U.S. patent application number 16/893203 was filed with the patent office on 2020-12-10 for sorting with counter selection using sequence similar peptides.
The applicant listed for this patent is Immatics Biotechnologies GmbH, Immatics US, Inc.. Invention is credited to Amir ALPERT, Sebastian BUNK, Dominik MAURER, Gisela SCHIMMACK, Heiko SCHUSTER, Claudia WAGNER, Sara YOUSEF.
Application Number | 20200384028 16/893203 |
Document ID | / |
Family ID | 1000004931282 |
Filed Date | 2020-12-10 |
United States Patent
Application |
20200384028 |
Kind Code |
A1 |
BUNK; Sebastian ; et
al. |
December 10, 2020 |
SORTING WITH COUNTER SELECTION USING SEQUENCE SIMILAR PEPTIDES
Abstract
The present invention relates to a method for selecting a cell
or a virus expressing on its surface an antigen-binding protein
specifically binding to a protein antigen of interest (PAI) while
counter selection using a similar protein antigen (SPA) is applied.
Further, the invention provides a method for determining the
sequence of a nucleic acid encoding an antigen-binding protein or
an antigen-binding part thereof and a method for producing a cell
expressing a nucleic acid encoding an antigen-binding protein or an
antigen-binding part thereof. The invention also relates to a
method for treating a subject with a selected cell population.
Inventors: |
BUNK; Sebastian; (Tuebingen,
DE) ; MAURER; Dominik; (Moessingen, DE) ;
SCHIMMACK; Gisela; (Tuebingen, DE) ; SCHUSTER;
Heiko; (Tuebingen, DE) ; WAGNER; Claudia;
(Tuebingen, DE) ; YOUSEF; Sara; (Tuebingen,
DE) ; ALPERT; Amir; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immatics Biotechnologies GmbH
Immatics US, Inc. |
Tuebingen
Houston |
TX |
DE
US |
|
|
Family ID: |
1000004931282 |
Appl. No.: |
16/893203 |
Filed: |
June 4, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62858167 |
Jun 6, 2019 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/7051 20130101;
C07K 14/47 20130101; A61K 35/17 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; C07K 14/47 20060101 C07K014/47; C07K 14/725 20060101
C07K014/725 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 30, 2019 |
DE |
102019129341.3 |
Claims
1. A method for selecting a cell or a virus expressing on its
surface an antigen-binding protein specifically and/or selectively
binding to a protein antigen of interest (PAI) comprising the
following steps: (i) providing a cell population or a virus
population; (ii) contacting the cell population or the virus
population of step (i) with a first antigen complex (1.sup.st AC)
comprising the PAI and a detectable label A or with the PAI
comprising a detectable label A; (iii) contacting the cell
population or the virus population of step (i) with at least a
second antigen complex (2.sup.nd AC) comprising a similar protein
antigen (SPA), wherein the amino acid sequence of the SPA differs
by at least 1 amino acid from the amino acid sequence of the PAI
and wherein the 2.sup.nd AC comprises a detectable label B; or with
the SPA and a detectable label B; and (iv) selecting at least one
cell or virus that specifically and/or selectively binds to the
1.sup.st AC, wherein the detectable label A and the detectable
label B are detectably different from each other.
2. The method according to claim 1, wherein (i) the selected cell
is an immune cell, preferably a T-cell, preferably a CD4 or CD8
T-cell; or a B-cell; or a mammalian or yeast cell expressing a
heterologous antigen binding protein; or (ii) the selected virus is
a bacteriophage.
3. The method according to claim 1, wherein the antigen-binding
protein is selected from the group comprising a T-cell receptor
(TCR) or antigen binding fragments thereof, a B-cell receptor (BCR)
or antigen binding fragments thereof, and a chimeric antigen
receptor (CAR) or antigen binding fragments thereof.
4. The method according to claim 1, wherein (a) the cell population
comprises: (i) immune cells preferably tumor-infiltrating
lymphocytes (TILs), T cell receptor libraries, peripheral blood of
healthy subjects, peripheral blood of diseased subjects or an
immune cell enriched fraction thereof; or (ii) eukaryotic cells,
preferably mammalian cells or yeast cells expressing a library of
heterologous antigen binding proteins; or (b) the virus population
comprises viruses expressing a library of heterologous antigen
binding proteins.
