0047] This document also provides for the use of the peptides,
polypeptides, nucleic acids, and cells disclosed herein in the
manufacture of medicaments (e.g., for activating self-reactive T
cells in a subject in a peptide-specific manner). The peptides,
polypeptides, nucleic acids, or cells can be admixed, encapsulated,
conjugated or otherwise associated with other molecules, molecular
structures, or mixtures of compounds, such as liposomes, receptor
or cell targeted molecules, or oral, topical or other formulations
for assisting in uptake, distribution and/or absorption. In some
embodiments, a composition can contain a peptide, polypeptide,
nucleic acid, or cell as provided herein in combination with a
pharmaceutically acceptable carrier and/or an adjuvant.
Pharmaceutically acceptable carriers include, for example,
pharmaceutically acceptable solvents, suspending agents, or any
other pharmacologically inert vehicles for delivering polypeptides,
nucleic acids, or cells to a subject. Pharmaceutically acceptable
carriers can be selected with the planned manner of administration
in mind so as to provide for the desired bulk, consistency, and
other pertinent transport and chemical properties, when combined
with one or more therapeutic compounds and any other components of
a given pharmaceutical composition. Exemplary pharmaceutically
acceptable carriers include, without limitation: water; saline
solution; binding agents (e.g., polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose or dextrose
and other sugars, gelatin, or calcium sulfate); lubricants (e.g.,
starch, polyethylene glycol, or sodium acetate); disintegrates
(e.g., starch or sodium starch glycolate); and wetting agents
(e.g., sodium lauryl sulfate). Exemplary adjuvants (e.g., that can
be used to increase an immunological response) depend on the host
species, and include, without limitation, Freund's adjuvant
(complete and incomplete), mineral gels such as aluminum hydroxide,
surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and
dinitrophenol. Suitable adjuvants also include, for example,
cytokines (e.g., interleukin-2 (IL-2), granulocyte--macrophage
colony-stimulating factor (GM-CSF), IL-12, and IL-4) and bacterial
products (e.g., lipopolysaccharides (LPS) and CpG). See, also, Finn
(2003) Nat. Rev. Immunol. 3:630-641.
[0048] Pharmaceutical compositions containing molecules described
herein can be administered by a number of methods. Administration
can be, for example, parenteral (e.g., by subcutaneous,
intrathecal, intraventricular, intramuscular, or intraperitoneal
injection, or by intravenous (i.v.) drip); oral; topical (e.g.,
transdermal, sublingual, ophthalmic, or intranasal); or pulmonary
(e.g., by inhalation or insufflation of powders or aerosols), or
can occur by a combination of such methods. Administration can be
rapid (e.g., by injection) or can occur over a period of time
(e.g., by slow infusion or administration of slow release
formulations).
[0049] This document also provides an article of manufacture that
can include one or more modified MHC polypeptides as provided
herein, nucleic acids encoding modified MHC polypeptides, and/or
cells expressing modified MHC polypeptides (e.g., RMAS cells, or
any other suitable type of cells). The article of manufacture can
include the one or more polypeptides, nucleic acids, and/or cells
formulated in a composition as described herein. In some
embodiments, an article of manufacture also can contain one or more
peptide epitopes to which modified MHC polypeptides can bind.
[0050] An article of manufacture can include, for example, a
composition containing (a) a cell expressing on its surface a
variant of an MHC molecule having one or more amino acid changes
from wild-type in the portion of the MHC molecule that interacts
with a T cell receptor, wherein the cell has been identified as
having the ability to activate a T cell in the presence of a
particular peptide epitope; and (b) a pharmaceutically acceptable
carrier. Optionally, the article of manufacture can further include
the peptide epitope. In some cases, the article of manufacture also
can include an adjuvant as described herein (e.g., one or more
cytokines, such as IL-2, IL-4, IL-5, IL-10, IL-12, IL-13,
IFN-.alpha., IFN-.beta., or IFN-.gamma.).
