Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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POLYPEPTIDES AND METHODS FOR THYMIC VACCINATION
RELATED APPLICATIONS
This application claims priority to U.S Provisional Application 60/168,167,
filed November 30, 1999 and to U.S. Provisional Application 60!167,378, filed
S November 24, 1999, the entire teachings of which are incorporated herein by
reference.
GOVERNMENT SUPPORT
The work described herein was supported in part by Grants AI19807,
GM56008, and AI45022 from the National Institutes of Health. The government
has
certain rights in the invention.
BACKGROUND OF THE INVENTION
T cell receptors (TCRs) are generated in the thymus through a stochastic
process involving rearrangement of V, D and J gene segment elements (Chien et
al.,
Nature, 312:31-35 (1984); Hendrick et al., Nature, 308:149-153 (1984); Saito
et al.,
Nature, 312:36-40 (1984); Yoshikai et al., Nature, 312:521-52 (1984); Davis,
M.M.
and Bjorkman, P.J., Nature, 334:395-402 (1988) and Fowlkes, B.J. and Pardon,
D.M., Adv. Immunol., 44:207-264 (1989)). Recombinatorial diversity resulting
from
the joining of the various gene segments and association of diverse a and ~3
subunits
coupled with functional diversity arising from N and P nucleotide additions
gives
rise to enormous diversity, approximately 10'6 TCR types. Many of these
receptor
specificities are useful to the organism in establishing protective cognate
immune
responses. On the other hand, some of the TCRs so created may be detrimental,
including ones with self reactive specificities able to mediate autoimmune
diseases.
It is the process of negative selection in the thymus that eliminates, in
large part,
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unwanted autoreactive specificities (Nossal, G.J.V., Cell, 76:229-239 (1994)).
In
addition, cells bearing TCRs unable to productively interact in any manner
with the
MHC of the host are lost through a death by neglect mechanism. Positive
selection
enriches for those useful TCRs which are restricted by self MHC molecules
expressed by the individual (Beven, M.J., Nature, 269:417-418 (1977); Berg et
al.,
Cell, 58:1035-1046 (1989); Zinkernagel et al., .I. Exp. Med., 147:882-896
(1978);
von Boehmer, H., Cell, 76:219-228 (1994); Fowlkes, B.J. and Schweighoffer, E.,
Curr. Opin. Immunol., 7:188-195 (1995); Jameson et al., Annu. Rev. Immunol.,
13:93-126 (1995)). These selection processes shape the T cell repertoire.
SUMMARY OF THE INVENTION
The present invention is drawn to methods of influencing the selection
processes of T cell receptors (TCRs) in order to influence the T cell
repertoire of an
host. In the methods, a polypeptide of interest or peptidomimetic is
administered;
the polypeptide of interest or peptidomimetic is a polypeptide that causes
selection
(either positive or negative) of thymocytes having a T cell receptor
specificity in the
thymus. The methods of influencing the TCR selection processes can be used for
thymic vaccination, which causes selection of thymocytes with TCR
specificities
that are designed to recognize disease antigens or foreign antigens, such as
to treat or
prevent cancers, autoimmune diseases, infections, or effects of biological
warfare
agents. Polypeptides, vaccine compositions, expanded thymic cell populations
produced according to the methods are described herein. Also described is a
synthetic thymus comprising stromal elements bearing relevant MHC molecules
and
loaded with the desired polypeptides suitable for carrying out the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 depicts the proliferation of splenic T cells to VSV8 and p4-substituted
VSV8 variant peptides.
Figs. 2A-2B demonstrate that L4, norvaline4, and y-methylleucine4 induce
positive selection of NlStg RAG-2-~-~32M-~- thymocytes phenotypically and
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functionally. Fig. 2A). The total number of CD8+ SP thymocytes (mean ~ SD of
four different lobes) after FTOC are shown for the indicated culture
conditions. Fig.
2B). Thymocytes selected on L4, norvaline4 and y-methylleucine4 are
functionally
responsive to V S V 8.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based on the discovery that the T cell repertoire of an
individual can be influenced by targeting the selection processes of TCRs. As
described herein, N15 TCR transgenic (tg) RAG-2-/- H-2b mice recognizing the
vesicular stomatitis virus (VSVB) octapeptide RGYVYQGL bound to Kb were
utilized in conjunction with VSV8 variants differing only at the central p4
position
to probe the specificity of TCR selection. The V4I mutant octamer, like VSVB,
induces negative selection of immature double positive (DP) thymocytes on the
(32M+/+ background and is a strong agonist for mature N15 T cells. In
contrast, V4L
or V4norvaline octamers promote positive selection in N15 tg ~i2M-/- RAG-2-/-
H-
2b FTOC and are weak agonists for N15 T cells. Hence, the absence of a p4 side
chain ~i-methyl group results in positive selection of the N15 TCR.
Hydrophobicity
of the p4 residues also modulates thymocyte fate: the positively selecting
norvaline
and leucine variants have one and two Cy-methyl groups, respectively, while
the
weakly selecting y-methylleucine p4 contains three Cy-methyl groups. Moreover,
the most hydrophobic octamer containing a p4 cyclohexylglycine substitution
fails to
select. Thus, for N15 and other class I MHC-restricted TCRs, there is a high
degree
of structural specificity to peptide-dependent thymic selection processes.
In addition, Applicants have determined that this structural specificity also
applies to Class II MHC -restricted TCRs. As described herein, the crystal
structure
of a complex involving the D10 T cell receptor (TCR), 16 residue foreign
peptide
antigen and the I-Ak self MHC class II molecule has been assessed at 3.2t~
resolution. The D10 TCR is oriented in an orthogonal mode relative to its
peptide-
MHC (pMHC) ligand, necessitated by the amino-terminal extension of peptide
residues projecting from the MHC class II antigen-binding groove as part of a
mini
(3-sheet. Consequently, the disposition of D10 CDR loops is altered relative
to that
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of most pMHCI-specific TCRs; the latter TCRs assume a diagonal orientation
although with substantial variability. Peptide recognition involves P-1 to P8
residues, being dominated by the Va domain which also binds to the class II
MHC
(31 helix. The fact that docking is limited to one segment of MHC-bound
peptide
explains the epitope recognition and altered peptide ligand effects, provides
a
structural basis for alloreactivity, and illustrates how bacterial
superantigens can
span the TCR-pMHCII surface. Furthermore, the opposing processes of positive
and
negative selection that are present in the T lineage repertoire can be probed
by
utilizing octapeptides designed based upon the limited segment of the MHC-
bound
peptide that is involved in docking.
As a result of these discoveries, methods are now available to influence the
selection processes of thymocytes bearing TCRs (both Class I and Class II MHC
restricted), and thereby to perform thymic vaccination. The term, "selection
process" refers to positive or negative selection of thymocytes with a
targeted TCR
1 S specificity in the thymus, to retain a desired specificity (positive) or
to eliminate an
undesired specificity (negative). "Thymic vaccination" refers to
administration of a
polypeptide which influences the selection processes of TCRs while still in
the
thymus, thereby altering cognate antigens in order to create variants which
positively
select desired TCR specificities at the level of repertoire development, or
which
negatively select undesired TCR specificities at the level of repertoire
development.
The invention provides a method for educating thymic cells to recognize
disease or foreign antigens not previously recognized by the immune system
(naive
preselected double positive (CD4+ CD8+) thymic cells). According to the
method,
naive thymic cells (functional thymus or synthetic thymus) are contacted with
a
polypeptide of interest that causes selection of a thymocytes with TCR
receptor
specificities in the thymus capable of recognizing disease antigens or foreign
antigens. The so produced "educated" thymic cells can then be induced to leave
the
thymus and be expanded into a population of antigen recognizing thymic cells
that is
sufficient to elicit an immune response against the disease antigens or
foreign
antigens. The methods described herein are applicable to both Class I and
Class II
MHC complex formation. In general, the antigens can be components such as
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bacterial, viruses and macro components of cells and soluble antigens such as
proteins, peptides, glycoproteins and carbohydrates. Antigens of particular
interest
are viral or bacterial antigens, allergens, tumor-associated antigens,
oncogene
products, parasite antigens, fungal antigens or fragments of these. Thus, the
peptides
and methods described herein can be used to treat cancer tumors and infections
in an
individual such as, but not limited to, infections caused by bacteria,
viruses, fungus
and parasites. Examples of human bacterial pathogens include, but are not
limited
to, Haemophilus influenzae, Escherichia coli, Neisseria meningitidis,
Streptococcus
pneumoniae, Streptococcus pyogenes, Branhamella catarrhalis, Vibrio cholerae,
Corynebacteria diphtheriae, Neisseria gonorrhoeae, Bordetella pertussis,
Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae and
Clostridium tetani. Examples of pathogenic virus include, but are not limited
to,
human immunodeficiency virus, human T cell leukemia virus, respiratory
syncyctial
virus, hepatitis A, hepatitis B, hepatitis C, non-A and non-B hepatitis virus,
herpes
simplex virus (types I and Il], cytomegalovirus, influenza virus,
parainfluenza virus,
poliovirus, rotavirus, coronavirus, rubella virus, measles virus, varicella,
Epstein
Barr virus, adenovirus, papilloma virus and yellow fever virus. Examples of
fungal
pathogens and opportunistic fungi include species of Cryptococcus, Candida,
Coccidioides, Histoplasma, Blastomyces, Sporothrix and Aspergillus.
The methods are particularly useful in vaccinating an individual that has a
genetic predisposition to disease and diseases caused by immune deficiency.
Thymic cells can be educated to distinguish between antigens of normal cells
which
are to be left unharmed and those of unwanted intruder which are to be
destroyed.
For example, a newborn can be genetically screened and if found to be at risk
for a
certain disease, the newborn's repertoire of T cells can be altered to
increase those
that are capable of recognizing disease associated antigens. The educated T
cells
can be induced to come out of the thymus by giving the infant a variation of
the
polypeptide recognizable by the educated T cells which the immune system could
be
tricked into recognizing as normal or self.
In one embodiment of the invention, a polypeptide of interest or
peptidomimetic is administered to an individual to eliminate (e.g., destroy)
thymic
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cells having TCRs of undesired or detrimental specificity (negative
selection). For
example, TCRs having self reactive specificities able to mediate autoimmune
diseases can be eliminated in the thymus before they leave and induce an
autoimmune response. According to this embodiment, thymic cells having TCRs of
undesired or detrimental specificity are contacted with polypeptides of
interest or
peptidomimetics that can reduce or eliminate cells bearing TCRs capable of
recognizing a self antigen. Once eliminated the detrimental TCRs are not
capable of
leaving the thymus and mediating autoimmune disease. Thus, the invention can
be
used to treat or prevent individuals predisposed to autoimmune diseases or
inflammatory diseases. Examples of acute and chronic immune and autoimmune
diseases include, but are not limited to, chronic hepatitis, systemic lupus
erythematosus, arthritis, thyroidosis, Scleroderma, diabetes mellitus, Graves'
disease, Beshet's disease and graft versus host disease (graft rejection).
In the methods of the invention, a polypeptide or peptidomimetic is
1 S administered to a host. The host can be any mammal; in a preferred
embodiment,
the host is a human. The host can be adult, child, infant or fetus. In a
preferred
embodiment, the host is an infant, because T cells are produced in the thymus
gland
during the first year of life, and during that time are "educated" to
distinguish
between antigens of normal cells, which are to be left unharmed, and those of
unwanted intruders, which are to be destroyed. When the host is a child or
adult, the
methods of the invention are carried out using a synthetic thymus in which
bone
marrow progenitor cells of the host are induced in an implanted thymus to
produce
thymocytes. An artificial or synthetic thymus can be implanted (e.g.,
intramuscularly, subcutaneously) into the host and comprises stromal elements
bearing relevant MHC molecules and loaded with the desired peptides or
peptidomimetic described herein.
The polypeptide of interest is a short polypeptide, having between
approximately 6 and 16 amino acids, preferably between 8 and 12 amino acids.
The
polypeptide of interest is a polypeptide which influences the TCR selection
process
in the thymus to select TCR specificities (either positive or negative). The
polypeptide can include natural amino acids, artificially created amino acids,
and/or
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amino acid analogs, and can also be modified, such as by substituted linkages,
glycosylations, acetylations, carboxylations, phosphorylations,
ubiquitination,
labeling (e.g., with radionuclides), enzymatic modifications, or other
modifications
known in the art, both naturally and non-naturally occurring. If desired, a
carrier
molecule, such as another polypeptide or other agent, can be used in
conjunction
with the polypeptide of interest.
Polypeptides useful for methods described herein are generated by
structurally altering the central recognition position of the peptide, i.e.,
p4 for Class I
MHC-restricted TCRs and p5 for Class II MHC-restricted TCRs, as described
herein. For example, the VSV8 peptide (RGYVYQGL) (SEQ. 117 NO:1) and
variants having other amino acids at position 4 (e.g., isoleucine, leucine,
norvaline,
y-methylleucine or cyclohexylglycine) can be used for Class I MHC-restricted
TCRs. However, other TCR contact residues of the peptide can be altered.
Polypeptides used herein can be isolated from naturally-occurnng sources,
chemically synthesized or recombinantly produced. For example, a nucleic acid
molecule can be used to produce a recombinant form of the encoded polypeptide
via
microbial or eukaryotic cellular processes. Ligating the polynucleotide
sequence
into a gene construct, such as an expression vector, and transforming or
transfecting
into hosts, either eukaryotic (yeast, avian, insect, plant or mammalian) or
prokaryotic
(bacterial cells), are standard procedures used in producing other well known
proteins. Similar procedures, or modifications thereof, can be employed to
prepare
recombinant polypeptides by microbial means or tissue-culture technology. The
polypeptides can be isolated or purified (e.g., to homogeneity) from cell
culture by a
variety of processes. These include, but are not limited to, anion or cation
exchange
chromatography, ethanol precipitation, affinity chromatography and high
performance liquid chromatography (HPLC). The particular method used will
depend upon the properties of the polypeptide; appropriate methods will be
readily
apparent to those skilled in the art. For example, with respect to protein or
polypeptide identification, bands identified by gel analysis can be isolated
and
purified by HPLC, and the resulting purified protein can be sequenced.