5. The method according to claim 4, wherein the immune cell
enriched fraction is enriched in stem cells; T-cells, preferably
CD8 T-cells or CD4 T-cells; B-cells; plasma cell.
6. The method according to claim 1, wherein the protein antigen of
interest (PAI) is a tumor associated antigen (TAA), a viral protein
or a bacterial protein.
7. The method according to claim 4, wherein the diseased subject
suffers from a disease selected from the group consisting of immune
diseases, neoplastic diseases, a disease caused by a virus or a
disease caused by bacteria.
8. The method according to claim 4, wherein the immune cell
enriched fraction is selected by detectably labeling one or more
immune cell specific surface marker.
9. The method according to claim 1, comprising the further step of
incubating the cell population in the presence of growth and/or
differentiation factors, preferably selected from the group
consisting of cytokines.
10. The method according to claim 1, wherein the AC is an
antigen-presenting cell, or a complex comprising a particle, the
PAI and the detectable label A or the SPA and the detectable label
B.
11. The method according to claim 1, comprising one or more of the
following further steps: (a) contacting the cell population of step
(i) with a third antigen complex (3.sup.rd AC) comprising the PAI
and a detectable label C that is detectably different from one or
more or all of the other detectable labels of the other ACs
contacted with the cell population, preferably detectably different
from at least the detectable label A, preferably from at least the
detectable label A and a detectable label D, if a detectable label
D is present; and/or (b) contacting the cell population of step (i)
with a fourth antigen complex (4.sup.th AC) comprising the PAI and
a detectable label D that is detectably different from one or more
or all of the other detectable labels of the other ACs contacted
with the cell population, preferably detectably different from at
least the detectable label A and, preferably from at least the
detectable label A and the detectable label C; and/or (c)
contacting the cell population of step (i) with a fifth antigen
complex (5.sup.th AC) comprising the SPA and a detectable label E
that is detectably different from one or more or all of the other
detectable labels of the other ACs contacted with the cell
population, preferably detectably different from at least the
detectable label B and, preferably from at least the detectable
label B and a detectable label F, if a detectable label F is
present; and/or (d) contacting the cell population of step (i) with
a sixth antigen complex (6.sup.th AC) comprising the SPA and a
detectable label F that is detectably different from one or more or
all of the other detectable labels of the other ACs contacted with
the cell population, preferably detectably different from at least
the detectable label B and, preferably from at least the detectable
label B and the detectable label E; and/or (e) contacting the cell
population of step (i) with one or more further antigen complexes
(AC) wherein each comprises a SPA that differs in at least one
amino acid sequence from the amino acid sequence of the SPA of the
2.sup.nd AC, and wherein each further AC comprises one or more
labels, wherein the one or more label is the same to or detectably
different from the one or more labels of the 2.sup.nd AC.
12. The method according to claim 1, wherein (i) the 1.sup.st AC
comprises at least one further detectable label and the 2.sup.nd AC
comprises at least one further detectable label, which are either
the same or different; and/or (ii) the one or more further AC
comprises at least one further detectable label; wherein the at
least one further label is selected in such that it allows to
distinguish the 1.sup.st AC from the 2.sup.nd AC and the one or
more further ACs.
13. The method according to claim 1, wherein the detectable labels
are independently selected from a fluorescent label, preferably
selected from the group consisting of xanthens, acridines,
oxazines, cyanines, styryl dyes, coumarines, porphines,
metal-ligand-complexes, fluorescent proteins, nanocrystals,
perylenes and phtalocyanines.
14. The method according to claim 1, wherein the 1.sup.st AC is a
complex of a MHC-I or MHC-II and the PAI, and wherein the PAI is a
target peptide (TP), preferably a tumor-specific target peptide
and/or the 2.sup.nd AC is a complex of a MHC-I or MHC-II and the
SPA, and wherein the SPA is a target similar peptide (TSP) and
wherein the TSP differs by at least 1 amino acid from the amino
acid sequence of the TP.
15. The method according to claim 14, wherein the amino acid
sequence of the at least one TSP is selected by one or more of the
following criteria: (a) presentation of the TSP on healthy tissue;
(b) derived from HLA typed source; and (c) binding to the
respective HLA.
