U.S. patent application number 14/417434 was filed with the patent office on 2015-07-09 for engineering t cell receptors.
This patent application is currently assigned to The Board of Trustees of the University of Illinoi. The applicant listed for this patent is The Board of Trustees of the University of Illinois. Invention is credited to David M. Kranz, Sheena N. Smith.
Application Number | 20150191524 14/417434 |
Document ID | / |
Family ID | 49997854 |
Filed Date | 2015-07-09 |
United States Patent
Application |
20150191524 |
Kind Code |
A1 |
Smith; Sheena N. ; et
al. |
July 9, 2015 |
ENGINEERING T CELL RECEPTORS
Abstract
The use of model T cell receptors (TCRs) as scaffolds for in
vitro engineering of novel specificities is provided. TCRs with de
novo binding to a specific peptide-major histocompatibility complex
(MHC) product can be isolated by: 1) mutagenizing a T cell receptor
protein coding sequence to generate a variegated population of
mutants (a library), 2) selection of the library of TCR mutants
with the specific peptide-MHC, using a process of directed
evolution and a "display" methodology (e.g., yeast, phage,
mammalian cell) and the peptide-MHC ligand. The process can be
repeated to identify TCR variants with improved affinity for the
selecting peptide-MHC ligand.
Inventors: |
Smith; Sheena N.;
(Champaign, IL) ; Kranz; David M.; (Champaign,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the University of Illinois |
Urbana |
IL |
US |
|
|
Assignee: |
The Board of Trustees of the
University of Illinoi
Urbana
IL
|
Family ID: |
49997854 |
Appl. No.: |
14/417434 |
Filed: |
July 26, 2013 |
PCT Filed: |
July 26, 2013 |
PCT NO: |
PCT/US13/52283 |
371 Date: |
January 26, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61676373 |
Jul 27, 2012 |
|
|
|
Current U.S.
Class: |
530/350 ; 506/9;
536/23.5 |
Current CPC
Class: |
C12N 15/1037 20130101;
C07K 14/70503 20130101; A61P 35/00 20180101; C07K 14/7051
20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This disclosure was made with U.S. Government support under
Grant numbers R01 GM55767 and T32 GM070421, awarded by the National
Institutes of Health. The U.S. Government has certain rights in the
disclosure.
Claims
1. A modified T cell receptor, or antigen binding fragment thereof,
comprising a V.alpha. and a V.beta. derived from a wild type T cell
receptor, wherein the V.alpha., the V.beta., or both, comprise a
mutation in one or more complementarity determining regions (CDRs)
relative to the wild type T cell receptor, wherein the modified T
cell receptor binds to a non-cognate peptide-MHC not bound by the
wild type T cell receptor.
2. The modified T cell receptor of claim 1, wherein the wild type T
cell receptor comprises the V.alpha. amino acid sequence set forth
in SEQ ID NO:1 and the V.beta. amino acid sequence set forth in SEQ
ID NO:2.
3. The modified T cell receptor of claim 2, comprising a modified
V.alpha. comprising an amino acid sequence having at least 80%
identity to the V.alpha. amino acid sequence set forth in SEQ ID
NO:1 and a modified V.beta. comprising an amino acid sequence
having at least 80% identity to the V.beta. amino acid sequence set
forth in SEQ ID NO:2, wherein the modified T cell receptor does not
bind to the cognate peptide-MHC bound by the wild type T cell
receptor.
4. The modified T cell receptor of claim 2, wherein the modified T
cell receptor comprises an amino acid substitution at one or more
of CDR1.alpha. 31, CDR3.alpha. 98, CDR3.beta. 99, CDR3.alpha. 97,
CDR3.beta. 102, CDR3.alpha. 99, CDR3.beta. 100, CDR3.beta. 101,
CDR1.alpha. 32, CDR1.beta. 30, CDR3.beta. 98.
5. The modified T cell receptor of claim 2, wherein the modified T
cell receptor comprises the wild type amino acid at position
CDR2.alpha. 51.
6. The modified T cell receptor of claim 5, wherein the modified T
cell receptor further comprises the wild type amino acid at
position CDR1.alpha. 31.
7. The modified T cell receptor of claim 6, wherein the modified T
cell receptor further comprises the wild type amino acid at
position CD1.alpha. 28 and CD1.alpha.52.
8. The modified T cell receptor of claim 1, wherein the wild type T
cell receptor is a single-chain T cell receptor A6-X15 comprising
the amino acid sequence set forth in SEQ ID NO:3.
9. The modified T cell receptor of claim 1, wherein the non-cognate
peptide-MHC comprises Mart1:HLA.A2, SL9 HIV:HLA.A2, WT-1:HLA.A2, or
SURV:HLA.A2.
10. The modified T cell receptor of claim 9, comprising 1) a
modified V.alpha. region comprising an amino acid sequence having
at least 90% identity to the V.alpha. region of the amino acid
sequence set forth in one of SEQ ID NOs:33, 41, or 42 and 2) a
modified V.beta. region comprising an amino acid sequence having at
least 90% identity to the V.beta. region of the amino acid sequence
set forth in one of SEQ ID NOs:33, 41, or 42.
11. The modified T cell receptor of claim 10, comprising the amino
acid sequence set forth in one of SEQ ID NOs:33, 41, or 42.
12. The modified T cell receptor of claim 1, wherein the modified T
cell receptor is generated by in vitro selection of a yeast display
library of mutant T cell receptors.
13. The modified T cell receptor of claim 1, wherein the wild type
T cell receptor is human.
14. The modified T cell receptor of claim 1, wherein the modified T
cell receptor is a single chain T cell receptor.
15. The modified T cell receptor of claim 1, wherein the wild type
T cell receptor binds HLA-A2.
16. A polypeptide encoding the modified T cell receptor of claim
1.
17. A polynucleotide encoding the polypeptide of claim 16.
18. A modified T cell receptor, or antigen binding fragment
thereof, comprising a V.alpha. and a V.beta. derived from a wild
type T cell receptor, wherein the V.alpha. comprises amino acid
residues 140 to 256 of SEQ ID NO:34, and wherein the V.beta.
comprises amino acid residues 1 to 122 of SEQ ID NO:34.
19. A modified T cell receptor, or antigen binding fragment
thereof, comprising a V.alpha. and a V.beta. derived from a wild
type T cell receptor, wherein the V.alpha. comprises amino acid
residues 140 to 255 of SEQ ID NO:43, and wherein the V.beta.
comprises amino acid residues 1 to 122 of SEQ ID NO:43.
20. A method for engineering a T cell receptor, or an antigen
binding fragment thereof, with a desired specificity comprising: a)
isolating a polynucleotide that encodes a wild type T cell
receptor, or an antigen binding fragment thereof; b) generating a
library of mutant T cell receptors, or antigen binding fragments
thereof, wherein the mutant T cell receptors, or antigen-binding
fragment thereof, comprise a mutation in one or more
complementarity determining regions relative to the wild type T
cell receptor; c) expressing the mutant T cell receptors in a
surface display system; and d) selecting mutant T cell receptors
that bind to a non-cognate peptide-MHC.
21. The method of claim 20 wherein the wild type T cell receptor
comprises the V.alpha. amino acid sequence set forth in SEQ ID NO:1
and the V.beta. amino acid sequence set forth in SEQ ID NO:2.
22. The method of claim 20 wherein the wild type T cell receptor is
a single-chain T cell receptor A6-X15 comprising the amino acid
sequence set forth in SEQ ID NO:3.
23. The method of claim 20 wherein the surface display system is a
yeast display system.
24. The method of claim 20 wherein the non-cognate peptide-MHC is
Mart1:HLA.A2, SL9 HIV:HLA.A2, WT-1:HLA.A2, or SURV:HLA.A2.
25. The method of claim 20, further comprising a step of affinity
maturation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Patent Application No. 61/676,373
filed Jul. 27, 2012, and this provisional application is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The disclosure relates to T cell receptor (TCR) scaffolds
and TCR libraries, as well as methods of producing modified TCRs
and single chain TCRs and the corresponding use of the TCRs for
therapeutic, diagnostic, and imaging methods.
STATEMENT REGARDING SEQUENCE LISTING
[0004] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
IMMU.sub.--002.sub.--01WO_ST25.txt. The text file is 65 KB, was
created on Jul. 26, 2013 and is being submitted electronically via
EFS-Web.
BACKGROUND
[0005] T cell receptors (TCRs) and antibodies are molecules that
have evolved to recognize different classes of antigens
(ligands)((Murphy (2012), xix, 868 p.)). TCRs are antigen-specific
molecules that are responsible for recognizing antigenic peptides
presented in the context of a product of the major
histocompatibility complex (MHC) on the surface of antigen
presenting cells (APCs) or any nucleated cell (e.g., all human
cells in the body, except red blood cells). In contrast, antibodies
typically recognize soluble or cell-surface antigens, and do not
require presentation of the antigen by an MHC. This system endows T
cells, via their TCRs, with the potential ability to recognize the
entire array of intracellular antigens expressed by a cell
(including virus proteins) that are processed intracellularly into
short peptides, bound to an intracellular MHC molecule, and
delivered to the surface as a peptide-MHC complex (pepMHC). This
system allows virtually any foreign protein (e.g., mutated cancer
antigen or virus protein) or aberrantly expressed protein to serve
a target for T cells (reviewed in (Davis and Bjorkman (1988)
Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16,
523-544; Murphy (2012), xix, 868 p.)).
[0006] The interaction of a TCR and a pepMHC can drive the T cell
into various states of activation, depending on the affinity (or
dissociation rate) of binding. The TCR recognition process allows a
T cell to discriminate between a normal, healthy cell and, e.g.,
one that has become transformed via a virus or malignancy, by
providing a diverse repertoire of TCRs, wherein there is a high
probability that one or more TCRs will be present with a binding
affinity for the foreign peptide bound to an MHC molecule that is
above the threshold for stimulating T cell activity (Manning and
Kranz (1999) Immunology Today, 20, 417-422).
[0007] To date, wild type TCRs isolated from either human or mouse
T cell clones that were identified by in vitro culturing have been
shown to have relatively low binding affinities (K.sub.D=1-300
.mu.M) (Davis et al. (1998) Annu Rev Immunol, 16, 523-544). Part of
the explanation for this seems to be that T cells that develop in
the thymus are negatively selected (tolerance induction) on
self-pepMHC ligands, such that T cells with too high of an affinity
are deleted (Starr et al. (2003) Annu Rev Immunol, 21, 139-76). To
compensate for these relatively low affinities, T cells have
evolved a co-receptor system in which the cell surface molecules
CD4 and CD8 bind to the MHC molecules (class II and class I,
respectively) and synergize with the TCR in mediating signaling
activity. CD8 is particularly effective in this process, allowing
TCRs with very low affinity (e.g., K.sub.D=300 .mu.M) to mediate
potent antigen-specific activity.
[0008] Directed evolution has been used to generate TCRs with
higher affinity for a specific pepMHC. The three different display
methods that have been used are yeast display (Holler et al. (2003)
Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA,
97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23,
349-54), and T cell display (Chervin et al. (2008) J Immunol
Methods, 339, 175-84). In all three approaches, the process
involves engineering, or modifying, a TCR that exhibits the normal,
low affinity of the wild-type TCR, so that affinity of mutants of
the TCR have increased affinity for the cognate pepMHC (the
original antigen that the T cells were specific for). Thus, the
wild-type TCR was used as a template for producing mutagenized
libraries in one or more of the CDRs, and mutants with higher
affinity were selected by binding to the cognate peptide-MHC
antigen.
[0009] A major problem with each of these TCR-engineering
approaches is that they require a different TCR isolated from a T
cell clone with reactivity towards a specific peptide antigen in
order to develop a higher affinity TCR mutant specific for the
peptide antigen (cognate antigen), or structurally similar variants
thereof. As there are over 300 defined peptide antigens from
various cancers, and many antigens from viruses, it would be
advantageous if the same TCR could be used as a platform to
generate TCRs against structurally very different antigens (called
non-cognate antigens), using in vitro engineering. The present
invention addresses these needs and more.
SUMMARY OF THE INVENTION
[0010] The present invention relates to T cell receptor (TCR)
scaffolds useful, for example and by way of example only, for the
generation of products having novel binding specificities. More
specifically, the present invention relates to a library of T cell
receptor proteins displayed on the surface of yeast, phage, or
mammalian cells; to TCR proteins that are selected from the library
for binding to a non-cognate antigen not recognized by the original
TCR; and to the use of the TCR proteins selected in vitro for
therapeutic, diagnostic, or imaging applications.
[0011] One aspect of the invention relates to a modified T cell
receptor, or antigen binding fragment thereof, comprising a
V.alpha. and a V.beta. derived from a wild type T cell receptor,
wherein the V.alpha., the V.beta., or both, comprise a mutation in
one or more complementarity determining regions (CDRs) relative to
the wild type T cell receptor, wherein the modified T cell receptor
binds to a non-cognate peptide-MHC not bound by the wild type T
cell receptor.
[0012] In one embodiment, the wild type T cell receptor comprises
the V.alpha. amino acid sequence set forth in SEQ ID NO:1 and the
V.beta. amino acid sequence set forth in SEQ ID NO:2. In a related
embodiment, the modified T cell receptor comprises a modified
V.alpha. comprising an amino acid sequence having at least 80%
identity to the V.alpha. amino acid sequence set forth in SEQ ID
NO:1 and a modified V.beta. comprising an amino acid sequence
having at least 80% identity to the V.beta. amino acid sequence set
forth in SEQ ID NO:2, wherein the modified T cell receptor does not
bind to the cognate peptide-MHC bound by the wild type T cell
receptor. In another embodiment, the modified T cell receptor
comprises an amino acid substitution at one or more of CDR1.alpha.
31, CDR3.alpha. 98, CDR3.beta. 99, CDR3.alpha. 97, CDR3.beta. 102,
CDR3.alpha. 99, CDR3.beta. 100, CDR3.beta. 101, CDR1.alpha. 32,
CDR1.beta. 30, CDR3.beta. 98. In yet another embodiment, the
modified T cell receptor comprises the wild type amino acid at
position CDR2.alpha. 51. In one embodiment, the modified T cell
receptor further comprises the wild type amino acid at position
CDR1.alpha. 31. In a related embodiment, the modified T cell
receptor further comprises the wild type amino acid at position
CD1.alpha. 28 and CD1.alpha.52.
[0013] In one embodiment, the wild type T cell receptor is a
single-chain T cell receptor A6-X15 comprising the amino acid
sequence set forth in SEQ ID NO:3. In another embodiment, the
non-cognate peptide-MHC comprises Mart1:HLA.A2, SL9 HIV:HLA.A2,
WT-1:HLA.A2, or SURV:HLA.A2. In a related embodiment, the modified
T cell receptor comprises 1) a modified V.alpha. region comprising
an amino acid sequence having at least 90% identity to the V.alpha.
region of the amino acid sequence set forth in one of SEQ ID
NOs:33, 41, or 42 and 2) a modified V.beta. region comprising an
amino acid sequence having at least 90% identity to the V.beta.
region of the amino acid sequence set forth in one of SEQ ID
NOs:33, 41, or 42. In certain embodiments, the amino acid sequence
set forth in one of SEQ ID NOs:33, 41, or 42.
[0014] In another embodiment, the modified T cell receptor is
generated by in vitro selection of a yeast display library of
mutant T cell receptors. In one embodiment, the wild type T cell
receptor is human. In another embodiment, the modified T cell
receptor is a single chain T cell receptor. In yet another
embodiment, the wild type T cell receptor binds HLA-A2. In one
embodiment, a polypeptide encoding the modified T cell receptor is
provided. In a related embodiment, a polynucleotide encoding the
polypeptide is provided.
[0015] Another aspect of the invention provides a modified T cell
receptor, or antigen binding fragment thereof, comprising a
V.alpha. and a V.beta. derived from a wild type T cell receptor,
wherein the V.alpha. comprises amino acid residues 140 to 256 of
SEQ ID NO:34, and wherein the V.beta. comprises amino acid residues
1 to 122 of SEQ ID NO:34.
[0016] Another aspect of the invention provides a modified T cell
receptor, or antigen binding fragment thereof, comprising a
V.alpha. and a V.beta. derived from a wild type T cell receptor,
wherein the V.alpha. comprises amino acid residues 140 to 255 of
SEQ ID NO:43, and wherein the V.beta. comprises amino acid residues
1 to 122 of SEQ ID NO:43.
[0017] One aspect of the invention provides a method for
engineering a T cell receptor, or an antigen binding fragment
thereof, with a desired specificity comprising: a) isolating a
polynucleotide that encodes a wild type T cell receptor, or an
antigen binding fragment thereof; b) generating a library of mutant
T cell receptors, or antigen binding fragments thereof, wherein the
mutant T cell receptors, or antigen-binding fragment thereof,
comprise a mutation in one or more complementarity determining
regions relative to the wild type T cell receptor; c) expressing
the mutant T cell receptors in a surface display system; and d)
selecting mutant T cell receptors that bind to a non-cognate
peptide-MHC.
[0018] In one embodiment, the wild type T cell receptor comprises
the V.alpha. amino acid sequence set forth in SEQ ID NO:1 and the
V.beta. amino acid sequence set forth in SEQ ID NO:2. In another
embodiment, the wild type T cell receptor is a single-chain T cell
receptor A6-X15 comprising the amino acid sequence set forth in SEQ
ID NO:3. In one embodiment, the surface display system is a yeast
display system. In another embodiment, the non-cognate peptide-MHC
is Mart1:HLA.A2, SL9 HIV:HLA.A2, WT-1:HLA.A2, or SURV:HLA.A2. In
one embodiment, the method further comprises a step of affinity
maturation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a diagram that shows a method for the rational
design of a single scaffold for engineering higher affinity TCRs
specific for non-cognate antigens.
[0020] FIG. 2A is a 3-dimensional diagram that shows a structural
view of the A6 TCR:pepMHC complex (A6; PDB:1AO7). The variable (V)
and constant (C) regions of the .alpha.-chain and .beta.-chain are
indicated. The structure shown does not include the C.alpha. region
of the A6 TCR. HLA-A2 (.alpha.1, .alpha.2, .alpha.3, and .beta.2m)
is shown in gray, and the Tax peptide (LLFGYPVYV; SEQ ID NO:5) is
shown in black.
[0021] FIG. 2B is a 3-dimensional diagram that shows the CDR
footprint over the peptide-MHC (Tax-HLA.A2).
[0022] FIG. 3 is an overlay of the footprint of the 5 most
encountered residues within 3.0 .ANG. of corresponding peptide in
the A6 wt and 5 predicted structures.
[0023] FIG. 4A depicts the crystal structures of
V.alpha.2-containing TCRs and shows predicted key MHC contact
positions in the TCR CDR1.alpha. and CDR2.alpha. loops (modified
from (Borbulevych et al. (2011) J Immunol, 187, 2453-63)).
[0024] FIG. 4B shows the binding of high-affinity A6 TCR X15 and 4
variants, each having an alanine substitution at one of four
residues, to various concentrations of Tax (LLFGYPVYV, SEQ ID
NO:5):HLA-A2 dimer (DimerX; obtained from BD Pharmingen).
[0025] FIG. 5 shows the amino acid sequence of the A6 V.beta. (SEQ
ID NO:2) and V.alpha. (SEQ ID NO:1) regions, and the positions
shaded in gray indicate where degenerate libraries were constructed
in the stabilized variant A6-X15 (SEQ ID NO:3). The CDRs of each V
domain are labeled, and the sequence of the linker that joins the
two V regions in the yeast display vector is also shown.
[0026] FIG. 6 is a schematic of a single-chain T cell receptor
(scTCR) using yeast display.
[0027] FIG. 7 shows the amino acid sequence alignment of ten clones
chosen from a degenerate library of the human A6 X15 scTCR, the RD1
library.
[0028] FIG. 8 shows a flow cytometry histogram of the RD1 library
after sorting with the cognate antigen (Tax:HLA.A2). Gray indicates
yeast cells stained with secondary antibody only.
[0029] FIG. 9 shows a flow cytometry histogram of the RD1 library
after sorting with the non-cognate antigen (Mart1:HLA.A2). Gray
indicates yeast cells stained with secondary antibody only.
[0030] FIG. 10A shows the sequence alignments of six clones
isolated following the 4th sort with the cognate ligand, Tax
(LLFGYPVYV; SEQ ID NO:5):HLA-A2 dimer.
[0031] FIG. 10B shows the DNA sequence alignments of the six RD1
scaffold variants at degenerate positions. Below each codon is the
amino acid encoded by that codon with the number of possible codon
combinations within an NNS library.
[0032] FIG. 11A is a histogram depicting positive staining with the
anti-HA antibody for the N-terminal tag, thus indicating surface
expression of the AGA2 fusion. Cells were stained with the anti-HA
antibody and goat anti-mouse IgG alexa 647 secondary antibody
(black line histogram) or secondary only as a control (gray filled
histogram). The negative peak for the HA stained cells (black line)
is observed in all yeast display experiments and is due to yeast
that have lost plasmid, and serves as an internal control for each
induced yeast sample.
[0033] FIG. 11B is a histogram that shows negative staining with
c-myc as this clone lacked the C-terminal c-myc tag. Cells were
stained with chicken anti-c-myc antibody and goat anti-chicken IgY
alexa 647 secondary antibody or secondary only as a control
(gray).
[0034] FIG. 11C is a histogram showing staining of the A6-X15 clone
with various concentrations of the selecting cognate antigen,
Tax:HLA.A2, dimer at the indicated concentrations.
[0035] FIG. 11D is a histogram showing staining of the A6-X15 clone
with various concentrations of the non-selecting non-cognate
antigen, Mart1:HLA.A2.
[0036] FIG. 11E is a plot of the mean fluorescence intensity (MFI)
from staining with various concentrations of the peptide:HLA.A2
dimers Tax or Mart1 peptide:HLA.A2 dimer at 4-500 nM.
[0037] FIG. 12A shows the sequence alignments of five clones
isolated following the 5th sort with a non-cognate ligand, Mart1
(ELAGIGILTV; SEQ ID NO:7):HLA-A2 dimer.
[0038] FIG. 12B shows the DNA sequence alignments of the five RD1
scaffold variants at degenerate positions. Below each codon is the
amino acid encoded by that codon with the number of possible codon
combinations within an NNS library.
[0039] FIG. 13A is a histogram that shows positive staining of the
clone with the anti-HA antibody for the N-terminal tag, and thus
indicating surface expression of the AGA2 fusion. Cells were
stained with anti-HA antibody and goat anti-mouse IgG alexa 647
secondary antibody (black line histogram) or secondary only as a
control (gray filled histogram).
[0040] FIG. 13B is a histogram that shows positive staining with
c-myc as this clone contained the C-terminal c-myc tag. Cells were
stained with chicken anti-c-myc antibody and goat anti-chicken IgY
Alexa 647 secondary antibody or secondary only as a control
(gray).
[0041] FIG. 13C is a histogram that shows staining of the A6-S5-4
clone with various concentrations of the selecting non-cognate
antigen, Mart1:HLA.A2 dimer, at the indicated concentrations.
[0042] FIG. 13D is a histogram that shows the staining of the
A6-S5-4 clone with various concentrations of the non-selecting
cognate antigen, Tax:HLA.A2.
[0043] FIG. 13E is a plot of the mean fluorescence intensity (MFI)
from staining with various concentrations of the peptide:HLA.A2
dimers Mart1 or Tax peptide:HLA.A2 dimer at 4-500 nM.
[0044] FIG. 14 shows the amino acid sequences of the scaffold A6
single-chain TCR wild type A6 V.alpha. and V.beta. regions and two
high-affinity variants isolated from the RD1 library, including the
five positions of degeneracy in the library. The two clones were
isolated from the selection with Tax (clone S4-3; identical to the
single-chain stabilized TCR A6-X15; SEQ ID NO:3), and Mart1 (clone
S5-4; SEQ ID NO:33). X represents any amino acid. The asterisk is
used to indicate where no linker is present in the wild-type A6
structure.
[0045] FIG. 15A shows flow cytometry histograms of the
RD1-MART1-S5-4 CDR3 libraries after sorting with the non-cognate,
selecting antigen, MART1 (ELAGIGILTV; SEQ ID NO:7)/HLA.A2.
[0046] FIG. 15B is a histogram that shows staining of the
RD1-MART1-S5-4 clone, which was used as a template for CDR3
affinity maturation libraries, with 200 nM, 1 .mu.M, and 5 .mu.M
MART1 (ELAGIGILTV; SEQ ID NO:7)/HLA.A2 UV-exchanged monomers
followed by PE-conjugated streptavidin.
[0047] FIG. 15C is a histogram that shows staining of the
RD1-MART1.sup.HIGH clone isolated after the second sort of the
RD1-MART1-S5-4 CDR3 libraries with 200 nM, 1 .mu.M, and 5 .mu.M
MART1 (ELAGIGILTV; SEQ ID NO:7)/HLA.A2 UV-exchanged monomers
followed by PE-conjugated streptavidin.
[0048] FIG. 16A is a histogram that shows the RD1-MART1.sup.HIGH
clone stained with 10 nM MART1/HLA-A2 dimer (DimerX; obtained from
BD Pharmingen) and APC-conjugated goat anti-mouse secondary
antibody as a positive control. Gray indicates yeast cells stained
with secondary antibody only.
[0049] FIG. 16B is a histogram that shows the RD1-MART1.sup.HIGH
clone stained with 500 nM null Tax/HLA-A2 dimer (DimerX; obtained
from BD Pharmingen) and APC-conjugated goat anti-mouse secondary
antibody.