[0051] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Enhancing T Cell Activation Using Altered MHC-Peptide Ligands
[0052] Altered peptide ligands for T cell receptors: There are two
widely accepted schemes for priming T cells expressing low affinity
receptors for self. First, the quantity of self-peptide presented
by MHC can be increased to enhance the avidity of the receptor
ligand interaction between T cells and antigen presenting cells
(England et al. (1995) J. Immunol. 155:4295-4306; and Berzofsky et
al. (1999) Immunol. Rev. 170:151-172). The second is to alter the
structure of the peptide subtly to enhance the affinity of the
MHC-peptide ligand for the T cell receptor (Maile et al. (2005) J.
Immunol. 174(2):619-627). Both of these approaches require
engineering each individual peptide antigen of interest, either to
increase binding to MHC molecules or binding of the MHC-peptide
complex to the T cell receptor. The methods described herein
provide the option for increasing affinity of the MHC-peptide
ligand for the T cell receptor for essentially all peptides, yet
still maintaining peptide specificity in the antigen recognition
process.
[0053] The three dimensional structure of the TCR interacting with
MHC-peptide ligand (FIG. 1A) reveals a common mode of interaction.
The complementarity determining regions (CDR, comprised of loops
connecting beta strands of the immunoglobulin fold) of the TCR
variable alpha (V.alpha.) and beta (V.beta.) chains interact with a
surface comprised of the amino terminal alpha helical regions of
the MHC proteins and the bound peptide (Garcia et al. (1998),
supra; and Garcia et al. (1999) Annu. Rev. Immunol. 17:369-397).
Key to the approach for generating altered MHC-peptide ligands as
described herein are the observations derived from these early
structural studies that the T cell receptor contact with the bound
peptide is largely determined by the CDR3 segments and that the
CDR1 and CDR2 segments of the T cell receptor interact mostly with
the alpha helices of the MHC peptide presenting molecule. It was
hypothesized that changes in the MHC molecule that primarily alter
the interactions between the alpha helices and the T cell receptor
in a way that generates a higher affinity interaction might enhance
the T cell response to peptide antigens during the priming stage.
Once these T cells achieve the primed state, they can be activated
by the native MHC molecule presenting the same peptide. By
generating a panel of altered MHC molecules that bind to the T cell
receptor outside the CDR3 regions, a set of reagents that can be
coupled with many different peptides can be assembled, without need
for further engineering of the peptide antigens. Having a panel of
"on the shelf" altered MHC ligands provides a practical alternative
to the formidable task of engineering sets of peptides for antigen
specific responses for each target of interest. This also provides
a significant advance in the development of vaccines designed to
break tolerance to weak cancer antigens, and might also be used to
mobilize T cells remaining after the establishment of a persistent
infection.
[0054] To demonstrate this principle, the following experiments
were conducted, taking advantage of available reagents. The mouse
OT-1 T cell expresses a defined T cell receptor that binds to the
mouse H-2K.sup.b MHC molecule when complexed with a specific
chicken ovalbumin peptide, SIINFEKL (SEQ ID NO:1). This T cell
receptor MHC-peptide ligand interaction is sufficient to activate
both the priming and effector stages of the T cell response. The
antibody known as 25-D1.16 also binds to H-2K.sup.b specifically
when the MHC molecule is complexed to the SIINFEKL (SEQ ID NO:1)
peptide. The degree that the 25-D1.16 antibody mimics T cell
receptor binding is evident in the resolved three dimensional
structure of the antibody:MHC:petide complex (FIG. 1B), which
demonstrates interactions of the antibody CDR regions and the
MHC-peptide ligand resembling those of reported T cell receptor
MHC-peptide three dimensional structures (Mareeva et al. (2008) J.
Biol. Chem. 283:29053-29059).
[0055] A variant of the SIINFEKL (SEQ ID NO:1) peptide containing a
glutamine substitution at position 7 [SIINFEQL (SEQ ID NO:2), also
referred to herein as "Q7"] bound H-2K.sup.b comparably to SIINFEKL
(SEQ ID NO:1) (Daniels et al. (2006) Nature 444:724-729), but the
H-2K.sup.b-Q7 ligand did not bind the OT-1 T cell receptor with
sufficient affinity to prime naive OT-1 T cells (FIG. 2). Once
primed, however, OT-1 T cells recognized cells expressing
H-2K.sup.b-Q7 ligand sufficiently to be reactivated, and lysis
occurred (FIG. 3). Similarly, binding of Ab 25-D1.16 to H-2K.sup.b
was substantially reduced when K.sup.b was complexed with Q7,
relative to K.sup.b complexed with SIINFEKL (SEQ ID NO:1) (Table
1). Reduction in binding also was seen, albeit to a lesser degree,
with other peptide variants of SIINFEKL (SEQ ID NO:1) (see K.sup.b
WT row in Table 1).