Alternatively, the purified polypeptide can be enzymatically digested by
methods
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known in the art to produce polypeptide fragments which can be sequenced. The
sequencing can be performed, for example, by the methods of Wilm et al.
(Nature
379(6564):466-469 (1996)). The polypeptide may be isolated by conventional
means of protein biochemistry and purification to obtain a substantially pure
S product, i.e., 80, 95 or 99% free of cell component contaminants, as
described in
Jacoby, Methods in Enzymology Volume 104, Academic Press, New York (1984);
Scopes, Protein Purification, Principles and Practice, 2nd Edition, Springer-
Verlag,
New York (1987); and Deutscher (ed), Guide to Protein Purification, Methods in
Enzymology, Vol. 182 (1990).
The sequence of the polypeptide can be determined, for example, by
assessing the foreign peptide antigen that binds in the complex of a T cell
receptor,
antigen and MHC Class I or Class II molecule, such as by examining the crystal
structure, as described below. The crystal structure indicates which amino
acids of
the polypeptide interact with the TCR. Those amino acids of the polypeptide
which
interact with TCR are referred to herein as "active amino acids," and their
position
in the polypeptide (e.g., as amino acid #1, #4, #5 or #6) is referred to as an
"active
amino acid position". Varying the type of amino acid at the active amino acid
position by substituting the original amino acid with other amino acids,
allows
determination of what type of amino acids contribute to positive or negative
selection of desired TCR specificities. The ability of a polypeptide of
interest to
stimulate proliferation of the desired TCR cells can be assessed by culturing
splenocytes with samples of the polypeptide of interest and assessing
activation of
the T cells, as described below. The ability of a polypeptide of interest to
interact
with TCRs on thymocytes can be assessed by performing thymocyte dulling
assays,
as described below. To assess the ability of a polypeptide of interest to
influence
TCR negative specificity, an assay can be performed, such as an in vivo assay
as
described below in which a host is inoculated with the peptide of interest,
and the
surviving subsets of thymocytes examined after an appropriate incubation time.
To
assess the ability of a polypeptide of interest to influence positive
specificity, an
assay can be performed, for example, using fetal thymic organ culture as
described
below in which the organ is cultured with a sample of the polypeptide of
interest,
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and the type of thymocytes produced are examined after an appropriate
incubation
time. As described herein, a mouse recipient has been used to exemplify the
invention. Similarly, a mouse model having a humanized immune system can be
used to appropriately predict human therapies.
Alternatively, a peptidomimetic having the same characteristics as a
polypeptide of interest (e.g., the peptidomimetic can influence the selection
processes of TCRs in the thymus, altering cognate antigens and creating
variants
which positively select desired TCR specificities or which negatively select
undesired TCR specificities) can be used. The peptidomimetic can be, for
example,
a complex carbohydrate or other oligomer of individual units or monomers which
binds specifically to its binding partner (e.g., the TCR). Peptidomimetics can
be
developed, for example, with the aid of computerized molecular modeling (see
e.g.,
Fauchere, J. Adv. Drug Res., 15:29 (1986); Veber and Freidinger, TINS 392
(1985);
and Evans et al., J. Med. Chem., 30:1229 (1987)). Peptidomimetics that are
structurally similar to the peptides described herein can be used to produce
an
equivalent therapeutic effect. Generally, peptiomimetics are structurally
similar to a
paradigm peptide (i.e., a peptide that has a biological or pharmacological
activity),
such as a peptide of interest herein, but have one or more peptide linkages
optionally
replaced by other organic linkages. Peptidomimetics may be generated, for
example,
by methods described in Spatola, A.F. in CHEMISTRY AND BIOCHEMISTRY OF
AMINO ACIDS, PEPTIDES, AND PROTEINS 267 (B. Weinstein, eds. 1983);
Spatola, A.F., Vega Data Vol. 1, Issue 3, "Peptide Backbone Modifications"
(March
1983)(general review); Moreley, J.S., Trends Pharm. Sci., pp. 463-468
(1980)(general review); Hudson, D. et al., Int. J. Pept. Prot. Res. 14:177-185
(1979);
Spatola, A.F. et al., Life Sci. 38:1243-1249 (1986); Hann, M., .I. Chem. Soc.
Perkin
Trans. 1307-314 (1982); Alnquist, R.G. et al., J. Med. Chem. (1980) 23:1392-
1398;
Jennings-White, C. et al., Tetrahedron Lett. 23:2533 (1982); Szelke, M. et
al.,
European Appln. EP 45665 (1982) CA:97:39405 (1982); Holladay, M.W. et al.,
Tetrahedron Lett. 24:4401-4404 (1983); and Hruby, V. J., Life Sci. 31:189-199
(1982).
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To perform the methods of the invention, a polypeptide of interest or
peptidomimetic, which influences the selection processes of thymocytes with
TCRs
of targeted specificities, is administered to the host animal. The polypeptide
of
interest or peptidomimetic is administered by a means which exposes it to the
immune system in the host animal. In a preferred embodiment, the polypeptide
of
interest can be administered in a pharmaceutical composition. For instance, a
polypeptide or peptidomimetic can be formulated with a physiologically
acceptable
Garner or excipient to prepare a pharmaceutical composition. The carrier and
composition can be sterile. The formulation should suit the mode of
administration.
Suitable pharmaceutically acceptable Garners include but are not limited to
water, salt solutions (e.g., NaCI), saline, buffered saline, alcohols,
glycerol, ethanol,
gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin,
carbohydrates such as lactose, amylose or starch, dextrose, magnesium
stearate, talc,
silicic acid, viscous paraffin, perfume oil, fatty acid esters,
hydroxymethylcellulose,
polyvinyl pyrolidone, etc., as well as combinations thereof. The
pharmaceutical
preparations can, if desired, be mixed with auxiliary agents, e.g.,
lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic
pressure, buffers, coloring, flavoring and/or aromatic substances and the like
which
do not deleteriously react with the active compounds.
The composition, if desired, can also contain minor amounts of wetting or
emulsifying agents, or pH buffering agents. The composition can be a liquid
solution, suspension, emulsion, tablet, pill, capsule, sustained release
formulation, or
powder. The composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can include
standard
carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium
carbonate,
etc.
Methods of introduction of these compositions include, but are not limited
to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous,
subcutaneous, oral and intranasal. Other suitable methods of introduction can
also
include gene therapy, rechargeable or biodegradable devices, particle
acceleration
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devises ("gene guns") and slow release polymeric devices. The pharmaceutical
compositions of this invention can also be administered as part of a
combinatorial
therapy with other agents.
The composition can be formulated in accordance with the routine
procedures as a pharmaceutical composition adapted for administration to human
beings. For example, compositions for intravenous administration typically are
solutions in sterile isotonic aqueous buffer. Where necessary, the composition
may
also include a solubilizing agent and a local anesthetic to ease pain at the
site of the
injection. Generally, the ingredients are supplied either separately or mixed
together
in unit dosage form, for example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the composition is to be
administered
by infusion, it can be dispensed with an infusion bottle containing sterile
pharmaceutical grade water, saline or dextrose/water. Where the composition is
administered by injection, an ampoule of sterile water for injection or saline
can be
provided so that the ingredients may be mixed prior to administration.
Agents described herein can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with free amino groups
such
as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc.,
and those formed with free carboxyl groups such as those derived from sodium,
potassium, ammonium, calcium, fernc hydroxides, isopropylamine, triethylamine,
2-
ethylamino ethanol, histidine, procaine, etc.
The agents are administered in an effective amount. The amount of agents
which will be effective in the generation of desired TCRs can be determined by
standard clinical techniques. In addition, in vitro or in vivo assays may
optionally be
employed to help identify optimal dosage ranges. The precise dose to be
employed
in the formulation will also depend on the route of administration and should
be
decided according to the judgment of an individual of ordinary skill in the
art.
Effective doses may be extrapolated from dose-response curves derived from in
vitro or animal model test systems.
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Administration of the polypeptide of interest or peptidomimetic alters TCRs
with specificity for cognate antigens and thereby positively selects desired
TCR
specificities at the level of repertoire development, or which negatively
select
undesired TCR specificities at the level of repertoire development. This
alteration,
also referred to as "thymic vaccination," can be used to generate specific
reactivities
in the thymus, in order to generate TCRs which are designed to recognize
disease
antigens or foreign antigens, such as to treat or prevent cancers, autoimmune
diseases, infections, or effects of biological warfare agents. For example, if
an
individual was at risk for a certain disease (e.g., childhood leukemia), a
polypeptide
of interest or peptidomimetic that generates positive selection for TCRs that
are
capable of recognizing cells affected by the disease can be administered.
Alternatively, if an individual was at risk for, or affected by, an autoimmune
disease,
a polypeptide of interest or peptidomimetic that generates negative selection
of
"self' antigens can be administered, so that TCRs that recognize the self
antigen are
reduced or eliminated.
The invention also provides a pharmaceutical pack or kit comprising one or
more containers filled with one or more of the ingredients (e.g., the
polypeptides of
interest or peptidomimetics) of the pharmaceutical compositions described
herein.
Optionally associated with such containers) can be a notice in the form
prescribed
by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals
or biological products, which notice reflects approval by the agency of
manufacture,
use of sale for human administration. The pack or kit can be labeled with
information regarding mode of administration, sequence of drug administration
(e.g.,
separately, sequentially or concurrently), or the like. The pack or kit may
also
include means for reminding the patient to take the therapy. The pack or kit
can be a
single unit dosage of the combination therapy or it can be a plurality of unit
dosages.
In particular, the agents can be separated, mixed together in any combination,
present in a single vial or tablet. Agents assembled in a blister pack or
other
dispensing means is preferred. For the purpose of this invention, unit dosage
is
intended to mean a dosage that is dependent on the individual pharmacodynamics
of
each agent and administered in FDA approved dosages in standard time courses.
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The invention is further illustrated by the following references, which are
not
intended to be limiting in any way. The teachings of all references cited
herein are
incorporated by reference in their entirety.
EXAMPLES
The examples set forth below describe in detail many diagrams of molecular
structures and interactions. Color photographs of the diagrams can be found in
the
following two references which describe Applicants' work and which were
published after the filing date of the priority applications: Sasada, T. et
al., Eur. .l.
Immunol. 30(5):1281-9 (May 2000), and in Reinherz, E.L. et al., Science 286
(5446):1913-21 (December 1999). The entire teachings of these references, and
the
references cited in the Specification, are incorporated herein by reference.
EXAMPLE 1 Influence of Subtle Structural Variation Involving the p4 Residue of
an MHC Class I-Bound Peptide on Thymic Selection
Recently, using TCR-transgenic (NlStg) (32-microglobulin deficient (~32M-~-)
RAG-2-~- H-2b mice specific for the VSV8 octapeptide bound to Kb, a single
weak
agonist peptide variant V4L (L4) was identified which induced phenotypic and
functional T cell maturation (Ghendler et al., Proc. Natl. Acad. Sci. USA,
95:10061-
10066 (1998)). The cognate VSV8 peptide, in contrast, triggered negative
selection.
The crystal structure of L4/Kb was determined and refined to 2.1~ for
comparison
with the VSV8/Kb structure at similar resolution. Aside from changes on the
side
chain of the p4 position of L4 and the resulting alteration of the exposed Kb
Lys66
side chain, these two structures are essentially identical. Hence, focal,
local
structural change in the pMHC can be readily discerned by the TCR.
An objective of the work described herein was to define a structure-activity
relationship between alteration of the p4 side chain constituent and thymic
selection.
The findings suggested that the survival outcome for thymocyte-bearing the N15
TCR can be reduced to simple chemical parameters involving the selecting
peptide.
Similar rules apply to at least some other class I MHC-restricted TCRs.
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EXPERIMENTAL PROCEDURES
Mice: N15 TCR tg (N15+/+) RAG-/- (H-2b) and N15 TCR tg
(N15+/+) RAG-/- ~32M-/- mice were generated as previously described (Ghendler
et
al., Eur. J. Immunol., 27:2279-2289 (1997); Ghendler et al., J. Exp. Med.,
187:1529-
1536 (1998)). The lack of RAG-2 or ~3zM gene expression in knockout animals
were
identified based on the fluorescence-activated cell sorter (FACS) analysis on
peripheral blood cells (Ghendler et al., Proc. Natl. Acad. Sci. USA, 95:10061-
10066
(1998)). The homozygosity of the N15 TCR transgenes was proved by subsequent
breeding analysis. TAP-/- (129/01a X C57BL/6) mice were purchased from Taconic
(NY). All lines were maintained and bred under sterile barner conditions at
the
animal facility of Dana-Farber Cancer Institute.
Peptides synthesis
VSV8 variant peptides were synthesized by standard solid phase methods on
an Applied Biosystems 430A synthesizer (Foster City, CA) at the Biopolymers
Laboratory of Massachusetts Institute of Technology. Norvaline (nV) and y-
methylleucine (mL) were obtained from Bachem Biosicence Inc. (King of Prussia,
PA) and cyclohexylglycine (chG) was from Sigma (St. Louis, MO). All peptides
were purified by reverse phase HPLC (Hewlett Packard HPLC 1100, Palo Alto, CA)
with a C4, 2 mm column. Peptides were analyzed for purity and correct
molecular
weight by electrospray mass spectrometry, amino acid analysis and HPLC.