[0050] FIG. 16C is a histogram that shows the RD1-MART1.sup.HIGH
clone stained with 500 nM null WT1/HLA-A2 dimer (DimerX; obtained
from BD Pharmingen) and APC-conjugated goat anti-mouse secondary
antibody.
[0051] FIG. 16D is a histogram that shows the RD1-MART1.sup.HIGH
clone stained with 500 nM null Survivin/HLA-A2 dimer (DimerX;
obtained from BD Pharmingen) and APC-conjugated goat anti-mouse
secondary antibody.
[0052] FIGS. 17A-D are a series of histograms that show flow
cytometry analysis of human T2 (HLA-A2+) cells incubated first with
no peptide (FIG. 17A), Tax (FIG. 17B), WTI (FIG. 17C), or MART1
(FIG. 17D), followed by incubation with biotin-labeled
RD1-MART1.sup.HIGH TCR.
[0053] FIG. 17E is a line graph that depicts the titration showing
that the RD1-MART1.sup.HIGH TCR had an affinity (K.sub.D value) of
at least 28 nM.
[0054] FIG. 18 shows the amino acid sequences of the scaffold A6
single-chain TCR (wild-type) and high-affinity variants isolated
and affinity matured from the RD1 library, including the five
positions of degeneracy in the library. Two of the clones were
isolated from the selection with Tax (clone RD1-Tax-S4-3; identical
to the single-chain stabilized TCR A6-X15) and MART1 (clone
RD1-MART1-S5-4). The high affinity clone selected from the
RD1-MART1-S5-4 CDR3 affinity maturation libraries is shown
(RD1-MART1.sup.HIGH). X represents any amino acid. The asterisk
indicates where no linker is present in the wild-type A6
structure.
[0055] FIG. 19A is 3-dimensional diagram of the A6:Tax (LLFGYPVYV;
SEQ ID NO:5)/HLA.A2 crystal structure (PDB: 1AO7) in close
proximity to an overlay of MART1 (ELAGIGILTV; SEQ ID NO:7)/HLA.A2
(PDB: 1JF1) (Sliz et al. (2001) J Immunol, 167, 3276-84) and WT1
(RMFPNAPYL; SEQ ID NO:9)/HLA.A2 (PDB: 3HPJ) (Borbulevych et al.
(2010) Mol Immunol, 47, 2519-24) crystal structures. Positions
labeled in bold (TCR.alpha. D27, G29, and S99; TCR.beta. L99 and
W100) were made degenerate based on NNK nucleic acid composition.
TCR.alpha. Q31 is a binary position where either the wild type
residue glutamine or threonine may be selected. Positions 100-103
in CDR3.beta. are binary where the four adjacent residues may be
selected as A6 wild type (AGGR, SEQ ID NO:44) or A6-X15 (MSAQ, SEQ
ID NO:45).
[0056] FIG. 19B is a flow cytometry histogram of the RD2 library
after sorting with the selecting cognate antigen Tax/HLA.A2. Gray
indicates yeast cells stained with secondary antibody only.
[0057] FIG. 19C is a flow cytometry histogram of the RD2 library
after sorting with the non-cognate antigen MART1/HLA.A2.
[0058] FIG. 20 shows the sequence alignment of five clones isolated
from the second generation degenerate library (RD2) of the human A6
scTCR that shows diversity prior to selection.
[0059] FIG. 21A is a histogram that shows the RD2-MART1-S3-3 clone,
selected following the 3.sup.rd sort of the RD2 library, stained
with 2 .mu.M MART1/HLA.A2 UV-exchanged monomers, PE-conjugated
streptavidin.
[0060] FIG. 21B is a histogram that shows the RD2-MART1-S3-4 clone,
selected following the 3.sup.rd sort of the RD2 library, stained
with 2 .mu.M MART1/HLA.A2 UV-exchanged monomers, PE-conjugated
streptavidin.
[0061] FIG. 21C is a histogram that shows the RD2-MART1-S3-3 clone,
selected following the 3.sup.rd sort of the RD2 library, stained
with 2 .mu.M null Tax/HLA.A2 UV-exchanged monomers, PE-conjugated
streptavidin.
[0062] FIG. 21D is a histogram that shows the RD2-MART1-S3-4 clone,
selected following the 3.sup.rd sort of the RD2 library, stained
with 2 .mu.M null Tax/HLA.A2 UV-exchanged monomers, PE-conjugated
streptavidin.
[0063] FIG. 21E shows the sequences of the scaffold A6 single-chain
TCR and high-affinity variants isolated from the RD2 library
selection with MART1. Sequences of the wild type A6 V.alpha. and
V.beta. regions of the A6 TCR (Garboczi et al. (1996) Nature, 384,
134-141), the high affinity single-chain variant A6-X15 (Aggen et
al. (2011) Protein Engineering, Design, & Selection, 24,
361-72), and two of the clones isolated from the selection with
MART1 (clone RD2-MART1-S3-3 and clone RD2-MART1-S3-4) are
shown.
[0064] FIG. 22 shows the amino acid sequence of an alternative
scaffold, human TCR T1-S18.45.
[0065] FIG. 23A is a histogram that shows the titration of
biotinylated T1-S18.45 scTv on antigen-presenting cell line T2
(HLA-A2+) pre-loaded with MART-1 peptide (1 .mu.M) or null peptide,
SL9 (1 .mu.M). Cells were stained with 3.9 nM, 7.8 nM, 15.2 nM,
31.1 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1 .mu.M, and 5 .mu.M
biotinylated T1-S18.45 scTv as indicated and followed by SA:PE.
Data shown is representative of 4 experiments.
[0066] FIG. 23B is a line graph showing the mean fluorescence unit
(MFU) values of histograms in FIG. 23A plotted versus scTv-biotin
concentration.
[0067] FIG. 24 is a diagram that illustrates exemplary therapeutic
applications of the high-affinity, single-chain TCRs isolated from
the scaffold libraries. FIG. 24A shows five examples of TCR formats
for use as soluble therapeutic products: 1) single-chain TCR in
either a V.alpha.-V.beta. orientation or V.beta.-V.alpha.
orientation (mutated high-affinity V domains are shown with an
asterisk); 2) single-chain TCR fused in frame with the constant
region domains of an antibody; 3) in-frame immunoglobulin fusion to
either the constant region of the light chain or the heavy chain;
4) single-chain TCR (or the immunoglobulin fusions shown in 2 and
3) directly coupled to a drug; and 5) single-chain TCR linked
in-frame with a single-chain Fv (VL-linker-VH).
[0068] FIG. 24B shows the variable domains (V) isolated by yeast
display for high-affinity binding using the TCR scaffold inserted
into mammalian cell vectors for expression by T cells in adoptive T
cell therapy as 1) single-chain receptors in chimeric antigen
receptors (CARs) and 2) full length .alpha. and .beta. TCRs.
BRIEF DESCRIPTION OF THE SEQUENCES
[0069] SEQ ID NO:1 is the amino acid sequence of the V.alpha.
region of the A6 TCR.
[0070] SEQ ID NO:2 is the amino acid sequence of the V.beta. region
of the A6 TCR.
[0071] SEQ ID NO:3 is the amino acid sequence of the single chain
TCR A6-X15 and the identical clones RD1-Tax-S4-3 and
RD1-Tax-S4-5.
[0072] SEQ ID NO:4 is the amino acid sequence of the RD1
library.
[0073] SEQ ID NO:5 is the amino acid sequence of the Tax
antigen.
[0074] SEQ ID NO:6 is the amino acid sequence of the Mart1-9 mer
antigen.
[0075] SEQ ID NO:7 is the amino acid sequence of the Mart1-10 mer
antigen.
[0076] SEQ ID NO:8 is the amino acid sequence of the SL9 HIV
antigen.
[0077] SEQ ID NO:9 is the amino acid sequence of the WT-1
antigen.
[0078] SEQ ID NO:10 is the amino acid sequence of the Survivin
antigen.
[0079] SEQ ID NO:11 is the amino acid sequence of the NY-ESO-1
antigen.
[0080] SEQ ID NO:12 is the amino acid sequence of the PPI
antigen.
[0081] SEQ ID NO:13 is the amino acid sequence of the MDM2
antigen.
[0082] SEQ ID NO:14 is the amino acid sequence of the HBE183
antigen.
[0083] SEQ ID NO:15 is the amino acid sequence of the gp100
antigen.
[0084] SEQ ID NO:16 is the amino acid sequence of the MUC1
antigen.
[0085] SEQ ID NO:17 is the amino acid sequence of the MAGE A3
antigen.
[0086] SEQ ID NO:18 is the amino acid sequence of the HER-2/neu
antigen.
[0087] SEQ ID NO:19 is the amino acid sequence of the EGFRvIII
antigen.
[0088] SEQ ID NO:20 is the amino acid sequence of the CEA
antigen.
[0089] SEQ ID NO:21 is the amino acid sequence of the linker of the
RD1 library.
[0090] SEQ ID NOs:22-31 are the amino acid sequences of clones
#1-10 of the RD1 library.
[0091] SEQ ID NO:32 is the amino acid sequence of the clone
RD1-Tax-S4-1, and the identical clones RD1-Tax-S4-2, RD1-Tax-S4-4,
and RD1-Tax-S4-6.
[0092] SEQ ID NO:33 is the amino acid sequence of the clone
RD1-Mart1-S5-1, and the identical clones RD1-Mart1-S5-2,
RD1-Mart1-S5-3, RD1-Mart1-S5-4, RD1-Mart1-S5-5, and
RD1-Mart1-S5-6.
[0093] SEQ ID NO:34 is the amino acid sequence of the clone
RD1-Mart1.sup.HIGH.
[0094] SEQ ID NO:35 is the amino acid sequence of the RD2
library.
[0095] SEQ ID NOs:36-40 are the amino acid sequences of clones #1-5
of the RD2 library.
[0096] SEQ ID NO:41 is the amino acid sequence of the clone
RD2-Mart1-S3-3.
[0097] SEQ ID NO:42 is the amino acid sequence of the clone
RD2-Mart1-S3-4.
[0098] SEQ ID NO:43 is the amino acid sequence of the clone
T1-S18.45.
[0099] SEQ ID NO:44 is the amino acid sequence of positions 100-103
in CDR3.beta. of the A6 wild type TCR.
[0100] SEQ ID NO:45 is the amino acid sequence of positions 100-103
in CDR3.beta. of A6-X15.
[0101] SEQ ID NO:46 is the amino acid sequence of the cognate
antigen of the TCR modified by Kessels et al. ((2000) Proc Natl
Acad Sci USA, 97, 14578-14583).
[0102] SEQ ID NO:47 is the amino acid sequence of the structurally
similar peptide of the TCR modified by Kessels et al. ((2000) Proc
Natl Acad Sci USA, 97, 14578-14583).
[0103] SEQ ID NO:48 is the polynucleotide sequence of the 5' region
of the RD1 gene optimized for both yeast and E. coli.
[0104] SEQ ID NO:49 is the polynucleotide sequence of the 3' region
of the RD1 gene optimized for both yeast and E. coli.
[0105] SEQ ID NO:50 is the polynucleotide sequence of the forward
primer used to add pCT302 overhangs.
[0106] SEQ ID NO:51 is the polynucleotide sequence of the reverse
primer used to add pCT302 overhangs.
[0107] SEQ ID NO:52 is the polynucleotide sequence of the forward
primer used to generate the CDR3.beta.1 library (Splice 4L).
[0108] SEQ ID NO:53 is the polynucleotide sequence of the reverse
primer used to generate a CDR3.beta.1 library (Splice 4L).
[0109] SEQ ID NO:54 is the polynucleotide sequence of the forward
primer used to generate a CDR3.beta.1 library (T7).
[0110] SEQ ID NO:55 is the polynucleotide sequence of the reverse
primer used to generate a CDR3.beta.1 library (T7).
[0111] SEQ ID NO:56 is the polynucleotide sequence of the forward
primer used to generate a CDR3.beta.2 library.
[0112] SEQ ID NO:57 is the polynucleotide sequence of the reverse
primer used to generate a CDR3.beta.2 library.
[0113] SEQ ID NO:58 is the polynucleotide sequence of the forward
primer used to generate a CDR3.alpha. library.
[0114] SEQ ID NO:59 is the polynucleotide sequence of the reverse
primer used to generate a CDR3.alpha. library.
[0115] SEQ ID NO:60 is the polynucleotide sequence of the
N-terminal DNA flanking sequence added to the RD2 library
sequence.
[0116] SEQ ID NO:61 is the polynucleotide sequence of the
C-terminal DNA flanking sequence added to the RD2 library
sequence.
DETAILED DESCRIPTION
[0117] The following description is intended to facilitate
understanding of the disclosure but is not intended to be
limiting.
[0118] In general, the terms and phrases used herein have their
art-recognized meaning, which can be found by reference to standard
texts, journal references and contexts known to those skilled in
the art. The following definitions are provided to clarify their
specific use in the context of the disclosure.
[0119] As used herein, "linked" refers to an association between
two groups, which can be a covalent or non-covalent association.
Groups may be linked using a variable length peptide chain, a
non-amino acid chemical group or other means as known in the art. A
linker region can be an amino acid sequence that operably links two
functional or structural domains of a protein or peptide.
[0120] As used herein, the term "chemotherapeutic agent" refers to
any substance capable of reducing or preventing the growth,
proliferation, or spread of a cancer cell, a population of cancer
cells, tumor, or other malignant tissue. The term is intended also
to encompass any antitumor or anticancer agent.
[0121] As used herein, the term "effective amount" is intended to
encompass contexts such as a pharmaceutically effective amount or
therapeutically effective amount. For example, in certain
embodiments, the effective amount is capable of achieving a
beneficial state, beneficial outcome, functional activity in a
screening assay, or improvement of a clinical condition.
[0122] As used herein, the term "cancer cell" is intended to
encompass definitions as broadly understood in the art. In one
embodiment, the term refers to an abnormally regulated cell that
can contribute to a clinical condition of cancer in a human or
animal. In one embodiment, the term can refer to a cultured cell
line or a cell within or derived from a human or animal body. A
cancer cell can be of a wide variety of differentiated cell,
tissue, or organ types as is understood in the art. Particular
examples of cancer cells include breast cancer, colon cancer, skin
cancer, ovarian cancer, leukemia, lung cancer, liver cancer,
testicular cancer, esophageal cancer, and other types of
cancer.
[0123] As used herein, the term "cognate antigen" refers to the
antigen for which the original TCR was shown to bind to and have
specificity for. Similarly, the term "non-cognate antigen" refers
to an antigen for which the TCR did not bind to nor have
specificity for. More specifically, the "cognate" peptide refers to
the antigenic peptide that the original TCR bound to, when it was
part of a complex with a protein encoded by the major
histocompatibilty complex (MHC). The "non-cognate" peptide refers
to a peptide that the original TCR did not bind to, when it was
part of a complex with a protein encoded by the MHC.
[0124] The terms "wild type" and "wt" are used interchangeably
herein and are used in reference to a TCR having an amino acid
sequence or a polynucleotide encoding the variable regions isolated
from a naturally occurring or non-modified TCR, e.g., the original
or parent T cell clone, with specificity for the cognate
antigen.
[0125] In the figures and tables that present amino acid sequences,
the wild type is designated "wt". In the sequences presented below
the top sequence, a dash indicates the amino acid is the same as
that present in the wt or top sequence of the alignment. A letter
indicates a substitution has been made in that position from the
top sequence.
[0126] As used herein, the terms "modified", "variant", "mutant",
"mutated" and "derived" T cell receptor refer to TCR sequences of
the variable regions as isolated from the original T cell clone
having one or more mutations. Examples of modified TCRs include
higher affinity TCRs and TCRs having binding specificity for a
non-cognate antigen.
[0127] A coding sequence is the part of a gene or cDNA which codes
for the amino acid sequence of a protein, or for a functional RNA
such as a tRNA or rRNA.
[0128] Complement or complementary sequence means a sequence of
nucleotides which forms a hydrogen-bonded duplex with another
sequence of nucleotides according to Watson-Crick base-pairing
rules.
[0129] Downstream refers to a relative position in DNA or RNA and
is the region toward the 3' end of a strand.
[0130] Expression refers to the transcription of a gene into
structural RNA (rRNA, tRNA) or messenger RNA (mRNA) and subsequent
translation of an mRNA into a protein.
[0131] Two nucleic acid sequences are heterologous to one another
if the sequences are derived from separate organisms, whether or
not such organisms are of different species, as long as the
sequences do not naturally occur together in the same arrangement
in the same organism.
[0132] Homology refers to the extent of identity between two
nucleotide or amino acid sequences.
[0133] An amino acid sequence that is functionally equivalent to a
specifically exemplified TCR sequence is an amino acid sequence
that has been modified by single or multiple amino acid
substitutions, by addition and/or deletion of amino acids, or where
one or more amino acids have been chemically modified, but which
nevertheless retains the binding specificity and high affinity
binding activity of a cell-bound or a soluble TCR protein of the
present disclosure. Functionally equivalent nucleotide sequences
are those that encode polypeptides having substantially the same
biological activity as a specifically exemplified cell-bound or
soluble TCR protein. In the context of the present disclosure, a
soluble TCR protein lacks the portions of a native cell-bound TCR
and is stable in solution (i.e., it does not generally aggregate in
solution when handled as described herein and under standard
conditions for protein solutions).
[0134] The term "isolated" refers to a composition, compound,
substance, or molecule altered by the hand of man from the natural
state. For example, a composition or substance that occurs in
nature is isolated if it has been changed or removed from its
original environment, or both. For example, a polynucleotide or a
polypeptide naturally present in a living animal is not isolated,
but the same polynucleotide or polypeptide separated from the
coexisting materials of its natural state is isolated, as the term
is employed herein.
[0135] A nucleic acid construct is a nucleic acid molecule which is
isolated from a naturally occurring gene or which has been modified
to contain segments of nucleic acid which are combined and
juxtaposed in a manner which would not otherwise exist in
nature.
[0136] Nucleic acid molecule means a single- or double-stranded
linear polynucleotide containing either deoxyribonucleotides or
ribonucleotides that are linked by 3'-5'-phosphodiester bonds.
[0137] Two DNA sequences are operably linked if the nature of the
linkage does not interfere with the ability of the sequences to
effect their normal functions relative to each other. For instance,
a promoter region would be operably linked to a coding sequence if
the promoter were capable of effecting transcription of that coding
sequence.
[0138] A polypeptide is a linear polymer of amino acids that are
linked by peptide bonds.
[0139] The term "promoter" refers to a cis-acting DNA sequence,
generally 80-120 base pairs long and located upstream of the
initiation site of a gene, to which RNA polymerase may bind and
initiate correct transcription. There can be associated additional
transcription regulatory sequences which provide on/off regulation
of transcription and/or which enhance (increase) expression of the
downstream coding sequence.
[0140] A recombinant nucleic acid molecule, for instance a
recombinant DNA molecule, is a novel nucleic acid sequence formed
in vitro through the ligation of two or more nonhomologous DNA
molecules (for example a recombinant plasmid containing one or more
inserts of foreign DNA cloned into at least one cloning site).
[0141] The terms "transformation" and "transfection" refer to the
directed modification of the genome of a cell by the external
application of purified recombinant DNA from another cell of
different genotype, leading to its uptake and integration into the
subject cell's genome. In bacteria, the recombinant DNA is not
typically integrated into the bacterial chromosome, but instead
replicates autonomously as a plasmid. The terms "transformed" and
"transfected" are used interchangeably herein.
[0142] Upstream means on the 5' side of any site in DNA or RNA.
[0143] A vector is a nucleic acid molecule that is able to
replicate autonomously in a host cell and can accept foreign DNA. A
vector carries its own origin of replication, one or more unique
recognition sites for restriction endonucleases which can be used
for the insertion of foreign DNA, and usually selectable markers
such as genes coding for antibiotic resistance, and often
recognition sequences (e.g., promoter) for the expression of the
inserted DNA. Common vectors include plasmid vectors and phage
vectors.
[0144] High affinity T cell receptor (TCR) is an engineered TCR
with stronger binding to a target ligand than the wild type TCR.
Some examples of high affinity include an equilibrium binding
constant for a target ligand of between about 10.sup.-6 M and
10.sup.-12 M and all individual values and ranges therein. This
range encompasses affinities between those reported to be wild type
affinities 10.sup.-4 to 10.sup.-6 M, and those which have been
isolated by directed evolution (about 10.sup.-12 M).
[0145] A cytokine is a protein, peptide or glycoprotein made by
cells that affect other cells.
[0146] Mammal includes both human and non-human mammals.
[0147] It will be appreciated by those of skill in the art that,
due to the degeneracy of the genetic code, numerous functionally
equivalent nucleotide sequences encode the same amino acid
sequence.
[0148] T Cell Receptors
[0149] The T cell receptor (TCR) is composed of two chains
(.alpha..beta. or .gamma..delta.) that pair on the surface of the T
cell to form a heterodimeric receptor. The .alpha..beta. TCR is
expressed on most T cells in the body and is known to be involved
in the recognition of MHC-restricted antigens. The molecular
genetics, structure, and biochemistry of .alpha..beta. TCRs have
now been studied thoroughly. Each .alpha. and .beta. chain is
composed of two domains: Constant domains (C) that anchor the
protein in the cell membrane and that associate with invariant
subunits of the CD3 signaling apparatus, and Variable domains (V)
that confer antigen recognition through six loops, called
complementarity determining regions (CDR). Each of the V domains
has three CDRs. These CDRs interact with a complex between an
antigenic peptide bound to a protein encoded by the major
histocompatibility complex (pepMHC) (Davis and Bjorkman (1988)
Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16,
523-544; Murphy (2012), xix, 868 p.).
[0150] The molecular genetics of the TCR have revealed a process of
genetic recombination between multiple genes that combine to form
the coding region of the V domains. The process is analogous to
antibody development in which the heavy and light chain genes
rearrange to generate the tremendous diversity exhibited by B
cell-derived antibodies (Tonegawa (1988) In Vitro Cell Dev Biol,
24, 253-65). In the case of T cells, the .alpha. chain V domain is
formed by the rearrangement of one V region (among about 75 in
humans) to one Joining (J) gene segment (among about 61 in humans)
(FIG. 5.8, Janeway, 8.sup.th edition). The 13 chain V domain is
formed by the rearrangement of one V region (among about 52 in
humans) to one Diversity (D) gene (among 2 in humans) to one
Joining (J) gene segment (among 13 in humans) (FIG. 5.8, (Murphy
(2012), xix, 868 p.)). The junctions of the V.alpha.J.alpha. and
J.beta.D.beta.J.beta. gene rearrangements encode the CDR3 loops of
each chain, and they contribute to the tremendous diversity of the
.alpha..beta. TCR, with a theoretical limit of over 10.sup.15
different TCRs (Davis and Bjorkman (1988) Nature, 334, 395-402),
well above the achievable diversity in a human because there are
only about 10.sup.11 T cells total (Mason (1998) Immunol Today, 19,
395-404). The possible CDR1 and CDR2 diversity of each chain is
represented by the number of V genes, as these loops are encoded
within the V gene, and TCRs do not undergo somatic mutation in
vivo. Although the diversity of CDR1 and CDR2 loops are relatively
limited compared to CDR3 loops, there have been a number of
examples shown where there has been selection for particular V
regions based on the peptide antigen and/or MHC product.
[0151] Class I MHC products bind to peptides of 8 to 10 amino acids
in length and they are expressed on all nucleated cells in the body
(reviewed by (Rock and Goldberg (1999) Annu Rev Immunol, 17,
739-79)). Whereas all the binding energy of an antibody-antigen
interaction is focused on the foreign antigen, a substantial
fraction of the binding energy of the TCR-peptide:MHC is directed
at the self-MHC molecule (Manning and Kranz (1999) Immunology
Today, 20, 417-422). In fact, more recent studies have suggested
that particular residues of the CDR1 and/or CDR2 loops have evolved
to interact with particular residues on the MHC helices, thereby
providing a basal affinity for MHC, accounting for the process of
MHC-restriction (Garcia et al. (2009) Nat Immunol, 10, 143-7;
Marrack et al. (2008) Annu Rev Immunol, 26, 171-203).
[0152] There has been interest in using TCRs that have affinities
for a peptide-MHC antigen (class I) above the normal range (so
called higher affinity TCRs) in order to: 1) drive the activity of
CD4 helper T cells (which lack the CD8 co-receptor) or 2) develop
soluble TCRs that could be used for direct targeting of a cell, by
attaching an "effector" molecule (e.g., antibody Fc regions, a
toxic drug, or an antibody scFv such as an anti-CD3 antibody, to
form a bispecific protein)((Ashfield and Jakobsen (2006) Drugs, 9,
554-9; Foote and Eisen (2000) Proc Natl Acad Sci USA, 97, 10679-81;
Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92; Molloy et
al. (2005) Curr Opin Pharmacol, 5, 438-43; Richman and Kranz (2007)
Biomol Eng, 24, 361-73). This approach also could overcome a
problem faced by some cancer patients, whereby their T cells do not
express TCRs with adequate specificity and binding affinity to
potential tumor antigens (in part due to the thymic and peripheral
processes of tolerance). For example, over 300 MHC-restricted, T
cell-defined tumor antigens have now been identified
(cancerimmunity.org/peptide/)(Boon and Old (1997) Curr Opin
Immunol, 9, 681-3; Cheever et al. (2009) Clin Cancer Res, 15,
5323-37). These tumor antigens include mutated peptides,
differentiation antigens, and overexpressed antigens, all of which
could serve as targets for therapies. Because the majority of the
cancer antigens described to date were derived from intracellular
proteins that can only be targeted at the cell surface in the
context of an MHC molecule, TCRs make the ideal candidate for
therapeutics as they have evolved to recognize this class of
antigen.