[0056] To assess whether mutations of the MHC protein could enhance
receptor affinity for a MHC-peptide ligand, a model was developed
for a peptide specific receptor-ligand interaction using the
antibody 25-D1.16 as the receptor, H-2K.sup.b as the MHC peptide
presenting molecule, and the SIINFEKL (SEQ ID NO:1) variants E1
(SEQ ID NO:3), G4 (SEQ DI NO:4), Q7 (SEQ ID NO:2), and Q4H7 (SEQ ID
NO:5) as the weak receptor binding peptide ligands. This model
emulates the low binding affinity that a T cell receptor retained
in the immune repertoire would have for a self peptide. A library
of cells expressing H-2K.sup.b and closely related variants
containing defined amino acid substitutions in the MHC peptide
presenting protein was screened. Antibody 25-D1.16 bound
substantially better to three of the MHC variants in complex with
variant peptides than it bound to K.sup.b WT in complex with the
same peptides (Table 1, and FIG. 2). Most dramatic was the variant
K.sup.bm3 containing an Asp to Ser amino acid substitution at
position 77 and a Lys to Ala substitution at position 89 of the
mature MHC glycoprotein (FIG. 1C). The K.sup.bm3 MHC variant bound
to other peptides was not bound appreciably by Ab 25-D1.16,
demonstrating the peptide-specific nature of interaction between
altered MHC-peptide ligand and receptor. Similarly, the increases
in binding seen with 65 Gln to Arg with the G4 peptide variant and
24 Glu to Ser with the Q4H7 peptide variant also were
peptide-specific. These experiments demonstrated that it is
possible to alter the MHC peptide presenting molecule in such a way
as to increase the affinity between the MHC ligand and a receptor
for that ligand, while retaining the peptide specificity of the
interaction.
[0057] Biological Significance: The example using 25-D1.16 antibody
as a model for the interaction between T cell receptors and altered
MHC-peptide ligands illustrated the principle that a mutation in
the MHC molecule can enhance binding of the MHC-peptide ligand to
peptide-specific receptors. Fundamental to this scheme for
activating an immune response against self peptide is the
oligoclonality of the repertoire of T cells expressing receptors
with sub-threshold affinity for the peptide self antigen of
interest. Only a subset of such T cells needs to be activated by
this scheme to induce a productive immune response. Presumably the
activated cells will undergo clonal expansion to generate a
population of primed T cells capable of lysing cells expressing the
self-peptide. T cells not activated or anergized by interaction
with the MHC variant ligands would become irrelevant to the ensuing
response.
[0058] To test whether the K.sup.bm3 mutation had any measurable
biological activity on the OT-1 T cell receptor, a sensitive model
for detecting autoimmune T cell activity in vivo was employed. Type
I diabetes (T1D) is a chronic autoimmune disease in which
pancreatic 13-cells (which secrete insulin) are selectively
destroyed. It is thought to be a T helper 1 mediated disease that
involves CD8.sup.+ T cells and innate immune cells. Individuals
with T1D develop hyperglycemia and can develop diabetes-associated
complications in several organ systems due to a lack of insulin
(Lehuen et al. (2010) Nat. Rev. Immunol. 10:501-513).
[0059] A mouse model of T1D is the RIP-OVA mouse, which expresses
OVA under the control of the rat insulin promoter. These mice were
crossed with OT-1 mice, which have class I-restricted OVA-specific
CD8.sup.+ T cells (Blanas et al., Science (1996) 274:1707-1709).
The mice spontaneously develop diabetes characterized by increased
urine glucose levels and infiltration of islets.