Peptides
are named to indicate the substituted amino acid and the position in the
sequence
(e.g., I4 denotes replacement of valine by isoleucine at the fourth residue of
the
VSV8 peptide).
Antibodies and flow cytometric analysis
The following mAbs were used: R-phycoerythrin (PE) anti-mouse CD4
(H129.19; Life Technologies, Grand Island, NY); Cychrome anti-mouse CDBa (53-
6.7; Pharmingen, San Diego, CA). For flow cytometry, single cell thymocyte
suspensions were prepared in phosphate-buffered saline (PBS) containing 2%
fetal
calf serum (FCS). Thymocytes were stained at 5 x 106 cells per ml in PBS-2%
FCS
containing the antibodies at saturating concentrations. Phenotypes and
proportions
of thymocyte subsets were analyzed by two-color flow cytometry using FACScan
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(Beckton Dickinson) and the Cell Quest program. Dead cells were excluded from
the analysis by forward and side scatter gating.
DP dulling assay
Peritoneal exudate cells (PEC) from TAP-/- (129/01a X C57BL/6) mice
(Taconic), induced 5 days previously with 2m1 of 3% thioglycollate, were
suspended
in AIM-V medium (Life Technologies) and plated at 1 x 106 per well in a 96-
well
microtiter plate. After adherence for 2 hr, monolayers were washed with AIM-V
medium four times. Thymocytes (5 x 105) from 4- to 6-week-old N15 tg
RAG-/-/ (32M-/- were co-cultured with the PEC for 18 hr at 37°C, and
stained for the
expression of CD4 and CDBa.
Peptide injection
Negative selection was examined by the reduction of CD4+CD8+ DP
thymocytes in N15 tg RAG-/- ~i2M-/- H-26 mice after injection of peptides as
described (Ghendler et al., Eur. J. Immunol., 27:2279-2289 (1997); Ghendler et
al.,
J. Exp. Med., 187:1529-1536 (1998)). Twenty-four ~,g of each peptide dissolved
in
200 ml PBS was injected into the tail vein of 4-week-old NlStg RAG-2-/- mice.
After 24 hr of peptide injection, thymocytes were stained for the expression
of CD4
and CD8a.
Fetal thymic organ culture (FTOC)
Fetuses of N15 tg RAG-/- (32M-/- H-2b mice were dissected at day 15.5 (plug
= day 1) and fetal thymic lobes were cultured with or without the indicated
peptides
as described (Clayton et al., EMBO J., 16:2282-2293 (1997)). Human ~3zM
(Calbiochem, San Diego, CA) was added to AIM-V medium at 5 pg/ml, and the
medium was replaced every 48 h. After 7 days, thymocytes were stained for the
expression of CD4 and CDBa, or were tested for their capacity to respond to
antigen
in a 2 day proliferation assay, as described below.
Proliferation assay
Thymocytes from the organ cultures or fresh splenocytes from N15 tg RAG-
2-/- H-2b mice (1 x 105/well) were incubated at 37°C with 2 x 104
irradiated EL-4
cells, which were pre-loaded for 2 h with 1 nM or 10 nM VSV8 in the presence
of
100 U/ml recombinant IL-2 in AIM-V medium containing 50 ~,M 2-ME. After 48 h .
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of incubation, 0.4 p,Ci per well of 3H-TdR (ICN Biomedicals, Aurora, OH) was
added, and after an additional 18 h of culture at 37°C, the cells were
harvested and
the incorporated radioactivity was measured.
RESULTS AND DISCUSSION
p4 variants of the VSV8 octapeptide
In the B6 mouse, the VSV8 octapeptide bound to the Kb MHC class I
molecule is a major determinant of the vesicular stomatitis virus nuclear
protein
recognized by CD8 CTL. When VSV8 is in complex with Kb, only p1 Arg, p4 Val
and p6 Gln peptide side chains point to solvent. Hence, three VSV8 residues
can
make contact with the TCR (Ghendler et al., Proc. Natl. Acad. Sci. USA,
95:10061-
10066 (1998)). An examination of the structure of the VSV8 octamer bound to H-
2Kb and variant p4 side chains shows upward pointing TCR contact p 1 R, p4V
and
p6Q residues. Substitutions at any of these positions dramatically and
adversely
affect T cell recognition as judged by cytolytic effector function (Ghendler
et al.,
Proc. Natl. Acad. Sci. USA, 95:10061-10066 (1998)) or other functional
measurements of T cell activation (Ghendler et al., Proc. Natl. Acad. Sci.
USA,
95:10061-10066 (1998)). The effects of alterations at the p4 position on
thymic
selection were studied in detail since this residue is centrally located on
the
recognition surface of the VSV8 Kb complex when bound to the N15 TCR
(Ghendler et al., Proc. Natl. Acad. Sci. USA, 95:10061-10066 (1998)).
Moreover,
earlier studies showed that even a conservative substitution, namely p4 Val
Leu,
altered the fate of DP thymocytes, leading to positive selection in contrast
to
negative selection resulting from in vivo injection of N15 tg Rag-2-~- H-2b
mice
with the cognate peptide (Ghendler et al., Proc. Natl. Acad. Sci. USA,
95:10061-
10066 ( 1998)).
In the present study, a series of altered peptide ligands (APL) of VSV8 were
created, harboring highly related yet distinct amino acid substitutions at the
p4
position. The amino acids used for the substitutions included valine,
isoleucine,
leucine, norvaline, gamma-methylleneine, and cyclohexylglycine. The structure
of
the side chains of these substitutions varied, relative to the valine R group
at the p4
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position of VSVB. In particular, for example, the Isoleucine4 variant (I4),
like the
p4 Val in VSVB, has a C~3 branch (methyl group) whereas the Leucine4 (L4)
variant
does not. The hydrophobicity of I4 and L4 are identical, however. Norvaline4,
L4
and y-methylleucine4 all lack a C(3 methyl group but differ in having 1, 2 and
3 Cy-
methyl groups, respectively. Cyclohexylglycine4 is the most hydrophobic of all
p4
side chain R groups. Given that the p3, p5 and p8 anchor residues have not
been
modified in these APL, it is not surprising that the Kb binding affinity for
these
variants relative to VSV8 are unaltered (Ghendler et al., Proc. Natl. Acad.
Sci. USA,
95:10061-10066 (1998)).
Activation of mature N15 splenic CD8+ T cells by p4 variants
To examine the ability of APL to stimulate proliferation of CD8+ N15 TCR-
bearing T cells, splenocytes (1 x 105) from N15 tg Rag-2-~- H-2b mice were
cultured
with varying molar concentrations of the individual peptides using irradiated
EL4
cells as Kb-bearing APC (2 x 104). After 48 h of stimulation, cells were
pulsed
with 3H-TdR and mean ~ SD of triplicate cultures determined. As shown in Fig.
1,
both VSV8 and I4 maximally stimulate 3H-TdR incorporation by 10-8 M. In
contrast, in norvaline4 and L4 maximal stimulation requires a 10-S M peptide
concentration. Thus, norvaline4 and L4 are weak agonists, differing by z 1000
fold
from I4 and VSVB. y-methylleucine4 and cyclohexylglycine4 are even weaker with
detectable stimulating activity present only at a peptide concentration of 10-
4 M.
Interaction of the NI S TCR with VSV8 and APL detected by DP thymocyte
dulling assay
To determine which of these APL when complexed to cell surface bound Kb
is able to interact with the N15 TCRs on immature thymocytes, a thymocyte
dulling
assay was performed. For this purpose, N15 tg Rag-2-~- (32M-~- thymocytes were
cultured in vitro for 18 h with peritoneal exudate cells (PEC) from TAP-- mice
at
varying peptide concentrations. In this assay (Barnden et al., Eur. J.
Immunol.,
24:2452-2456 (1994); Hogquist et al., Immunity, 6:389-399 (1997); Vasquez et
al.,
J. Exp. Med., 175:1307-1316 (1992)), TCR interaction with pMHC ligand is
detected as a reduction in the intensity of CD4 and CD8 expression on the
surface of
the DP thymocytes. Alterations in the expression of CD4 and CDBa on DP
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thymocytes were detected by flow cytometry after gating on 10,000 live cells.
Thus,
the DP dulling assay results detected the interactions of NlStg thymocytes
with
VSV8 and altered peptide ligands of VSV8. A negative control was used that
contained thymocytes plus PEC cultured in the absence of any exogenous peptide
additions; for the negative control, in the absence of peptide, 90% of the
cells are DP
thymocytes whose CD4 and CD8 expression falls in an expected range. In
contrast,
in the VSV8 exposed cultures, only 10-26% of thymocytes fall in this range at
peptide concentrations from 10 ~,M to 1 nM. The remaining thymocytes have
clearly reduced co-receptor expression levels. Even at a VSV8 concentration of
10
pM, there is dulling of a fraction (~5%) of DP thymocytes. An identical result
was
observed with I4. Dulling is also observed with L4 at 10 ~,M and 100 nM
concentrations. With norvaline4, dulling is seen at 10 ~M and minimally at 100
nM
whereas with y-methylleucine4 or cyclohexylglycine4, activity is only observed
at a
peptide concentration of 10 mM. Thus, interaction is observed with all VSV8-
related peptides. The potency of the dulling effects vary with the individual
peptides, roughly corresponding to the strength of agonist activity observed
with the
mature peripheral T cells, hence VSV8 > L4/norvaline4 > y-
methylleucine4/cyclohexylglycine4.
Negative selection of N15 TCR-bearing DP thymocytes by octamers with a
p4 residue containing a Cb methyl group
While the above dulling assay offers a sensitive means to detect TCR-pMHC
interaction involving thymocytes and APCs, it does not provide information
about
the ability of peptides to mediate positive vs. negative selection. To examine
the
capacity of the various peptides to induce negative selection of DP
thymocytes, an in
vivo assay was performed. Individual N15 tg Rag-2-/- H-2b mice were injected
i.v.
with 24 ~,g of each peptide and the surviving subsets of thymocytes examined
after
24 h. The expression of CD4 and CDBa in thymocytes were detected by two-color
flow cytometry after gating on live cells. Without injection, the % of DP
thymocytes
is 81. This number is virtually the same for L4, norvaline4, y-methylleucine4
and
cyclohexylglycine4 (64, 72, 75 and 84%, respectively). By contrast, with VSVB
and
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I4, the % of DP thymocytes was diminished (14 and 18, respectively). Prior
studies
showed that the majority of DP thymocytes were deleted by a caspase-dependent
apoptotic mechanism during this time (Ghendler et al., J. Exp. Med., 187:1529-
1536
(1998); Clayton et al., EMBO J., 16:2282-2293 (1997)). Consistent with this
observation, <10% of the surviving thymocytes were DP in VSV8 injected animals
compared with 81% in uninfected animals. A similar deletion was observed
following administration of the I4 peptide. In contrast, L4, norvaline4, ~y-
methylleucine4 and cyclohexylglycine4 induced no deletion. Careful titration
analysis of varying molar concentrations of each peptide in FTOC failed to
detect
significant reduction in DP thymocytes at any concentration of L4, norvaline
4, y-
methyleucine4 and cyclohexylglycine4 tested. In view of the chemical
differences
among these VSV8.variants, it would appear that a Chi methyl group needs to be
present on the p4 side chain side chain to induce negative selection.
Positive selection by octamers lacking a p4 C~ methyl group and further
modulation by side chain hydrophobicity
To investigate which other APL in addition to L4 might induce positive
selection, FTOC was performed with N15 tg Rag-2-~- (3zM-~- thymus lobes using
synthetic media and human (32M with and without VSV8 or other individual APL.
FTOC was performed by using NlStg RAG-2-~- ~iZM-~- thymic lobes in AIM-V
medium containing 5 mg/ml human ~3zM with or without the indicated peptides
(VSVB, I4, L4: 10 ~,M; norvaline4, y-methylleucine4, Cyclohexylglycine4: 100
wM). After 7 days, thymocytes were released from the lobes by passing through
a
steel mesh and cell numbers were counted by flow cytometry to detect CD4
versus
CDBa staining profiles of total thymocytes after FTOC.. With VSV8 in I4, only
1%
of CD8 SP thymocytes were generated compared to 9% for the control culture
(minus peptide). With 100 ~,M L4 and norvaline4, 57% and 63%, respectively, of
thymocytes were CD8+ after 7 days of culture. Positive selection was also
detected
using 10 ~M concentrations of these two peptides. Although some positive
selection was also induced by 100 ~.M y-methylleucine, no increase in positive
selection was observed at a 10 wM peptide concentration.
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Moreover, positive selection was not augmented by cyclohexylglycine4 even
at a 100 ~.M peptide concentration. Fig. 2A offers information on the absolute
number of surviving CD8 SP thymocytes generated. The total number of CD8+ SP
thymocytes (mean ~ SD of four different lobes) after FTOC are shown for the
indicated culture conditions. The numbers of CD8 SP thymocytes were calculated
by quantifying the total numbers of thymocytes and percentages of CD8+ SP
subsets
determined above by FACS. Number of CD8 SP thymocytes positive selected by
L4 and norvaline4 are quite comparable and each ~10 fold more than the
"peptide
minus" control.
Perhaps more importantly, thymocytes from the above organ cultures or fresh
thymocytes from an adult NlStg RAG-2-/- (H-2b) mice (1 x 105/well) were
assayed
for their proliferative response to 2 x 104 irradiated EL-4 cells, in the
presence of 1
nM or 10 nM VSV8 or no peptide. [3H] thymidine incorporation was determined
after 48 hr. Results are shown as mean ~ SD of triplicate cultures in Fig. 2B,
which
demonstrates that the phenotypic increase in CD8 SP thymocytes induced by L4
and
norvaline4 and, to a lesser extent, by y-methylleucine4, is accompanied by
functional maturation.