[0153] Similarly, TCRs can detect peptides derived from viral
proteins that have been naturally processed in infected cells and
displayed by an MHC molecule on the cell surface. Many viral
antigen targets have been identified over the past 25 years,
including peptides derived from viral genomes in HIV and HTLV
(e.g., Addo et al. (2007) PLoS ONE, 2, e321; Tsomides et al. (1994)
J Exp Med, 180, 1283-93; Utz et al. (1996) J Virol, 70, 843-51).
However, patients with these diseases may lack the optimal TCRs for
binding and destruction of the infected cells. Finally, it is
possible that TCRs could be used as receptor antagonists of
autoimmune targets, or as delivery agents to immunosuppress the
local immune cell response, in a process that would be highly
specific, thereby avoiding general immune suppression ((Molloy et
al. (2005) Curr Opin Pharmacol, 5, 438-43; Stone et al. (2012)
Protein Engineering)).
Modified T Cell Receptors
[0154] Directed evolution has been used to generate TCRs with
higher affinity for a specific pepMHC. The three different display
methods that have been used are yeast display (Holler et al. (2003)
Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA,
97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23,
349-54), and T cell display (Chervin et al. (2008) J Immunol
Methods, 339, 175-84). In all three approaches, the process
involves the engineering of a TCR that exhibits the normal, low
affinity of the wild-type TCR, so that affinity of mutants of the
TCR had increased affinity for the cognate pepMHC (i.e., the
original antigen that the T cells were specific for). Thus, the
wild-type TCR was used as a template for producing mutagenized
libraries in one or more of the CDRs, followed by selection of
mutants with higher affinity, by binding to the cognate peptide-MHC
antigen.
[0155] Yeast display allows for the protein of interest to be
expressed on the surface as an Aga2-fusion (Boder and Wittrup
(1997) Nat. Biotech., 15, 553-557; Boder and Wittrup (2000) Methods
Enzymol, 328, 430-44). This system has been used successfully in
the engineering of higher affinity TCRs, single-chain antibodies,
fibronectin, and other proteins. In the yeast display system, the
TCR has been displayed as a stabilized single-chain protein, in
V.beta.-linker-V.alpha. or V.alpha.-linker-V.beta. forms (Aggen et
al. (2011) Protein Engineering, Design, & Selection, 24,
361-72; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92;
Kieke et al. (1999) Proc Natl Acad Sci USA, 96, 5651-6; Richman et
al. (2009) Mol Immunol, 46, 902-16; Weber et al. (2005) Proc Natl
Acad Sci USA, 102, 19033-8), or as a two-chain heterodimer (Aggen
et al. (2011) Protein Engineering, Design, & Selection, 24,
361-72; Richman et al. (2009) Mol Immunol, 46, 902-16). Two mouse
TCRs have been engineered for higher affinity using this system: 2C
(MHC class-I restricted) and 3.L2 (MHC class-II restricted) (Holler
et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92; Weber et al.
(2005) Proc Natl Acad Sci USA, 102, 19033-8). Human TCR
single-chain V.alpha.V.beta. fragments (called scTv or scTCR) have
also recently been developed by taking advantage of the exceptional
stability of the human V.alpha. region called V.alpha.2 (Aggen et
al. (2011) Protein Engineering, Design, & Selection, 24,
361-72). In this case, in vitro engineered, high-affinity T cell
receptors in a single-chain format were used to isolate human
stabilized scTv fragments (V.beta.-linker-V.alpha.), which could be
expressed as stable proteins, both on the surface of yeast and in
soluble form from E. coli. The TCRs included two stabilized, human
scTv fragments, the A6 scTv that is specific for a peptide derived
from the human T cell lymphotrophic virus Tax protein (peptide:
Tax.sub.11-19, SEQ ID NO:5), and the 868 scTv that is specific for
a peptide derived from the human immunodeficiency virus Gag protein
(peptide: SL9.sub.77-85, SEQ ID NO:8). Both of these TCRs used the
V.alpha.2 gene (IMGT: TRAV12 family), but they had CDR3.alpha.,
CDR1.beta., CDR2.beta., and CDR3.beta. residues derived from the
original T cell clone from which the TCRs were isolated. Thus, the
higher affinity mutants of these scTCRs were each derived from
their original (parental) TCR against their cognate peptide-MHC
antigens.
[0156] In a second system, phage display, the protein of interest
is fused to the N-terminus of a viral coat protein (Scott and Smith
(1990) Science, 249, 386-90). Various TCRs, including those called
A6, 868, and 1G4 (MHC class-I restricted), have been engineered for
higher affinity using this method (Li et al. (2005) Nat Biotechnol,
23, 349-54; Sami et al. (2007) Protein Eng Des Sel, 20, 397-403;
Varela-Rohena et al. (2008) Nat Med, 14, 1390-5). Phage display of
these TCRs was enabled by introduction of a non-native disulfide
bond between the two C domains in order to promote pairing of the
.alpha. and .beta. chains. This system thus uses full-length
(V.alpha.C.alpha./V.beta.C.beta.) heterodimeric proteins derived
from the original T cell clones for engineering against their
cognate peptide-MHC.
[0157] A third system that has been reported for the engineering of
TCRs is mammalian cell display (Chervin et al. (2008) J Immunol
Methods, 339, 175-84; Kessels et al. (2000) Proc Natl Acad Sci USA,
97, 14578-83). This system uses a retroviral vector to introduce
the TCR .alpha. and .beta.-chains into a TCR-negative T cell
hybridoma. In one study (Kessels et al. (2000) Proc Natl Acad Sci
USA, 97, 14578-83), the selected mutant TCR was shown to bind to a
peptide that was structurally very similar to the cognate peptide
(ASNENMDAM, SEQ ID NO:46, versus ASNENMETM, SEQ ID NO:47). In the
other study, the affinity of the mutant TCR was shown to be
increased for the cognate pepMHC (Chervin et al. (2008) J Immunol
Methods, 339, 175-84). It has been shown in many studies that such
higher affinity TCRs also exhibit higher affinities against
structurally similar variants of the cognate peptide (e.g., (Holler
et al. (2003) Nat Immunol, 4, 55-62)). In the mammalian cell
display system, introduced TCRs were expressed on the surface in
its native conformation, complexed with CD3 subunits, allowing for
a fully functional T cell (signaling competent). Full-length,
heterodimeric TCRs in their native host were thus engineered using
this method.
[0158] TCR Scaffold
[0159] The present invention provides for the use of a single,
e.g., human TCR as a "platform" for engineering higher affinity
TCRs against desired antigens (e.g., cognate or non-cognate
antigens). In certain embodiments, the TCR scaffold-based TCR
engineering methods described herein can include, for example,
generating site-directed, mutated libraries of the single TCR,
followed by selections for binding to a non-cognate antigen.
Engineering is guided by structural analysis of the original,
single, or parent TCR. In certain embodiments, the engineered TCRs
can be used in soluble form for targeted delivery in vivo, or as
recombinantly expressed by T cells in an adoptive transfer method
or treatment.
[0160] Generally, a TCR scaffold that can be used to engineer TCR
mutants against specific antigens is provided. The TCRs are useful
for many purposes including, e.g., but not limited to, the
treatment of cancer, viral diseases and autoimmune diseases. In a
particular embodiment, a single-chain V.alpha.V.beta. TCR (scTCR)
scaffold can be prepared and used with a payload such as a
cytokine, toxin, radioisotope, chemotherapeutic agent, or drug
(similar to antibody-drug conjugates) to deliver the effector
molecule to the location where the TCR binds (e.g., tumor). The TCR
can also be used in cell therapies, such as adoptive transfer of
CD4+ T cells, CD8+ T cells, and/or natural killer (NK) cells, to
mediate a response against, e.g., a cancer cell or virus-infected
cell. The scTCR scaffolds provided herein can also be used for
diagnosis of, e.g., malignant or viral-infected cells through
identification of, e.g., neoplastic or viral-associated
cell-surface antigens by covalent linkage, for example through
amine-reactive or sulfhydryl-reactive amino acid side chains of the
TCR, to a detectable group, such as a radioisotope or fluorescent
moiety.
[0161] In one embodiment, the scTCR scaffold described herein is
displayable on the surface of yeast, phage, or mammalian cells and
can be used to engineer TCRs with higher affinity to a non-cognate
antigen. In one embodiment, the scTCR scaffold described herein can
be expressed in a prokaryotic cell, such as Escherichia coli,
Aspergillus niger, Aspergillus ficuum, Aspergillus awamori,
Aspergillus oryzae, Trichoderma reesei, Mucor miehei, Kluyveromyces
lactis, Pichia pastoris, Saccharomyces cerevisiae, Bacillus
subtilis or Bacillus licheniformis, insect cells (e.g.,
Drosophila), mammalian cells including cell lines such as Chinese
hamster ovary cell lines (CHO), or plant species (e.g., canola,
soybean, corn, potato, barley, rye, wheat) for example, or other
art-known protein expression sources and produced in large
quantities. The TCR scaffold can be generated against a particular
antigen, and used, for example and by way of example only, to
detect a specific peptide/MHC on the surface of a cell. In one
embodiment, the scTCR genes disclosed can be linked by use of
suitable peptide sequences, encoded within the DNA construct, to
the genes for signaling domains and introduced into T cells that
can eliminate the targeted cells. These constructs have been termed
chimeric antigen receptors (CARs), which are now widely used in the
field, including the use of CARs that contain a scTCR.
[0162] In another embodiment, the current disclosure provides the
amino acid sequences and the form of a single-chain V.alpha.V.beta.
T cell receptor (sc V.alpha.V.beta. TCR) scaffold. In the sc
V.alpha.V.beta. TCR scaffold provided, the variable alpha and
variable beta chains are connected using any suitable peptide
linker, including those known in the art such as with antibody
single-chain Fv linkages (Bird et al. (1988) Science, 242, 423-426;
Holliger et al. (1993) Proc Natl Acad Sci USA, 90, 6444-8;
Hoogenboom (2005) Nat Biotechnol, 23, 1105-16; Turner et al. (1997)
J Immunol Methods, 205, 43-54). In one embodiment, a soluble human
single-chain TCR having the structure: V.alpha.-L-V.beta. or
V.beta.-L-V.alpha., wherein L is a linker peptide that links
V.beta. with V.alpha., V.beta. is a TCR variable .beta. region, and
V.alpha. is a TCR variable .alpha. region is provided. In one
embodiment, the model V.alpha.V.beta. TCR is called A6 where
V.beta. is a TCR variable .beta. region of group 13, and V.alpha.2
is a TCR variable .alpha. region of group 2 (Utz, U., et al.,
1996). In one embodiment, the model V.alpha.V.beta. TCR is a
stabilized single-chain variant of A6 known as A6 X15 (Aggen, D.
A., et al., 2011). In one embodiment, the linker peptide contains
more than 5 lysine residues. In one embodiment, the linker peptide
contains between 5 and 30 amino acids. In one embodiment, the
linker peptide has an amino acid sequence of GSADDAKKDAAKKDGKS (SEQ
ID NO:21). In one embodiment, the sc V.alpha.V.beta. TCR scaffold
provided does not contain a constant region. When the terminology
sc V.alpha.V.beta. TCR scaffold is used herein, it is understood
that sc V.beta.V.alpha. TCR scaffold is also included as the
terminology is understood and used in the art. Thus, the V.alpha.
and V.beta. chains can be connected to each other in any
configuration through the linker.
[0163] In an aspect of the disclosure, the scV.alpha.V.beta. TCR
scaffold of the disclosure binds specifically to a ligand with an
equilibrium binding constant K.sub.D of between about 10.sup.-6 M
and 10.sup.-12 M. In one embodiment of this aspect of the
disclosure, the ligand is a peptide/MHC ligand. In one embodiment,
the sc V.alpha.V.beta. TCR of the disclosure has enhanced affinity
toward a ligand compared to the affinities of normal, wild type
TCRs.
[0164] TCRs that bind to a collection of HLA-A, B, and C alleles
could be used to treat diseases that encompass a large fraction of
the human population. For example, the frequency of many HLA
alleles in the population has been determined, and there are many
cancer peptide antigens that have been described in association
with these alleles (Marsh, Parham, and Barber, The HLA Facts Book,
copyright 2000 by Academic Press, ISBN 0-12-545025-7).
[0165] By way of example, the average frequency (and range) among
Caucasian populations are: HLA-A1, 14%; HLA-A2, 25%; HLA-A3, 12%;
HLA-A11, 7%; HLA-A24, 10%; HLA-B7, 9%; HLA-B44, 11%; HLA-Cw4, 12%;
HLA-Cw7, 23%. The range found within these populations are: HLA-A1
(5-28%); HLA-A2 (7-40%); HLA-A3 (3-20%); HLA-A11 (2-25%); HLA-A24
(5-18%); HLA-B7 (1-16%); HLA-B44 (5-22%); HLA-Cw4 (6-19%); HLA-Cw7
(13-39%).
[0166] The TCR scaffold approach can be extended to other human HLA
alleles in various ways. For example, using structure-based design
of the TCR scaffolds described here, it is possible to focus
mutated libraries in the CDR loops that contact the MHC helices in
order to generate leads against other alleles. For example, from
the structure of the A6 TCR in complex with HLA-A2, it is known
that CDR2alpha libraries would generate variants that bind in the
region of the alpha2 helix of HLA-A2. The TCR A 6 residue Y51
resides near HLA-A2 alpha2 helix position(s) E154, Q155, and A158.
The HLA-A1 allele differs only at position 158, with a valine
rather than an alanine. Thus, the A6 TCR may have a basal affinity
for the HLA-A1 allele, which could be improved by generating
libraries of CDR2 mutants that encompass position 51, followed by
selections for higher affinity binding to the HLA-A1 allele.
[0167] Another example makes use of a scaffold that is derived from
a TCR specific for a different allele (i.e., peptide bound to the
product of that MHC allele). Here, it is possible to generate CDR
libraries, as shown for the A6 TCR scaffold, which will react with
alternative non-cognate peptide-MHC complexes of that allele. For
example, a cancer antigen peptide from MAGE-A3 binds to HLA-A1 and
this could be used for selection of the TR libraries.
[0168] Biologically Active Groups
[0169] Also provided is a sc V.alpha.V.beta. TCR scaffold as
described herein which includes a biologically active group. As
used herein, "biologically active group" is a group that causes a
measurable or detectable effect in a biological system. In one
embodiment, the biologically active group is selected from: an
anti-tumor agent such as, but not limited to, angiogenesis
inhibitors, enzyme inhibitors, microtubule inhibitors, DNA
intercalators or cross-linkers, DNA synthesis inhibitors; a
cytokine such as, but not limited to IL-2, IL-15, GM-CSF, IL-12,
TNF-.alpha., IFN-.gamma. or LT-.alpha. (Schrama et al. (2006) Nat
Rev Drug Discov, 5, 147-59; Wong et al. (2011) Protein Eng Des Sel,
24, 373-83); an anti-inflammatory group such as, but not limited
to, TGF-.beta., IL-37, IL-10 (Nold et al. (2010) Nat Immunol, 11,
1014-22; Stone et al. (2012) Protein Engineering), a radioisotope
such as, but not limited to, .sup.90Y or .sup.131I (Reichert and
Valge-Archer (2007) Nat Rev Drug Discov, 6, 349-56); a toxin such
as, but not limited to, Pseudomonas exotoxin A, diphtheria toxin,
or the A chain of ricin (Pastan et al. (2006) Nat Rev Cancer, 6,
559-65; Schrama et al. (2006) Nat Rev Drug Discov, 5, 147-59); a
drug, or an antibody such as a single-chain Fv.
[0170] In one embodiment of this aspect of the disclosure, the
biologically active group is a cytotoxic molecule, sometimes
referred to as a drug (e.g., in the term "antibody drug
conjugate"). As used herein, "cytotoxic" means toxic to cells.
Examples of cytotoxic molecules include, but are not limited to,
doxorubicin, methotrexate, mitomycin, 5-fluorouracil, duocarmycin,
auristatins, maytansines, calicheamicins and analogs of the above
molecules (Jarvis (2012) Chemical and Engineering News, 90, 12-18;
Litvak-Greenfeld and Benhar (2012) Adv Drug Deliv Rev; Ricart and
Tolcher (2007) Nat Clin Pract Oncol, 4, 245-55). Cytotoxic
molecules do not need to cause complete cell death, but rather, a
measurable or detectable inhibition of growth or decrease in cell
activity.
[0171] In one embodiment, a TCR described herein is linked to an
enzyme capable of converting a prodrug into a drug. This is useful,
for example, by allowing the active form of the drug to be created
at the location targeted by the TCR (e.g., at the site of a
tumor).
[0172] In one embodiment, the biologically active group is bound to
the single-chain TCR through a linker, which may be accomplished
through standard chemical reactions such as with free amine groups
or sulfhydryl groups of the TCR.
[0173] In another embodiment, the TCR is attached to a single-chain
antibody fragment (scFv) to generate a bispecific agent. Bispecific
antibodies that contain one scFv against a tumor antigen, and one
against the CD3 molecule of the T cell have now been used
successfully in the clinic (Bargou et al. (2008) Science, 321,
974-7). In addition, a bispecific agent containing a TCR and a scFv
against CD3 has also been reported (Liddy et al. (2012) Nat Med,
18, 980-7).
[0174] Also provided is a single-chain V.alpha.V.beta. TCR as
described herein which includes a detectable group. In one
embodiment, the detectable group is one that can be detected by
spectroscopic or enzyme-based methods. In one embodiment, the
detectable group is a fluorescent group, such as, but not limited
to fluorescein, R-phycoerythrin (PE), PE-Cy5, PE-Cy7, Texas red, or
allophycocyanin (APC); a radiolabeled group such as, but not
limited to, .sup.125I, .sup.32P, .sup.99mTc; an absorbing group, or
an enzyme with properties that generate detectable products such
as, but not limited to, horseradish peroxidase, or alkaline
phosphatase.
[0175] As known in the art, a biologically active group, detectable
group or other group attached to the TCR can be attached using a
flexible peptide linker or by chemical conjugation, and can be
covalently or noncovalently attached to the TCR.
[0176] Antigen Specificity
[0177] Also provided herein are sc V.alpha.V.beta. TCR scaffolds
that recognize (or target) a specific antigen. In one embodiment,
the TCR is specific for recognition of a virus or fragment thereof.
In one embodiment, the TCR is specific for recognition of a
cancer-specific epitope. In one embodiment, the TCR is specific for
recognition of autoimmune associated epitope. Other targets include
those listed in The HLA Factsbook (Marsh et al. (2000)) and others
known in the art. Specific target antigens include tumor-associated
antigens (van der Bruggen P et al. Peptide database: T cell-defined
tumor antigens. Cancer Immun 2013. cancerimmunity.org/peptide/;
(Cheever et al. (2009) Clin Cancer Res, 15, 5323-37), viral
antigens ((Addo et al. (2007) PLoS ONE, 2, e321; Anikeeva et al.
(2009) Clin Immunol, 130, 98-109)), and autoimmune associated
epitopes ((Bulek et al. (2012) Nat Immunol, 13, 283-9; Harkiolaki
et al. (2009) Immunity, 30, 348-57; Skowera et al. (2008) J Clin
Invest, 118, 3390-402). In one embodiment, the target antigen is
one of MART-1 (Kawakami Y et al. J Exp Med 1994; 180:347-352;
Romero et al. 2002. Immunol. Rev. 188, 81-96), WT-1 (Gessler et al.
Nature 343 (6260), 774-778 (1990)), SURV (Schmidt S M et al. Blood
2003; 102: 571-6; Schmitz M et al. Cancer Res 2000; 60: 4845-9),
NY-ESO-1 (Barbed A M et al. Scand J Immunol 2000; 51: 128-33.), PPI
(Bulek 2012 Nature Immunology), MDM2 (Asai et al 2002 Cancer
Immunity), MDM4, HBE183, gp100 (Bakker A B et al. Int J Cancer
1995; 62: 97-102; Kawakami Y et al. J Immunol 1995; 154:
3961-3968), MUC1 (Brossart P et al. Blood 1999; 93: 4309-17), MAGE
A3 (van der Bruggen P et al. Eur J Immunol 1994; 24: 3038-43),
HER-2/neu (Fisk B et al. J Exp Med 1995; 181: 2109-2117), EGVFvIII
(Sampson Semin Immunol 2008; 20:267-75), CEA (Tsang K Y et al. J
Natl Cancer Inst 1995; 87: 982-990), and SL9/HIV gag (Altfeld et
al. 2001 J. Virol. 75:1301) listed in Table 1 below. The ranking
score in Table 1 is from Cheever et al. (2009) Clin Cancer Res, 15,
5323-37. Also provided is a scV.alpha.V.beta. TCR mutant derived
from the A6 scaffold that recognizes the specific non-cognate
antigen called MART-1/HLA-A2 (peptide ELAGIGILTV, SEQ ID NO:7,
bound to the HLA molecule A2).
TABLE-US-00001 TABLE 1 Target Antigens Peptide Sequence SEQ Antigen
Type 0 1 2 3 4 5 6 7 8 9 ID NO Ranking MART-1 Cancer E L A G I G I
L T V SEQ ID 14 NO: 7 WT-1 Cancer -- R M F P N A P Y L SEQ ID 1 NO:
9 SURV Cancer -- L T L G E F L K L SEQ ID 21 NO: 10 NY-ESO-1 Cancer
-- S L L M W I T Q C SEQ ID 10 NO: 11 PPI Auto- -- A L W G P D A A
A SEQ ID -- immune NO: 12 MDM2 Cancer -- V L F Y L G Q Y -- SEQ ID
-- (also in NO: 13 mdm4) HBE183 Viral -- F L L T R I L T I SEQ ID
-- NO: 14 gp100 Cancer -- K T W G Q Y W Q V SEQ ID 16 NO: 15 MUC1
Cancer -- S T A P P V H N V SEQ ID 2 NO: 16 MAGE A3 Cancer -- F L W
G P R A L V SEQ ID 8 NO: 17 HER-2/neu Cancer -- K I F G S L A F L
SEQ ID 6 NO: 18 EGFRvIII Cancer -- L E E K K G N Y V SEQ ID 5 NO:
19 CEA Cancer -- Y L S G A N L N L SEQ ID 13 NO: 20 SL9/ Viral -- S
L Y N T V A T L SEQ ID -- HIVgag NO: 8
[0178] Also provided herein is a human TCR for use in a method of
treating or preventing a disease or disorder in a mammal,
comprising administering an effective amount of a modified TCR
linked to a therapeutically effective molecule to a mammal. In a
particular embodiment, the mammal is human. In another embodiment,
the mammal is a companion animal (e.g., a dog, cat, rabbit, rodent,
horse) or a livestock animal (e.g., a cow, horse, pig).
[0179] As used herein a "disease state" is an abnormal function or
condition of an organism. In one embodiment, the disease state is
selected from the group consisting of: cancer, viral, bacterial or
autoimmune disease. Also provided is an isolated single-chain TCR
as described herein, and a method for producing the single-chain
TCR in E. coli. Also provided is a pharmaceutical composition
comprising a scTCR as described herein and a pharmaceutically
acceptable carrier. Also provided is the sc V.alpha.V.beta. TCR
described herein which has been linked to signaling domains that
yields an active TCR on the surface of a T cell. In one embodiment,
this scTCR can be used in a method of treating a disease state in a
mammal, comprising: cloning the TCR into a vector, introducing the
vector into T cells of a patient, and adoptive transferring of the
T cells back into a patient.
[0180] Modified TCR Polypeptides and Polynucleotides
[0181] The disclosure contemplates a DNA vector that includes at
least one DNA segment encoding a single-chain T cell receptor
(scTCR).
[0182] Those of skill in the art, through standard mutagenesis
techniques, in conjunction with the assays described herein, can
obtain altered TCR sequences and test them for particular binding
affinity and/or specificity. Useful mutagenesis techniques known in
the art include, without limitation, de novo gene synthesis,
oligonucleotide-directed mutagenesis, region-specific mutagenesis,
linker-scanning mutagenesis, and site-directed mutagenesis by PCR
(see e.g., Sambrook et al. (1989) and Ausubel et al. (1999)).
[0183] In obtaining variant TCR coding sequences, those of ordinary
skill in the art will recognize that TCR-derived proteins may be
modified by certain amino acid substitutions, additions, deletions,
and post-translational modifications, without loss or reduction of
biological activity. In particular, it is well known that
conservative amino acid substitutions, that is, substitution of one
amino acid for another amino acid of similar size, charge, polarity
and conformation, are unlikely to significantly alter protein
function. The 20 standard amino acids that are the constituents of
proteins can be broadly categorized into four groups of
conservative amino acids as follows: the nonpolar (hydrophobic)
group includes alanine, isoleucine, leucine, methionine,
phenylalanine, proline, tryptophan and valine; the polar
(uncharged, neutral) group includes asparagine, cysteine,
glutamine, glycine, serine, threonine and tyrosine; the positively
charged (basic) group contains arginine, histidine and lysine; and
the negatively charged (acidic) group contains aspartic acid and
glutamic acid. Substitution in a protein of one amino acid for
another within the same group is unlikely to have an adverse effect
on the biological activity of the protein.
[0184] In one embodiment, a scTCR of the disclosure may contain
additional mutations in any region or regions of the variable
domain that results in a stabilized protein. In one embodiment, one
or more additional mutations is in one or more of CDR1, CDR2, HV4,
CDR3, FR2, and FR3. The regions used for mutagenesis can be
determined by directed evolution, where crystal structures or
molecular models are used to generate regions of the TCR which
interact with the ligand of interest (antigen, for example). In
other examples, the variable region can be reshaped, by adding or
deleting amino acids to engineer a desired interaction between the
scTCR and the ligand.