16. The method according to claim 14, wherein the amino acid
sequence of the TSP has a length of 8 to 16 amino acids and
wherein: (1) the amino acid sequence of the TSP differs from the
amino acid sequence of the TP
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8 (i)
at position X.sub.1, X.sub.2 and X.sub.3, and wherein position
X.sub.4 to X.sub.8 are identical or similar to the TP; (ii) at
position X.sub.4, X.sub.5 and X.sub.6, and wherein positions
X.sub.1 to X.sub.3 and X.sub.7 and X.sub.9 are identical or similar
to the TP; or (iii) at position X.sub.7 and X.sub.8, and wherein
position X.sub.1 to X.sub.6 are identical or similar to the TP; or
if the TP has a length of 8 amino acids; or (2) the amino acid
sequence of the TSP differs from the amino acid sequence of the TP
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9
(i) at position X.sub.1, X.sub.2 and X.sub.3, and wherein position
X.sub.4 to X.sub.9 are identical or similar to the TP; (ii) at
position X.sub.4, X.sub.5 and X.sub.6, and wherein position X.sub.1
to X.sub.3 and positions X.sub.7 to X.sub.9 are identical or
similar to the TP; or (iii) at position X.sub.4, X.sub.5, X.sub.6
and X.sub.7, and wherein position X.sub.1 to X.sub.3 and positions
X.sub.8 to X.sub.9 are identical or similar to the TP; or (iv) at
position X.sub.7 X.sub.8 and X.sub.9, and wherein position X.sub.1
to X.sub.6 are identical or similar to the TP; or if the TP has a
length of 8-9 amino acids; or (3) the amino acid sequence of the
TSP differs from the amino acid sequence of the TP
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10 (i) at position X.sub.1, X.sub.2 and X.sub.3, wherein
position X.sub.4 to X.sub.10 are identical or similar to the TP;
(ii) at position X.sub.4, X.sub.5, X.sub.6 and X.sub.7, wherein
position X.sub.1 to X.sub.3 and positions X.sub.8 to X.sub.10 are
identical or similar to the TP; or (iii) at position X.sub.4,
X.sub.5 and X.sub.6, and wherein position X.sub.1 to X.sub.3 and
positions X.sub.7 to X.sub.10 are identical or similar to the TP;
or (iv) at position X.sub.8, X.sub.9 and X.sub.10, wherein position
X.sub.1 to X.sub.7 are identical or similar to the TP; or if the TP
has a length of 8-10 amino acids; or (4) the amino acid sequence of
the TSP differs from the amino acid sequence of the TP
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10X.sub.11 (i) at position X.sub.1, X.sub.2 and X.sub.3,
wherein position X.sub.4 to X.sub.11 are identical or similar to
the TP; (ii) at position X.sub.4, X.sub.5, X.sub.6 and X.sub.7,
wherein position X.sub.1 to X.sub.3 and positions X.sub.8 to
X.sub.11 are identical or similar to the TP; or (iii) at position
X.sub.4, X.sub.5 and X.sub.6, and wherein position X.sub.1 to
X.sub.3 and positions X.sub.7 to X.sub.11 are identical or similar
to the TP; or (iv) at position X.sub.8, X.sub.9, X.sub.10 and
X.sub.11, wherein position X.sub.1 to X.sub.7 are identical or
similar to the TP; or (v) at position X.sub.9, X.sub.10 and
X.sub.11, wherein position X.sub.1 to X.sub.8 are identical or
similar to the TP; if the TP has a length of 8-11 amino acids; or
(5) the amino acid sequence of the TSP differs from the amino acid
sequence of the TP
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10X.sub.11 X.sub.12 (i) at position X.sub.1, X.sub.2 and
X.sub.3, wherein position X.sub.4 to X.sub.12 are identical or
similar to the TP; (ii) at position X.sub.4, X.sub.5, X.sub.6 and
X.sub.7, wherein position X.sub.1 to X.sub.3 and positions X.sub.8
to X.sub.12 are identical or similar to the TP; or (iii) at
position X.sub.4, X.sub.5 and X.sub.6, and wherein position X.sub.1
to X.sub.3 and positions X.sub.7 to X.sub.12 are identical or
similar to the TP; or (iv) at position X.sub.8, X.sub.9, X.sub.10,
X.sub.11 and X.sub.12, wherein position X.sub.1 to X.sub.7 are
identical or similar to the TP; or (v) at position X.sub.9,
X.sub.10, X.sub.11 and X.sub.12, wherein position X.sub.1 to
X.sub.8 are identical or similar to the TP; if the TP has a length
of 8-12 amino acids.