Hence, if FTOC are established in the presence of L4 or norvaline4 for 7
days, the subsequent immune response of the harvested thymocytes to the VSV8
cognate peptide is dramatically increased as judged by cellular proliferation
of
VSV8 at 1 or 10 nM. In fact, the proliferation of thymocytes obtained from the
L4
or norvaline4 culture at FTOCs is comparable to that of adult N1 S tg Rag-2-/-
H-2b
thymocyte controls. By contrast, little proliferation is observed to VSV8 by
thymocytes harvested from FTOC lacking exogenous peptide addition. Consistent
with the data discussed above, moreover, exposure of FTOC to ~y-methylleucine4
for
7 days augments subsequent proliferation of fetal thymocytes to VSV8 whereas
comparable culture with cyclohexylglycine4 is without effect.
Implications
Recently, three experimental approaches toward characterization of the role
of peptides in positive selection have been utilized: 1 ) analysis of FTOC in
non-TCR
tg or TCR tg mouse strains carrying mutations that interfere with peptide
loading
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and surface expression of class I MHC molecules ((32M-~-, TAP1-~-) (Ashton-
Rickardt et al., Cell, 76:651-663 (1994); Hogquist et al., J. Exp. Med.,
177:1469-
1473 (1993); Hogquist et a.1., Cell, 76:17-27 (1994); Hogquist et al.,
Immunity,
3:79-86 (1995); 2) analysis of the T cell repertoire of class II-restricted
responses in
mice expressing a single pMHC class II complex generated from a transgenic
construct of class II (3 chain covalently linked to a peptide, or in H-2M null
mutant
mice where class II molecules are filled with an invariant chain (Ii) peptide
(Ignatowicz et al., Cell, 84:521-529 (1996); Ignatowicz et al., Immunity,
7:179-186
(1997); Tourne et al., Immunity, 7:187-195 (1997); Surh et al., Immunity,
7:209-219
(1997); Grubin et al., Immunity, 7:197-208 (1997); or 3) analysis of T cell
responses
in mice intrathymically infected by an adenoviral-based vector-mediated
delivery of
invariant chain-peptide fusion proteins (Nakano et al., Science, 275:678-683
(1997)). From such studies, it is clear that peptides are essential to foster
positive
selection, that peptides bound to a given MHC allele mediate positive
selection in a
manner which is quite specific for a given TCR but that a single pMHC complex
can
facilitate differentiation of a substantial number of TCRs. For example, in
non-tg
~32M-~- FTOC, the size of the selected CD8 SP repertoire increases with the
greater
complexity of exposed peptides. Analysis of FTOC for TCR tg TAP1-~- and TCR
~32M-~- is also consistent with the role for peptides in induction of positive
selection.
Although initial results suggested that in the case of the TCR tg (32M-~-
model,
antagonistic peptides induced positive selection while the cognate peptide
induced
negative selection, the relationship between agonist and antagonist vis-a-vis
selection has become less clear (Spain et al., Immunol., 152:1709-1717 (1994);
Page
et al., Proc. Natl. Acad. Sci. USA, 91:4057-4061 (1994); Sebzda et al., J.
Exp. Med.,
183:1093-1104 (1996)). Other studies, particularly those utilizing TCR tg TAP1-
~-
FTOC have shown that positive selection can be driven by low concentrations of
cognate antigenic peptide whereas negative selection can be induced by high
concentrations of the same peptide (Ashton-Rickardt et al., Cell, 76:651-663
(1994)). Moreover, in animals exposed to the adenovirus delivery system,
cognate
peptide fosters positive selection in a class II MHC-based system (Nakano et
al.,
Science, 275:678-683 (1997)).
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For the N15 TCR, no concentration of VSV8 or I4 agonists was observed to
induce positive selection in FTOC. The specificity of the selection process is
striking. Hence, L4 and I4 VSV8 variant peptides induced positive and negative
selection, respectively, despite the fact that all residues except p4 are the
same.
S Moreover, the hydrophobicity of the two p4 substituents is identical. These
findings
indicate that the [3 branch of the isoleucine results in negative selection
while the
absence of the ~i branch facilitates development, i.e. positive selection.
That VSV8
itself induces negative selection of N1 S TCR bearing DP thymocytes is
consistent
with this notion since valine has a C(3 methyl group as well. Crystallographic
data
further supports this hypothesis. In the case of the L4/Kb structure, the
aliphatic
portion (C~3 and Cy atoms) of the exposed side chain of Lys66 on the a 1 helix
of Kb
swings about 2.5~ toward the antigen binding groove in the absence of p4 side
chain
C(3 methyl group (Ghendler et al., Proc. Natl. Acad. Sci. USA, 95:10061-10066
(1998)). By contrast, in VSV8/Kb there is van der Waal's contact between the
Cy2
atom of the p4 Val of VSV8 and the Cd atom of Lys66 (Ghendler et al., Proc.
Natl.
Acad. Sci. USA, 95:10061-10066 (1998)). This interaction helps to "prop up"
the
Lys66 side chain. Likewise, in the I4/Kb structure which has been recently
obtained
(Ghendler et al., Proc. Natl. Acad. Sci. USA, 95:10061-10066 (1998)) the side
chain
of I4 makes close contact with the C8 atom of Lys66. The movement of the Lys66
side chain towards the MHC groove in the L4/Kb complex may increase the off
rate
of the TCR-pMHC interaction sufficiently to permit the N15 TCR tg DP
thymocytes
to escape from TCR-triggered negative selection. Conversely, by directing the
Lys66 side chain upwards toward the TCR, it is postulated that the p4 ~3-
methyl
groups of VSV8 and its I4 variant augment the TCR-pMHC interaction. The latter
would facilitate negative selection. Given that the monomeric affinity of the
N15
TCR for VSV8/Kb is >200~.M, the predictably worse affinity of the N15 TCR for
L4/Kb is not measurable by existing BIAcore technology.
Another determinant of selection outcome is the hydrophobicity of the p4
side chain. Thus, while norvaline4 and L4 peptides with one and two Cy-methyl
groups, respectively, induced equivalent levels of positive selection, the
three Cy
methyl group containing y-methylleucine p4 R group was a weak positive
selector.
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The larger and hydrophobic cyclohexylglycine was without any discernible
positively selecting effect. As none of these four variants induced negative
selection, it seems unlikely that the hydrophobicity enhanced TCR contact.
More
likely, the increasing side chain bulk serves to reduce interface
complementarity,
making contacts less favorable for either negative or positive selection.
It seems clear that p1 and p6 residues of the VSV8 octamer participate in the
selection process as well. For example, a p1 Arg Lys mutation in VSV8 results
in a
peptide with neither the ability to negatively or positively select in the N15
TCR tg
system (Ghendler et al., Proc. Natl. Acad. Sci. USA, 95:10061-10066 (1998)).
Nonetheless, of more than 4 dozen APL variants at p1, p4 and p6 tested, the
L4,
norvaline4 and y-methylleucine4 peptides are the only ones able to induce
positive
selection. Given these observations and the central position of the p4 residue
in the
TCR-pMHC interaction surface, these results suggest that alteration at p4 is
pivotal
for regulating the balance between positive and negative selection. In view of
the
common docking orientation of TCRs to pMHCl molecules, moreover, it seems
likely that these results are applicable to other class I-restricted TCRs as
well.
EXAMPLE 2 Specific Recognition Function of T Lymphocytes
The adaptive immune response is dependent on the specific recognition
function of a~i T lymphocytes (1). Each T cell detects a protein fragment
(i.e.
peptide) of a self protein or cell-associated pathogen derived from either
viral,
bacterial, fungal, parasitic or tumor cell origin bound to a major
histocompatibility
complex (MHC) molecule. The physical binding of the peptide-MHC (pMHC)
complex to the TCR then initiates a series of signal transduction events. Once
triggered, T lymphocytes release cytotoxic molecules and/or inflammatory
cytokines
which destroy the infected or otherwise altered cells through various effector
mechanisms. For a given a~3 T lymphocyte, immune recognition is mediated via a
clonotypic a~i heterodimeric structure (Ti) non-covalently associated with the
monomorphic CD3 signaling components.
Sequence analysis of TCR a~3 heterodimers first suggested that they would
share with antibodies a common structure (2); however, direct evidence
supporting
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this notion has been provided only in the last several years, initially from
crystal
structures of a~i TCR components (3) and subsequently through analysis of
intact a~3
TCR heterodimers alone (4) or in complex with pMHC (5). As anticipated, aside
from the Ca domain, the three dimensional structure of the TCR resembles an
antibody Fa~3 fragment such that each of the a and (3 chains consists of
canonical
immunoglobulin (Ig)-like variable and constant domains with the hypervariable
complementarity-determining regions (CDRs) from the two variable domains (Va
and Vii) forming the ligand binding site for pMHC within the immunorecognition
module.
Class I and class II MHC molecules have evolved to facilitate T cell
detection of pathogens residing in distinct intracellular compartments (6-8).
Although the domain organization of the class I and class II MHC extracellular
segments is different, these molecules possess a very similar overall antigen
presenting groove consisting of a 1 plus a2 domains and a 1 plus ~i 1 domains
for
1 S class I and class II MHC, respectively (9-11 ). For both molecules, the a-
helices of
these two domains form the sides of the antigen binding groove with the floor
created by an eight-stranded (3-sheet arising from both domains. However,
unique
structural features of the two MHC classes dictate the binding of peptides
differing
in length and composition (reviewed in 12). The bipartite nature of the immune
recognition molecules expressed on antigen presenting cells is reflected at
the level
of a~i T lymphocytes by the evolution of two subsets bearing specialized MHC
binding structures, termed CD4 and CD8 (13, 14). CD8 cells are cytolytic
precursor/effector cells, whereas CD4 cells comprise the helper T cell subset
which
initiates inflammatory responses. CD4 and CD8 molecules have been termed co-
receptors since CD4 binds to the membrane proximal X32 domain of class II MHC,
while CD8 (aa and a(3) isoforms bind to the corresponding a3 domain of class I
MHC (15, 16).
At present, four distinct class I-restricted TCRs have been crystallized in
complex with their specific pMHCI ligands (5). Rather extensive interactions
with
the pMHC a-helices has suggested a common "diagonal" docking mode, regardless
of TCR specificity or species origin, in which the TCR Va domain overlies the
class
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I MHC a2 helix and the V[i domain overlies the MHC al helix. As a result, the
CDR1 and CDR3 loops of the TCR Va and V~3 domains make the major contacts
with the peptide while the two CDR2 loops interact primarily with the MHC.
Given
the distinct nature of class II vs. I MHC expression, peptide binding and the
differential interactions with CD4 and CD8 T cell subsets, the TCR-pMHCII
interaction was structurally defined. The first x-ray crystal structures of a
TCR-
pMHCII ternary complex are described herein. The complex contains the V module
of the D10 TCR [single chain (sc) D10] derived from AKR/J (H-2k) mouse T cell
clone D10.G4 and a fragment of conalbumin (CA) bound to the self I-Ak molecule
(17, 18). A striking difference in TCR docking topology relative to TCR-pMHCI
complexes is noted.
EXAMPLE 3 Overview of the Complex Structure
The crystal structure of the scDlO-CA/I-Ak complex was determined with
molecular replacement and alternative cycles of model building and refinement.
Crystals of the ternary complex were grown using the conventional hanging
droplet
vapor diffusion method at room temperature.
The scDlO TCR, constructed by PCR, consists of 237 residues and was
organized from - to C-terminus as follows: V~38.2 (residues 3-110)-linker
(GSADDAKKDAAKKDG)-Va2 (AV225) (residues 1-112) with a Cys235Ser
mutation. This linker (SEQ ID N0:2) was modified from that previously utilized
for
NMR studies (20) since the longer linker failed to give rise to I-Ak co-
crystals of
diffraction quality. For bacterial expression, the T7 promoter expression
vector
pET-1 la was used. An overnight preculture of transformant (20 ml) was
inoculated
into 1L fresh Luria broth supplemented with 50 pg/ml carbenicillin at
37°C and
grown until the OD600 reached 0.6. Il'TG was added to a final concentration of
1
mM and a further 4 h incubation was performed. Cells were harvested by
centrifugation and lysed by sonication. The inclusion bodies were washed and
dissolved in 100 mM Tris-HCl (pH 8.0) containing 6 M guanidine chloride, 10 mM
EDTA and 10 mM DTT. An efficient refolding was achieved by diluting rapidly
into a refolding buffer (50 mM Tris-HCI, pH 8.0, 400 mM arginine, 2 M urea, 2
mM
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EDTA, 4 mM reduced glutathione and 0.4 mM oxidized glutathione). The refolded
material was then applied to a 3D3 affinity column followed by gel filtration
on
Superdex 75 and exchanged to crystallization buffer (20 mM sodium acetate, pH
S.0
with 0.025% sodium azide).
S The undeglycosylated CA/I-Ak from CHO Lec3.2.8.1 cells were prepared as
follows: a 13 residue hen egg CA peptide (residues 134-146) which is
recognized by
D10 TCR was fused (48) to the N-terminus of the mature I-Ak (3 chain via a
flexible
linker. The 37 residue leucine zipper (LZ) sequences (49) were attached to
both the
a and (3 chains, with AC>D-p 1 to the (3 chain and BASE-p 1 to the a chain via
flexible thrombin-cleavable linkers. The cDNA constructions were subcloned
into
the pEEl4 vector and expressed in Lec3.2.8.1 CHO cells. The screening of
secreted
recombinant protein in the culture supernatant was performed by both sandwich
ELISA and BIAcore using antibodies specific for I-Ak (10.2.16) and the LZ
epitope
(2H11 or 13A12). The yield was ~0.7 mg of I-Ak/1 supernatant. The production
supernatant was applied to 2H11 affinity column and I-Ak protein was eluted by
50
mM citrate, 20 mM Tris, 0.5 M NaCI, 10% glycerol, pH 4Ø The eluted protein
was
then exchanged to 50 mM Tris-HCI, pH 8.0 and cleaved by thrombin (2u/SO~,g I-
Ak) at 4°C for 4 h. Thrombin was then removed by passage through
benzamidine
Sepharose 6B beads and gel filtration on Superdex 75. Subsequently, the
purified I-
Ak was exchanged to 10 mM HEPES, pH 7.0, 0.025% sodium azide for
crystallization.