[0185] Polypeptides of the invention include modified TCRs, and
antigen-binding fragments thereof (e.g., scTCR), and chimeric
antigen receptors (CARs). The terms "polypeptide" "protein" and
"peptide" and "glycoprotein" are used interchangeably and mean a
polymer of amino acids not limited to any particular length. The
term does not exclude modifications such as myristylation,
sulfation, glycosylation, phosphorylation and addition or deletion
of signal sequences. The terms "polypeptide" or "protein" means one
or more chains of amino acids, wherein each chain comprises amino
acids covalently linked by peptide bonds, and wherein said
polypeptide or protein can comprise a plurality of chains
non-covalently and/or covalently linked together by peptide bonds,
having the sequence of native proteins, that is, proteins produced
by naturally-occurring and specifically non-recombinant cells, or
genetically-engineered or recombinant cells, and comprise molecules
having the amino acid sequence of the native protein, or molecules
having deletions from, additions to, and/or substitutions of one or
more amino acids of the native sequence. The terms "polypeptide"
and "protein" specifically encompass the modified TCRs, or
antigen-binding fragments thereof, of the present disclosure, or
sequences that have deletions from, additions to, and/or
substitutions of one or more amino acid of a modified TCR, or
antigen binding fragment thereof. Thus, a "polypeptide" or a
"protein" can comprise one (termed "a monomer") or a plurality
(termed "a multimer") of amino acid chains.
[0186] The term "isolated protein" referred to herein means that a
subject protein (1) is free of at least some other proteins with
which it would typically be found in nature, (2) is essentially
free of other proteins from the same source, e.g., from the same
species, (3) is expressed by a cell from a different species, (4)
has been separated from at least about 50 percent of
polynucleotides, lipids, carbohydrates, or other materials with
which it is associated in nature, (5) is not associated (by
covalent or noncovalent interaction) with portions of a protein
with which the "isolated protein" is associated in nature, (6) is
operably associated (by covalent or noncovalent interaction) with a
polypeptide with which it is not associated in nature, or (7) does
not occur in nature. Such an isolated protein can be encoded by
genomic DNA, cDNA, mRNA or other RNA, of may be of synthetic
origin, or any combination thereof. In certain embodiments, the
isolated protein is substantially free from proteins or
polypeptides or other contaminants that are found in its natural
environment that would interfere with its use (therapeutic,
diagnostic, prophylactic, research or otherwise).
[0187] In particular embodiments, a subject modified TCR may have:
a) a TCR alpha chain variable region having an amino acid sequence
that is at least 80% identical, at least 85% identical, at least
90%, at least 95% or at least 98% or 99% identical, to the alpha
chain variable region of a modified TCR described herein; and b) a
beta chain variable region having an amino acid sequence that is at
least 80% identical, at least 85%, at least 90%, at least 95% or at
least 98% or 99% identical, to the light chain variable region of a
modified TCR described herein.
[0188] In particular embodiments, the modified TCR may comprise: a)
a TCR alpha chain variable region comprising: i. a CDR1 region that
is identical in amino acid sequence to the alpha chain CDR1 region
of a selected TCR described herein; ii. a CDR2 region that is
identical in amino acid sequence to the alpha chain CDR2 region of
the selected TCR; and iii. a CDR3 region that is identical in amino
acid sequence to the alpha chain CDR3 region of the selected TCR;
and b) a beta chain variable region comprising: i. a CDR1 region
that is identical in amino acid sequence to the beta chain CDR1
region of the selected TCR; ii. a CDR2 region that is identical in
amino acid sequence to the beta chain CDR2 region of the selected
TCR; and iii. a CDR3 region that is identical in amino acid
sequence to the beta chain CDR3 region of the selected TCR; wherein
the TCR specifically binds a selected non-cognate antigen. In a
further embodiment, the modified TCR, or antigen-binding fragment
thereof, is a variant modified TCR wherein the variant comprises an
alpha chain and a beta chain identical to the selected modified TCR
except for up to 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid
substitutions in the CDR regions of the V alpha and V beta regions.
In this regard, there may be 1, 2, 3, 4, 5, 6, 7, 8, or in certain
embodiments, 9, 10, 11, 12, 13, 14, 15 more amino acid
substitutions in the CDR regions of the selected variant modified
TCR. Substitutions may be in CDRs either in the V alpha and/or the
V beta regions. (See e.g., Muller, 1998, Structure
6:1153-1167).
[0189] In one embodiment, a polynucleotide encoding a modified TCR,
or an antigen-binding fragment thereof, is provided. In other
related embodiments, the polynucleotide may be a variant of a
polynucleotide encoding the modified TCR. Polynucleotide variants
may have substantial identity to a polynucleotide sequence encoding
a modified TCR described herein. For example, a polynucleotide may
be a polynucleotide comprising at least 70% sequence identity,
preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
or higher, sequence identity compared to a reference polynucleotide
sequence such as a sequence encoding an antibody described herein,
using the methods described herein, (e.g., BLAST analysis using
standard parameters, as described below). One skilled in this art
will recognize that these values can be appropriately adjusted to
determine corresponding identity of proteins encoded by two
nucleotide sequences by taking into account codon degeneracy, amino
acid similarity, reading frame positioning and the like.
[0190] Typically, polynucleotide variants will contain one or more
substitutions, additions, deletions and/or insertions, preferably
such that the binding affinity of the antibody encoded by the
variant polynucleotide is not substantially diminished relative to
an antibody encoded by a polynucleotide sequence specifically set
forth herein.
[0191] When comparing polynucleotide sequences, two sequences are
said to be "identical" if the sequence of nucleotides in the two
sequences is the same when aligned for maximum correspondence, as
described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, 40 to about
50, in which a sequence may be compared to a reference sequence of
the same number of contiguous positions after the two sequences are
optimally aligned.
[0192] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O. (1978) A
model of evolutionary change in proteins--Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure, National Biomedical Research Foundation,
Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., Unified
Approach to Alignment and Phylogenes, pp. 626-645 (1990); Methods
in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;
Higgins, D. G. and Sharp, P. M., CABIOS 5:151-153 (1989); Myers, E.
W. and Muller W., CABIOS 4:11-17 (1988); Robinson, E. D., Comb.
Theor 11:105 (1971); Santou, N. Nes, M., Mol. Biol. Evol. 4:406-425
(1987); Sneath, P. H. A. and Sokal, R. R., Numerical Taxonomy--the
Principles and Practice of Numerical Taxonomy, Freeman Press, San
Francisco, Calif. (1973); Wilbur, W. J. and Lipman, D. J., Proc.
Natl. Acad., Sci. USA 80:726-730 (1983).
[0193] Alternatively, optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman, Add. APL. Math 2:482 (1981), by the identity alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by
the search for similarity methods of Pearson and Lipman, Proc.
Natl. Acad. Sci. USA 85: 2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by
inspection.
[0194] One preferred example of algorithms that are suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nucl. Acids Res. 25:3389-3402 (1977), and Altschul et al.,
J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0
can be used, for example with the parameters described herein, to
determine percent sequence identity among two or more the
polynucleotides. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology
Information. In one illustrative example, cumulative scores can be
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed
of the alignment. The BLASTN program (for nucleotide sequences)
uses as defaults a wordlength (W) of 11, and expectation (E) of 10,
and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc.
Natl. Acad. Sci. USA 89:10915 (1989)) alignments, (B) of 50,
expectation (E) of 10, M=5, N=-4 and a comparison of both
strands.
[0195] In certain embodiments, the "percentage of sequence
identity" is determined by comparing two optimally aligned
sequences over a window of comparison of at least 20 positions,
wherein the portion of the polynucleotide sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
of 20 percent or less, usually 5 to 15 percent, or 10 to 12
percent, as compared to the reference sequences (which does not
comprise additions or deletions) for optimal alignment of the two
sequences. The percentage is calculated by determining the number
of positions at which the identical nucleic acid bases occurs in
both sequences to yield the number of matched positions, dividing
the number of matched positions by the total number of positions in
the reference sequence (i.e., the window size) and multiplying the
results by 100 to yield the percentage of sequence identity.
[0196] It will be appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
many nucleotide sequences that encode an antibody as described
herein. Some of these polynucleotides bear minimal sequence
identity to the nucleotide sequence of the native or original
polynucleotide sequence that encode modified TCRs that bind to,
e.g., the same non-cognate antigen. Nonetheless, polynucleotides
that vary due to differences in codon usage are expressly
contemplated by the present disclosure. In certain embodiments,
sequences that have been codon-optimized for mammalian expression
are specifically contemplated.
[0197] Standard techniques for cloning, DNA isolation,
amplification and purification, for enzymatic reactions involving
DNA ligase, DNA polymerase, restriction endonucleases and the like,
and various separation techniques are those known and commonly
employed by those skilled in the art. A number of standard
techniques are described in Sambrook et al. (1989) Molecular
Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview,
N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor
Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218,
Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al. (eds.) (1983)
Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth.
Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics,
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Old
and Primrose (1981) Principles of Gene Manipulation, University of
California Press, Berkeley; Schleif and Wensink (1982) Practical
Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol.
I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985)
Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and
Hollaender (1979) Genetic Engineering: Principles and Methods,
Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature,
where employed, are deemed standard in the field and commonly used
in professional journals such as those cited herein.
[0198] Homology between nucleotide sequences can be determined by
DNA hybridization analysis, wherein the stability of the
double-stranded DNA hybrid is dependent on the extent of base
pairing that occurs. Conditions of high temperature and/or low salt
content reduce the stability of the hybrid, and can be varied to
prevent annealing of sequences having less than a selected degree
of homology. For instance, for sequences with about 55% G-C
content, hybridization and wash conditions of 40-50.degree. C.,
6.times.SSC (sodium chloride/sodium citrate buffer) and 0.1% SDS
(sodium dodecyl sulfate) indicate about 60-70% homology,
hybridization and wash conditions of 50-65.degree. C., 1.times.SSC
and 0.1% SDS indicate about 82-97% homology, and hybridization and
wash conditions of 52.degree. C., 0.1.times.SSC and 0.1% SDS
indicate about 99-100% homology. A wide range of computer programs
for comparing nucleotide and amino acid sequences (and measuring
the degree of homology) are also available, and a list providing
sources of both commercially available and free software is found
in Ausubel et al. (1999). Readily available sequence comparison and
multiple sequence alignment algorithms are, respectively, the Basic
Local Alignment Search Tool (BLAST) (Altschul et al., 1997) and
ClustalW programs. BLAST is available on the Internet at
ncbi.nlm.nih.gov and a version of ClustalW is available at
www2.ebi.ac.uk.
[0199] Industrial strains of microorganisms (e.g., Aspergillus
niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae,
Trichoderma reesei, Mucor miehei, Kluyveromyces lactis, Pichia
pastoris, Saccharomyces cerevisiae, Escherichia coli, Bacillus
subtilis or Bacillus licheniformis), insect (Drosophila), mammalian
(e.g., Chinese hamster ovary cell lines, CHO), or plant species
(e.g., canola, soybean, corn, potato, barley, rye, wheat) may be
used as host cells for the recombinant production of the TCR
proteins. In certain embodiments, the first step in the
heterologous expression of a high affinity TCR protein or soluble
protein, an expression construct is assembled to include the TCR or
soluble TCR coding sequence and control sequences such as
promoters, enhancers and terminators. Other sequences such as
signal sequences and selectable markers may also be included. To
achieve extracellular expression of the TCR, the expression
construct may include a secretory signal sequence. In embodiments,
the signal sequence is not included on the expression construct if
cytoplasmic expression is desired. In embodiments, the promoter and
signal sequence are functional in the host cell and provide for
expression and secretion of the TCR or soluble TCR protein.
Transcriptional terminators may be included to ensure efficient
transcription. Ancillary sequences enhancing expression or protein
purification may also be included in the expression construct.
[0200] Various promoters (transcriptional initiation regulatory
region) may be used according to the disclosure. The selection of
the appropriate promoter may be dependent upon the proposed
expression host. Promoters from heterologous sources may be used as
long as they are functional in the chosen host.
[0201] Promoter selection is also dependent upon the desired
efficiency and level of peptide or protein production. Inducible
promoters such as tac are often employed in order to dramatically
increase the level of protein expression in E. coli. Overexpression
of proteins may be harmful to the host cells. Consequently, host
cell growth may be limited. The use of inducible promoter systems
allows the host cells to be cultivated to acceptable densities
prior to induction of gene expression, thereby facilitating higher
product yields.
[0202] Various signal sequences may be used according to the
disclosure. A signal sequence which is homologous to the TCR coding
sequence may be used. Alternatively, a signal sequence which has
been selected or designed for efficient secretion and processing in
the expression host may also be used. For example, suitable signal
sequence/host cell pairs include the B. subtilis sacB signal
sequence for secretion in B. subtilis, and the Saccharomyces
cerevisiae .alpha.-mating factor or P. pastoris acid phosphatase
phoI signal sequences for P. pastoris secretion. The signal
sequence may be joined directly through the sequence encoding the
signal peptidase cleavage site to the protein coding sequence, or
through a short nucleotide bridge consisting of usually fewer than
ten codons, where the bridge ensures correct reading frame of the
downstream TCR sequence.
[0203] Elements for enhancing transcription and translation have
been identified for eukaryotic protein expression systems. For
example, positioning the cauliflower mosaic virus (CaMV) promoter
1000 bp on either side of a heterologous promoter may elevate
transcriptional levels by 10- to 400-fold in plant cells. The
expression construct should also include the appropriate
translational initiation sequences. Modification of the expression
construct to include a Kozak consensus sequence for proper
translational initiation may increase the level of translation by
10 fold.
[0204] A selective marker is often employed, which may be part of
the expression construct or separate from it (e.g., carried by the
expression vector), so that the marker may integrate at a site
different from the gene of interest. Examples include markers that
confer resistance to antibiotics (e.g., bla confers resistance to
ampicillin for E. coli host cells, nptII confers kanamycin
resistance to a wide variety of prokaryotic and eukaryotic cells)
or that permit the host to grow on minimal medium (e.g., HIS4
enables P. pastoris or His.sup.- S. cerevisiae to grow in the
absence of histidine). The selectable marker has its own
transcriptional and translational initiation and termination
regulatory regions to allow for independent expression of the
marker. If antibiotic resistance is employed as a marker, the
concentration of the antibiotic for selection will vary depending
upon the antibiotic, generally ranging from 10 to 600 .mu.g of the
antibiotic/mL of medium.
[0205] The expression construct is assembled by employing known
recombinant DNA techniques (Sambrook et al., 1989; Ausubel et al.,
1999). Restriction enzyme digestion and ligation are the basic
steps employed to join two fragments of DNA. The ends of the DNA
fragment may require modification prior to ligation, and this may
be accomplished by filling in overhangs, deleting terminal portions
of the fragment(s) with nucleases (e.g., ExoIII), site directed
mutagenesis, or by adding new base pairs by PCR. Polylinkers and
adaptors may be employed to facilitate joining of selected
fragments. The expression construct is typically assembled in
stages employing rounds of restriction, ligation, and
transformation of E. coli. Numerous cloning vectors suitable for
construction of the expression construct are known in the art
(.lamda.ZAP and pBLUESCRIPT SK-1, Stratagene, LaJolla, Calif.; pET,
Novagen Inc., Madison, Wis.--cited in Ausubel et al., 1999) and the
particular choice is not critical to the disclosure. The selection
of cloning vector will be influenced by the gene transfer system
selected for introduction of the expression construct into the host
cell. At the end of each stage, the resulting construct may be
analyzed by restriction, DNA sequence, hybridization and PCR
analyses.
[0206] The expression construct may be transformed into the host as
the cloning vector construct, either linear or circular, or may be
removed from the cloning vector and used as is or introduced onto a
delivery vector. The delivery vector facilitates the introduction
and maintenance of the expression construct in the selected host
cell type. The expression construct is introduced into the host
cells by any of a number of known gene transfer systems (e.g.,
natural competence, chemically mediated transformation, protoplast
transformation, electroporation, biolistic transformation,
transfection, or conjugation) (Ausubel et al., 1999; Sambrook et
al., 1989). The gene transfer system selected depends upon the host
cells and vector systems used.
[0207] For instance, the expression construct can be introduced
into S. cerevisiae cells by protoplast transformation or
electroporation. Electroporation of S. cerevisiae is readily
accomplished, and yields transformation efficiencies comparable to
spheroplast transformation.
[0208] Monoclonal or polyclonal antibodies, preferably monoclonal,
specifically reacting with a TCR protein at a site other than the
ligand binding site may be made by methods known in the art, and
many are commercially available. See, e.g., Harlow and Lane (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories;
Goding (1986) Monoclonal Antibodies: Principles and Practice, 2d
ed., Academic Press, New York; and Ausubel et al. (1999) Current
Protocols in Molecular Biology, John Wiley & Sons, Inc., New
York.
[0209] TCRs in cell-bound or soluble form which are specific for a
particular target ligand are useful, for example, as diagnostic
probes for screening biological samples (such as cells, tissue
samples, biopsy material, bodily fluids and the like) or for
detecting the presence of the target ligand in a test sample.
Frequently, the TCRs are labeled by joining, either covalently or
noncovalently, a substance which provides a detectable signal.
Suitable labels include but are not limited to radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent agents,
chemiluminescent agents, magnetic particles and the like.
Additionally the TCR can be coupled to a ligand for a second
binding molecules: for example, the TCR can be biotinylated.
Detection of the TCR bound to a target cell or molecule can then be
effected by binding of a detectable streptavidin (a streptavidin to
which a fluorescent, radioactive, chemiluminescent, or other
detectable molecule is attached or to which an enzyme for which
there is a chromophoric substrate available). United States patents
describing the use of such labels and/or toxic compounds to be
covalently bound to the scTCR include but are not limited to Nos.
3,817,837; 3,850,752; 3,927,193; 3,939,350; 3,996,345; 4,277,437;
4,275,149; 4,331,647; 4,348,376; 4,361,544; 4,468,457; 4,444,744;
4,640,561; 4,366,241; RE 35,500; 5,299,253; 5,101,827;
5,059,413.
[0210] Labeled TCRs can be detected using a monitoring device or
method appropriate to the label used. Fluorescence microscopy or
fluorescence activated cell sorting can be used where the label is
a fluorescent moiety, and where the label is a radionuclide, gamma
counting, autoradiography or liquid scintillation counting, for
example, can be used with the proviso that the method is
appropriate to the sample being analyzed and the radionuclide used.
In addition, there can be secondary detection molecules or particle
employed where there is a detectable molecule or particle which
recognized the portion of the TCR which is not part of the binding
site for the target ligand in the absence of a MHC component as
noted herein. The art knows useful compounds for diagnostic imaging
in situ; see, e.g., U.S. Pat. Nos. 5,101,827; 5,059,413.
Radionuclides useful for therapy and/or imaging in vivo include
.sup.111Indium, .sup.97Rubidium, .sup.125Iodine, .sup.131Iodine,
.sup.123Iodine, .sup.67Gallium, .sup.99Technetium. Toxins include
diphtheria toxin, ricin and castor bean toxin, among others, with
the proviso that once the TCR-toxin complex is bound to the cell,
the toxic moiety is internalized so that it can exert its cytotoxic
effect. Immunotoxin technology is well known to the art, and
suitable toxic molecules include, without limitation,
chemotherapeutic drugs such as vindesine, antifolates, e.g.,
methotrexate, cisplatin, mitomycin, .anthrocyclines such as
daunomycin, daunorubicin or adriamycin, and cytotoxic proteins such
as ribosome inactivating proteins (e.g., diphtheria toxin, pokeweed
antiviral protein, abrin, ricin, pseudomonas exotoxin A or their
recombinant derivatives. See, generally, e.g., Olsnes and Pihl
(1982) Pharmac. Ther. 25:355-381 and Monoclonal Antibodies for
Cancer Detection and Therapy, Eds. Baldwin and Byers, pp. 159-179,
Academic Press, 1985.
[0211] The general structure of TCR molecules and methods of making
and using, including binding to a peptide:Major Histocompatibility
Complex have been disclosed. See, for example PCT/US98/04274;
PCT/US98/20263; WO99/60120.
[0212] Pharmaceutical Compositions and Therapeutic Agents
[0213] scTCRs specific for a particular target ligand are useful in
treating animals and mammals, including humans believed to be
suffering from a disease associated with the particular
antigen.
[0214] Therapeutic products can be made using the materials shown
herein. Effective amounts of therapeutic products are the minimum
dose that produces a measurable effect in a subject. Therapeutic
products are easily prepared by one of ordinary skill in the art.
In one embodiment, a scTCR of the disclosure is administered
directly to a patient. In one embodiment, a scTCR of the disclosure
is linked to PEG or to immunoglobulin constant regions, as known in
the art. This embodiment lengthens the serum clearance. In one
embodiment, the scTCR is linked to a chemotherapeutic agent or drug
in order to deliver the drug to a target cell such as a cancer
cell. In one embodiment, the scTCR is linked to a biologic effector
molecule such as a cytokine (Tayal and Kalra (2008) Eur J
Pharmacol, 579, 1-12). In one embodiment, the scTCR is linked to a
cytokine with anti-tumor activity, such as IL-2, IL-12, or
TNF.alpha. (Wong et al. (2011) Protein Eng Des Sel, 24, 373-83). In
one embodiment, the scTCR is linked to an immune-inhibitory
cytokine, such as IL-10 or IL-13 (Stone et al. (2012) Protein
Engineering). In one embodiment, the scTCR is linked to another
antigen binding molecule to form a bispecific agent (Miller et al.
(2010) Protein Eng Des Sel, 23, 549-57; Thakur and Lum (2010) Curr
Opin Mol Ther, 12, 340-9). In one embodiment, the bispecific
molecule is comprised of a scTCR linked to a single chain Fv, such
as an anti-CD3 ((Bargou et al. (2008) Science, 321, 974-7; Liddy et
al. (2012) Nat Med, 18, 980-7), to crosslink T cells and diseased
cells. In one embodiment, the scTCR is linked to TCR signaling
domains, such as CD3, to form a chimeric antigen receptor ((Porter
et al. (2011) N Engl J Med, 365, 725-33; Sadelain et al. (2009)
Curr Opin Immunol, 21, 215-23; Stroncek et al. (2012) J Transl Med,
10, 48). These methods and other methods of administering, such as
intravenously, are known in the art. Useful dosages can be
determined by one of ordinary skill in the art.
[0215] The scTCR compositions can be formulated by any of the means
known in the art. They can be typically prepared as injectables,
especially for intravenous, intraperitoneal or synovial
administration (with the route determined by the particular
disease) or as formulations for intranasal or oral administration,
either as liquid solutions or suspensions. Solid forms suitable for
solution in, or suspension in, liquid prior to injection or other
administration may also be prepared. The preparation may also, for
example, be emulsified, or the protein(s)/peptide(s) encapsulated
in liposomes.
[0216] The active ingredients are often mixed with optional
pharmaceutical additives such as excipients or carriers which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients include but are not limited to
water, saline, dextrose, glycerol, ethanol, or the like and
combinations thereof. The concentration of the scTCR in injectable,
aerosol or nasal formulations is usually in the range of 0.05 to 5
mg/ml. The selection of the particular effective dosages is known
and performed without undue experimentation by one of ordinary
skill in the art. Similar dosages can be administered to other
mucosal surfaces.
[0217] In addition, if desired, vaccines that could include a scTCR
may contain minor amounts of pharmaceutical additives such as
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, and/or adjuvants which enhance the effectiveness
of the vaccine. Examples of adjuvants which may be effective
include but are not limited to: aluminum hydroxide;
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP);
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dip-
almitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine (CGP 19835A,
referred to as MTP-PE); and RIBI, which contains three components
extracted from bacteria: monophosphoryl lipid A, trehalose
dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%
squalene/Tween.RTM. 80 emulsion. Such additional formulations and
modes of administration as are known in the art may also be
used.
[0218] The scTCRs of the present disclosure and/or binding
fragments having primary structure similar (more than 90% identity)
to the TCR variable regions and which maintain the high affinity
for the target ligand may be formulated into vaccines as neutral or
salt forms. Pharmaceutically acceptable salts include but are not
limited to the acid addition salts (formed with free amino groups
of the peptide) which are formed with inorganic acids, e.g.,
hydrochloric acid or phosphoric acids; and organic acids, e.g.,
acetic, oxalic, tartaric, or maleic acid. Salts formed with the
free carboxyl groups may also be derived from inorganic bases,
e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides,
and organic bases, e.g., isopropylamine, trimethylamine,
2-ethylamino-ethanol, histidine, and procaine.
[0219] scTCRs for therapeutic use are administered in a manner
compatible with the dosage formulation, and in such amount and
manner as are prophylactically and/or therapeutically effective,
according to what is known to the art. The quantity to be
administered, which is generally in the range of about 100 to
20,000 .mu.g of protein per dose, more generally in the range of
about 1000 to 10,000 .mu.g of protein per dose. Similar
compositions can be administered in similar ways using labeled
scTCRs for use in imaging, for example, to detect cells to which a
target ligand is bound. Precise amounts of the active ingredient
required to be administered may depend on the judgment of the
physician or veterinarian and may be peculiar to each individual,
but such a determination is within the skill of such a
practitioner.