[0060] B6-RIP-OVA mice are functionally normal and are not
responsive to the SIINFEKL (SEQ ID NO:1) peptide antigen, as this
fragment of ovalbumin is a self peptide in this model. When
B6-RIP-OVA.sup.hi mice received 5.times.10.sup.5 naive OT-1 spleen
cells to fill the hole in the T cell repertoire caused by
endogenous expression of soluble chicken ovalbumin in the pancreas,
thymus, and kidney (and possibly other unknown tissues), they did
not develop autoimmune diabetes unless treated further. Challenge
of mice harboring OT-1 cells with a Thyleirs murine
encephalomyelitis virus (TMEV) expressing SIINFEKL (SEQ ID NO:1)
induced diabetes within ten days. As few as 100 naive OT-1 cells
can transfer the ability to respond to the virus as judged by
diabetes onset.
[0061] The initial test for enhanced antigen presentation by the
K.sup.bm3 mutant MHC peptide presenting molecule was to transfer
5.times.10.sup.5 naiveOT-1 T cells into the B6-RIP-OVA.sup.hi hosts
and immunize them intravenously with engineered antigen presenting
cells expressing wild type K.sup.b or a K.sup.b variant containing
the 77 Asp to Ser mutation. The engineered antigen presenting cells
were prepared from the TAP deficient RMAS cell (Attay et al.,
Nature (1992) 355:647-649), a B cell line which expresses K.sup.b
and the co-stimulatory molecule CD80. CD80, CD86, and K.sup.bm3
were introduced into the RMAS cells by DNA mediated gene transfer.
Cell lines expressing CD80 and CD86 with or without K.sup.bm3 were
established. 10.sup.6 Q7 (SEQ ID NO:2) peptide-pulsed, TAP
deficient RMAS cells expressing transfected CD80/CD86 with or
without H-2K.sup.bm3 were monitored for 2 weeks; none developed
diabetes (Table 2). As a positive control, RMAS cells pulsed with
SIINFEKL (SEQ ID NO:1) were included in the experiment. These mice
also did not develop diabetes. This result was interpreted to
indicate that the engineered antigen presenting cells were not able
to prime the T cell response to the extent needed to induce
diabetes in this model.
[0062] In the context of the model, the K.sup.bm3 mutation may not
optimize, from a functional perspective, the affinity of the
altered MHC-peptide ligand for the OT-1 T cell receptor. It was
hypothesized that while the enhanced affinity of the alter
MHC-peptide ligand may not provide a fully activating signal to the
OT-1 T cells, it may still provide a signal sufficient to enhance
the ability of the OT-1 cells to persist functionally in the
toleragenic B6-RIP-OVA.sup.hi host environment. To test for the
persistence of adoptively transferred OT-1 T cells, the mice in
Table 2 were challenged intraperitoneally with a picornavirus
expressing SIINFEKL (SEQ ID NO:1) (on day 15 after the original
treatment with RMAS-derived antigen presenting cells). Whereas none
of the mice pretreated 15 days prior with RMAS-K.sup.b-Q7 pulsed
cells developed diabetes, 75% of the mice treated with
RMAS-K.sup.bm3-Q7 rapidly developed disease, as did the mice
treated with RMAS-K.sup.b-SIINFEKL (SEQ ID NO:1). This result was
interpreted to mean that the Q7 peptide-pulsed RMAS-K.sup.bm3
antigen presenting cells provided a functional signal to the
adoptively transferred OT-1 cells, enhancing their ability to
respond to subsequent challenge with a pircornavirus expressing a
self antigen. In contrast, RMAS cells expressing the wild type MHC
molecule K.sup.b pulsed with the Q7 (SEQ ID NO:2) peptide were not
able to respond to self antigen (SIINFEKL; SEQ ID NO:1) presented
by this same virus. This result demonstrated that modification of
the MHC protein sequence can alter the quality of antigen
presentation in vivo, enhancing the potential to develop a T cell
response against a self antigen.