The E. coli expressed scD 10 (52) and undeglycosylated CA/I-Ak from CHO
Lec3.2.8.1 cells (53), prepared as described above, were mixed at a 1:1 molar
ratio
to a final concentration of 23 mg/ml in O.1M Tris-HCl buffer at pH 8.5. The
protein
solution was further mixed with a crystallization buffer of 8% PEG 8K/O.1M
Tris
pH 8.5/0.01M KCI, and then sealed against a reservoir with the same buffer.
These
crystals belong to the space group P21212 with unit cell parameters a=97.6,
b=345.3, and c=97.7A. There are two complexes in asymmetric unit with 78%
solvent. Crystals were stepwisely transferred to cryoprotectant solution that
contains
30% glycerol in addition to the crystallization buffer before freezing. One
data set
was collected at the SBC-CAT of Advanced Photon Source (APS) at the Argonne
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National Laboratory with an APS 1 mosaic 3x3 CCD detector under
100°K. The
wavelength used was 1.069t~. Data were processed using programs DENZO and
SCALEPACK (54). The structure was solved with molecular replacement using
AMoRe (55). The refined structure of CA/I-Ak (56) was taken as the search
model.
S At the beginning, only one of the CA/I-Ak pMHC (molecule A) was identified.
The
CA/I-Ak molecule B was located only after the first one was rigid body refined
and
fixed. The rigid body refinement of the two I-Ak molecules was then carried
out,
each of the Ig-like domains and the bound peptide being treated as one rigid
body.
A few degrees of rotations were seen for the a2 and X32 domains. After
positional
and individual B-factor refinement, the Rfree dropped, and the Ig-like domains
of
scDlO, especially the one in complex-A, were already visible in the calculated
2Fo
F~ difference map. Cycles of model building and refinement gradually improved
the
density, allowing the correct sidechain assignment and eventually the
completion of
the model building and refinement. All the refinement was done using the
program
X-PLOR (57), and model building with program O (50). Ten percent of
reflections
were set aside for Rfree calculation. In the current model, each complex
contains
residues 1-182 and 2-190 of I-Ak a and ~3 chains, respectively, all 16
residues of
the bound peptide (3 leader derived and 13 CA-derived), as well as residues 2-
117
and 3-116A of D10 Va and V(3 domains, respectively. Ten carbohydrate moieties
were modeled in three potential glycosylation sites in I-Ak molecules. At this
resolution no water molecules were included. The final 2Fo FC map is of
excellent
quality, particularly in the TCR regions and the interface between TCR and
pMHC.
There are very few density breaks, mainly in the BC loops of the I-Ak (32
domains.
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TABLE 1: CRYSTALLOGRAPHIC ANALYSIS
Data collection
Resolution limit(last shell) 30.0 - 3.2A (3.31A-3.200
Reflections
total number 501,406
unique 52,592
I/o(I) 10.0 (2.2)
Completeness 95.2% (87.1 %)
Rmerge 7.0% (29.2%)
Refinement statistics
Resolution range 15.0-3.20t~
Number of reflection* 46,332 (F>0)
Rwork 24.7%
Rfree 29.3%
Rms deviations
Bonds 0.007 ~
Angles 1.4°
Dihedrals 30.2°
Impropers 0.7°
Ramachandran plot
Favored 71.4%
Allowed 21.7%
Generous 6.9%
Unfavored 0%
Rmerge=E(~li(hklJ-<Ii(hklJ>~)l Eli(hkl)
*in working set.
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In the asymmetric unit there are two complexes related to each other by a
115° rotation. In fact, the complex-A and D10-B pack together to form
layers
perpendicular to the longest Y axis, whereas the I-Ak molecules B connect
layers in
a fashion analogous to pillars between different floors in a building, thus
leaving
spaces filled with large amounts of solvent. The structures of the two
complexes are
very similar. The root-mean-square deviation (Rmsd) value of Ca superposition
is
only 0.8~ for the whole complex. Consequently, only complex A is discussed
further.
Analysis of the scD 10-CA/I-Ak complex was performed on a model
generated using the program MOLSCRIPT (56). The secondary structures, ~3-
strands
and a-helices of all component domains were defined by the program DSSP (58).
CA/I-Ak was crystallized with the hanging droplet vapor diffusion method by
equilibrating 7.2 mg/ml protein solution against a buffer of 16%
isopropanol/18%
PEG 4K/O.1M sodium citrate at pH 5.6 in reservoir. The crystals belong to
space
group P2~2~2, with unit cell parameters a=99.1A, b=122.4, and c=68.4. One
single crystal was transferred to the cryoprotectant solution of 30%
glycerol/18%
PEG 4K/O.1M sodium citrate for half an hour before dipping into liquid
nitrogen for
freezing. A 95% complete data set was collected at Brookhaven NSLS X12C
beamline using wavelength 1.072A with Brandeis CCD detector. The data were
integrated and scaled by DENZO and SCALPACK (50). The Rmerge of the data
was 9.2% to 3A resolution for 19,854 unique reflections. The structure was
solved
by molecular replacement method with AMoRe (51), using a structure of I-Ak
complexed with a peptide from hen egg lysozyme residues 50-62 (PDB code liak)
as a search model. The structure was refined with X-PLOR. The final Rfree is
29.5%, and Rwork is 23.1%.
The immediately striking observation on the structure was that the scDlO
molecule sits on top of the MHC with its longer dimension crossing the bound
peptide in an orthogonal manner, rather than the "diagonal" mode commonly
recognized in structures of TCR-pMHCI complexes (5). The Va domain of scDlO
contacts the X31 helical region of I-Ak, whereas the V~3 domain touches the al
helical
region. Table 2 lists all contacts between the D10 TCR and the CA/I-Ak pMHC
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ligand. In contrast to the class I pMHC-TCR ternary structures, the much
longer
peptide stretches out both sides of the TCR-MHC complex. In particular, the C-
terminal three residues have no interaction with either TCR or MHC. The
orthogonal orientation for the TCR-pMHCII interaction noted herein excludes
the
possibility that direct TCR contact with C-terminal peptide flanking residues
is the
basis for any observed functional dependence on this peptide segment in T cell
recognition ( 19).
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TABLE 2: ATOMIC CONTACTS BETWEEN THE D10 TCR AND THE CA/I-Ak
pMHC LIGAND
CDR TCR residue pMHC residue
Hydrogen bond van der Waals contacts
CDRla Asp26 081 P-1-Ser
Ser27 O Thr77~i
Thr28 Oy 1 P2-Arg
Cy2 P2-Arg ,
O P2-Arg Nr~ 1 P2-Arg
Phe29 O Thr77(3
Asp30 C(3 P2-Arg, Thr77~3,
A1a73~i
Cy Arg70(3, A1a73~i
081 Arg70~i Nr~l Arg70(3, A1a73(3
082 Arg70(3 Nrl Arg Nrl l Arg70(3
l, P2-
Tyr31 C(3 Arg70(3
Cy Arg70~3
Cs1 Arg7op
CDR2a Ser50 Oy G1u69[3
Leu51 C81 Thr77(3
Va152 C~3 G1u69~3
Cy 1 Arg72~i, G1u69~3
Cy2 G1u69~3, A1a73~3,
Arg72(3
HV4a Lys68 N~ Asp76~3 082
25CDR3a Thr93 Cy2 Arg70~i
G1y99 Ca P2-Arg, Arg70[3
C P5-Ile
O P2-Arg, PS-Ile
Ser100 N PS-Ile
Ca PS-Ile
C PS-Ile
O G1n61 a
Phe101 N Arg70~3 Nrl2 PS-Ile
Ca Arg70(3
3 C ~3 P 5-Ile
S
Cy PS-Ile
C81 PS-Ile, P6-Glu, P7-Trp
CE P7-Trp, P8-Glu
C~ P8-Glu
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O Gln61 a OE 1
CDR 1 (3 Asn31 081 G1n61 a OE
1
CDR2(3 Tyr48 CE2 G1n57a
C~ G1n57a
Orl G1n57a OE
1
Gln57a NE2 G1n57a
Tyr50 C81 Leu60a
CE 1 Leu60a, G1n57a,
*G1n61 a
10C82 G1n61 a
CE2 G1n61 a
C~ Gln57a, G1n61 a
Orb Gln57a NE2
G1n57a O G1n57a, Gln6la
15Thr55 C *Lys39a
O Lys39a N~
G1u56 C(3 Gln57a
C8 Lys39a
OE 1 Lys39a N~ Lys39a
20Lys57 O G1n57a
CDR3 ~i G1y96 O P8-Glu OE
1
G1n97 Ca *Tyr67~3, P8-Glu
C(3 Tyr67~3, P8-Glu
OE 1 Tyr67~i Orb
25C Tyr67(3
G1y98 N Tyr67~3 Orb Tyr67(3
Ca Tyr67~i, P7-Trp
C Tyr67 ~3
Arg99 Cy Tyr67~3
30NE G1n64~i O
C~ G1n64(3
Nr~ 1 G1n64(30E
1
Nr~2 G1n64~3 O G1n64(3
35*These residues are
<$0% conserved. All
other MHC residues
are >60% conserved
in 100 murine (3 MHC murine a MHC class II
class II molecules
and ~30
molecules according Bonds were determined
to Kabat tables of using the
variability.
program CONTACTSYM (25).Given the
resolution
of the current
structure,
the
atomic contacts cannot
be determined with
absolute precision.
40 An omit map in the bound peptide region showed the core of CA (P-1 to P8)
to be involved in TCR-based immune recognition. A sigma A weighted 2Fo-F~ omit
electron density map contoured at 1.0a was generated using the program O (50)
and
prepared using a cover radius around the atoms. The omit map was generated by
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omitting the CA peptide entirely and after a round of torsion angle dynamics
calculation.
While there have been several structures of class I-restricted a ~i TCRs or
derivative fragments (3-5), our scDlO represents the crystal structure of a
class II
restricted a ~i TCR V module in complex with its cognate pMHC partner. The
structure of the Va-V(3 heteiodimer is very similar to the recently published
NMR
structure of an unligated scD 10 (20). Rmsd's for all of the backbone atoms of
residues in ~3-strands between structures in the NMR ensemble and the crystal
structure are 1.3~ and 1.4~ for Va and V(3 domains, respectively. Notably,
there
does not appear to be any significant three-dimensional structural difference
between
TCRs that recognize peptides bound to class I vs. II MHC molecules. The human
class I HLA-A2/Tax-specific B7 TCR is by far the most structurally similar to
the
murine class II-specific scDlO described herein. Virtually the entire V module
of
these two TCRs can be superimposed. The Rmsd values of the superposition for
the
entire Va domain's 110 Ca atoms (excluding the first residue which is not seen
in
the density map of our scDlO structure) and 107 Ca atoms of the Vii domain
(excluding part of the CDR3) are only 0.98 and 0.72t~, respectively. Moreover,
if
the two Va domains are superimposed, then the orientations of two Vii domains
differ only by a 3.7° rotation, indicating that Va-V~3 dimerization is
very similar for
these two TCRs as well.
EXAMPLE 4 The Orthogonal Binding Mode
The orthogonal docking mode was not correctly predicted by either extensive
mutagenesis studies (17) or modeling using a scDlO NMR structure in
conjunction
with a CA/I-Ak crystal structure as a starting point (20). To establish a
quantitative
and comparative measurement of binding orientation among TCRs and their pMHC
ligands, an angle was defined between two vectors. One vector passes through
the
mass centers of the Va and V(3 domains of the TCR, while the other is drawn
from
the N-terminal Ca atom at the P 1 position (the first residue bound in the P 1
pocket
of the MHC) to the C-terminal Ca atom (the P9 position, the last buried
residue) of
the bound peptide. The angle for scDlO-CA/I-Ak complex is 80°, very
close to a
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right angle. Table 3 lists the orientation angle calculated for all known TCR-
pMHC
complex structures.
TABLE 3: THE ORIENTATION ANGLE OF A TCR ONTO A pMHC
LIGAND*
S TCR-peptide/MHC Complex Orientation angle (°) MHC Class
D 10-CA/I-Ak 80 II
2C-dEV8/H-2Kb 45 I
N15-VSV8/H-2Kb 54 I
A6-Tax//HLA-A2 S 6 I
B7-Tax/HLA-A2 70 I
*The orientation angle of a TCR on MHC is defined as the angle between
two vectors determined for the orientation of the TCR and pMHC, respectively.
The
vector representing the TCR direction is drawn from the mass center of Va to
the
mass center of V~3 . The vector representing the pMHC complex direction is
drawn
from the N-terminal Ca atom to the C-terminal Ca atom of the peptide in the
case of
MHC class I. In the case of MHC class II, the vector is drawn from the P 1
residue to
the P9 residue of the peptide. Note that in Teng, et al. (5), twist and tilt
were used
for semi-quantitative comparison among different TCR-MHC complexes.
Essentially, the twist and tilt angles are two projections of the orientation
angle more
accurately defined here. While the twist is a top view from the TCR towards
the
MHC, the tilt is a side view, perpendicular to the bound peptide.
Note that for class I complexes, the peptide vector is defined between the
anchoring residues at the two termini. The angles for TCR-pMHCI complexes span
a broad range, from diagonal (45°) to close to orthogonal (70°).