17.-18. (canceled)
19. The method according to claim 1, wherein the cell population of
step (i) is contacted with not more than 10 antigen complexes (AC)
each comprising a different similar protein antigen (SPA), not more
than nine different SPAs, not more than eight different SPAs, not
more than seven different SPAs, not more than six different SPAs,
not more than five different SPAs, not more than four different
SPAs, not more than three different SPAs, not more than two
different SPAs, or not more than one SPA, is used.
20. The method according to claim 19, wherein the SPA is a TSP.
21. The method according to claim 20, wherein the number of
different TSPs is between 1-10; between 2-8; between 3-5 or between
1-3, preferably three TSPs are used.
22. (canceled)
23. The method according to claim 1, wherein step (iv) comprises:
a) positively selecting (selecting) cells bound to the 1.sup.st AC,
1.sup.st and 3.sup.rd or 1.sup.st, 3.sup.rd and 4.sup.th AC; and/or
b) negatively selecting (excluding) cells bound to the 2.sup.nd AC,
the 2.sup.nd and 5.sup.th or the 2.sup.nd, 5.sup.th and 6.sup.th
AC.
24.-39. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/858,167, filed Jun. 6, 2019, and German
Application No. 10 2019 129 341.3, filed Oct. 30, 2019, the content
of each of these applications is herein incorporated by reference
in their entireties.
REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT
FILE (.txt)
[0002] Pursuant to the EFS-Web legal framework and 37 CFR
.sctn..sctn. 1.821-825 (see MPEP .sctn. 2442.03(a)), a Sequence
Listing in the form of an ASCII-compliant text file (entitled
"Sequence_Listing_3000058-017000_ST25.txt" created on 4 Jun. 2020,
and 31,154 bytes in size) is submitted concurrently with the
instant application, and the entire contents of the Sequence
Listing are incorporated herein by reference.
[0003] The present invention relates to a method for selecting a
cell or a virus expressing on its surface an antigen-binding
protein specifically binding to a protein antigen of interest (PAI)
while counter selection using a similar protein antigen (SPA) is
applied. Further, the invention provides a method for determining
the sequence of a nucleic acid encoding an antigen-binding protein
or an antigen-binding part thereof and a method for producing a
cell expressing a nucleic acid encoding an antigen-binding protein
or an antigen-binding part thereof. The invention also relates to a
method for treating a subject with a selected cell population.
BACKGROUND OF THE INVENTION
[0004] The field of adoptive cell transfer (ACT) has become one of
the most promising and innovative approaches to treat cancer, viral
infections and other immune-modulated disease. To support the
broader clinical application of T-cell receptor (TCR)-modified
T-cells, it is important that risks can be appropriately identified
and mitigated, preferably at the pre-clinical level. The toxicity
observed to date with the administration of TCR-modified T-cells is
similar to that observed during standard ACT and can be grossly
divided into three main groups: toxicity due to the lymph depleting
preparation regimen, cytokine-related toxicity and immune-related
toxicity. Immune-related toxicity can be classified into two
subcategories: so-called "off-tumor/on-target" effects and
"off-tumor/off-target" effects. The optimal gene-engineered T-cell
therapy target antigen is one that is only present on the tumor
cell and absent in healthy cells; however, in most cases the
selected tumor target antigens are over-expressed or aberrantly
expressed proteins that may be present to varying extent in normal
cells (Johnson L A et al., Gene therapy with human and mouse T-cell
receptors mediate cancer regression and targets normal tissues
expressing cognate antigen, Blood 2009; 114:535-46).
"Off-Tumor/On-Target" Toxicity
[0005] Gene-engineered T-cell therapies may, therefore, trigger a
potent cellular immune response against normal cells, even those
that express the target antigens at low levels. This type of
toxicity is known as "off-tumor/on-target" and is due to, for
example, the engineered T-cells being unable to distinguish between
normal cells and cancer cells that express the targeted antigen.