[0220] The vaccine or other immunogenic composition may be given in
a single dose; two dose schedule, for example two to eight weeks
apart; or a multiple dose schedule. A multiple dose schedule is one
in which a primary course of vaccination may include 1 to 10 or
more separate doses, followed by other doses administered at
subsequent time intervals as required to maintain and/or reinforce
the immune response, e.g., at 1 to 4 months for a second dose, and
if needed, a subsequent dose(s) after several months. Humans (or
other animals) immunized with the retrovirus-like particles of the
present disclosure are protected from infection by the cognate
retrovirus.
[0221] Autoimmune diseases are those diseases in which the immune
system produces an immune response against an antigen that is
normally present in the host. Autoimmune diseases include
rheumatoid arthritis, adjuvant arthritis, myasthenia gravis,
encephalomyelitis, multiple sclerosis, thyroiditis, inflammatory
bowel disease or systemic lupus erythematosus, type I diabetes,
non-obese diabetes, Grave's disease, Hashimoto's disease,
osteoarthritis, dermatitis, hepatitis, pemphigus vulgaris, celiac
disease, Sjogren's syndrome, Addison's disease, primary myxedema,
Goodpasture's syndrome, tuberculoid leprosy, ankylosing
spondylitis, Reiter's disease, uveitis, amyloidosis, psoriasis
vulgaris, idiopathic hemochromatosis and psorasis.
[0222] Every formulation or combination of components described or
exemplified can be used to practice the disclosure, unless
otherwise stated. Specific names of substances are intended to be
exemplary, as it is known that one of ordinary skill in the art can
name the same substances differently. When a compound is described
herein such that a particular isomer or enantiomer of the compound
is not specified, for example, in a formula or in a chemical name,
that description is intended to include each isomers and enantiomer
of the compound described individual or in any combination. One of
ordinary skill in the art will appreciate that methods, target
ligands, biologically active groups, starting materials, and
synthetic methods other than those specifically exemplified can be
employed in the practice of the disclosure without resort to undue
experimentation. All art-known functional equivalents, of any such
methods, target ligands, biologically active groups, starting
materials, and synthetic methods are intended to be included in
this disclosure. Whenever a range is given in the specification,
for example, a temperature range, a time range, or a composition
range, all intermediate ranges and subranges, as well as all
individual values included in the ranges given are intended to be
included in the disclosure.
[0223] The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition (see e.g., Fingl et. al., in The Pharmacological Basis of
Therapeutics, 1975, Ch. 1 p. 1).
[0224] It should be noted that the attending physician would know
how to and when to terminate, interrupt, or adjust administration
due to toxicity, or to organ dysfunctions. Conversely, the
attending physician would also know to adjust treatment to higher
levels if the clinical response were not adequate (precluding
toxicity). The magnitude of an administered dose in the management
of the disorder of interest will vary with the severity of the
condition to be treated and to the route of administration. The
severity of the condition may, for example, be evaluated, in part,
by standard prognostic evaluation methods. Further, the dose and
perhaps dose frequency, will also vary according to the age, body
weight, and response of the individual patient. A program
comparable to that discussed above also may be used in veterinary
medicine.
[0225] Depending on the specific conditions being treated and the
targeting method selected, such agents may be formulated and
administered systemically or locally. Techniques for formulation
and administration may be found in Alfonso and Gennaro (1995).
Suitable routes may include, for example, oral, rectal,
transdermal, vaginal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous, or
intramedullary injections, as well as intrathecal, intravenous, or
intraperitoneal injections.
[0226] For injection, the agents of the disclosure may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks' solution, Ringer's solution, or
physiological saline buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0227] Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the disclosure into
dosages suitable for systemic administration is within the scope of
the disclosure. With proper choice of carrier and suitable
manufacturing practice, the compositions of the present disclosure,
in particular those formulated as solutions, may be administered
parenterally, such as by intravenous injection. Appropriate
compounds can be formulated readily using pharmaceutically
acceptable carriers well known in the art into dosages suitable for
oral administration. Such carriers enable the compounds of the
disclosure to be formulated as tablets, pills, capsules, liquids,
gels, syrups, slurries, suspensions and the like, for oral
ingestion by a patient to be treated.
[0228] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents may be encapsulated into
liposomes, and then administered as described above. Liposomes are
spherical lipid bilayers with aqueous interiors. All molecules
present in an aqueous solution at the time of liposome formation
are incorporated into the aqueous interior. The liposomal contents
are both protected from the external microenvironment and, because
liposomes fuse with cell membranes, are efficiently delivered into
the cell cytoplasm. Additionally, due to their hydrophobicity,
small organic molecules may be directly administered
intracellularly.
[0229] Pharmaceutical compositions suitable for use in the present
disclosure include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein.
[0230] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, dragees, capsules, or
solutions, including those formulated for delayed release or only
to be released when the pharmaceutical reaches the small or large
intestine.
[0231] The pharmaceutical compositions of the present disclosure
may be manufactured in a manner that is itself known, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levitating, emulsifying, encapsulating, entrapping
or lyophilizing processes.
[0232] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0233] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0234] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0235] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added.
EXAMPLES
[0236] The following examples further describe non-limiting
examples of the disclosure.
Example 1
Selection of a Model TCR
[0237] TCRs all adopt a similar Ig-fold and docking angle, and TCR
recognition of pepMHC is mediated entirely by specific residues on
CDR loops (Garcia et al. (2009) Nat Immunol, 10, 143-7; Marrack et
al. (2008) Annu Rev Immunol, 26, 171-203; Rudolph et al. (2006)
Annu Rev Immunol, 24, 419-66)). Hence, according to the present
invention, a single TCR with known structure provides a scaffold
for in vitro engineering with specificity and high affinity against
non-cognate peptides displayed on MHC. That is, by generating
mutants with degenerate residues within CDR loop residues that are
most likely to directly contact peptide, libraries of mutants
within a single TCR were generated in order to provide TCRs that
can be developed having high affinity against non-cognate
peptide-MHC antigens.
[0238] The general strategy used to discover, or generate, novel
TCRs against non-cognate antigens from a single scaffold is shown
in FIG. 1. The process involves: selecting a single TCR with a
known structure. In this example, the human TCR called A6 was used.
The A6 TCR was the first human TCR whose structure was solved
(Garboczi et al. (1996) Nature, 384, 134-141), and it has been the
topic of a long series of structural and biochemical studies (e.g.,
(Armstrong et al. (2008) Biochem J, 415, 183-96; Ding et al. (1999)
Immunity, 11, 45-56; Hawse et al. (2012) J Immunol, 188, 5819-23).
In addition, it has been used in heterodimer form for engineering
higher affinity mutants against it's cognate antigen Tax:HLA.A2 (Li
et al. (2005) Nat Biotechnol, 23, 349-54). Also, a stable version
of the single-chain TCR (V.beta.-linker-V.alpha.) has been
displayed on the surface in the yeast display system (Aggen et al.
(2011) Protein Engineering, Design, & Selection, 24,
361-72).
[0239] Another step in the process is identifying residues within
the TCR (e.g., A6) binding site that are most likely to contribute
to peptide specificity. This step involved modeling a collection of
peptides into the HLA.A2 molecule, docking of the A6 TCR, analysis
of the frequency by which A6 TCR residues reside within 3 angstroms
of each peptide.
[0240] Cloning libraries of mutants that have variability at the
positions identified by structure-based analysis, as described
above, is the next step. These libraries can be cloned into various
display systems, such as yeast display. Phage display vectors and
cloning have yielded library sizes of 10.sup.11, whereas yeast
display vectors and homologous recombination steps have yielded
library sizes of 10.sup.10 ((Benatuil et al. (2010) Protein Eng Des
Sel, 23, 155-9).
[0241] Next, mutants that bind to specific, non-cognate pep-MHC
ligands are selected. Various methods have been used for selecting
variants, including affinity-based binding to immobilized ligands
(phage display) or magnetic particle selections with antigens
(yeast display), or fluorescent activated cell sorting with
labeled-peptide-MHC antigens (yeast display). Examples of each of
these steps are described further below.
Example 2
Analysis of the Human TCR A6 in Complex with Tax:HLA.A2 as a
Scaffold for TCR Engineering
[0242] For illustrative purposes, the A6 TCR was selected as the
single TCR having a known structure. The structure of the A6:Tax
peptide:HLA-A2 complex (PDB: 1AO7) (Garboczi et al. (1996) Nature,
384, 134-141), was published in 1996. The side view of the complex
showed that the ends of the variable domains that contained the six
CDRs docked onto the Tax:HLA.A2 molecule, with the central region
of the binding site positioned over the peptide Tax (FIG. 2A) The
top down view of the Tax:HLA.A2 complex, with the TCR "removed",
except for the six CDR loops. This view shows that the TCR adopts a
diagonal position over the peptide-MHC, a finding which has now
been observed for all TCR:peptide-MHC structures. In this
orientation, the two CDR3 loops are positioned over the peptide,
while there are various residues from CDR1 and CDR2 loops that
interact predominantly with the helices of the MHC molecule. This
diagonal docking orientation, with the V.alpha. region positioned
over the .alpha.2 MHC helix and the N-terminal end of the peptide,
and the V.beta. region positioned over the .alpha.1 MHC helix and
C-terminal end of the peptide has been observed in virtually all
complexes to date. The conserved features of these interactions
suggested that it may be possible to use a single TCR as a
scaffold, in which mutagenesis of various key "peptide-interacting"
residues allowed the generation and discovery of novel TCR
specificities (FIG. 2B).
[0243] Recent studies have used the A6 TCR for engineering higher
affinity TCRs against the cognate antigen Tax/HLA.A2 by 1) directed
evolution (Li et al. (2005) Nat Biotechnol, 23, 349-54), and 2)
predictive algorithms for site-directed design (Pierce et al.
(2010) Biochemistry, 49, 7050-9). According to the present
invention, it is shown for the first time that it is possible to
use structure-based, rational design of degenerate TCR libraries
with mutations in key positions, combined with high-throughput
screening to discover TCRs against non-cognate peptides bound to
HLA products.
Example 3
Analysis of the CDR Loop Residues Most Likely to Contribute to
Peptide Binding and Specificity
[0244] In order to identify potential contact and
specificity-determining residues, various approaches were used to
determine which residues of the A6 CDR loops would be most likely
to accommodate, and provide binding energy to, a wide array of
peptides in the HLA.A2 peptide-binding groove. First, a panel of
other HLA.A2 restricted peptides was modeled into the A6 crystal
structure (FIG. 3). Using the A6:Tax peptide:HLA.A2 crystal
structure (PDB:1AO7) as a starting point (Garboczi et al. (1996)
Nature, 384, 134-141), the Rosetta Backrub modeling program was
used to model the HLA.A2 restricted peptides (i.e., Tax, Mart1-9
mer, Mart1-10 mer, SL9 HIV, WT1, and Survivin) into the HLA.A2
groove using Rosetta Backrub flexible backbone modeling algorithms
(FIG. 3) ((Lauck et al. (2010) Nucleic Acids Res, 38, W569-75);
kortemmelab.ucsf.edu/backrub/). The peptides that were modeled into
the binding groove of the A6:tax:HLA-A2 structure in order to
determine the A6 TCR residues that are within hydrogen boding
distance (2.5-3.5 .ANG.). Candidate residues for degeneracy were
then determined by measuring which CDR loop positions would be most
likely to allow for contacts with these peptides in the lowest
energy conformation model for each peptide.
TABLE-US-00002 TABLE 2 HLA.A2 Restricted Polypeptides Peptide 0 1 2
3 4 5 6 7 8 9 Tax (wt) (SEQ ID NO: 5) L L F G Y P V Y V Mart1 9 mer
(SEQ ID NO: 6) -- A A G I G I L T V Mart1 10 mer (SEQ ID NO: 7) E L
A G I G I L T V SL9 HIV (SEQ ID NO: 8) S L Y N T V A T L WT-1 (SEQ
ID NO: 9) R M F P N A P Y L SURV (SEQ ID NO: 10) L T L G E F L K
L
[0245] Two related peptides for Mart1 are listed, as both have been
studied for their binding to HLA-A2. For modeling purposes, the
residue in position "0" of the 10 mer was omitted from the
prediction.
[0246] CDR loop residues that were within 3 .ANG. of peptide
residues were determined for each of the six models. This analysis
revealed that six residues (Table3) were within 3 angstroms in the
majority (4/6 or 5/6) of the models. These codon positions thus
served as the focus for development of A6 TCR libraries to be used
for discovery of novel mutants that bind to non-cognate peptide-HLA
antigens.
TABLE-US-00003 TABLE 3 CDR Loop Residues within 3 .ANG. of Peptide
Loop Contact position Percent of structures CDR1.alpha. Q31 83%
CDR3.alpha. D98 83% CDR3.beta. L99 83% CDR3.alpha. T97 67%
CDR3.beta. A102/G102 67% CDR3.alpha. S99 67% CDR3.beta. A100/M100
33% CDR3.beta. G101/S101 33% CDR1.alpha. S32 17% CDR1.beta. E30 17%
CDR3.beta. G98 17%
[0247] Table 3 lists the A6 TCR residues found within 3.0 .ANG. of
peptide in the 6 structures and the percentage of structures in
which it was found. Positions in bold type were used to construct a
degenerate library, but other positions can be used either
separately or in combination to construct additional libraries for
developing TCRs against many different peptide/HLA complexes.
[0248] The positions of five residues in the A6 TCR are shown in
the sequence of the single-chain form of the TCR (FIG. 5). In
addition, the sequence of the stabilized, A6 single-chain TCR
called A6-X15 (SEQ ID NO:3), which was engineered previously for
high-affinity binding to the cognate antigen Tax:HLA.A2, is shown.
This sequence also includes five framework mutations (S33A, E59D,
N63D, N66K, K121I, all in the V.beta. domain) and two CDR mutations
(A52V and Q106L, both in the V.beta. domain) that were isolated
previously in a stability screen of the scTCR. This mutant also
contained four CDR3.beta. mutations (A100M, G101S, G102A, and
R103Q) that yielded higher affinity binding to Tax:HLA.A2 (Li et
al. (2005) Nat Biotechnol, 23, 349-54). A6-X15 also uses the highly
stable V.alpha.2 segment (IMGT: TRAV12-2), but contains a Phe49Ser
V.alpha. mutation for improved stability.
Example 4
Analysis of Binding Contributions Several CDR Residues
[0249] Several CDR residues were predicted to be involved in
binding to the HLA.A2 helices and/or the Tax peptide (Borbulevych
et al. (2011) J Immunol, 187, 2453-63; Marrack et al. (2008) Annu
Rev Immunol, 26, 171-203). The process of engineering A6 TCR
mutants that bind to different peptides bound to HLA.A2 would
benefit from retaining those amino acid side chains that provide
binding energy to their interaction with the HLA.A2 helices.
Crystal structures of A6:pepHLA-A2 with both Tax peptide and tax
peptide variants have shown pepMHC-contacts mediated predominately
by .alpha.-chain residues (Ding et al. (1999) Immunity, 11, 45-56;
Garboczi et al. (1996) Nature, 384, 134-141). Recent studies of
V.alpha.2-containing TCRs have described in detail putative
conserved resides in the V.alpha.2 segment that have evolved to
recognize the HLA.A2 helices.
[0250] FIG. 4 shows the analysis of selected A6 TCR residues for
their contribution to binding of Tax:HLA.A2. In accordance with
studies that have suggested these residues in CDR1 or CDR2 are
important in maintaining MHC restriction, four residues of the
(Aggen et al. (2011) Protein Engineering, Design, & Selection,
24, 361-72) were substituted with alanine and tested for binding.
Table3 shows approximate affinities of A6 X15 alanine mutants and
fold changes in binding affinity relative to wild type. The results
showed that the tyrosine at position 51 of CDR2.alpha., which
contacts the .alpha. helix of HLA.A2, was most important in
binding. Thus, this residue was maintained in the library described
here.
TABLE-US-00004 TABLE 4 Alanine Substitution Apparent Fold Mutant
K.sub.D Change wt 44 nM -- S52A 27 nM 0.6X R28A 68 nM 1.6X Q31A
>4 .mu.M >100X Y51A >4 .mu.M >100X
[0251] CDR2.alpha. residues Y51 and S52 are conserved across
different .alpha.-chains and frequently bind HLA.A2 at the same
position (Marrack et al. (2008) Annu Rev Immunol, 26, 171-203).
Crystal structure analysis of the V.alpha.2-containing TCRs A6,
DMF4, and DMF5 have shown that CDR1.alpha. residues R28 and Q31 are
important in making MHC contacts, although the exact HLA residues
contacted vary. Q31 has also been shown to make contacts with
peptide in addition to HLA-A2 (Borbulevych et al. (2011) J Immunol,
187, 2453-63).
[0252] Although these V.alpha.2 residues (R28, Q31, Y51, and S52)
are important in contacting the HLA.A2 molecule, the binding energy
contribution of each contact had not been previously described. In
order to determine which residues contributed significant binding
energy and should therefore be retained in the library,
site-directed alanine mutants were made in the A6 X15 construct at
each position and stained with Tax (LLFGYPVYV, SEQ ID NO:5):HLA-A2
dimer (FIG. 4B). The residue that contributed the most significant
binding energy was CDR2.alpha. position Y51. Since this residue
exclusively contacted MHC helices in the A6 wt structure, this
residue was retained as wild type in the library. The residue that
contributed the next highest binding energy was CDR1.alpha. residue
Q31; however, since this residue also made contacts with the
peptide in the A6 wt structure, it was randomized in the RD1
library to prevent any cross-reactivity with the cognate ligand.
The other two residues examined, R28 and S52, did not contribute
substantial binding energy to the A6:tax:HLA.A2 interaction, but
were retained as wild type in order to prevent increased
peptide-independent reactivity towards the MHC helices.
Example 5
Yeast Display and Library Construction
[0253] This example describes the preparation of a library of
mutant TCRs. In order to identify novel TCRs from the single
scaffold, it is necessary to use a display system in which a
library of TCR mutants can be screened for the rare mutant(s) that
bind to the non-cognate antigen. Three display systems have been
used for engineering TCRs for higher affinity, and could be used
for this process: yeast display, phage display, and T cell
(mammalian cell) display. Alternative display methods, such as
ribosome, RNA, DNA, and CIS display, may also be suitable for this
process. In all of these cases, the wild type TCR with low affinity
for the cognate antigen was cloned into the system, and used as a
template for engineering higher affinity TCRs against the cognate
antigen. Any of these systems could be applied to the approach
described here, in which a single scaffold TCR (A6 in the present
case) is used as a template for rational design of libraries and
the selection of TCRs against non-cognate antigens.
[0254] In the present example, yeast display was used as the
platform. The single-chain A6-X15 TCR was used as the template as
it is stably expressed in properly folded form on the surface of
yeast (FIG. 6). The gene containing degenerate codons at each of
the top five positions shown in FIG. 3C and FIG. 4 was synthesized
by Genscript (Piscataway, N.J., USA), and was designated the RD1
library. Genes with complete or partial codon degeneracy can now be
readily synthesized by commercial sources, or can be generated by
PCR using multiple synthetic oligonucleotide primers. The synthetic
genes can be used as templates after cloning into a common plasmid
(e.g., pET vectors) or directly as PCR product templates. Two
flanking oligonucleotide primers with homology to the linearized
yeast display vector were synthesized and PCR was performed in
order to generate double stranded PCR products that could be
inserted by homologous recombination into the yeast display vector,
pCT302. The human A6-X15 scTCR library was thus introduced into the
yeast display vector by combining the linearized pCT302 vector,
A6-X15 RD1 library PCR product, and competent EBY100 yeast cells.
The resultant RD1 library contained about 6.times.10.sup.6
independent clones, was judged by plating limiting dilution
aliquots of yeast after electroporation. As expected due to the
diversity at each of the residues that are near the Tax peptide in
the complex, the resultant library did not show positive staining
with either the cognate antigen Tax:HLA-A2 or the Mart1:HLA-A2
complex as determined using peptide-HLA.A2 DimerX preparations.
[0255] In FIG. 6, a V.beta.-Linker-V.alpha. scTCR is shown with an
N-terminal hemagglutinin (HA) and C-terminal c-myc tags, used to
detect expression of the scTCR on the surface of yeast (Stone et
al. (2012) Methods Enzymol, 503, 189-222). The scTCR construct was
expressed as a fusion with the AGA-2 yeast mating protein which
allows the scTCR to be expressed on the surface of yeast and
rapidly analyzed via flow cytometry and screened by fluorescent
activated cell sorting (FACS).
[0256] In order to verify the diversity of the RD1 library at each
of the five codon positions, ten colonies from the plated library
were sequenced (FIG. 7). Each of the five positions showed
extensive diversity, indicating that the library contained diverse
potential binding sites within the regions that were predicted to
be able to contact the peptides bound to HLA.A2.
Example 6
Selection of the Yeast-Display A6 TCR Library by Cell Sorting
[0257] This example describes selecting mutant TCRs that bind to
target ligands. In order to determine if the A6 scaffold library
could be used to identify TCR mutants with binding to a non-cognate
peptide antigen, the library was selected with the non-cognate
antigen Mart1-10 mer:HLA.A2, in addition to the cognate antigen,
Tax:HLA.A2. These two peptides differ not only in length, but when
aligned only the leucine at position 2 of Mart1-10 mer (position 1
of Tax) and the valine at position 10 of Mart1 (position 9 of Tax)
are identical. In addition, the valine is considered an HLA.A2
anchor residue, such that the side chains exposed to the TCR are
distinctly different between the two cases.
[0258] To verify that the library contained mutants that bound the
cognate antigen, fluorescent-activated cell sorting (FACS) was used
with the Tax:HLA-A2 dimer (FIG. 8). The RD1 library was sorted
sequentially with 10-100 nM Tax (LLFGYPVYV, SEQ ID NO:5):HLA-A2
dimer (DimerX; obtained from BD Pharmingen), APC-conjugated goat
anti-mouse secondary antibody, for a total of four sorts according
to Table 5. Aliquots of yeast cells after each sort were then
incubated with 100 nM Tax (LLFGYPVYV, SEQ ID NO:5):HLA-A2 dimer
(DimerX; obtained from BD Pharmingen), followed by APC-conjugated
goat anti-mouse secondary antibody. Because the nucleotide
sequences vary between clones of the same amino acid sequence, it
suggests a strong selective pressure for these residues.
TABLE-US-00005 TABLE 4 Sorting Conditions Sort Conditions 1 20 nM
Tax (SEQ ID NO: 5): HLA-A2 dimer APC-conjugated goat anti-mouse
secondary antibody 2 100 nM Tax (SEQ ID NO: 5): HLA-A2 dimer
APC-conjugated goat anti-mouse secondary antibody 3 10 nM Tax (SEQ
ID NO: 5): HLA-A2 dimer APC-conjugated goat anti-mouse secondary
antibody 4 10 nM Tax (SEQ ID NO: 5): HLA-A2 dimer APC-conjugated
goat anti-mouse secondary antibody
[0259] As indicated the RD1 library did not show a detectable
positive peak, but after the second sorting, a positive population
began to emerge, and cells were plated after the fourth sorting for
additional analyses. Six clones revealed that 2 of 6 had identical
amino acid sequences to A6-X15 (although the nucleotide
sequences/codon usage varied) and 4 of 6 had a threonine
substitution at position 31 in CDR1.alpha.. All clones had similar
staining profiles. The amino acid and nucleotide sequences of the
six clones analyzed are in FIG. 10.
[0260] To determine whether the scaffold approach is capable of
generating TCRs with non-cognate specificities,
fluorescent-activated cell sorting (FACS) was used with the
Mart1-10 mer:HLA-A2 dimer (FIG. 9). The RD1 library was sorted
sequentially with 20-500 nM Mart1 (ELAGIGILTV, SEQ ID NO:7):HLA-A2
dimer (DimerX; obtained from BD Pharmingen), APC-conjugated goat
anti-mouse secondary antibody, for a total of five sorts according
to Tale 5. During the 3.sup.rd sort, yeast cells were also stained
with chicken anti-c-myc antibody, goat anti-chicken IgY alexa 647
secondary antibody and double positives were isolated in order to
exclude truncated clones. (B) Aliquots of yeast cells after each
sort were then incubated with 100 nM Mart1 (ELAGIGILTV, SEQ ID
NO:7):HLA-A2 dimer (DimerX; obtained from BD Pharmingen), followed
by APC-conjugated goat anti-mouse secondary antibody.
TABLE-US-00006 TABLE 6 Sorting Conditions Sort Conditions 1 500 nM
Mart1 (SEQ ID NO: 7): HLA-A2 dimer APC-conjugated goat anti-mouse
secondary antibody 2 500 nM Mart1 (SEQ ID NO: 7): HLA-A2 dimer
APC-conjugated goat anti-mouse secondary antibody 3 100 nM Mart1
(SEQ ID NO: 7): HLA-A2 dimer APC-conjugated goat anti-mouse
secondary antibody AND 1:100 Anti-Cmyc epitope antibody
FITC-conjugated goat anti-chicken secondary antibody 4 100 nM Mart1
(SEQ ID NO: 7): HLA-A2 dimer APC-conjugated goat anti-mouse
secondary antibody 5 20 nM Mart1 (SEQ ID NO: 7): HLA-A2 dimer
APC-conjugated goat anti-mouse secondary antibody
[0261] The RD1 library and the first three sorts did not show
detectable positive peaks, but after the fourth sorting, a positive
population began to emerge, and cells were plated after the fifth
sorting for additional analyses. Five clones revealed that they all
had identical amino acid sequences, indicating that these residues
were important in conferring high-affinity binding. Although the
nucleotide sequences do not vary between clones of the same amino
acid sequence, only a small amount of possible codon combinations
are possible with this amino acid sequence.
[0262] The amino acid and nucleotide sequences are shown in FIG.