Differences
between orthogonal and diagonal docking were seen in a comparison of the scDlO-
CA/I-Ak structure and the 2C-dEVB/H-2Kb (pMHCl) complex, by comparing a
stereo view of scDlO (VaV~3) on CA/I-Ak with a stereo view of 2C (VaV~3) on
dEVB/H-2Kb.
Garboczi et al. (S) argue that in pMHCI structures, there are two high
"peaks" near the N-termini of the a-helical regions forming the side wall of
the
peptide binding groove. These two "peaks" limit the TCR-pMHC class I binding
to
a diagonal mode such that the TCR can fit at a low enough point on the MHC
surface to contact the entire complexed antigenic peptide. Teng et al. (5)
have
compared three TCR-pMHC class I complex structures, and identified a common
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docking mode of the TCR relative to the MHC with substantial variation of
twist, tilt
and shift.
Furthermore, it was noticed that the inherent left-handed twist of the eight-
stranded ~3 -sheet that forms the platform of the binding groove is the
structural basis
for the breaks in the two helical regions, resulting in the formation of high
"peaks".
In this context, an MHC class II molecule is similar to an MHC class I
molecule
because all MHC molecules have the same platform. However, there are distinct
features to the mode of peptide binding between the two classes. In the class
I
system, the 8-10 residue peptide has its termini anchored into two binding
pockets
whose unique chemical environments determine the polarity of the bound
peptide.
In addition, the bulky sidechains of the conserved Trp 167a and Tyr84a from
the
MHC molecule occlude the peptide-binding groove at both ends. In class II MHC,
these blocking sidechains are replaced by smaller ones and/or reoriented; the
open
ends eliminate the peptide length restriction. Moreover, the peptide (15-20 as
in
1 S length) binds to the class II MHC molecule with hydrogen bonds not just at
the
termini, but throughout the entire peptide via mainchain atoms (for review,
see 12).
The hydrogen-bonding pattern between the CA peptide and the I-Ak
molecule which is conserved in other pMHC class II structures was analyzed.
Ten
hydrogen bonds between the CA and I-Ak are conserved in known pMHCII
structures. Compared with the class I system, the P-3 to P-1 segment is an
extension. This extension plays a unique role in the orthogonal docking mode.
The
peptide binding groove is much wider in the middle relative to its tapered
ends so
that the MHC class II molecule needs to use sidechains of multiple conserved
residues from a 1 and ~3 1 helical regions to reach the peptide mainchain
atoms. The
residues include asparagine and glutamine which form bidentate hydrogen bonds
to
the peptide backbone. This H-bonding pattern determines the peptide binding
polarity in the class II MHC system (9-12). An important characterization of
class II
MHC molecules is that the al helix is two turns shorter in the N-terminus than
the
corresponding class I MHC molecule a 1 helix. In particular, from Arg52a to
G1u55a, the helix is replaced by an extended strand that reaches close enough
to the
N-terminal extension segment of the bound peptide to form a mini parallel (3-
sheet
using mainchain atoms. The pair of mainchain-mainchain hydrogen bonds between
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Arg53a of the MHC class II molecule and the P-2 and P1 residues at the N-
terminal
part of the peptide are conserved among all known pMHCII structures. The
beginning of the al helix, G1n57a, is at the high "peak", so from Glu55a to
Arg52a
toward the N-terminus the chain runs down, away from the TCR binding surface.
However, the left-handed twist of the mini ~3 -sheet then forces the N-
terminus of the
peptide to point in the opposite direction, curving up toward the TCR binding
surface. Together, the extended N-terminus of the bound peptide and the MHC
molecule now form a broader high "peak", or a small protruding "ridge". A
stereo
view of the molecular surface of I-Ak (a 1 X31) together with the CA peptide
shows
the surface topology of the D10 docking platform on the CA/I-Ak ligand, which
can
be compared with the same view of pMHCI taken from the 2C-dEV8/H-2Kb
structure, demonstrating the stereo view of the molecular surface of H-2Kb (a
1/a2)
together with the dEV8 peptide (5) and showing the smaller high point on the
left
side of the docking platform for the MHC class I-restricted TCR molecule. The
peptide is mostly buried and makes little, if any, contribution to the
elevated points.
The scD 10-CA/I-Ak structure shows that a diagonal TCR docking would
result in a collision between the Va domain of TCR and the pMHCII on the
"left"
side. Moreover, the tilt angle of a TCR relative to an MHC molecule (see Table
3
legend for the definition of tilt angle) exacerbates this potential clash by
maintaining
the Va domain in close proximity to MHC. It is proposed that while the TCR-
pMHC class I docking may have more variation in terms of the orientation angle
as
demonstrated in Table 3, the topology of TCR binding to pMHC class II may be
more closely restricted to an orthogonal mode due to the "ridge" described
above. It
is interesting to note that the protrusion of the peptide's N-terminus has
been
suggested as a site for disruption by DM in the process of exchanging CLIP for
an
antigenic peptide in the MHC class II molecule (21).
EXAMPLE S The Interface
The interaction between D 10 and CA/I-Ak buries 1718~z~'of surface area,
8612 from the pMHC and 857Az from the TCR using a 1.7t~ probe (22). Twenty-
three percent of the pMHC buried surface involves the peptide. In general, the
size
of the buried surface is comparable to that previously reported for three
class I
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ternary structures (1700-1880~z). However, the Sc value (the shape correlation
statistic, a measurement of the degree of geometric match between two
juxtaposed
surfaces, where interfaces with Sc = 1 fit perfectly whereas interfaces with
Sc = 0
effectively define topologically uncorrelated surfaces (see Lawrence &
Coleman, ref.
22) of the interface between the scDlO VaV~i module and the CA/I-Ak ligand is
0.70, higher than for class I TCR-pMHC interfaces whose Sc values range from
0.45
to 0.64 (23, 24). Moreover, the number of atomic contacts (25) in our class II
complex structure is about twice as many as those for the class I complexes.
For I-
Ak, 68 atomic contacts exist with D10. By contrast, there are just 27 H-2Kb
contacts with the 2C TCR, and 27 and 34 HLA-A2 contacts for the A6 and B7
TCRs, respectively. These results suggest a much better shape complementarity
of
the scD 10-CA/I-Ak interface, and agree well with the higher affinity of D 10
for its
pMHC ligand relative to that of 2C, for example (1-2 ~.M vs. 100 ~,M) (5, 26).
Of
particular relevance is the fording that the additional interface atomic
contacts can
largely be ascribed to contacts between the TCR and the I-Ak molecule rather
than
between the TCR and the CA peptide (Table 2). Assuming that these results are
representative for other class II MHC-specific TCRs, the dominance of this TCR-
MHC class II contact may explain why expression of a single pMHC class II
complex in the thymus can select many different TCRs (27). The data also show
that despite having roughly the same buried surface area, the complementarity
of
TCR-pMHC recognition surfaces can vary substantially from a low extreme to one
even better than that of antigen-Fab complex, as is the case for the scDlO-
CA/I-Ak
complex. In complexes like 2C-dEV8/H-2Kb, a few large cavities (5) contribute
to
poor shape complementarity. The presence or absence of such cavities may vary
for
different TCR-pMHC complexes, thereby influencing the shape of
complementarity.
Of the total buried surface area, Va accounts for 5192 while V(3 accounts
for 3382 of the TCR buried surfaces. This result is consistent with the notion
that
Va dominates in the interaction which is generally true for the class I system
as well.
The calculations showed that the buried surface areas of Va and V(3 are 4802
and
4302 for the 2C-dEVB/H-2Kb complex, 576A2 and 319~Z for A6-Tax/HLA-A2,
and S55~z and 26012 for B7-Tax/HLA-A2, respectively. Perhaps more importantly,
amongst different TCR-pMHC complexes, the variation in buried surfaces is
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significantly smaller for Va than for V(3. Given that the rotation angle of
known
TCRs relative to their MHC ligands varies as much as 35° (Table 3),
these data
suggest that the pivot point is closer to Va so that the Va domain location on
the
pMHC will not change as much as the V~3 domain which can alter dramatically.
Differences in the disposition of CDR loops reflect this pivot point; these
differences
were seen in models prepared using the program GRASP (51).
Variability in TCR docking also arises from differences in the tilt angle as
described by Teng et al. (5) and noted in the Table 3 legend. The extreme is
the A6-
Tax/HLA-A2 structure, where the large tilt essentially precludes CDRIb and
CDR2b from making contact with the MHC molecule (S). Given that Va is critical
for TCR selection in thymic development as well as mature T cell activation
(28),
this Va dominance in immune recognition is not unexpected.
Comparison between the scDlO TCR interaction with CA/I-Ak analyzed
herein and the 2C TCR interaction with dEV8/Kb (5) shows how a single TCR
V~38.2 domain can bind in distinct orientations to class I and class II pMHC
ligands.
In the 2C-dEV8/Kb complex, the germline V~i8.2 segment recognizes the Kb al
helical MHC residues G1n72, Va176 and Arg79 via CDR2, and the Kb a2 helical
residues Lys146, G1n149 and A1a150 via CDR1 (S). In the scDlO-CA/I-Ak
complex, the identical germline V~i8.2 segment recognizes the I-Ak al helical
residues Lys39, G1n57 and Leu60 via CDR2, and G1n61 via CDR1. Given that these
two docking interactions are to highly conserved MHC class I and to highly
conserved class II residues, respectively, it appears that the Vii domain
plays a major
role in MHC recognition by both classes of TCRs and perhaps in pre-TCRs as
well
(29).
EXAMPLE 6 Antigenic Peptide Recognition
Although the peptide in the ternary structure is 16 residues in length,
designated as from P-3 to P13, the TCR interaction is restricted to the P-1 to
P8
segment. Table 2 lists all the contacts to the peptide. It is noteworthy that
of 27
atomic contacts with the peptide, 23 involve Va and only four involve V(3.
This
dominance of the Va domain in peptide recognition was not appreciated
previously,
although early molecular modeling efforts correctly suggested that an
orthogonal
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0
TCR docking mode was possible (Davis & Bjorkman, ref. 2). The spiral
conformation of bound peptide (12) dictates that of the deeply buried peptide
residues, only those at positions P2, PS and P8 are accessible to the TCR
molecule.
The Trp at the P7 position is an exception due to its bulky indole ring, which
is
partially exposed on the TCR binding surface. As for the rest of the peptide,
the
backbone of the P-2 residue is engaged in a mini parallel (3-sheet with the
MHC
molecule as discussed above, while the P-3 and the C-terminal three residues
(P11-
P13) have no contacts with either MHC or TCR whatsoever, though well defined
by
unambiguous densities.
The P2 residue is an Arg. It forms multiple salt bridges with both Asp30a in
the D10 CDRIa and I-Ak G1u74~i, respectively. The same TCR Asp30a also
interacts with I-Ak Arg70~3. Moreover, the upward-pointing P2-Arg is within
van
der Waals contacts to backbone of CDR3a Gly99a and CDRla Thr28a (see Table
2). This knitted local structure packs closely onto the sidechain of Ile at
the PS
position from the N-terminal side of the peptide. The PS residue is important
structurally and biologically. Alteration of this residue adversely affects
D10-TCR
recognition of CA/I-Ak (17). The sidechain of Ile at PS fits extremely well
into a
hydrophobic pocket. Apart from the neutralized network discussed above, on the
C-
terminal peptide side is the indole ring of the Trp at P7 stacking onto the
isobutyl
group of the PS-Ile. On the top, from the TCR direction, the PS-Ile contacts
the
backbone of the tip of CDR3a which consists of Gly99a-Ser100a-PhelOla. The
phenolic ring of PhelOla bends towards the P7-Trp position. The exposed tip of
the
indole ring of P7-Trp makes contacts with the PhelOla aromatic ring. The other
peptide residue engaged in recognition is the P8-Glu residue. P8-Glu forms
bifurcated hydrogen bonds to sidechains of Tyr60(3 and Tyr67~3 of the I-Ak
molecule. Only the aliphatic portion of the P8-Glu sidechain makes van der
Waals
interactions with CDR3a PhelOla and the aliphatic part of CDR3~i G1n97~3. In
addition, there is one H-bond between the carbonyl oxygen of G1y96~3 and the
sidechain of P8-Glu. Together, it appears that the TCR recognition of the
particular
antigenic peptide in question is largely hydrophobic and involves a number of
backbone associations with the TCR molecule. Although scD 10 TCR recognition
of
CA/I-Ak is centered at the PS position, it is also coordinated with
interactions to
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peptide residues at P2, P7 and P8. The observed contacts are consistent with
studies
mapping the D 10 footprint onto CA/I-Ak ( 18). For example, immunization of D
10
TCR Va2 tg mice with the GIuB~AIaCA peptide variant gives rise to T cell
hybridomas, all of which use V(38.2 but with variation in CDR3[3, consistent
with
the view that CDR3[3 is involved in recognition of the P8 position.
Immunization of
D 10 TCR a tg or D 10 TCR [3 tg with the Ile~Lys CA variant both failed to
generate
specific hybridomas, implying that CDR3a and/or (3 may be important for
IIeSPro
recognition. Immunization of D 10 TCR [3 tg mice with the Arg2~Asp CA variant
resulted in a switch in Va usage from Va2 to VaB, suggesting that the germline
CDR1 and/or 2 loops interact with this peptide residue.
EXAMPLE 7 Implications for Class II MHC-based Immune T Cell Recognition
The current scDlO-CA/I-Ak complex offers several insights into immune
recognition of other pMHC class II ligands by other TCRs. First, the size of a
TCR
footprint on the MHC covers maximally nine peptide residues (~25~). Hence,
while
MHC class II molecules capture peptides of substantially larger length, only a
subset
of residues is "read out" by the bound TCR. Second, the PS residue of the MHC-
bound peptide occupies the central position [corresponding to the P4 position
of the
MHC class I-bound peptide (28)]. As such, this central, solvent exposed
residue is
critically important for the TCR binding process. Therefore, even a minor
conservative substitution at this residue can destroy binding (i.e. null
ligand) or lead
to altered peptide ligands with very weak agonist or in fact, antagonist
activity (31-
33). Third, for all class II molecules examined, there appear to be 3-4 pMHC
binding pockets (at P1, P4, P6 and P9 for I-Ak, I-Ek and DR and P1, P4 and P9
for
I-Ad). Only several upward pointing peptide residues can serve as direct TCR
contacts.