Targeting of Melan A (MLA; also referred to as "melanoma antigen
recognized by T-cells 1" (MART-1)) has been associated with
significant "off-tumor/on-target" side effects (Johnson L A et al.,
Gene therapy with and mouse T-cell receptors mediates cancer
regression and targets normal tissue expressing cognate antigens,
Blood 2009, 114:535-46; van den Berg J H et al., Case report of a
Fatal Serious Adverse event upon Administration of T-cells
transduced with a MART-1 specific T-cell Receptor, Mol. Ther. 2015;
23:1541-50). Specifically, a case report has been published
describing a fatal serious adverse event 3 days after transduced
T-cell administration with a MART-1 specific TCR to a patient with
metastatic melanoma. Infused T-cells were recovered from blood,
broncho-alveolar lavage, ascites, tumor sites and heart tissue, and
although no cross-reactivity of the modified T-cells toward a 3-D
beating cardiomyocyte culture was observed, the authors were not
able to exclude the possibility of cross-reactivity with an
allogeneic MHC-peptide complex. Additionally, multiple-organ
failure was found to be due to on-target cytokine release.
Off-tumor/on target toxicity can be avoided by selecting target
antigens that show a sufficiently low expression off-tumor to lead
to an acceptable toxicity upon application of doses that are
therapeutically effective on the tumor.
"Off-Tumor/Off-Target" Toxicity
[0006] Because most tumor antigens are derived from self-proteins
(tumor-associated antigens), the isolation of high-affinity
tumor-specific T-cells is effectively precluded by thymic
selection. TCR affinity can, nevertheless, be considerably enhanced
through mutation of specific regions within the
complementarity-determining regions (CDRs). Although useful to
promote modified T-cell efficacy, due to TCR degeneracy, this
approach carries the risk that a TCR might recognize other related
peptide antigens present on normal tissue through cross-reactivity.
Previously published results have shown lethal toxicities in two
patients, who were infused with T-cells engineered to express a TCR
targeting melanoma-associated antigen A3 (MAGE-A3) cross-reacting
with a peptide from the muscle protein Titin, even though no
cross-reactivities had been predicted in the pre-clinical studies
(Linette, G P et al., Cardiovascular toxicity and titin
cross-reactivity of affinity enhanced T-cells in myeloma and
melanoma, Blood 2013; 122:863-71; Cameron, B J et al.,
Identification of a Titin-derived HLA-A1-presented peptide as a
cross-reactive target for engineered MAGE-A3 directed T-cells, Sci.
Transl. Med. 2013; 5:197-103). These patients demonstrated that
TCR-engineered T-cells can have serious and not readily predictable
off-target and organ-specific toxicities and highlight the need for
improved methods to define the specificity of engineered TCRs.
Strategies such as peptide scanning and the use of more complex
cell structures are therefore recommended in pre-clinical studies
to mitigate the risk of off-target toxicities in future clinical
investigations. Therefore, there is still an unmet medical need to
develop and provide TCRs with low off-tumor/off-target toxicity.
The present invention provides methods to rapidly identify antigen
binding molecules, in particular TCRs that specifically and
selectively bind to their target antigens and, thus provide
enhanced safety profiles and reduced cross-reactivity to sequence
similar target antigens, in particular sequence similar peptides on
healthy tissues. The rapid, preferably one step, selection method
of the present invention is particularly useful in the
identification of patient-derived T-cells expressing TCRs with
desired anti-tumor activity.
SUMMARY OF THE INVENTION
[0007] A first aspect of the invention relates to a method for
selecting a cell or a virus expressing on its surface an
antigen-binding protein specifically and/or selectively binding to
a protein antigen of interest (PAI) comprising the following steps:
[0008] (i) providing a cell population comprising cells or a virus
population; [0009] (ii) contacting the cell population or the virus
population of step (i) with a first antigen complex (1.sup.st AC)
comprising the PAI and a detectable label A or with the PAI
comprising a detectable label A; [0010] (iii) contacting the cell
population or the virus population of step (i) with at least a
second antigen complex (2.sup.nd AC) comprising a similar protein
antigen (SPA), wherein the amino acid sequence of the SPA differs
by at least 1 amino acid from the amino acid sequence of the PAI
and wherein the 2.sup.nd AC comprises a detectable label B; or with
the SPA and a detectable label B; and [0011] (iv) selecting at
least one cell or virus that specifically and/or selectively binds
to the 1.sup.st AC, wherein the detectable label A and the
detectable label B are detectably different from each other.