12. As would be expected due to the distinctly different sequences
of the Tax and Mart1 peptides, all five TCR residues, derived by
sorting of the A6 library, differed between the original
high-affinity TCR and the Mart1 selection. In the A6 library
selection with tax:HLA.A2, the wild type sequence was encountered
in 2 of 6 sequences (e.g., Tax-S4-3) and a variant containing
threonine at position 31 occurred in 4 of 6 sequences (e.g.,
Tax-S4-1). In the Tax-specific TCR variant the five residues were:
Leu99.beta., Ala102.beta., Gln31.alpha. or Thr31.alpha.,
Thr97.alpha., and Asp98.alpha.. In the Mart1-specific TCR the
residues were: Trp99.beta., Gly102.beta., Thr31.alpha.,
Lys97.alpha., and Tyr98.alpha.. Although the Thr31.alpha. residue
was found in both the Mart1-specific TCR and one variant of the
Tax-specific TCR (e.g., Tax-S4-1), the A6 crystal structure shows
this position as being important in contacting both the peptide and
MHC (see FIG. 4). Due to its structural similarity to glutamine,
threonine may allow for MHC contacts to be maintained without
leading to cross-reactivity with other peptides.
Example 7
Binding and Specificity Analysis of Selected High-Affinity TCRs
[0263] In order to use TCRs in the present disclosure for specific
targeting of cells that express the antigens, it is critical that
they exhibit peptide specificity. To determine if the TCRs selected
for binding to the Tax (SEQ ID NO:5) and Mart1 (SEQ ID NO:7)
peptides exhibited specificity in their reaction with the selecting
peptides, representative clones for each were titrated with various
concentrations of both the Tax and Mart1:HLA.A2 dimers. Clone
RD1-Mart1-S5-4 which had been selected for binding to the
Mart1:HLA-A2 dimer was subjected to binding analysis by titrating
yeast with Mart1:HLA-A2 DimerX and tax:HLA-A2 DimerX at
concentrations ranging from 4 to 500 nM (FIGS. 11 and 13).
[0264] After four sorts of the library with Tax:HLA.A2 as described
in FIG. 8, individual yeast clones were cultured, induced, and
analyzed for cell surface levels, and peptide:HLA.A2 binding. The
A6 high-affinity mutant called X15, which was identical in amino
acid sequence to two of six clones isolated after the fourth sort
(data not shown), was analyzed.
[0265] After five sorts of the library with Mart1:HLA.A2 as
described in FIG. 9, individual yeast clones were cultured,
induced, and analyzed for cell surface levels, and peptide:HLA.A2
binding. The A6 high-affinity mutant called S5-4 (SEQ ID NO:33),
which was identical in amino acid sequence to all clones isolated
after the fourth sort (data not shown), was analyzed.
[0266] The Mart1-specific TCR bound only to the Mart1 complex, and
not to the Tax complex, with a half-maximal concentration of
binding in the low nanomolar range. Conversely, the Tax-specific
TCR bound only to the Tax complex, and not to the Mart1 complex,
with a half-maximal concentration of binding also in the low
nanomolar range. The lack of binding even at the highest
concentration indicated that the high-affinity TCR variant
maintained specificity for the selecting ligand, Tax:HLA.A2.
Similarly, the lack of binding even at the highest concentration
indicated that the high-affinity TCR variant maintained specificity
for the selecting ligand, Mart1:HLA.A2.
Example 8
Affinity Maturation of the MART1-Selected RD1 Scaffold Variant,
RD1-MART1-S5-4 scTv Via Site Directed Mutagenesis
[0267] In order to increase the affinity of the RD1-MART1-S5-4
isolated scTv (SEQ ID NO:33) for the selecting ligand MART1 (SEQ ID
NO:7), degenerate libraries were made in the CDR3 loops of
RD1-MART1-S5-4 in order to select for mutants for increased
affinity to peptide MART1/HLA-A2/Ig dimers. The RD1-MART1-S5-4 CDR3
libraries were sorted sequentially with 1-200 nM MART1/HLA-A2 dimer
(DimerX; obtained from BD Pharmingen), APC-conjugated goat
anti-mouse secondary antibody, for a total of two sorts. Following
two rounds of selection by FACS with MART1/HLA-A2/Ig dimers, a
positively staining population of yeast that bound strongly to
MART1/HLA.A2 emerged and various clones were isolated and examined
for binding (FIG. 15A). Aliquots of yeast cells after each sort
were then incubated with 50 nM MART1/HLA-A2 dimer (DimerX; obtained
from BD Pharmingen), followed by APC-conjugated goat anti-mouse
secondary antibody. One clone, RD1-MART1.sup.HIGH (SEQ ID NO:34),
showed a significant binding increase from the template clone,
RD1-MART1-S5-4, when stained with MART1/HLA.A2 monomers (FIGS. 15B
and 15C).
Example 9
Specificity and Sequence of Selected RD1-Mart1.sup.HIGH scTv
[0268] In order to determine that the isolated TCR was specific
only for the selecting ligand MART1, staining RD1-MART1.sup.HIGH
with non-selecting cognate peptide Tax/HLA.A2, non-selecting
non-cognate peptide WT1/HLA.A2, and non-selecting non-cognate
peptide Survivin/HLA.A2 were performed. Staining showed no
detectable signal with 500 nM peptide/HLA.A2 dimers suggesting the
scTv was highly specific for the selecting antigen, MART1 (FIG.
16).
[0269] Sequences of the wild type A6 V.alpha. and V.beta. regions
of the A6 TCR are shown (Garboczi et al. (1996) Nature, 384,
134-141) and the high affinity single-chain variant A6-X15 (Aggen
et al. (2011) Protein Engineering, Design, & Selection, 24,
361-72) are shown in FIG. 18. Sequencing revealed that the
RD1-MART1.sup.HIGH clone contained three mutations in CDR3.beta.,
from RD1-MART1-S5-4 template (S101A, Q103G, and P104V) (FIG. 18).
Thus, compared to A6, RD1-MART1.sup.HIGH contained TCR.alpha.
mutations Q31T, T97K, and D98Y, and TCR.beta. mutations L99W,
A100M, G101A, R103G, P104V (SEQ ID NO:34).
[0270] To further show that the RD1-MART1.sup.HIGH scTv
specifically bound MART1/HLA-A2 with high-affinity, a soluble form
of RD1-MART1.sup.HIGH scTv was expressed and refolded from E. coli
inclusion bodies and biotinylated via a C-terminal BirA tag (Aggen
et al. (2011) Protein Engineering, Design, & Selection, 24,
361-72; Zhang et al. (2007) J Exp Med, 204, 49-55). The human cell
line T2 (HLA-A2+) was incubated with 1 .mu.M MART1, Tax, or WT1
peptides and washed. Biotinylated RD1-MART1.sup.HIGH scTv was
titrated on T2 cells pre-loaded with MART-1 peptide (1 .mu.M), null
peptide, tax (1 .mu.M), or without peptide. The cells were washed
and incubated with SA-PE and analyzed by flow cytometry. Only cells
loaded with MART1 peptide were bound by the RD1-MART1.sup.HIGH TCR.
The results showed that the soluble TCR was specific for MART-1 and
that it exhibited low nanomolar binding affinity (FIG. 17).
Example 10
Design and Selection of a Second Generation A6 Scaffold Library,
RD2
[0271] To show that the scaffold approach could work with other
libraries using the A6 TCR template, the Rosetta modeling
information used to generate the RD1 library (FIG. 3), and the
A6:Tax/HLA.A2 crystal structure (PDB: 1AO7) overlaid with the
crystal structures of MART1/HLA.A2 (PDB: 1JF1) (Sliz et al. (2001)
J Immunol, 167, 3276-84) and WT1/HLA.A2 (PDB: 3HPJ) (Borbulevych et
al. (2010) Mol Immunol, 47, 2519-24) (FIG. 19A), were used to guide
the generation of a second generation library, called RD2. This
library included 5 degenerate positions (TCR.alpha. D27, G29, and
S99; TCR.beta. L99 and W100) based on NNK nucleic acid composition,
one binary position at TCR.alpha. Q31 where either the wild type
residue glutamine or threonine could be selected, and a binary
sequence in position s100-103 in CDR3.beta. where the four adjacent
residues could be selected as A6 wild type (AGGR, SEQ ID NO:44) or
A6-X15 (MSAQ, SEQ ID NO:45) (FIG. 20). In addition, the glutamine
at position 1 of V.alpha.2 was omitted. In order to verify the
diversity of the RD2 library at each of diverse positions, five
clones from the plated library were sequenced (FIG. 20).
[0272] Two sequential magnetic bead selections of the RD2 library
were performed following incubation with 5 .mu.M Tax/HLA.A2
UV-exchanged monomers and streptavidin MACS beads (obtained from
Miltenyi Biotec) (FIG. 19B). Following the second selection, yeast
cells were incubated with 1 nM Tax/HLA-A2 dimer (DimerX; obtained
from BD Pharmingen) and APC-conjugated goat anti-mouse secondary
antibody. Aliquots of yeast cells after each selection were then
incubated with 50 nM Tax/HLA-A2 dimer (DimerX; obtained from BD
Pharmingen), APC-conjugated goat anti-mouse secondary antibody.
[0273] Two sequential magnetic bead selections of the RD2 library
were also performed following incubation with 5 .mu.M MART1/HLA-A2
UV-exchanged monomers and streptavidin MACS beads (obtained from
Miltenyi Biotec) (FIG. 19C). Following the second selection, yeast
cells were incubated with 100 nM MART1/HLA-A2 dimer (DimerX;
obtained from BD Pharmingen) and APC-conjugated goat anti-mouse
secondary antibody. Aliquots of yeast cells after each selection
were then incubated with 50 nM MART1/HLA-A2 dimer (DimerX; obtained
from BD Pharmingen), APC-conjugated goat anti-mouse secondary
antibody.
[0274] The RD2 library was selected with two peptide/MHC ligands,
Tax/HLA.A2 (cognate) and MART1/HLA.A2 (non-cognate) via two MACS
magnetic bead selections followed by one round of FACS. Selections
with the cognate antigen, Tax, showed the emergence of a positively
staining population after the first magnetic selection with
Tax/HLA.A2 monomers (FIG. 19B). Selections with MART1/HLA.A2
revealed the emergence of a positively staining population
following the third FACS selections (FIG. 19C).
Example 11
Isolation and Characterization of RD2 Variants that Bind to
MART1/HLA.A2
[0275] Following the third selection of the RD2 library with
MART1/HLA.A2, six colonies were isolated and analyzed for improved
staining for MART1/HLA.A2 (data not shown). Individual yeast clones
were cultured, induced, and analyzed for peptide/HLA.A2 binding.
Two clones, called RD2-MART1-S3-3 (SEQ ID NO:41) and RD2-MART1-S3-4
(SEQ ID NO:42), showed increased binding to MART1/HLA.A2 (FIGS. 21A
and 21B), and did not bind to the cognate non-selecting Tax/HLA.A2
(FIGS. 21C and 21D). Sequencing analysis revealed the selection of
the A6 wild-type CDR3.beta. loop sequence AGGR (SEQ ID NO:44) for
both RD2-MART1-S3-3 and RD2-MART1-S3-4. Additionally, both
RD2-MART1-S3-3 and RD2-MART1-S3-4 selected TCR-.beta. M99,
TCR-.alpha. S27 and H29, and two PCR-based mutations in TCR-.alpha.
(S34 and P40). Whereas RD2-MART1-S3-3 selected threonine at
position 31 in CDR1.alpha. binary position, the RD2-MART1-S3-4
clone retained the wild-type Q31. Additionally, RD2-MART1-S3-3
selected TCR.alpha. R99 and S100, whereas RD2-MART1-S3-4 selected
L99 and W100 (FIG. 21E).
[0276] Thus, RD2-MART1-S3-3 contained TCR.alpha. mutations D27S,
G29H, Q31T, F34S, 540P, S99R, W100S, and TCR.beta. mutation L99M
(SEQ ID NO:41) and RD2-MART1-S3-4 contained TCR.alpha. mutations
D27S, G29H, F34S, S40P, S99L, and TCR.beta. mutation L99M (SEQ ID
NO:42).
Example 12
Use of an Alternative Scaffold
[0277] To show that other single-chain TCRs, in addition to the
wild type TCR A6 and the stabilized variant of A6 known as A6 X15,
another engineered high affinity scTCR known as T1-S18.45 was used
as a template TCR for a single chain scaffold in the yeast display
system. T1-518.45 uses V.alpha.2 and V.beta.16 and was isolated
against the MART1/HLA-A2 antigen (Fleischer et al. (2004) J
Immunol, 172, 162-9). The wild-type single-chain TCR was used as a
template for CDR3 libraries, and affinity maturation was performed
as described above. The high-affinity mutant T1-518.45 was
sequenced (FIG. 22), showing that it contained the TCR.alpha.
mutations N92S, D99S, N100S, A101D, and R102F (SEQ ID NO:43).
[0278] To further show that the T1-518.45 scTv bound MART1/HLA-A2
with high-affinity, a soluble form of T1-518.45 scTv was produced
in E. coli and biotinylated (Aggen et al. (2011) Protein
Engineering, Design, & Selection, 24, 361-72; Zhang et al.
(2007) J Exp Med, 204, 49-55). Biotinylated T1-S18.45 scTv was
titrated on antigen-presenting cell line T2 (HLA-A2+) pre-loaded
with MART-1 peptide (1 .mu.M) or null peptide, 5L9 (1 .mu.M). The
results showed that the soluble TCR was specific for MART-1 and
that it exhibited low nanomolar binding affinity (FIG. 23).
Example 13
Therapeutic Formats of TCRs Engineered Using the Scaffold
Process
[0279] It is now well known that higher affinity TCRs can be used
in various formats for targeting cells that express the
corresponding antigen. Thus, it is clear that the TCRs generated
from the scaffold strategy could be used either in soluble form or
in TCR gene therapy for adoptive T cell therapies.
[0280] As summarized in FIG. 24, the TCRs can be readily formatted
for use as soluble therapeutic products, which carry a "payload" to
the target cell expressing the specific peptide-MHC antigen. The
formats include those already practiced in the art, including
immunoglobulin fusions, chemotherapeutic or drug conjugates, and
bispecific antibodies: 1) single-chain TCRs in either a
V.alpha.-V.beta. orientation or V.beta.-V.alpha. orientation,
expressed in soluble form for binding applications or as a platform
for the other applications shown; mutated, high-affinity V domains
are shown with an asterisk (*); 2) the single-chain TCR can be
fused in frame with the constant regions domains of an antibody to
produce an immunoglobulin fusion with effector functions and other
properties of the Fc regions, as has been done with the
extracellular domain of the TNF-.alpha. receptor in the product
Enbrel (Brower (1997) Nat Biotechnol, 15, 1240); 3) The individual
V.alpha. and V.beta. domains can be configured like a conventional
antibody, as in-frame fusions to either the constant region of the
light chain or the constant regions of the heavy chain, to produce
an immunoglobulin fusion; 4) the single-chain TCR (or the
immunoglobulin fusions shown in 2 and 3) can be directly coupled to
a drug in order to endow the peptide-MHC targeting domain with a
toxic compound for killing the target cell; 5) the single-chain TCR
can be linked as a single-chain in-frame with a single-chain Fv
(VL-linker-VH) of an antibody to produce a bispecific single-chain;
the scFv can be directed against the CD3 subunits of the TCR/CD3
complex in order to recruit the activity of T cells, as has now
been done with scFv-based bispecific antibodies, or more recently,
a TCR-based bispecific.
[0281] Additionally, FIG. 24B shows the variable domains (V)
isolated by yeast display for high-affinity binding using the TCR
scaffold can be inserted into mammalian cell vectors for expression
in T cells in an adoptive T cell therapies. The TCRs can be used
either as 1) single-chain receptors in chimeric antigen receptors
(CAR) as is now well known, or 2) they can be cloned as full length
.alpha. and .beta. TCRs for conventional TCR gene therapy in
adoptive T cell formats.
Antibodies, Peptide:HLA-A2, and Flow Cytometry
[0282] Antibodies used to detect yeast surface expression included:
anti-HA epitope tag (Clone HA.11; Covance), anti-Cmyc epitope tag
(Clone 9E10; Molecular Probes), Goat-anti-mouse IgG F(ab').sub.2
AlexaFluor 647 secondary antibody (Invitrogen), and
Goat-anti-chicken IgG F(ab').sub.2 AlexaFluor 647 secondary
antibody (Invitrogen). Peptides that bind to HLA-A2 [Tax.sub.11-19:
SEQ ID NO:5, Mart1.sub.26-35 A27L: SEQ ID NO:7] were synthesized by
standard F-moc (N-(9-fluorenyl)methoxycarbonyl) chemistry at the
Macromolecular Core Facility at Penn State University College of
Medicine (Hershey, Pa., USA). For FACS and flow cytometry analysis,
recombinant soluble dimeric HLA-A2:Ig fusion protein (BD DimerX)
was used.
A6 RD1 Library Design
[0283] Candidate residues for degeneracy were determined by
measuring which CDR loop positions would be most likely to allow
for contacts with a variety of peptides using Rosetta Backrub
flexible backbone modeling algorithms ((Lauck et al. (2010) Nucleic
Acids Res, 38, W569-75; Smith and Kortemme (2008) J Mol Biol, 380,
742-56), kortemmelab.ucsf.edu/backrub/). Using the A6:Tax
peptide:HLA-A2 crystal structure (PDB: 1AO7) (Garboczi et al.
(1996) Nature, 384, 134-141) as input, Rosetta was used to model in
a variety of HLA-A2 restricted peptides of interest (SL9, Mart1,
WT1, Survivin) into the peptide binding groove of HLA-A2 by using
the Multiple Mutation Mutagenesis module. Next, CDR loop residues
that were within 3 .ANG. of peptide residues were determined for
the lowest energy conformation of each model, and the 5 most
frequently encountered positions were made degenerate using A6 X15
scTCR, which contains stabilizing mutations as previously
described, as a template (Aggen et al. (2011) Protein Engineering,
Design, & Selection, 24, 361-72).
[0284] Computational design methodology to improve affinities of T
cell receptors for their cognate peptide-MHC has been described
(Haidar et al. (2009) Proteins, 74, 948-60; Hawse et al. (2012) J
Immunol, 188, 5819-23). In this design method, called ZAFFI, single
point mutations are modeled and analyzed for improved binding.
Next, point mutations that increase binding were combined and
analyzed for additive effects. This algorithm has been used to
describe an A6 variant containing 4 mutations that bound 99 times
more tightly than the wild type TCR (Haidar et al. (2009) Proteins,
74, 948-60). In a later study, the same methodology was used to
design a higher affinity variant of the Mart-1 specific TCR DMF5.
The high affinity DMF5 variant contained 2 mutations that increased
the affinity 250-fold (Hawse et al. (2012) J Immunol, 188,
5819-23). The use of computational approaches to engineer T cell
receptors with de novo affinities for non-cognate ligands has not
been described. The present invention provides for the first time
the use of computational modeling to guide scaffold generation to
be used for directed evolution.
[0285] Candidate residues for degeneracy were determined by
measuring which CDR loop positions would be most likely to allow
for contacts with a variety of peptides using Rosetta Backrub
flexible backbone modeling algorithms (Lauck, 2010 #7691};
kortemmelab.ucsf.edu/backrub/). Using the A6:Tax peptide:HLA-A2
crystal structure (PDB: 1AO7), Rosetta was used to model in a
variety of HLA-A2 restricted peptides of interest (SL9, Mart1, WT1,
Survivin). Next, CDR loop residues that were within 3 .ANG. of
peptide residues were determined for each model, and the 5 most
frequently encountered positions were made degenerate using A6 X15
scTCR, which contains stabilizing mutations as previously
described, as a template (Aggen et al. (2011) Protein Engineering,
Design, & Selection, 24, 361-72).
Library Generation, Display, and Selection
[0286] The A6 RD1 Library was expressed in yeast display plasmid
pCT302 (V.beta.-L-V.alpha.) (Boder and Wittrup (1997) Nat.
Biotech., 15, 553-557; Boder and Wittrup (2000) Methods Enzymol,
328, 430-44), which contains a galactose-inducable AGA2 fusion
allowing for growth in Trp media. Induction of the scTv gene
involves growth of the transformed EBY100 yeast cells to stationary
phase in selection media followed by transfer to
galactose-containing media.
[0287] The A6 RD1 Library was synthesized by Genscript (Piscataway,
N.J., USA) using A6 X15 as a template (Aggen et al. (2011) Protein
Engineering, Design, & Selection, 24, 361-72; Li et al. (2005)
Nat Biotechnol, 23, 349-54). The construct consisted of the
variable fragments attached by the linker region GSADDAKKDAAKKDGKS
(SEQ ID NO: 21) (Aggen et al. (2011) Protein Engineering, Design,
& Selection, 24, 361-72; Kieke et al. (1999) Proc Natl Acad Sci
USA, 96, 5651-6; Soo Hoo et al. (1992) Proc. Natl. Acad. Sci., 89,
4759-4763; Weber et al. (2005) Proc Natl Acad Sci USA, 102,
19033-8), and N-terminal HA and C-terminal Cmyc epitope tags. The
following gene was synthesized where regions indicated by "X" were
made degenerate by NNS codons:
NAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMAWYRQDPGMGLRLIHYSVGVGI
TDQGDVPDGYKVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGXMSXQPELYFGPG
TRLTVTEDLINGSADDAKKDAAKKDGKSQKEVEQNSGPLSVPEGAIASLNCTYSDR
GSXSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDS
ATYLCAVTXXSWGKLQFGAGTQVVVTPDEQKLISEEDL** (SEQ ID NO:4). The gene
was codon optimized for both yeast and E. coli with 5' sequence TCT
GCT AGC (SEQ ID NO:48) and 3' sequence CTC GAG ATC TGA (SEQ ID
NO:49).
[0288] In order to do homologous recombination in yeast, pCT302
overhangs were added to the synthesized library using forward
primer 5'-CAGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTC
TGCTAGCAATGCTGGTGTAACACAAACGCCAA-3' (SEQ ID NO: 50) and reverse
primer 5'-GGAACAAAGTCGATTTTGTTACATCTACACTGTTGTTAACAGATCTCG
AGTCATTATAAATCTTCTTCAGAGATC-3' (SEQ ID NO: 51). Yeast libraries
were generated by homologous recombination in EBY100 yeast by
electroporating PCR products along with NheI and XhoI digested
pCT302 (Benatuil et al. (2010) Protein Eng Des Sel, 23, 155-9;
Colby et al. (2004) Methods Enzymol, 388, 348-58; Starwalt et al.
(2003) Protein Eng, 16, 147-56; Swers et al. (2004) Nucleic Acids
Res, 32, e36). The resultant library size was 6.times.10.sup.6. The
library was induced in galactose-containing media (SG-CAA) for 48
h, washed with 1 mL 1% PBS/BSA, and stained with the following:
anti-HA epitope tag (1:50), anti-V.alpha.2 antibody (1:50), and tax
or Mart1 peptide:HLA-A2 DimerX (100 nM) along with goat-anti-mouse
IgG F(ab').sub.2 AlexaFluor 647 secondary antibody (1:100), and
anti-cmyc (1:50) along with goat-anti-chicken IgG F(ab').sub.2
AlexaFluor 647 secondary antibody (1:100). Cells were washed (1 ml,
1% PBS/BSA), and the most fluorescent cells were selected using a
FACS Aria (BD Bioscience) high-speed sorter. Selection was
performed with tax:HLA-A2 dimer (10-20 nM) and Mart1:HLA-A2 dimer
(20-500 nM).
Generation, Display, and Selection of RD-MART1 CDR3 Yeast Display
Libraries
[0289] CDR3 libraries were generated by splicing by overlap
extension (SOE) PCR spanning 5 adjacent codons at a time (2
libraries in the CDR3.beta. loop; 1 in the CDR3.alpha. loop)
(Horton et al. (1990) Biotechniques, 8, 528-35) using the RD1-MART1
scTV clone selected from the RD1 library as a template. Pre-SOE PCR
products were generated for each of the four libraries utilizing
the following primer pairs.
TABLE-US-00007 .beta.1: (SEQ ID NO: 52) 5'-GGC AGC CCC ATA AAC ACA
CAG TAT-3' (Splice 4L) and (SEQ ID NO: 53) 5'-CGG ACG GGA AGC GCA
GAA ATA CAC TGA GGT TTG AGA AGG TGC AGC GCT TAA CAG ACG CAG CGG-3',
and (SEQ ID NO: 54) 5'-ACC TCA GTG TAT TTC TGC GCT TCC CGT CCG NNK
NNK NNK NNK NNK CAG CCT GAA CTG TAC TTT GGT CCA GGC ACT AGA C-3'
and (SEQ ID NO: 55) 5'-TAA TAC GAC TCA CTA TAG GG-3' (T7); .beta.2:
Splice 4L and (SEQ ID NO: 56) 5'-CGG ACG GGA AGC GCA GAA ATA CAC
TGA GGT TTG AGA AGG TGC AGC GCT TAA CAG ACG CAG CGG-3', and (SEQ ID
NO: 57) 5'-ACC TCA GTG TAT TTC TGC GCT TCC CGT CCG GGT TGG NNK NNK
NNK NNK NNK GAA CTG TAC TTT GGT CCA GGC ACT AGA CTG ACC G-3' and
T7; .alpha.: Splice 4L and (SEQ ID NO: 58) 5'-CGT AAC CGC GCA CAA
GTA TGT GGC CGA ATC GGA AGG CTG GGA GTC ACG AAT CAG CAA ACT AAC ATA
CTG GC-3', and (SEQ ID NO: 59) 5'-TCC GAT TCG GCC ACA TAC TTG TGC
GCG GTT ACG NNK NNK NNK NNK NNK AAA CTG CAA TTT GGT GCG GGC ACC CAG
GTT GTG G-3' and T7.