Based on the observed molecular envelope of the TCR and the observed
orthogonal orientation for class II MHC-restricted TCR interaction, it appears
that
these basic principles apply in a general way to recognition of multiple
pMHCII
ligands including HEL4g-62/I-Ak (10, 34), Hb64-76~I-Ek (35), moth cytochrome C
(MCC)93-103-Ek (31) and DR2-restricted myelin basic protein (MBP)gs-99 (36,
37). Moreover, it has been suggested that a single TCR can recognize multiple
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pMHCII ligands (36, 37). As the class II specific TCR focuses on the central
PS
residue, mutations which affect non-P5 positions may be less detrimental to
the
recognition process.
Despite overall structural similarity, CDR3 conformations appear to differ
between free and complexed scDlO. In the x-ray structure of the complex, the
CDR3 loops are close to one another. Packing among the sidechain of G1n106(3,
A1a104~3 and Leu104(3 forms a hydrophobic core between the CDR3 loops. In
contrast, there is no evidence that CDR3a packs with CDR3~i in unligated
scDlO.
Numerous NOES are observed between the methyl group of A1a104~3 and other
CDR3(3 residues (G1y96, G1n197, Arg99, G1u105), but contacts to Leu104a or
other
CDR3a residues are not observed. In all of the calculated NMR structures, the
CDR3a and CDR3(3 loops are well separated. The backbones of both CDR3s are
also highly mobile on the picosecond timescale in the free protein (20),
suggesting
that they are not tightly packed. Hence, during immune recognition, the mobile
CDR3 loops of scD 10 assume their pMHCII binding conformation, clamping down
on the central peptide region.
EXAMPLE 8 Structural Basis of Alloreactivity
Approximately 1-10% of peripheral T cells are able to recognize allogeneic
MHC molecules to which they were never exposed (38). The precise molecular
basis of alloreactivity is yet to be fully defined. In this regard,
the.complex of
scDlO-CA/I-Ak is informative since the D10 TCR not only recognizes the
antigenic
CA peptide bound to I-Ak but also responds to all MHC class II molecules whose
I
A ~3 chain contains the sequence "PEI" at positions 65-67, including I-Ab' v,
p, a, a ( 17).
In comparison, MHC class II molecules having a Tyr at this position such as I-
Af k, r,
5' °' g', cannot stimulate D 10 cells in the absence of the CA peptide.
Various
mutagenesis studies conducted on D10 showed that a hybrid I-A''a/I-Ab~3 MHCII
molecule can stimulate D10 cells in the absence of exogenous antigen,
suggesting
that polymorphic residues critical for alloreactivity are located on the I-A
(3 chain.
In order to elucidate this source of alloreactivity from the structural
perspective, the CA/I-Ak ligand and the alloreactive I-Ad molecule were
compared.
The latter was taken from the recently solved x-ray structure of ovalbumin
(323-339)
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complexed with I-Ad (11). Since all residues from I-Ak involved in the
interaction
with D10 as listed in Table 2 are conserved in I-Ad with the exception of
residues
Tyr67 in I-Ak and Pro65-G1u66-I1e67 (PEI) in I-Ad, it is likely that D10 docks
onto
I-Ad in the same way as onto I-Ak. A model using the I-Ad superimposed onto
the
S scDlO-CA/I-Ak complex was assessed for differences.
The major structural difference involves the (3 chain residues Pro65, G1u66
and I1e67 in I-Ad which form a protrusion interrupting the ~3 chain a helix.
As a
consequence, in I-Ad, residue I1e67 assumes a similar position to residue
Tyr67 in I-
Ak. The sidechains and mainchain atoms on the CDR1 and 2 loops of D10 Va
make no hydrogen bonds or salt bridges to I-Ak (31 helix residues. A model of
scD 10 bound to I-Ad was also assessed, based upon superposition of I-Ad and I-
Ak.
The a 1 H2 helix and the ~i 1 region between and including H2a-H2(3 of the two
class
II MHC molecules were superimposed (46 Ca's, Rmsd = 0.550. The potential
interactions between G1u66 from the PEI65-67 motif of I-Ad to Tyr31 of CDRl
and
A1a48, Ser50 and Lys56 of CDR2 of D10 Va were examined.
The aliphatic sidechain of the I1e67 in I-Ad can replace the aromatic ring on
the sidechain of Tyr67 in I-Ak, forming van der Waal's contacts with the V~3
CDR3
loop. To avoid steric clashes, side chains from residue Arg99 of D10 V(3 and
residue G1u66 of I-Ad (31 are rotated and the mainchain conformation around
PEI
on I-Ad is slightly modified. The backbone NH vectors of residues directly
adjacent
to Arg99 are among the most mobile in scDlO (20).
G1u66 can form multiple potential interactions with CDR1 and CDR2 of D10
Va. Additionally, the hydrogen bond between G1n64 and Arg99 from CDR3 of Vii
is preserved. Therefore, despite loss of one hydrogen bond of Tyr67 to D10 Vii
G1y98, these potential additional contacts between the CDR loops of D 10 Va
and
the inserted PEI residues can enhance the affinity between MHC and D10. Other
TCR allo-pMHCII interactions cannot be excluded. Consistent with this view, it
has
also been suggested that in the case of the 2C allo-MHCI response (Ld),
allorecognition results from increased interaction between the 2C TCR V~3
domain
and the allostimulus (39). The ability of exposed MHC helical polymorphic
residues
to permute the number and nature of contacts with the TCR is a feature of
other
class II MHC-restricted allogeneic responses (40, 41). For example, the
naturally
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occurnng I-Ab mutant H-2bm~12 generates a strong alloresponse in H-2b mice.
This
molecule differs from I-Ab at only three position: 673, 70[3 and 71 ~3.
While self peptides bound to MHC have been shown to play a critical role in
alloreactivity against MHC class I molecules (rev. in 41), less is known about
the
nature of peptide ligands in class II MHC-based alloreactivity (42). Given the
additional contacts between the D10 TCR and I-Ad, it appears that there are
fewer
interactions required between the peptides) associated with I-Ad molecules and
the
D10 TCR. However, a peptides) must also be involved since replacement of the
PEI sequence in lieu of Tyr67 in the I-Ak (3 chain is not sufficient to create
an
allostimulatory molecule for the D10 T cell clone (17).
EXAMPLE 9 Superantigen Binding
Superantigens (SAG) are a family of immunostimulatory and disease-causing
proteins derived from bacterial or endogenous retroviral genes which are
capable of
activating a large fraction of the T cell population (43). In general, the
activation
appears to require a bridging interaction between the V~3 domain of the TCR
and an
MHC class II molecule. Although crystal structures (44) showing the detailed
interactions between SEB, a representative bacterial SAG, and a TCR V~i8.2
chain
or SEB and the HLA-DR1 class II MHC molecule have been determined, the
physiologically relevant tripartite TCR-SAG-pMHC complex has not yet been
characterized. A structural model of TCR-SAG-pMHC complex was previously
generated (44) based on least squares superposition of 1) the 14.3.d V(3C(3-
SEB
complex, 2) the SEB-HLA-DR1 complex and 3) the 2C TCR a ~3 heterodimer.
However, since the docking mode of TCR on the class II MHC was structurally
unknown and presumed to be similar to the observed diagonal mode of TCR on
class I MHC, it was noted that the rotational orientation of the TCR and MHC
molecules in the predicted TCR-SEB-pMHC complex was substantially different
(~40°) from the 2C-dEV8/Kb complex. The structural determination of the
D10-
CA/I-Ak complex herein enables us to offer additional insight into the nature
of the
SAG binding to TCR and pMHC.
A study of the 14.3.d V(38.2 Cb-SEB complex superimposed onto the D10
CA/I-Ak complex included least-square fitting of the V(3 domains from each
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complex (92 Ca's from residues Val3-G1y94 of Vii, Rmsd = 0.670. Because the
TCR docks on the MHC molecule in a nearly perpendicular manner, the SEB
directly interacts with the MHC al helix without any requirement for TCR
rotation.
However, certain segments of SEB and the al helix collide. Since there is no
significant conformational change observed for either of the component domains
involved in this interaction, it was reasoned that a relative domain movement
could
alleviate any steric clash. To test this idea, I-Ak was removed from the
complex and
then superimposed the SEB HLA-DRl complex onto the 14.3.d V(3C~i-SEB
complex and scDlO (VaV~3) module. The two SEB superantigen molecules were
used for least-square fitting (83 Ca's, Rmsd = 0.630. From this second model,
it
was observed that the direct interaction between V(3 and the MHC al helix is
disrupted by SAG. The key interaction site for SEB involves CDR2 (Tyr50,
A1a52,
G1y53, Ser54, Thr55) and certain other residues (G1u56, Lys57, Tyr65, Lys66,
A1a67) as reported by Li et al. (44). In this way, the superantigen wedges
itself
between V~3 and the MHC class II al helix, forcing the MHC to swing away from
V
~3 and toward Va while preserving the direct interaction between the Va domain
of
the TCR and MHC class II (3 1 helix. This latter interaction has been proposed
to be
critical in stabilizing the TCR-SAG-pMHC complex. In fact, T cell activation
by
SAG is believed to be dependent upon the interaction between a given TCR Va
domain and the MHC class II ~i 1 helix (45). When the HLA-DR was replaced with
the I=Ak molecule based on the structural alignment of residues of the two
helices of
each MHC molecule (43 Ca's, Rmsd = 1.02t~), it was estimated that the relative
swing angle between TCR and MHC in the TCR-SAG-pMHCII complex compared
to the TCR-pMHCII complex is ~17°.
EXAMPLE 10 Differential TCR Binding and Co-receptor Selection in the
Thymus
Given that there are no intrinsic structural differences between class I vs.
class II MHC restricted TCR V modules as shown above, it is questioned what
directs expression of a TCR to the proper CD4 or CD8 subset. During thymocyte
development, progenitor cells transit from a CD4-CD8- double negative (DID
stage
through a CD4+CD8+ double positive (DP) stage and then into a CD4+CD8- or
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CD4-CD8+ single positive (SP) stage (46). Selection for maturation occurs upon
the interaction of thymocytes with stromal cells expressing self pMHCI or self
pMHCII ligands within the thymus, beginning at the DP stage where the TCR
first
appears. Differentiation to the SP thymocyte stage, however, requires a match
between the MHC class specificity of the TCR which a thymocyte bears and the
CD4 or CD8 co-receptor it expresses. To explain how a thymocyte precisely
coordinates co-receptor expression and TCR specificity, two models have been
proposed (47). The "instruction model" argues that co-engagement of TCR and
CD4
or CD8 on a DP thymocyte specifically signals the cell to move down one
pathway
while extinguishing the expression of the inappropriate co-receptor. On the
other
hand, the "selection model" postulates that cells initiate stochastically or
otherwise a
process which terminates expression of one of the two co-receptors. If the
correct
match was chosen, then the cell further differentiates but if not,
differentiation is
stalled.
1 S Distinctions between class I vs. II pMHC complexes and variation in TCR
docking observed herein offers strong support for the "instruction model". It
is
suggested that depending on the degree of complementarity of a given TCR
recognition surface and a self pMHCI or self pMHCII complex, binding occurs,
and
a diagonal docking mode with substantial variability onto pMHCI or a preferred
orthogonal docking mode onto pMHCII is established. Subsequently, CD8
preferentially co-engages with the former and CD4 with the latter. Expression
of the
irrelevant co-receptor is then extinguished. Based on CD8-MHC class I crystal
structures and on mapping of MHC class II residues involved in CD4 binding,
the
two co-receptors likely occupy an "homologous" orientation relative to the TCR
(15,
16). Thus, it appears that the differential TCR docking to self pMHCI vs. self
pMHCII contributes specificity for coordination of appropriate co-receptor
selection.
REFERENCES
1. E. L. Reinherz, S. C. Meuer, S. F. Schlossman, Immunol Rev 74, 83-112
(1983); P. Marrack, J. Kappler, Adv Immunol 38, 1-30 (1986); H. Clevers, B.
Alarcon, T. Wileman, C. Terhorst, Ann Rev Immunol 6, 629-662 (1988); J. D.
Ashwell, R. D. Klausner, Ann Rev Immunol 8, 139-167 (1990).
CA 02392387 2002-05-22
WO 01/38394 PCT/US00/31502
-46-
2. O. Acuto, et al., Proc Natl Acad Sci USA 81, 3851-3855 (1984); M. Fabbi,
O. Acuto, J. E. Smart, E. L. Reinherz, Nature 312, 269-271 (1984); G. K. Sim,
et al.,
Nature 312, 771-775 (1984); Y. Chien, et al., Nature 312, 31-35 (1984); N. R.
Gascoigne, Y. Chien, D. M. Becker, J. Kavaler, M. M. Davis, Nature 310, 387-
391
(1984); S. M. Hedrick, E. A. Nielsen, J. Kavaler, D. I. Cohen, M. M. Davis,
Nature
308, 153-158 (1984); S. M. Hedrick, D. I. Cohen, E. A. Nielsen, M. M. Davis,
Nature 308, 149-153 (1984); P. Patten, et al., Nature 312, 40-46 (1984); H.
Saito, et
al., Nature 312, 36-40 (1984); Y. Yanagi, et al., Nature 308, 145-149 (1984);
A. C.
Hayday, et al., Nature 316, 828-832 (1985); J. Novotny, S. Tonegawa, H. Saito,
D.