TABLE-US-00001 TABLE 1 25-D1.16 Binding MHC heavy chain amino acid
replacements influence the ability of MHC: peptide complexes to
function as ligands for the 25-D1.16 antibody ##STR00001## LTK
cells were transfected with K.sup.b wild type (WT) or mutant genes
encoding MHCs with the indicated amino acid substitutions (Column
1). The ability of the variant molecules relative to the WT
molecule when complexed with SIINFEKL (SEQ ID NO:1) to be bound by
antibody 25-D1.16 (Column 2) was determined by pulsing the cultured
cells with 10 .mu.g/m1 peptide for one hour prior to washing and
staining with antibody for analysis by flow cytometry. Median
fluorescent intensity (stained - unstained) was used as an estimate
of binding. The ability of each variant MHC complexed with the
SIINFEKL (SEQ ID NO:1) variants (E replacement at position 1, G
replacement at position 4, Q replacement at position 7, and Q at 4
and H at 7 double replacement) to bind antibody 25-D1.16, relative
to the ability of the same variant complexed with SIINFEKL (SEQ ID
NO:1) to bind 25-D1.16 are shown in Columns 3-6. Comparisons were
drawn only for variants that bound SIINFEKL (SEQ ID NO:1) at least
25% as well as WT K.sup.b bound the peptide. Substantially
increased binding to 25-D1.16 antibody by a MHC heavy chain variant
relative to the WT molecule when complexed to a given peptide is
represented in the shaded cells.
TABLE-US-00002 TABLE 2 The K.sup.bm3-Q7 ligand provides an
activation signal to OT-1 T cells Diabetes Onset in B6-RIP-OVA mice
Day 15 Challenge with Vaccine Day 14 TMEV-SIINFEKL Day 21 Day 1
Diabetes (SEQ ID NO:1) Diabetes RMAS(K.sup.b)- 0/2 Yes 2/2 SIINFEKL
(SEQ ID NO: 1) RMAS(K.sup.b)-Q7 0/4 Yes 0/4 RMAS-K.sup.bm3-Q7 0/4
Yes 3/4 B6-RIP-OVA mice received 5 .times. 10.sup.5 naive OT-1
spleen cells followed by 10.sup.6 of the indicated RMAS cells
pulsed with 10 .mu.g/ml of SIINFEKL (SEQ ID NO: 1) or Q7 (SEQ ID
NO: 2) peptide intravenously. Blood sugar readings were taken daily
for 30 days. Animals were judged to have developed diabetes when
two successive blood sugar readings exceeded 300 .mu.g/ml on two
successive days. On day 15 all mice were challenged
intraperitoneally with TMEV expressing the SIINFEKL peptide in its
amino terminal leader sequence.
[0063] In further experiments, B6-RIP OVA mice received
5.times.10.sup.5 OT-1 T cells and were immunized with RMAS
(K.sup.b) or RMAS-K.sup.bm3 cells, each pulsed with SIINEFQL (SEQ
ID NO:2; Q7) peptide as antigen, on day 1. On day 15, all mice
received 7.times.10.sup.4 TMEV-L/OVA virus challenge ip. Animals
were monitored for diabetes (>300 mg/dL blood glucose) for 30
days from the time of OT-1 T cell adoptive transfer. None of the
mice pretreated 15 days prior with RMAS-K.sup.b-Q7 pulsed cells
developed diabetes, but about 65% of the mice treated with
RMAS-K.sup.bm3-Q7 developed disease (FIG. 4).
[0064] In summary, the experiments described above suggested the
following: [0065] That TMEV-L/OVA specifically drove diabetes in
RIP-OVA mice adoptively transferred with OT-1 splenocytes. [0066]
That elevated blood glucose levels correlated with pancreatic islet
cell invasion. [0067] That varying the amounts of OT-1 splenocytes
transferred into RIP-OVA mice along with TMEV-L/OVA correlated with
T1D development.
Example 2
Assessing the Ability of Altered MHC Ligands to Induce Protective
Immunity Against Native Tumors
[0068] Experiments showed that EL4 lymphoma tumor grafts mismatched
by a single MHC class I mutation that altered peptide binding
relative to the host MHC were rejected by the host (FIG. 5). In
these experiments, 5.times.10.sup.5 lymphoma cells were introduced
subcutaneously into mice genetically matched with the tumor, with
the exception of amino acid substitutions in the peptide binding
domain of the H-2 K (bm 3 and 8) or D (bm14) class I antigen
presenting molecules. The bm3, bm8, and bm14 spontaneous mutations
occurred in genetically defined mouse strains and were described
previously (Pullen et al. (1989) J. Immunol. 143:1674-1679; Hunt et
al. (1990) J. Immunol. 145:1456-1462; and Hemmi et al. (1988) J.