SOE PCR was performed with each corresponding Pre-SOE along with
both T7 and Splice 4L for each library.
[0290] Yeast libraries were generated by homologous recombination
in EBY100 yeast by electroporation of PCR products along with NheI
and XhoI digested yeast display vector pCT302 (Benatuil et al.
(2010) Protein Eng Des Sel, 23, 155-9; Colby et al. (2004) Methods
Enzymol, 388, 348-58; Starwalt et al. (2003) Protein Eng, 16,
147-56; Swers et al. (2004) Nucleic Acids Res, 32, e36). The
resultant library sizes were .beta.1: 2.1.times.10.sup.7, .beta.2:
1.7.times.10.sup.7, and .alpha.: 1.1.times.10.sup.7. Libraries were
pooled in equal cell numbers in ratios reflecting relative
diversity, and expanded in SD-CAA media.
[0291] The combined library was induced in galactose-containing
media (SG-CAA) for 48 hours, washed with 1 mL 1% PBS/BSA, and
stained with MART1/HLA.A2 DimerX, goat-anti-mouse IgG F(ab')2
AlexaFluor 647 secondary antibody (1:100). Cells were washed (1 ml,
1% PBS/BSA), and the most fluorescent cells were selected using a
FACS Aria (BD Bioscience) high-speed sorter. Selection was
performed with MART1/HLA-A2 dimer (1-200 nM). Expression was
monitored with anti-HA epitope tag (1:50), goat-anti-mouse IgG
F(ab')2 AlexaFluor 647 secondary antibody (1:100), and anti-cmyc
(1:50), goat-anti-chicken IgG F(ab')2 AlexaFluor 647 secondary
antibody (1:100).
A6 RD2 Library Design
[0292] PyMOL software (The PyMOL Molecular Graphics System, Version
1.5.0.4 Schrodinger, LLC) was used to overlay crystal structures of
the A6:Tax/HLA.A2 complex (PDB: 1AO7) with crystal structures of
the MART1/HLA.A2 (PDB: 1JF1) (Sliz et al. (2001) J Immunol, 167,
3276-84) and WT1/HLA.A2 (PDB: 3HPJ) (Borbulevych et al. (2010) Mol
Immunol, 47, 2519-24). Visual inspection and rational design were
used to select residue positions in the A6:Tax/HLA.A2 crystal
structure that were in close proximity with MART1/HLA.A2 and
WT1/HLA.A2 in the overlaid crystal structures. Five positions
(TCR.alpha. D27, G29, and S99; TCR.beta. L99 and W100) were made
degenerate based on NNK nucleic acid composition. The glutamine at
the first position of V.alpha.2 was omitted from synthesis. The
TCR.alpha. Position Q31 was a binary position where either the wild
type residue glutamine or threonine could be selected, and the
positions 100-103 in CDR3.beta., were binary where the four
adjacent residues could be selected as A6 wild type (AGGR, SEQ ID
NO:44) or A6-X15 (MSAQ, SEQ ID NO:45).
Generation, Display, and Selection of RD2 Yeast Display Library
[0293] The A6 RD2 library was expressed in yeast display plasmid
pCT302 (V.beta.-L-V.alpha.) (Boder and Wittrup (1997) Nat.
Biotech., 15, 553-557; Boder and Wittrup (2000) Methods Enzymol,
328, 430-44), which contains a galactose-inducable AGA2 fusion
allowing for growth in Trp media. Induction of the scTv gene
involves growth of the transformed EBY100 yeast cells to stationary
phase in selection media followed by transfer to
galactose-containing media. The A6 RD2 Library was synthesized by
DNA2.0 (Menlo Park, Calif., USA) using the A6-X15 as a template.
The construct consisted of the variable fragments attached by the
linker region GSADDAKKDAAKKDGKS (SEQ ID NO:21) and N-terminal HA
and C-terminal Cmyc epitope tags. The following gene was
synthesized where positions indicated by "X" were made degenerate
by NNK codons, the positions labeled "1234" were binary allowing
for A6 wild type CDR3.beta. loop AGGR (SEQ ID NO:44) or A6-X15
CDR3.beta. loop MSAQ (SEQ ID NO:45), the position indicated by "#"
was binary allowing for either wild type residue Q or mutated T,
and positions indicated by "*" were stop codons:
NAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMAWYRQDPGMGLRLIHYSVGVGI
TDQGDVPDGYKVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGX1234PELYFGPGT
RLTVTEDLINGSADDAKKDAAKKDGKSKEVEQNSGPLSVPEGAIASLNCTYSXRXS
#SFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSAT
YLCAVTTDXXGKLQFGAGTQWVTPDIEQKLISEEDL** (SEQ ID NO:35). The gene was
codon optimized for yeast, and the following flanking DNA sequences
were added which contained overlap with the T7 and Splice4L cloning
primers: N-terminal DNA sequence: 5'-GGC AGC CCC ATA AAC ACA CAG
TAT GTT TTT AAG GAO AAT AGC TCG ACG ATT GAA GGT AGA TAO CCA TAO GAO
GTT CCA GAO TAO GCT CTG CAG GCT AGT GGT GGT GGT GGT TCT GGT GGT GGT
GGT TCT GGT GGT GGT GGT TCT GCT AGC-3' (SEQ ID NO:60), and
C-terminal DNA sequence: 5'-CTC GAG ATC TGT TAA CAA CAG TGT AGA TGT
AAC AAA ATC GAO TTT GTT CCC ACT GTA CTT TTA GCT CGT ACA AAA TAO AAT
ATA CTT TTC ATT TCT CCG TAA ACA ACA TGT TTT CCC ATG TAA TAT CCT TTT
CTA TTT TTC GTT CCG TTA CCA ACT TTA CAC ATA CTT TAT ATA GCT ATT CAC
TTC TAT ACA CTA AAA AAC TAA GAO AAT TTT AAT TTT GCT GCC TGC CAT ATT
TCA ATT TGT TAT AAA TTC CTA TAA TTT ATC CTA TTA GTA GCT AAA AAA AGA
TGA ATG TGA ATC GAA TCC TAA GAG AAT TGA GCT CCA ATT CGC CCT ATA GTG
AGT CGT ATT A-3' (SEQ ID NO:61). The delivered PCR product was
amplified via PCR using the Splice4L and T7 primers, and yeast
libraries were generated by homologous recombination in EBY100
yeast by electroporation of amplified PCR products along with NheI
and XhoI digested yeast display vector pCT302 (Benatuil et al.
(2010) Protein Eng Des Sel, 23, 155-9; Colby et al. (2004) Methods
Enzymol, 388, 348-58; Starwalt et al. (2003) Protein Eng, 16,
147-56; Swers et al. (2004) Nucleic Acids Res, 32, e36). The
resultant library size was 2.4.times.10.sup.8.
[0294] The RD2 library was induced in galactose-containing media
(SG-CAA) for 48 hours, washed with 1 mL 1% PBS/BSA, and stained
with 5 .mu.m Tax (SEQ ID NO:5) or MART1 (SEQ ID NO:7)
peptide/HLA.A2 UV-exchanged HLA.A2 monomers (Rodenko et al. (2006)
Nat Protoc, 1, 1120-32; Toebes et al. (2006) Nat Med, 12, 246-51).
Magnetic bead selections were performed utilizing streptavidin MACS
microbeads (Miltenyl Biotec), for a total of two selections using
MACS LS columns on a QuadroMACS.TM. Separator (Miltenyl Biotec).
Following two selections, the selected libraries was stained with
the following: selecting peptide/HLA.A2 DimerX, goat-anti-mouse IgG
F(ab')2 AlexaFluor 647 secondary antibody (1:100). Cells were
washed (1 ml, 1% PBS/BSA), and the most fluorescent cells were
selected using a FAGS Aria (BD Bioscience) high-speed sorter.
Selections were performed with 1 nM and 100 nM peptide/HLA.A2 for
selecting cognate antigen Tax, and selecting, non-cognate antigen
MART1, respectively. Expression was monitored with anti-HA epitope
tag (1:50), goat-anti-mouse IgG F(ab')2 AlexaFluor 647 secondary
antibody (1:100), and anti-cmyc (1:50), goat-anti-chicken IgG
F(ab')2 AlexaFluor 647 secondary antibody (1:100).
Isolation and Staining of High Affinity Clones
[0295] Following the fourth sort with Tax:HLA-A2 and the fifth sort
with Mart1:HLA-A2, single colonies were isolated by plating
limiting dilutions. Colonies were expanded and induced in
galactose-containing media (SG-CAA) for 48 h, washed with 1 mL 1%
PBS/BSA, and stained with the following: anti-HA epitope tag
(1:50), anti-V.alpha.2 antibody (1:50), and tax or Mart1
peptide:HLA-A2 DimerX (100 nM) along with goat-anti-mouse IgG
F(ab').sub.2 AlexaFluor 647 secondary antibody (1:100), and
anti-cmyc (1:50) along with goat-anti-chicken IgG F(ab').sub.2
AlexaFluor 647 secondary antibody (1:100). Cells were washed (1 ml,
1% PBS/BSA) and analyzed on an Accuri C6 flow cytometer.
[0296] Plasmids were recovered using Zymoprep.TM. Yeast Plasmid
Miniprep II (Zymo Research) and introduced back into E. coli via
heat shock transformation into Subcloning Efficiency.TM.
DH5.alpha..TM. Competent Cells (Invitrogen). E. coli cells were
expanded and plasmids were isolated using QIAprep Spin Miniprep Kit
(Qiagen). Sequences of individual clones were determined by Sanger
sequencing.
STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS
[0297] All references cited herein, for example patent documents
including issued or granted patents or equivalents; patent
application publications; and non-patent literature documents or
other source material; are hereby incorporated by reference herein
in their entireties, as though individually incorporated by
reference, to the extent each reference is at least partially not
inconsistent with the disclosure in this application (for example,
a reference that is partially inconsistent is incorporated by
reference except for the partially inconsistent portion of the
reference).
[0298] All patents and publications mentioned in the specification
are indicative of the levels of skill of those skilled in the art
to which the disclosure pertains. References cited herein are
incorporated by reference herein in their entirety to indicate the
state of the art, in some cases as of their filing date, and it is
intended that this information can be employed herein, if needed,
to exclude (for example, to disclaim) specific embodiments that are
in the prior art or to use methods or materials that are in the
state of the art without the specific inclusion of the methods or
materials in the disclosure herein. For example, when a compound is
claimed, it should be understood that compounds known in the prior
art, including certain compounds disclosed in the references
disclosed herein (particularly in referenced patent documents), are
not intended to be included in the claim.
[0299] When a Markush group or other grouping is used herein, all
individual members of the group and all combinations and
subcombinations possible of the group are intended to be
individually included in the disclosure.
[0300] Where the terms "comprise", "comprises", "comprised", or
"comprising" are used herein, they are to be interpreted as
specifying the presence of the stated features, integers, steps, or
components referred to, but not to preclude the presence or
addition of one or more other feature, integer, step, component, or
group thereof. Separate embodiments of the disclosure are also
intended to be encompassed wherein the terms "comprising" or
"comprise(s)" or "comprised" are optionally replaced with the
terms, analogous in grammar, e.g.; "consisting/consist(s)" or
"consisting essentially of/consist(s) essentially of" to thereby
describe further embodiments that are not necessarily coextensive.
For clarification, as used herein "comprising" is synonymous with
"having," "including," "containing," or "characterized by," and is
inclusive or open-ended and does not exclude additional, unrecited
elements or method steps. As used herein, "consisting of" excludes
any element, step, component, or ingredient not specified in the
claim element. As used herein, "consisting essentially of" does not
exclude materials or steps that do not materially affect the basic
and novel characteristics of the claim (e.g., not affecting an
active ingredient). In each instance herein any of the terms
"comprising", "consisting essentially of" and "consisting of" may
be replaced with either of the other two terms. The disclosure
illustratively described herein suitably may be practiced in the
absence of any element or elements, limitation or limitations which
is not specifically disclosed herein.
[0301] The disclosure has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the disclosure.
It will be appreciated by one of ordinary skill in the art that
compositions, methods, devices, device elements, materials,
optional features, procedures and techniques other than those
specifically described herein can be applied to the practice of the
disclosure as broadly disclosed herein without resort to undue
experimentation. All art-known functional equivalents of
compositions, methods, devices, device elements, materials,
procedures and techniques described herein; and portions thereof;
are intended to be encompassed by this disclosure. Whenever a range
is disclosed, all subranges and individual values are intended to
be encompassed. This disclosure is not to be limited by the
embodiments disclosed, including any shown in the drawings or
exemplified in the specification, which are given by way of example
or illustration and not of limitation. Some references provided
herein are incorporated by reference herein to provide details
concerning additional starting materials, additional methods of
synthesis, and additional methods of analysis and additional uses
of the disclosure.
[0302] One skilled in the art would readily appreciate that the
present disclosure is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The compositions and methods and accessory methods
described herein as presently representative of preferred
embodiments are exemplary and are not intended as limitations on
the scope of the disclosure. Changes therein and other uses will
occur to those skilled in the art, which are encompassed within the
spirit of the disclosure.
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Turner D. J., Ritter M. A. and George A. J. (1997) Importance of
the linker in expression of single-chain Fv antibody fragments:
optimisation of peptide sequence using phage display technology. J
Immunol Methods 205, 43-54. [0381] 79. Utz U., Banks D., Jacobson
S. and Biddison W. E. (1996) Analysis of the T-cell receptor
repertoire of human T-cell leukemia virus type 1 (HTLV-1)
Tax-specific CD8+ cytotoxic T lymphocytes from patients with
HTLV-1-associated disease: evidence for oligoclonal expansion. J
Virol 70, 843-51. [0382] 80. Varela-Rohena A., Molloy P. E., Dunn
S. M., Li Y., Suhoski M. M., Carroll R. G., Milicic A., Mahon T.,
Sutton D. H., Laugel B., Moysey R., Cameron B. J., Vuidepot A.,
Purbhoo M. A., Cole D. K., Phillips R. E., June C. H., Jakobsen B.
K., Sewell A. K. and Riley J. L. (2008) Control of HIV-1 immune
escape by CD8 T cells expressing enhanced T-cell receptor. Nat Med
14, 1390-5. [0383] 81. Weber K. S., Donermeyer D. L., Allen P. M.
and Kranz D. M. (2005) Class II-restricted T cell receptor
engineered in vitro for higher affinity retains peptide specificity
and function. Proc Natl Acad Sci USA 102, 19033-8. [0384] 82. Wong
R. L., Liu B., Zhu X., You L., Kong L., Han K. P., Lee H. I.,
Chavaillaz P. A., Jin M., Wang Y., Rhode P. R. and Wong H. C.
(2011) Interleukin-15:Interleukin-15 receptor alpha scaffold for
creation of multivalent targeted immune molecules. Protein Eng Des
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Schmidt H., Spiotto M. T., Schietinger A., Yu P., Fu Y. X.,
Weichselbaum R. R., Rowley D. A., Kranz D. M. and Schreiber H.
(2007) Induced sensitization of tumor stroma leads to eradication
of established cancer by T cells. J Exp Med 204, 49-55.
U.S. Patents
[0385] [0386] U.S. Pat. No. 7,569,357; Filed 20 Feb. 2004; Issued 4
Aug. 2009; Board of Trustees University of Illinois. High affinity
TCR proteins and methods. [0387] U.S. Pat. No. 7,465,787; Filed 16
Dec. 2003; Issued 16 Dec. 2008; Board of Trustees University of
Illinois. Yeast cell surface display of proteins and uses thereof.
[0388] U.S. Pat. No. 6,759,243; Filed 6 Dec. 2000; Issued 6 Jul.
2004; Board of Trustees University of Illinois. High affinity TCR
proteins and methods. [0389] U.S. Pat. No. 6,699,658; Filed 20 Jan.
1998; Issued 2 Mar. 2004; Board of Trustees University of Illinois.
Yeast cell surface display of proteins and uses thereof. [0390]
U.S. Pat. No. 6,696,251; Filed 28 Nov. 2000; Issued 24 Feb. 2004;
Board of Trustees University of Illinois. Yeast cell surface
display of proteins and uses thereof. [0391] U.S. Pat. No.
6,423,538; Filed 28 Nov. 2000; Issued 23 Jul. 2002; Board of
Trustees University of Illinois. Yeast cell surface display of
proteins and uses thereof. [0392] U.S. Pat. No. 6,300,065; Filed 26
Aug. 1998; Issued 9 Oct. 2001; Board of Trustees University of
Illinois. Yeast cell surface display of proteins and uses thereof.
[0393] U.S. Pat. No. 8,143,376; Filed 18 May 2005; Issued 27 Mar.
2012; Immunocore Limited; High affinity NY-ESO T cell receptor.
[0394] U.S. Pat. No. 8,088,379; Filed 26 Sep. 2007; Issued 3 Jan.
2012; Immunocore Limited; Modified T cell receptors and related
materials and methods. [0395] U.S. Pat. No. 8,017,730; Filed 19 May
2006; Issued 13 Sep. 2011; Immunocore Limited; T cell receptors
which bind to antigen-HLA-A24. [0396] U.S. Pat. No. 7,763,718;
Filed 29 Oct. 2007; Issued 27 Jul. 2010; Immunocore Limited;
Soluble T cell receptors. [0397] U.S. Pat. No. 7,666,604; Filed 9
Jul. 2003; Issued 23 Feb. 2010; Immunocore Limited; Modified
soluble T cell receptor. [0398] U.S. Pat. No. 7,608,410; Filed 7
Oct. 2008; Issued 27 Oct. 2009; Immunocore Limited; Method of
improving T cell receptors. [0399] U.S. Pat. No. 7,569,664; Filed 3
Oct. 2003; Issued 4 Aug. 2009; Immunocore Limited; Single chain
recombinant T cell receptors. [0400] U.S. Pat. No. 8,105,830; Filed
5 Nov. 2002; Issued 31 Jan. 2012; Altor Bioscience Corporation;
Polyspecific binding molecules and uses thereof. [0401] U.S. Pat.
No. 6,534,633; Filed 21 Oct. 1999; 18 Mar. 2003; Altor Bioscience
Corporation; Polyspecific binding molecules and uses thereof.