M. Kranz, H. N. Eisen, Proc Natl Acad Sci USA 83, 742-746 (1986); C. Chothia,
D.
R. Boswell, A. M. Lesk, EMBO J7, 3745-3755 (1988); M. M. Davis, P. J.
Bjorkman, Nature 334, 395-402 (1988).
3. G. A. Bentley, G. Boulot, K. Karjalainen, R. A. Mariuzza, Science 267,
1984-1987 (1995); B. A. Fields, et al., Science 270, 1821-1824 (1995); D.
Housset,
et al., EMBO J 16, 4205-4216 ( 1997).
4. K. C. Garcia, et al., Science 274, 209-219 (1996); J. Wang, et al., EMBO J
17, 10-26 (1998).
5. D. N. Garboczi, et al., Nature 384, 134-141 (1996); K. C. Garcia, et al.,
Science 279, 1166-1172 (1998); M.-K. Teng, et al., Curr Biol 8, 409-412
(1998); Y.
H. Ding, et al., Immunity 8, 403-411 (1998); Y. H. Ding, B. M. Baker, D. N.
Garboczi, W. E. Biddison, D. C. Wiley, Immunity II, 45-56 (1999).
6. B. Benacerraf, Harvey Lect 67, 109-141 (1973); B. Benacerraf, Jlmmunol
120, 1809-1812 (1978); M. J. Bevan, Nature 269, 417-418 (1977); R. M.
Zinkernagel, P. C. Doherty, Nature 248, 701-702 (1974); R. M. Zinkernagel, et
al., J
Exp Med 147, 882-896 (1978); R. N. Germain, Cell 76, 287-299 (1994).
7. F. Momburg, J. Roelse, G. J. Hammerling, J. J. Neefjes, JExp Med 179,
1613-1623 (1994); M. J. Androlewicz, P. Cresswell, Immunity 1-14 (1994).
8. A. Rudensky, P. Preston-Hurlburt, S. C. Hong, A. Barlow, C. A. J. Janeway,
Nature 353, 622-627 (1991); R. M. Chicz, et al., Nature 358, 764-768 (1992);
D. F.
Hunt, et al., Science 256, 1817-1820 (1992).
9. D. H. Fremont, M. Matsumura, E. A. Stura, P. A. Peterson, I. A. Wilson,
Science 257, 919-927 (1992); D. R. Madden, J. C. Gorga, J. L. Strominger, D.
C.
CA 02392387 2002-05-22
WO 01/38394 PCT/US00/31502
-47-
Wiley, Cell 70, 1035-1048 (1992); M. Matsumura, D. H. Fremont, P. A. Peterson,
I.
A. Wilson, Science 257, 927-934 (1992); J. H. Brown, et al., Nature 364, 33-39
(1993); L. J. Stern, et al., Nature 368, 215-221 (1994); D. H. Fremont, W. A.
Hendrickson, P. Marrack, J. W. Kappler, Science 272, 1001-1004 (1996); A.
Dessan, C. M. Lawrence, S. Cupo, D.M. Zaller, D.C. Wiley, Immunity 7, 473-481
(1997); V. L. Murthy, L. J. Stern, Structure 5, 1385-1396 (1997).
10. D. H. Fremont, D. Monnaie, C. A. Nelson, W. A. Hendrickson, E. R.
Unanue, Immunity 8, 305-317 (1998).
11. C. A. Scott, P. A. Peterson, L. Teyton, I. A. Wilson, Immunity 8, 319-329
(1998).
12. L. J. Stern, D. C. Wiley, Structure 2, 245-251 (1994); D. R. Madden, Annu
Rev Immunol 13, 587-622 (1995).
13. E. L. Reinherz, S. F. Schlossman, Cell 19, 821-827 (1980).
14. S. Meuer, S. F. Schlossman, E. L. Reinherz, Proc Natl Acad Sci USA 79,
4395-4399 (1982); W. E. Biddison, P. E. Rao, M. A. Talle, G. Goldstein, S.
Shaw, J
Exp Med 156, 1065-1076 (1982); A. M. Krensky, C. S. Reiss, J. W. Mier, J. L.
Strominger, S. J. Burakoff, Proc Natl Acad Sci USA 79, 2365-2369 (1982).
15. C. A. Doyle, J. L. Strominger, Nature 330, 256-259 (1987); R. Konig, L. Y.
Huang, R. Germain, Nature 356, 796-798 (1992); U. Moebius, P. Pallai, S. C.
Harrison, E. L. Reinherz, Proc Natl Acad Sci USA 90, 8259-8263 (1993); J. M.
Connolly, T. H. Hansen, A. L. Ingold, T. A. Potter, Proc Natl Acad Sci USA 87,
2137-2141 (1990); R. D. Salter, et al., Nature 345, 41-46 (1990).
16. G. F. Gao, et al., Nature 387, 630-634 (1997); P. Kern, et al., Immunity
9,
519-530 (1998).
17. J. Kaye, S. Porcelli, J. Tite, B. Jones, C. A. J. Janeway, JExp Med 158,
836-
856 (1983); P. Portoles, J. M. Rojo, C. A. J. Janeway, JMol Cell Immunol 4,
129-
137 (1989); S. C. Hong, et al., Cell 69, 999-1009 (1992); S.-C. Hong, et al.,
J
Immunol 159, 4395-4402 (1997).
18. D. B. SanfAngelo, et al., Immunity 4, 367-376 (1996).
19. R. T. Carson, K. M. Vignali, D. L. Woodland, D. A. Vignali, Immunity 7,
387-399 (1997).
20. B. J. Hare, et al., Nat Struct Biol 6, 574-581 (1999).
CA 02392387 2002-05-22
WO 01/38394 PCT/US00/31502
-48-
21. L. Mosyak, D. M. Zaller, D. C. Wiley, Immunity 9, 377-383 (1998).
22. C. Chothia, J. Janin, Nature 256:705-708 (1975); M. L. Connolly, JAppl
Crystallgr 16, 548-558 (1983); M. C. Lawrence, P. M. Coleman, JMolec Biol 234,
946-950 (1993).
23. X. Ysern, H. Li, R. A. Mariuza, Nat Struct Biol 5, 412-414 (1998).
24. K. C. Garcia, L. Teyton, I. A. Wilson, Annu Rev Immunol 17, 369-397
(1999).
25. S. Sheriff, W. A. Hendrickson, J. L. Smith, JMolec Biol 197, 273-296
(1987).
26. Our unpublished results.
27. CP. Liu, D. Parker, J. Kappler, P. Marrack, JExp Med 186, 1441-1450
(1997); L. Ignatowicz, et al., Immunity 7, 179-186 (1997); S. Tourne, et al.,
Immunity 7, 187-195 (1997); C. D. Surly D. S. Lee, W. Fung Leung, L. Karlsson,
J.
Sprent, Immunity 7, 209-219 (1997).
28. Y. Ghendler, et al., Proc Natl Acad Sci USA 95, 10061-10066 (1998); B. T.
,
Backstrom, U. Muller, B Hausmann, E. Palmer, Science 281, 835-838 (1998).
29. Y. Shinkai, et al., Science 259, 822-825 (1993); H. J. Fehling, A.
Krotkova,
C. Ruf Saint, H. von Boehmer, Nature 375, 795-798 (1995); C. N. Levelt, B.
Wang,
A. Ehrfeld, C. Terhorst, K. Eichmann, Eur Jlmmunol 25, 1257-1261 (1995); Y.
Xu,
L. Davidson, F.W. Alt, D. Baltimore, Proc Natl Acad Sci USA 93, 2169-2173
(1996); S. Koyasu, et al. Int Immunol 9, 1475-1480 (1997).
30. Results from specificity and sequence analysis of T cell hybridomas
derived
from mice carrying a given V2 or V~i8.2 D10 TCR transgene immunized with
altered peptide ligands of CA (18) are consistent with the structural
information
reported here.
31. P. A. Reay, R. M. Kantor, M. M. Davis, Jlmmunol 152, 3946-3957 (1994).
32. G. J. Kersh, P. M. Allen, JExp Med 184, 1259-1268 (1996).
33. K. Matsui, J. J. Boniface, P. Steffner, P. A. Reay, M. M. Davis, Proc Natl
Acad Sci USA 91, 12862-12866 (1994); D. S. Lyons, et al., Immunity 5, 53-61
(1996); G. J. Kersh, E. N. Kersh, D. H. Fremont, P. M. Allen, Immunity 9, 817-
826
(1998).
34. P. M. Allen, et al., Nature 327, 713-715 (1987).
CA 02392387 2002-05-22
WO 01/38394 PCT/US00/31502
-49-
35. B. D. Evavold, J. Sloan-Lancaster, K. J. Wilson, J. B. Rothbard, P. M.
Allen,
Immunity 2, 655-663 (1995).
36. K. J. Smith, J. Pyrdol, L. Gauthier, D. C. Wiley, K. W. Wucherpfennig, J
Exp Med 188, 1511-1520 (1998).
37. K. W. Wucherpfennig, J. L. Strominger, Cell 80, 695-705 (1995).
38. L. A. Sherman, S. Chattopadhyay, Annu Rev Immunol 11, 385-402 (1993).
39. J. A. Speir, et al., Immunity 8, 553-562 (1998).
40. I. F. McKenzie, et al., JExp Med 150, 1323-1328 (1979); J. Bill, et al.,
JExp
Med 169, 115-133 (1989).
41. J.A. Frelinger, M. McMillan, Immunol Rev 154, 45-58 (1996).
42. C. Daniel, S. Horvath, P. M. Allen, Immunity 8, 543-552 (1998).
43. M. T. Scherer, L. Ignatowicz, G. M. Window, J. W. Kappler, P. Marrack,
Annu Rev Immunol 9, 101-128 (1993); S. R. Webb, N. R. J. Gascoigne, Curr Opin
Immunol 6, 467-475 (1994).
44. H. Li, et al., Immunity 9, 807-816 (1998); T. S. Jardetzky, et al., Nature
368,
711-718 (1994).
45. D. L. Woodland, M. A. Blackman, Immunol Today 14, 208-212 (1993); K.
Daly, et al., Jlmmunol ISS, 27-34 (1995); M. A. Blackman, D. L. Woodland,
Immunol Res 15, 98-113 (1996); D. Donson, et al., Jlmmunol 158, 5229-5236
(1997); A. M. Deckhut, Y. Chien, M. A. Blackman, D. L. Woodland, JExp Med
180, 1931-1935 (1994); N. Labrecque, J. Thibodeau, W. Mourad, R. P. Sekaly, J
Exp Med 180, 1921-1929 (1994).
46. E. L. Reinherz, P. C. Kung, G. Goldstein, R. H. Levey, S. F. Schlossman,
Proc Natl Acad Sci USA 77, 1588-1592 (1980); B. J. Fowlkes, D. M. Pardoll, Adv
Immunol 44, 207-264 (1989); A. Kruisbeek, Curr Opin Immunol 5, 227-234 (1993).
47. S. H. Chan, D. Cosgrove, C. Waltzinger, C. Benoist, D. Mathis, Cell 73,
225-236 (1993); S. H. Chan, C. Benoist, D. Mathis, Immunol Rev 135, 119-131
(1993); C. B. Davis, N. Kileen, M. E. Casey Crooks, D. Raulet, D. R. Littman,
Cell
73, 237-247 (1993); H. S. Teh, et al., Nature 335, 229-233 (1988); J. P. M.
van
Meerwijk, R. N. Germain, Science 261, 911-915 (1993); J. P. M. van Meerwijk,
E.
M. O'Connel, R. N. Germain, Semin Immunol 6, 231-239 (1994).
CA 02392387 2002-05-22
WO 01/38394 PCT/US00/31502
-50-
48. H. Kozono, J. White, J. Clements, P. Marrack, J. W. Kappler, Nature 369,
151-154 (1994).
49. H.-C. Chang, et al., Proc Natl Acad Sci USA 91, 11408-11412 (1994); J.
Liu,
et al., JBiol Chem 271, 33639-33646 (1996).
50. T. A. Jones, J-Y. Zou, S. W. Cowtan, M. Kjeldgaard, Acta Crystallogr A47,
110-119 (1991).
51. A. Nicholls, K. A. Sharp, B. Honig, Proteins: Struct. Funct. Genet. 11,
281-
296 (1991).
52. Production and purification of scDlO TCR.
53. Production and purification of MHC class II I-Ak.
54. Z. Ortwinowski, W. Minor, Meth Enzymol 276, 307-326 (1997).
54. J. Navaza, Acta CrystallogrA50, 157-163 (1994).
56. CA/I-Ak was crystallized with the hanging-droplet vapor diffusion method.
57. A. T. Briinger, Yale Univ Press, New Haven, CT (1992).
58. W. Kabsch, C. Sander, Biopolymers 22, 2577-2637 (1983).
59. P. J. Kraulis, JAppl Crystallogr 24, 946-950 (1991).
While this invention has been particularly shown and described with references
to
preferred embodiments thereof, it will be understood by those skilled in the
art that
various changes in form and details may be made therein without departing from
the
scope of the invention encompassed by the appended claims.
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SEQUENCE LISTING
<110> Dana-Farber Cancer Institute, Inc.
Ellis L. Reinherz
Tetsuro Sasada
Jia-huai Wang
<120> POLYPEPTIDES AND METHODS FOR THYMIC
VACCINATION
<130> 1062.2001003
<150> US 60/168,167
<151> 1999-11-30
<150> US 60/167,378
<151> 1999-11-24
<160> 2
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 8
<212> PRT
<213> Peptide
<400> 1
Arg Gly Tyr Val Tyr Gln Gly Leu
1 5
<210> 2
<211> 15
<212> PRT
<213> Peptide
<400> 2
Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Gly
1 5 10 15