[0012] A second aspect of the invention further relates to a method
for determining the sequence of a nucleic acid encoding an
antigen-binding protein or an antigen-binding part thereof
comprising the steps of: [0013] (i) isolating the nucleic acid
encoding the antigen-binding protein or the antigen-binding part
thereof from the cell selected in the method of the first aspect of
the invention; and [0014] (ii) determining the sequence of the
nucleic acid.
[0015] A third aspect of the invention relates to a method for
producing a cell expressing a nucleic acid encoding an
antigen-binding protein or an antigen-binding part thereof
comprising the steps of: [0016] (i) providing the nucleic acid
sequence encoding the antigen-binding protein or an antigen-binding
part thereof from the cell selected in the method of the first
aspect of the invention; [0017] (ii) producing a nucleic acid
vector comprising the nucleic acid sequence provided in step (i)
optionally under the control of an expression control element; and
[0018] (iii) introducing the nucleic acid vector of step (ii) into
a host cell.
[0019] A fourth aspect of the invention relates to a method for
treating a subject in need thereof comprising the steps of: [0020]
(i) providing a cell population of the subject comprising immune
cells; [0021] (ii) contacting the cell population of step (i) with
a first antigen complex (1.sup.st AC) comprising a PAI and a
detectable label A; [0022] (iii) contacting the cell population of
step (i) with at least a second antigen complex (2.sup.nd AC)
comprising a SPA, wherein the amino acid sequence of the SPA
differs by at least 1 amino acid from the amino acid sequence of
the PAI and wherein the 2.sup.nd AC comprises a detectable label B;
and [0023] (iv) selecting at least one cell that specifically binds
to the 1.sup.st AC, wherein the detectable label A and the
detectable label B are detectably different from each other [0024]
(v) increasing the number of the at least one selected cell
selected in step (iv) by cultivation; and [0025] (vi) reintroducing
the cultivated cells into the subject.
[0026] A fifth aspect of the invention relates to a method for
selecting an immune cell expressing on its surface an
antigen-binding protein specifically binding to a protein antigen
of interest (PAI) comprising the following steps: [0027] (i)
providing a cell population comprising immune cells; [0028] (ii)
contacting the cell population of step (i) with a first antigen
complex (1.sup.st AC) comprising the PAI and a detectable label A
or with the PAI comprising a detectable label A; [0029] (iii)
contacting the cell population of step (i) with at least a second
antigen complex (2.sup.nd AC) comprising an irrelevant protein
antigen (IPA), wherein the amino acid sequence of the IPA when
aligned with the amino acid sequence of the PAI is identical to the
PAI at two amino acids positions or less and wherein the IAC
comprises a detectable label G; or with the IPA and a detectable
label G; and [0030] (iv) selecting at least one cell that
specifically binds to the 1.sup.st AC, wherein the detectable label
A and the detectable label G are detectably different from each
other.
LIST OF FIGURES
[0031] In the following, the content of the Figures comprised in
this specification is described. In this context please also refer
to the detailed description of the invention above and/or
below.
[0032] FIG. 1 shows a schematic presentation of two exemplifying
applications of the invention. The upper pathway of the figure
represents the use of the gating strategy when applied to primed
T-cells that underwent an individual T cell culturing step, the
lower part of the figure shows the use of the gating strategy in a
direct sorting approach, wherein a heterogenous T cell population
obtained from a natural repertoire is enriched with target specific
T cells using the gating strategy. In both examples the positively
sorted cell fraction represents immune cells expressing on their
surface an antigen-binding protein specifically and/or selectively
binding to a protein antigen of interest. Abbreviations used in the
figure: SPA: similar protein antigen, PAI: protein antigen of
interest, APC: antigen-presenting cell.
[0033] FIG. 2 shows an exemplary gating strategy of non-amplified
target-specific T cells. To enhance the frequency of low-frequency
target-specific T cells in the test sample, the cells have been
enriched by fluorochrome-tetramer specific magnetic bead isolation.
Subsequently, cells were stained for surface markers and assessed
by flow cytometry. Individual 2D-color tetramer combinations were
used to stain target-specific and similar peptide-specific T cells.
In this example 1.65% of CD8 T cells bind to target-peptide
tetramer and of those target-specific CD8 T cells 29.4% also bind
to similar peptide-tetramer (Target.sup.+/SIM.sup.+), which is
comprised of 3 different similar peptide-HLAs. By including similar
peptide tetramers in the sorting procedure, a high proportion of
cross-reactive T cells (Target.sup.+/SIM.sup.+) can be
excluded.