Exp. Med. 168(6):2319-2335). While the lymphoma cells grew out in
the wild type B6 hosts, the tumor did not grow in the mutant mice.
This demonstrated that alterations in the structure of MHC
molecules with respect to the host resulted in potent anti-tumor
resistance. The present approach seeks to use this host response
against the variant tumor cells to incite anti-tumor immunity that
will cross react back onto the native tumor.
[0069] A subsequent experimental scheme is to introduce engineered
.sup.altMHC ligands into native tumors, and to use these modified
tumors as vaccines against the native tumor. If enhanced TcR-MHC
affinity is achieved by mutagenesis of the class I .alpha. helices,
a strong allo reaction is expected such that the .sup.altMHC-tumor
will fail to grow in the A2 animal. It has been shown, using the
spontaneous variant of the K.sup.dm5 mutant with threonine
substituted for alanine at amino acid 158, that alloreactivity was
developed in the context of self peptides presented in common by
the parental and mutant MHC molecules (Pullen et al. (1994) J.
Immunol. 152:3445-3452). .sup.altMHC-EL4 cells were generated by
transfecting a pCI-vector (Promega Corp., Madison, Wis.) encoding
the .sup.altMHC gene into EL4 cells followed by selection of stable
transformants expressing the introduced MHC protein on their cell
surface by drug selection with G418 (Gibco/Invitrogen; Carlsbad,
Calif.). To test whether cross reactive T cells specific for tumor
associated peptides are stimulated by the tumors expressing
.sup.altMHC molecules, .sup.altMHC-expressing EL4 cells are
introduced into B6 mice bearing wild type tumor cells, either by
simultaneously challenging with tumors expressing .sup.altMHC on
one flank and wild type tumor on the opposite flank, or by
introducing wild type tumors into the hosts prior to treatment with
tumors expressing .sup.altMHC. The presence of protective cross
reactive immunity is detected by comparing the growth of the wild
type tumors in mice receiving tumor vaccines bearing .sup.altMHC
with the growth of wild type tumors in mice receiving a sham wild
type vaccine.
[0070] To translate this concept from mice to humans, the MHC class
I antigen presenting molecule HLA-A0201 is used for initial
studies. A0201 is a common MHC class I allele expressed by more
than 40% of Caucasians. A guide for generating .sup.altA0201
mutations is provided by the contact regions of the .alpha. helices
of the mouse K.sup.b MHC peptide presentation domain defined by
interactions with the 2C TcR (MHC aa 72, 76, and 79 for CDR2.beta.
and MHC aa 154, 158, 162, and 166 for CDR2.alpha.; FIG. 6 and
Garcia et al. (1998), supra), as well as the contacts determined
for human HLA-A2 that contact A6 TcR (residues 155, 158, 166; FIG.
6). A range of mutations representing changes in size, polarity,
and charge are evaluated for their ability to enhance binding
(Table 3), as amino acid changes at any or all of these positions
may provide optimal stimulation of host T cells while preserving
recognition of tumor associated peptides. Efforts are focused on
two of these amino acid positions on the wild type HLA-A0201
molecule: V.sup.76 and A.sup.158 (i.e., one residue in position to
contact the .alpha. chain of the T cell receptor and another
residue in position to contact the .beta. chain of the T cell
receptor). The Ala.sup.158 to Thr replacement is equivalent to the
dm5 mutant mouse, and Val.sup.76 is in the same area as the A77S
mutation found in bm3. Substitution of Ala often is useful to probe
structure, as it is considered to be the least disruptive
replacement with respect to secondary structure. In the case of
A.sup.158, Val is the next most similar amino acid, since Gly tends
to disrupt secondary structure. Otherwise, amino acids sharing
charge, polarity and similar sizes (by length and mass) are
selected as the most similar. The last category of substitutions
would result in a change in charge.