Sequence CWU 1
1
611113PRTHomo sapiens 1Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu
Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr
Ser Asp Arg Gly Ser Gln Ser 20 25 30 Phe Phe Trp Tyr Arg Gln Tyr
Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Ser Ile Tyr Ser Asn
Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 50 55 60 Leu Asn Lys
Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 65 70 75 80 Pro
Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser Trp 85 90
95 Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro Asp
100 105 110 Ile 2120PRTHomo sapiens 2Asn Ala Gly Val Thr Gln Thr
Pro Lys Phe Gln Val Leu Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu
Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met 20 25 30 Ser Trp Tyr
Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser
Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr 50 55
60 Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser
65 70 75 80 Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg
Pro Gly 85 90 95 Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro
Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu Asp Leu Lys Asn 115 120
3250PRTHomo sapiens 3Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln
Val Leu Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln
Asp Met Asn His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro
Gly Met Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly
Ile Thr Asp Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser
Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala
Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90
95 Leu Met Ser Ala Gln Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu
100 105 110 Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala
Lys Lys 115 120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Gln Lys Glu
Val Glu Gln Asn 130 135 140 Ser Gly Pro Leu Ser Val Pro Glu Gly Ala
Ile Ala Ser Leu Asn Cys 145 150 155 160 Thr Tyr Ser Asp Arg Gly Ser
Gln Ser Phe Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser Gly Lys Ser Pro
Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp 180 185 190 Lys Glu Asp
Gly Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr 195 200 205 Val
Ser Leu Leu Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr 210 215
220 Leu Cys Ala Val Thr Thr Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala
225 230 235 240 Gly Thr Gln Val Val Val Thr Pro Asp Ile 245 250
4250PRTArtificial SequenceSynthesized amino acid sequence of the
RD1 library 4Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu
Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met
Asn His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met
Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr
Asp Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser
Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro
Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Xaa
Met Ser Xaa Gln Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105
110 Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys
115 120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Gln Lys Glu Val Glu
Gln Asn 130 135 140 Ser Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala
Ser Leu Asn Cys 145 150 155 160 Thr Tyr Ser Asp Arg Gly Ser Xaa Ser
Phe Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser Gly Lys Ser Pro Glu Leu
Ile Met Ser Ile Tyr Ser Asn Gly Asp 180 185 190 Lys Glu Asp Gly Arg
Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr 195 200 205 Val Ser Leu
Leu Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr 210 215 220 Leu
Cys Ala Val Thr Xaa Xaa Ser Trp Gly Lys Leu Gln Phe Gly Ala 225 230
235 240 Gly Thr Gln Val Val Val Thr Pro Asp Ile 245 250 59PRTHuman
T cell lymphotrophic virus 5Leu Leu Phe Gly Tyr Pro Val Tyr Val 1 5
69PRTHomo sapiens 6Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5
710PRTHomo sapiens 7Glu Leu Ala Gly Ile Gly Ile Leu Thr Val 1 5 10
89PRTHuman immunodeficiency virus 8Ser Leu Tyr Asn Thr Val Ala Thr
Leu 1 5 99PRTHomo sapiens 9Arg Met Phe Pro Asn Ala Pro Tyr Leu 1 5
109PRTHomo sapiens 10Leu Thr Leu Gly Glu Phe Leu Lys Leu 1 5
119PRTHomo sapiens 11Ser Leu Leu Met Trp Ile Thr Gln Cys 1 5
129PRTHomo sapiens 12Ala Leu Trp Gly Pro Asp Ala Ala Ala 1 5
138PRTHomo sapiens 13Val Leu Phe Tyr Leu Gly Gln Tyr 1 5
149PRTHepatitis B virus 14Phe Leu Leu Thr Arg Ile Leu Thr Ile 1 5
159PRTHomo sapiens 15Lys Thr Trp Gly Gln Tyr Trp Gln Val 1 5
169PRTHomo sapiens 16Ser Thr Ala Pro Pro Val His Asn Val 1 5
179PRTHomo sapiens 17Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5
189PRTHomo sapiens 18Lys Ile Phe Gly Ser Leu Ala Phe Leu 1 5
199PRTHomo sapiens 19Leu Glu Glu Lys Lys Gly Asn Tyr Val 1 5
209PRTHomo sapiens 20Tyr Leu Ser Gly Ala Asn Leu Asn Leu 1 5
2117PRTArtificial SequenceLinker sequence 21Gly Ser Ala Asp Asp Ala
Lys Lys Asp Ala Ala Lys Lys Asp Gly Lys 1 5 10 15 Ser
22250PRTArtificial SequenceAmino acid sequence of clone from RD1
library 22Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys
Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn
His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly
Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp
Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr
Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser
Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Ile Met
Ser Glu Gln Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110
Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115
120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Gln Lys Glu Val Glu Gln
Asn 130 135 140 Ser Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser
Leu Asn Cys 145 150 155 160 Thr Tyr Ser Asp Arg Gly Ser Ser Ser Phe
Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser Gly Lys Ser Pro Glu Leu Ile
Met Ser Ile Tyr Ser Asn Gly Asp 180 185 190 Lys Glu Asp Gly Arg Phe
Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr 195 200 205 Val Ser Leu Leu
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr 210 215 220 Leu Cys
Ala Val Thr Pro Pro Ser Trp Gly Lys Leu Gln Phe Gly Ala 225 230 235
240 Gly Thr Gln Val Val Val Thr Pro Asp Ile 245 250
23250PRTArtificial SequenceAmino acid sequence of clone from RD1
library 23Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys
Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn
His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly
Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp
Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr
Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser
Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Arg Met
Ser Met Gln Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110
Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115
120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Gln Lys Glu Val Glu Gln
Asn 130 135 140 Ser Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser
Leu Asn Cys 145 150 155 160 Thr Tyr Ser Asp Arg Gly Ser Arg Ser Phe
Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser Gly Lys Ser Pro Glu Leu Ile
Met Ser Ile Tyr Ser Asn Gly Asp 180 185 190 Lys Glu Asp Gly Arg Phe
Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr 195 200 205 Val Ser Leu Leu
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr 210 215 220 Leu Cys
Ala Val Thr Pro Cys Ser Trp Gly Lys Leu Gln Phe Gly Ala 225 230 235
240 Gly Thr Gln Val Val Val Thr Pro Asp Ile 245 250
24250PRTArtificial SequenceAmino acid sequence of clone from RD1
library 24Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys
Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn
His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly
Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp
Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr
Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser
Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Ser Met
Ser Ala Gln Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110
Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115
120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Gln Lys Glu Val Glu Gln
Asn 130 135 140 Ser Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser
Leu Asn Cys 145 150 155 160 Thr Tyr Ser Asp Arg Gly Ser Ala Ser Phe
Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser Gly Lys Ser Pro Glu Leu Ile
Met Ser Ile Tyr Ser Asn Gly Asp 180 185 190 Lys Glu Asp Gly Arg Phe
Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr 195 200 205 Val Ser Leu Leu
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr 210 215 220 Leu Cys
Ala Val Thr Ile Val Ser Trp Gly Lys Leu Gln Phe Gly Ala 225 230 235
240 Gly Thr Gln Val Val Val Thr Pro Asp Ile 245 250
25250PRTArtificial SequenceAmino acid sequence of clone from RD1
library 25Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys
Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn
His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly
Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp
Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr
Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser
Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Thr Met
Ser Arg Gln Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110
Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115
120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Gln Lys Glu Val Glu Gln
Asn 130 135 140 Ser Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser
Leu Asn Cys 145 150 155 160 Thr Tyr Ser Asp Arg Gly Ser Gly Ser Phe
Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser Gly Lys Ser Pro Glu Leu Ile
Met Ser Ile Tyr Ser Asn Gly Asp 180 185 190 Lys Glu Asp Gly Arg Phe
Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr 195 200 205 Val Ser Leu Leu
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr 210 215 220 Leu Cys
Ala Val Thr Ser Leu Ser Trp Gly Lys Leu Gln Phe Gly Ala 225 230 235
240 Gly Thr Gln Val Val Val Thr Pro Asp Ile 245 250
26250PRTArtificial SequenceAmino acid sequence of clone from RD1
library 26Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys
Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn
His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly
Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp
Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr
Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser
Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Ser Met
Ser His Gln Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110
Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115
120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Gln Lys Glu Val Glu Gln
Asn 130 135 140 Ser Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser
Leu Asn Cys 145 150 155 160 Thr Tyr Ser Asp Arg Gly Ser Phe Ser Phe
Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser Gly Lys Ser Pro Glu Leu Ile
Met Ser Ile Tyr Ser Asn Gly Asp 180 185 190 Lys Glu Asp Gly Arg Phe
Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr 195 200 205 Val Ser Leu Leu
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr 210 215 220 Leu Cys
Ala Val Thr Leu His Ser Trp Gly Lys Leu Gln Phe Gly Ala 225 230 235
240 Gly Thr Gln Val Val Val Thr Pro Asp Ile 245 250
27249PRTArtificial SequenceAmino acid sequence of clone from RD1
library 27Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys
Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn
His Glu Tyr Met
20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile
His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp Gln Gly Asp Val
Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr Thr Glu Asp Phe
Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser Gln Thr Ser Val
Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Ala Met Ser Gln Pro Glu
Leu Tyr Phe Gly Pro Gly Thr Arg Leu Thr 100 105 110 Val Thr Glu Asp
Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys Asp 115 120 125 Ala Ala
Lys Lys Asp Gly Lys Ser Gln Lys Glu Val Glu Gln Asn Ser 130 135 140
Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr 145
150 155 160 Tyr Ser Asp Arg Gly Ser Tyr Ser Phe Phe Trp Tyr Arg Gln
Tyr Ser 165 170 175 Gly Lys Ser Pro Glu Leu Ile Met Ser Ile Tyr Ser
Asn Gly Asp Lys 180 185 190 Glu Asp Gly Arg Phe Thr Ala Gln Leu Asn
Lys Ala Ser Gln Tyr Val 195 200 205 Ser Leu Leu Ile Arg Asp Ser Gln
Pro Ser Asp Ser Ala Thr Tyr Leu 210 215 220 Cys Ala Val Thr Asn Phe
Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly 225 230 235 240 Thr Gln Val
Val Val Thr Pro Asp Ile 245 28250PRTArtificial SequenceAmino acid
sequence of clone from RD1 library 28Asn Ala Gly Val Thr Gln Thr
Pro Lys Phe Gln Val Leu Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu
Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met 20 25 30 Ala Trp Tyr
Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser
Val Gly Val Gly Ile Thr Asp Gln Gly Asp Val Pro Asp Gly Tyr 50 55
60 Lys Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser
65 70 75 80 Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg
Pro Gly 85 90 95 Ser Met Ser Arg Gln Pro Glu Leu Tyr Phe Gly Pro
Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu Asp Leu Ile Asn Gly Ser
Ala Asp Asp Ala Lys Lys 115 120 125 Asp Ala Ala Lys Lys Asp Gly Lys
Ser Gln Lys Glu Val Glu Gln Asn 130 135 140 Ser Gly Pro Leu Ser Val
Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys 145 150 155 160 Thr Tyr Ser
Asp Arg Gly Ser Ala Ser Phe Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser
Gly Lys Ser Pro Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp 180 185
190 Lys Glu Asp Gly Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr
195 200 205 Val Ser Leu Leu Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala
Thr Tyr 210 215 220 Leu Cys Ala Val Thr Arg Thr Ser Trp Gly Lys Leu
Gln Phe Gly Ala 225 230 235 240 Gly Thr Gln Val Val Val Thr Pro Asp
Ile 245 250 29250PRTArtificial SequenceAmino acid sequence of clone
from RD1 library 29Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val
Leu Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp
Met Asn His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly
Met Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile
Thr Asp Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg
Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala
Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95
Arg Met Ser Gln Gln Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100
105 110 Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys
Lys 115 120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Gln Lys Glu Val
Glu Gln Asn 130 135 140 Ser Gly Pro Leu Ser Val Pro Glu Gly Ala Ile
Ala Ser Leu Asn Cys 145 150 155 160 Thr Tyr Ser Asp Arg Gly Ser Trp
Ser Phe Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser Gly Lys Ser Pro Glu
Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp 180 185 190 Lys Glu Asp Gly
Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr 195 200 205 Val Ser
Leu Leu Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr 210 215 220
Leu Cys Ala Val Thr Ser Cys Ser Trp Gly Lys Leu Gln Phe Gly Ala 225
230 235 240 Gly Thr Gln Val Val Val Thr Pro Asp Ile 245 250
30250PRTArtificial SequenceAmino acid sequence of clone from RD1
library 30Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys
Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn
His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly
Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp
Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr
Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser
Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Tyr Met
Ser Pro Gln Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110
Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115
120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Gln Lys Glu Val Glu Gln
Asn 130 135 140 Ser Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser
Leu Asn Cys 145 150 155 160 Thr Tyr Ser Asp Arg Gly Ser Asn Ser Phe
Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser Gly Lys Ser Pro Glu Leu Ile
Met Ser Ile Tyr Ser Asn Gly Asp 180 185 190 Lys Glu Asp Gly Arg Phe
Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr 195 200 205 Val Ser Leu Leu
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr 210 215 220 Leu Cys
Ala Val Thr Phe Leu Ser Trp Gly Lys Leu Gln Phe Gly Ala 225 230 235
240 Gly Thr Gln Val Val Val Thr Pro Asp Ile 245 250
31250PRTArtificial SequenceAmino acid sequence of clone from RD1
library 31Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys
Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn
His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly
Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp
Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr
Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser
Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Leu Met
Ser Leu Gln Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110
Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115
120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Gln Lys Glu Val Glu Gln
Asn 130 135 140 Ser Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser
Leu Asn Cys 145 150 155 160 Thr Tyr Ser Asp Arg Gly Ser Gln Ser Phe
Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser Gly Lys Ser Pro Glu Leu Ile
Met Ser Ile Tyr Ser Asn Gly Asp 180 185 190 Lys Glu Asp Gly Arg Phe
Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr 195 200 205 Val Ser Leu Leu
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr 210 215 220 Leu Cys
Ala Val Thr Arg Ala Ser Trp Gly Lys Leu Gln Phe Gly Ala 225 230 235
240 Gly Thr Gln Val Val Val Thr Pro Asp Ile 245 250
32250PRTArtificial SequenceRD1-Tax-S4-1 clone sequence 32Asn Ala
Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly 1 5 10 15
Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met 20
25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His
Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp Gln Gly Asp Val Pro
Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr Thr Glu Asp Phe Pro
Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser Gln Thr Ser Val Tyr
Phe Cys Ala Ser Arg Pro Gly 85 90 95 Leu Met Ser Ala Gln Pro Glu
Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu Asp
Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115 120 125 Asp Ala Ala
Lys Lys Asp Gly Lys Ser Gln Lys Glu Val Glu Gln Asn 130 135 140 Ser
Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys 145 150
155 160 Thr Tyr Ser Asp Arg Gly Ser Thr Ser Phe Phe Trp Tyr Arg Gln
Tyr 165 170 175 Ser Gly Lys Ser Pro Glu Leu Ile Met Ser Ile Tyr Ser
Asn Gly Asp 180 185 190 Lys Glu Asp Gly Arg Phe Thr Ala Gln Leu Asn
Lys Ala Ser Gln Tyr 195 200 205 Val Ser Leu Leu Ile Arg Asp Ser Gln
Pro Ser Asp Ser Ala Thr Tyr 210 215 220 Leu Cys Ala Val Thr Thr Asp
Ser Trp Gly Lys Leu Gln Phe Gly Ala 225 230 235 240 Gly Thr Gln Val
Val Val Thr Pro Asp Ile 245 250 33250PRTArtificial
SequenceRD1-Mart1-S5-5 clone sequence 33Asn Ala Gly Val Thr Gln Thr
Pro Lys Phe Gln Val Leu Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu
Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met 20 25 30 Ala Trp Tyr
Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser
Val Gly Val Gly Ile Thr Asp Gln Gly Asp Val Pro Asp Gly Tyr 50 55
60 Lys Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser
65 70 75 80 Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg
Pro Gly 85 90 95 Trp Met Ser Gly Gln Pro Glu Leu Tyr Phe Gly Pro
Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu Asp Leu Ile Asn Gly Ser
Ala Asp Asp Ala Lys Lys 115 120 125 Asp Ala Ala Lys Lys Asp Gly Lys
Ser Gln Lys Glu Val Glu Gln Asn 130 135 140 Ser Gly Pro Leu Ser Val
Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys 145 150 155 160 Thr Tyr Ser
Asp Arg Gly Ser Thr Ser Phe Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser
Gly Lys Ser Pro Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp 180 185
190 Lys Glu Asp Gly Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr
195 200 205 Val Ser Leu Leu Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala
Thr Tyr 210 215 220 Leu Cys Ala Val Thr Lys Tyr Ser Trp Gly Lys Leu
Gln Phe Gly Ala 225 230 235 240 Gly Thr Gln Val Val Val Thr Pro Asp
Ile 245 250 34250PRTArtificial SequenceRD1-Mart1-High clone
sequence 34Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys
Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn
His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly
Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp
Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr
Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser
Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Trp Met
Ala Gly Gly Val Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110
Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115
120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Gln Lys Glu Val Glu Gln
Asn 130 135 140 Ser Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser
Leu Asn Cys 145 150 155 160 Thr Tyr Ser Asp Arg Gly Ser Thr Ser Phe
Phe Trp Tyr Arg Gln Tyr 165 170 175 Ser Gly Lys Ser Pro Glu Leu Ile
Met Ser Ile Tyr Ser Asn Gly Asp 180 185 190 Lys Glu Asp Gly Arg Phe
Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr 195 200 205 Val Ser Leu Leu
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr 210 215 220 Leu Cys
Ala Val Thr Lys Tyr Ser Trp Gly Lys Leu Gln Phe Gly Ala 225 230 235
240 Gly Thr Gln Val Val Val Thr Pro Asp Ile 245 250
35249PRTArtificial SequenceDegenerate amino acid sequence for RD2
library 35Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys
Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn
His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly
Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp
Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr
Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser
Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Xaa Xaa
Xaa Xaa Xaa Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110
Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115
120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Lys Glu Val Glu Gln Asn
Ser 130 135 140 Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser Leu
Asn Cys Thr 145 150 155 160 Tyr Ser Xaa Arg Xaa Ser Xaa Ser Phe Phe
Trp Tyr Arg Gln Tyr Ser 165 170 175 Gly Lys Ser Pro Glu Leu Ile Met
Ser Ile Tyr Ser Asn Gly Asp Lys 180 185 190 Glu Asp Gly Arg Phe Thr
Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val 195 200 205 Ser Leu Leu Ile
Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu 210 215 220 Cys Ala
Val Thr Thr Asp Xaa Xaa Gly Lys Leu Gln Phe Gly Ala Gly 225 230 235
240 Thr Gln Val Val Val Thr Pro Asp Ile 245 36249PRTArtificial
SequenceAmino acid
sequence of clone from RD2 library 36Asn Ala Gly Val Thr Gln Thr
Pro Lys Phe Gln Val Leu Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu
Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met 20 25 30 Ala Trp Tyr
Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser
Val Gly Val Gly Ile Thr Asp Gln Gly Asp Val Pro Asp Gly Tyr 50 55
60 Lys Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser
65 70 75 80 Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg
Pro Gly 85 90 95 Leu Met Ser Ala Gln Pro Glu Leu Tyr Phe Gly Pro
Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu Asp Leu Ile Asn Gly Ser
Ala Asp Asp Ala Lys Lys 115 120 125 Asp Ala Ala Lys Lys Asp Gly Lys
Ser Lys Glu Val Glu Gln Asn Ser 130 135 140 Gly Pro Leu Ser Val Pro
Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr 145 150 155 160 Tyr Ser Cys
Arg Met Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser 165 170 175 Gly
Lys Ser Pro Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp Lys 180 185
190 Glu Asp Gly Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val
195 200 205 Ser Leu Leu Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr
Tyr Leu 210 215 220 Cys Ala Val Thr Thr Asp Tyr Ser Gly Lys Leu Gln
Phe Gly Ala Gly 225 230 235 240 Thr Gln Val Val Val Thr Pro Asp Ile
245 37249PRTArtificial SequenceAmino acid sequence of clone from
RD2 library 37Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu
Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met
Asn His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met
Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr
Asp Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser
Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro
Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Asp
Ala Gly Gly Arg Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105
110 Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys
115 120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Lys Glu Val Glu Gln
Asn Ser 130 135 140 Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser
Leu Asn Cys Thr 145 150 155 160 Tyr Ser Pro Arg Arg Ser Thr Ser Phe
Phe Trp Tyr Arg Gln Tyr Ser 165 170 175 Gly Lys Ser Pro Glu Leu Ile
Met Ser Ile Tyr Ser Asn Gly Asp Lys 180 185 190 Glu Asp Gly Arg Phe
Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val 195 200 205 Ser Leu Leu
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu 210 215 220 Cys
Ala Val Thr Thr Asp Thr Asn Gly Lys Leu Gln Phe Gly Ala Gly 225 230
235 240 Thr Gln Val Val Val Thr Pro Asp Ile 245 38249PRTArtificial
SequenceAmino acid sequence of clone from RD2 library 38Asn Ala Gly
Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly 1 5 10 15 Gln
Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met 20 25
30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr
35 40 45 Ser Val Gly Val Gly Ile Thr Asp Gln Gly Asp Val Pro Asp
Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu
Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe
Cys Ala Ser Arg Pro Gly 85 90 95 Cys Met Ser Ala Gln Pro Glu Leu
Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu Asp Leu
Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115 120 125 Asp Ala Ala Lys
Lys Asp Gly Lys Ser Lys Glu Val Glu Gln Asn Ser 130 135 140 Gly Pro
Leu Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr 145 150 155
160 Tyr Ser Cys Arg Phe Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser
165 170 175 Gly Lys Ser Pro Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly
Asp Lys 180 185 190 Glu Asp Gly Arg Phe Thr Ala Gln Leu Asn Lys Ala
Ser Gln Tyr Val 195 200 205 Ser Leu Leu Ile Arg Asp Ser Gln Pro Ser
Asp Ser Ala Thr Tyr Leu 210 215 220 Cys Ala Val Thr Thr Asp Glu Val
Gly Lys Leu Gln Phe Gly Ala Gly 225 230 235 240 Thr Gln Val Val Val
Thr Pro Asp Ile 245 39249PRTArtificial SequenceAmino acid sequence
of clone from RD2 library 39Asn Ala Gly Val Thr Gln Thr Pro Lys Phe
Gln Val Leu Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala
Gln Asp Met Asn His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp
Pro Gly Met Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val
Gly Ile Thr Asp Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val
Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80
Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85
90 95 Leu Met Ser Ala Gln Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg
Leu 100 105 110 Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp
Ala Lys Lys 115 120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Lys Glu
Val Glu Gln Asn Ser 130 135 140 Gly Pro Leu Ser Val Pro Glu Gly Ala
Ile Ala Ser Leu Asn Cys Thr 145 150 155 160 Tyr Ser Thr Arg Tyr Ser
Thr Ser Phe Phe Trp Tyr Arg Gln Tyr Ser 165 170 175 Gly Lys Ser Pro
Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp Lys 180 185 190 Glu Asp
Gly Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val 195 200 205
Ser Leu Leu Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu 210
215 220 Cys Ala Val Thr Thr Asp Pro Leu Gly Lys Leu Gln Phe Gly Ala
Gly 225 230 235 240 Thr Gln Val Val Val Thr Pro Asp Ile 245
40249PRTArtificial SequenceAmino acid sequence of clone from RD2
library 40Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys
Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn
His Glu Tyr Met 20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly
Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp
Gln Gly Asp Val Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr
Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser
Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Arg Ala
Gly Gly Arg Pro Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110
Thr Val Thr Glu Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115
120 125 Asp Ala Ala Lys Lys Asp Gly Lys Ser Lys Glu Val Glu Gln Asn
Ser 130 135 140 Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser Leu
Asn Cys Thr 145 150 155 160 Tyr Ser Asn Arg Ser Ser Gln Ser Phe Phe
Trp Tyr Arg Gln Tyr Ser 165 170 175 Gly Lys Ser Pro Glu Leu Ile Met
Ser Ile Tyr Ser Asn Gly Asp Lys 180 185 190 Glu Asp Gly Arg Phe Thr
Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val 195 200 205 Ser Leu Leu Ile
Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu 210 215 220 Cys Ala
Val Thr Thr Asp Asn His Gly Lys Leu Gln Phe Gly Ala Gly 225 230 235
240 Thr Gln Val Val Val Thr Pro Asp Ile 245 41249PRTArtificial
SequenceRD2-Mart1-S3-3 clone sequence 41Asn Ala Gly Val Thr Gln Thr
Pro Lys Phe Gln Val Leu Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu
Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met 20 25 30 Ala Trp Tyr
Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser
Val Gly Val Gly Ile Thr Asp Gln Gly Asp Val Pro Asp Gly Tyr 50 55
60 Lys Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser
65 70 75 80 Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg
Pro Gly 85 90 95 Met Ala Gly Gly Arg Pro Glu Leu Tyr Phe Gly Pro
Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu Asp Leu Ile Asn Gly Ser
Ala Asp Asp Ala Lys Lys 115 120 125 Asp Ala Ala Lys Lys Asp Gly Lys
Ser Lys Glu Val Glu Gln Asn Ser 130 135 140 Gly Pro Leu Ser Val Pro
Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr 145 150 155 160 Tyr Ser Ser
Arg His Ser Thr Ser Phe Ser Trp Tyr Arg Gln Tyr Pro 165 170 175 Gly
Lys Ser Pro Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp Lys 180 185
190 Glu Asp Gly Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val
195 200 205 Ser Leu Leu Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr
Tyr Leu 210 215 220 Cys Ala Val Thr Thr Asp Arg Ser Gly Lys Leu Gln
Phe Gly Ala Gly 225 230 235 240 Thr Gln Val Val Val Thr Pro Asp Ile
245 42249PRTArtificial SequenceRD2-Mart1-S3-4 clone sequence 42Asn
Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly 1 5 10
15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met
20 25 30 Ala Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile
His Tyr 35 40 45 Ser Val Gly Val Gly Ile Thr Asp Gln Gly Asp Val
Pro Asp Gly Tyr 50 55 60 Lys Val Ser Arg Ser Thr Thr Glu Asp Phe
Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser Gln Thr Ser Val
Tyr Phe Cys Ala Ser Arg Pro Gly 85 90 95 Met Ala Gly Gly Arg Pro
Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu
Asp Leu Ile Asn Gly Ser Ala Asp Asp Ala Lys Lys 115 120 125 Asp Ala
Ala Lys Lys Asp Gly Lys Ser Lys Glu Val Glu Gln Asn Ser 130 135 140
Gly Pro Leu Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr 145
150 155 160 Tyr Ser Ser Arg His Ser Gln Ser Phe Ser Trp Tyr Arg Gln
Tyr Pro 165 170 175 Gly Lys Ser Pro Glu Leu Ile Met Ser Ile Tyr Ser
Asn Gly Asp Lys 180 185 190 Glu Asp Gly Arg Phe Thr Ala Gln Leu Asn
Lys Ala Ser Gln Tyr Val 195 200 205 Ser Leu Leu Ile Arg Asp Ser Gln
Pro Ser Asp Ser Ala Thr Tyr Leu 210 215 220 Cys Ala Val Thr Thr Asp
Leu Trp Gly Lys Leu Gln Phe Gly Ala Gly 225 230 235 240 Thr Gln Val
Val Val Thr Pro Asp Ile 245 43247PRTArtificial SequenceClone
T1-S18.45 amino acid sequence 43Glu Ala Gly Val Thr Gln Phe Pro Ser
His Ser Val Ile Glu Lys Gly 1 5 10 15 Gln Thr Val Thr Leu Arg Cys
Asp Pro Ile Ser Gly His Asp Asn Leu 20 25 30 Tyr Trp Tyr Arg Arg
Val Met Gly Lys Glu Ile Lys Phe Leu Leu His 35 40 45 Phe Val Lys
Glu Ser Lys Gln Asp Glu Ser Gly Met Pro Asn Asn Arg 50 55 60 Phe
Leu Ala Glu Arg Thr Gly Gly Thr Tyr Ser Thr Leu Lys Ile Gln 65 70
75 80 Pro Ala Glu Leu Glu Asp Ser Gly Val Tyr Phe Cys Ala Ser Ser
His 85 90 95 Ala Gly Leu Gly Val Glu Gln Tyr Phe Gly Pro Gly Thr
Arg Leu Thr 100 105 110 Val Thr Glu Asp Leu Lys Asn Gly Ser Ala Asp
Asp Ala Lys Glu Asp 115 120 125 Ala Ala Lys Lys Asp Gly Lys Ser Gln
Lys Glu Val Glu Gln Asn Ser 130 135 140 Gly Pro Leu Ser Val Pro Glu
Gly Ala Ile Ala Ser Leu Asn Cys Thr 145 150 155 160 Tyr Ser Asp Arg
Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Pro 165 170 175 Gly Lys
Ser Pro Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp Lys 180 185 190
Glu Asp Gly Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val 195
200 205 Ser Leu Leu Ile Arg Asp Ser Arg Pro Ser Asp Ser Ala Thr Tyr
Leu 210 215 220 Cys Ala Val Ser Ser Ser Asp Phe Leu Met Phe Gly Asp
Gly Thr Gln 225 230 235 240 Leu Val Val Lys Pro Asn Ile 245
444PRTHomo sapiens 44Ala Gly Gly Arg 1 454PRTHomo sapiens 45Met Ser
Ala Gln 1 469PRTHomo sapiens 46Ala Ser Asn Glu Asn Met Asp Ala Met
1 5 479PRTHomo sapiens 47Ala Ser Asn Glu Asn Met Glu Thr Met 1 5
489DNAArtificial Sequence5' region of RD1 gene optimized for yeast
and E. coli 48tctgctagc 94912DNAArtificial Sequence3' region of RD1
gene optimized for yeast and E.coli 49ctcgagatct ga
125085DNAArtificial SequenceForward primer used to add pCT302
overhangs 50caggctagtg gtggtggtgg ttctggtggt ggtggttctg gtggtggtgg
ttctgctagc 60aatgctggtg taacacaaac gccaa 855175DNAArtificial
SequenceReverse primer used to add pCT302 overhangs 51ggaacaaagt
cgattttgtt acatctacac tgttgttaac agatctcgag tcattataaa 60tcttcttcag
agatc 755224DNAArtificial SequenceForward primer to generate the
CDR3 1 library (Splice 4L) 52ggcagcccca taaacacaca gtat
245363DNAArtificial SequenceReverse primer to generate a CDR3 1
library (Splice 4L) 53cggacgggaa gcgcagaaat acactgaggt ttgagaaggt
gcagcgctta acagacgcag 60cgg 635478DNAArtificial Sequenceforward
primer to generate a CDR3 1 library (T7) 54acctcagtgt atttctgcgc
ttcccgtccg nnknnknnkn nknnkcagcc tgaactgtac 60tttggtccag gcactaga
785520DNAArtificial SequenceReverse primer to generate a CDR3 1
library (T7) 55taatacgact cactataggg 205663DNAArtificial
SequenceForward primer to generate a CDR3 2 library
56cggacgggaa gcgcagaaat acactgaggt ttgagaaggt gcagcgctta acagacgcag
60cgg 635785DNAArtificial SequencePrimer to generate a CDR3beta2
library 57acctcagtgt atttctgcgc ttcccgtccg ggttggnnkn nknnknnknn
kgaactgtac 60tttggtccag gcactagact gaccg 855871DNAArtificial
SequencePrimer to generate a CDR3alpha library 58cgtaaccgcg
cacaagtatg tggccgaatc ggaaggctgg gagtcacgaa tcagcaaact 60aacatactgg
c 715982DNAArtificial Sequenceprimer to generate a CDR3alpha
library 59tccgattcgg ccacatactt gtgcgcggtt acgnnknnkn nknnknnkaa
actgcaattt 60ggtgcgggca cccaggttgt gg 8260150DNAArtificial
SequenceFlanking N-terminal DNA from yeast codon optimized RD2 gene
60ggcagcccca taaacacaca gtatgttttt aaggacaata gctcgacgat tgaaggtaga
60tacccatacg acgttccaga ctacgctctg caggctagtg gtggtggtgg ttctggtggt
120ggtggttctg gtggtggtgg ttctgctagc 15061346DNAArtificial
SequenceFlanking C-terminal DNA from yeast codon optimized RD2 gene
61ctcgagatct gttaacaaca gtgtagatgt aacaaaatcg actttgttcc cactgtactt
60ttagctcgta caaaatacaa tatacttttc atttctccgt aaacaacatg ttttcccatg
120taatatcctt ttctattttt cgttccgtta ccaactttac acatacttta
tatagctatt 180cacttctata cactaaaaaa ctaagacaat tttaattttg
ctgcctgcca tatttcaatt 240tgttataaat tcctataatt tatcctatta
gtagctaaaa aaagatgaat gtgaatcgaa 300tcctaagaga attgagctcc
aattcgccct atagtgagtc gtatta 346
* * * * *