[0071] As shown in FIG. 6, three amino acids from the CDR2 loop of
the TcR.alpha. interact closely with amino acids on the MHC heavy
chain. Many Va genes in both the mouse and human systems express
different amino acids in these three positions, providing a
diversity of potential salt bridges, hydrogen bonding, van der Waal
forces, and hydrophobic interactions at TcR:MHC interfaces formed
with altered A2 molecules. Comparison of atomic contacts of three
different mouse TcR(s) specific for the same peptide epitope
presented by single MHC molecule has documented this point (Feng et
al. (2007) Nat. Immunol. 8(9):975-983). Substitutions are made at
any of the conserved positions on the MHC heavy chain along the
contact region with CDR2.alpha. (E.sup.154, A.sup.158,G.sup.162,
and E.sup.166) to determine whether the ability of TcR(s) to
interact with the .sup.altMHC-ligand is altered. Some substitutions
are expected to be destabilizing, but other mutations are expected
to enhance binding Variation in the contact positions for CDR2 of
TcR V.beta. with MHC al helix amino acids Q.sup.72, V.sup.76, and
R.sup.79 is just as extensive, providing two regions of the MHC:TcR
interface to manipulate while leaving peptide binding to the MHC
antigen presenting molecule and the TcR unchanged.
[0072] To begin these studies, the eight MHC mutants listed in
Table 3 for positions 76 and 158 were generated by creating genes
encoding the HLA-A0201 protein with single amino acid substitutions
at those positions (Val to Ala, Gly, Thr, or Glu at residue 76 and
Ala to Val, Gly, Thr, or Glu at residue 158). A mouse MHC
expression system is used to build HLA-A2 constructs so that the
portions of the encoded class I protein that are needed to interact
with species specific accessory molecules (e.g., CD8) and
intracellular regions of the molecule will function in
appropriately in mouse hosts. As with the mouse MHC, the engineered
A2 molecules are tagged with antibody epitopes (which do not
influence the structure of the peptide and TcR binding domain) and
stably-expressing RMAS and EL4 cells are generated (Kuhns et al.
(2000) Proc. Natl. Acad. Sci. USA, 97(2):756-760; and Pullen et al.
(supra)). To test how well the .sup.altMHC molecules activate naive
T cells in a normal immune repertoire, enhanced allogeneic T cell
activation is evaluated in vivo using B6 transgenic host animals
expressing wild type HLA-A0201 (i.e., "humanized" A2 mice).
.sup.wTMHC- or .sup.altMHC-expressing EL4 mouse tumor cells are
grafted onto the A2 mice in a manner similar to the studies for the
.sup.altK.sup.b expressing cells.
TABLE-US-00003 TABLE 3 WT Ala Most Different Amino Acid scan
Similar Smaller Larger Charge Q.sup.72 A N S R E V.sup.76 A A G T E
G.sup.79 A A -- R (as in B27) E E.sup.154 A D S R R Q.sup.155 A N S
R E A.sup.158 -- V G T E G.sup.162 A A -- T E E.sup.166 A D S R
R
[0073] Delivery of .sup.altMHC molecules using virus vectors: Using
the current strategy, the .sup.altMHC molecule is introduced
directly into the tumor cell line by gene mediated transfer. This
requires immune recognition to occur by direct recognition of the
tumor cells, and minimizes indirect recognition mediated by
professional antigen presenting cells such as dendritic cells or
macrophages. A vaccination strategy to be examined involves
introduction of the .sup.altMHC gene directly into tumor cells
using virus-based delivery system. For example, the cDNA encoding
the .sup.altMHC molecule is introduced into an adenovirus vector,
high titer virus prepared, and the virus is used to infect tumor
cells in situ. This delivery strategy provides an approach for
developing a tumor vaccine in patients where the virus is
introduced into cancer cells by injection directly into tumor.
Immunity induced by the vaccine provides anti-tumor protection
systemically, eliminating tumors throughout the body.
Other Embodiments
[0074] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
518PRTArtificial Sequencesynthetic peptide 1Ser Ile Ile Asn Phe Glu
Lys Leu1 528PRTArtificial Sequencesynthetic peptide 2Ser Ile Ile
Asn Phe Glu Gln Leu1 538PRTArtificial Sequencesynthetic peptide
3Glu Ile Ile Asn Phe Glu Lys Leu1 548PRTArtificial
Sequencesynthetic peptide 4Ser Ile Ile Gly Phe Glu Lys Leu1
558PRTArtificial Sequencesynthetic peptide 5Ser Ile Ile Gln Phe Glu
His Leu1 5
* * * * *