[0034] FIG. 3 shows an exemplary gating strategy of primed T cell
populations. Individual T cell cultures were repeatedly stimulated
with target peptide HLA-coated artificial presenting cells to
enhance low-frequency target-specific CD8 T cells. After 4 weeks in
culture those primed T cell cultures were stained for surface
markers and individual 2D-color tetramer combinations for
target-HLA and 3 similar peptide-HLAs. The upper panel shows a
monoclonally enriched T cell population binding to both target- and
similar-peptide tetramers (Target.sup.+/SIM.sup.+). The lower panel
shows a monoclonally enriched T cell population binding only to the
target- but not similar-peptide tetramer. By including similar
peptide tetramers in the staining procedure cross-reactive T cells
(Target.sup.+/SIM.sup.+) can be excluded from sorting.
[0035] FIG. 4 shows that TCRs from T cells sorted using
target-peptide tetramers only, can be cross-reactive to
target-similar peptides. TCRs identified using target-peptide
tetramers were assessed for cross-reactivity against 10 target
similar peptides after mRNA electroporation into healthy donor T
cells. As measure for reactivity, IFN.gamma. secretion upon
co-culture with peptide-loaded T2 cells was assessed. All TCRs in
this example react against the target peptide (positive control)
and not against controls, which are unrelated/irrelevant peptide
loaded T2 cells, unloaded T2 cells or effector only cells. However,
the TCRs in FIG. 4A and FIG. 4B also show reactivity to similar
peptides, namely similar peptide 1 and 10 for TCR in FIG. 4A and
similar peptide 9 and 10 for the TCR in FIG. 4B. Only the TCR in
FIG. 4C shows no cross-reactivity and is therefore selected for
further characterization.
[0036] FIG. 5 shows the functional assessment of a TCR isolated
from T cells binding to target-peptide tetramers only (TCR
PAI+/SPA-), as well as a control TCR specific for a control peptide
("control peptide"), and a no TCR control ("no peptide"). For this
end, TCR-mRNA was electroporated into NFAT-luciferase Jurkat
reporter cells and their activation assessed after co-culture with
peptide/target similar peptides ("SIM 1, SIM 2, and SIM 3") loaded
T2 antigen-presenting cells. The TCR derived from PAI+/SPA- sorted
T cells triggers activation only when co-cultured with target
peptide-loaded T2 cells. The control TCRs shows reactivity in the
presence of control-peptide and Jurkat cells without TCR mRNA
electroporation do not respond to peptide-loaded T2 cells. This
example shows that TCRs binding to target-peptide tetramers also
show reactivity toward those peptides on a functional level.
[0037] FIGS. 6, 7 and 8 show peptide presentation profiles of a
target similar peptide 1 (TSP1) (FIG. 6), TSP2 (FIG. 7) and an
irrelevant peptide (IP; FIG. 8) from Example 4 based on XPRESIDENT
mass spectrometry data. Upper part: Median relative MS signal
intensities from technical replicate measurements are plotted as
colored dots for single HLA-A*02 normal samples on which the
peptide was detected. Normal samples are grouped according to organ
of origin. Box-and-whisker plots represent normalized signal
intensities over multiple samples and have been defined in the log
space. Boxes display median, 25.sup.th and 75.sup.th percentile.
Whiskers extend to the lowest data point still within 1.5
interquartile range (IQR) of the lower quartile, and the highest
data point still within 1.5 IQR of the upper quartile. Lower part:
The peptide detection frequency in every organ is shown as a bar
plot. Numbers below the panel indicate number of samples on which
the peptide was detected out of the total number of samples
analyzed for each organ (N.gtoreq.628 for normal samples across all
organs). If the peptide has been detected on a sample but could not
be quantified for technical reasons, the sample is included in this
representation of detection frequency, but no dot is shown in the
upper part of the figure. adipose: adipose tissue; adrenal gl:
adrenal gland; bladder: urinary bladder; bloodvess: blood vessel;
esoph: esophagus; gall bl:gallbladder; intest. la: large intestine;
intest. sm: small intestine; nerve cent: central nerve; nerve
periph: peripheral nerve; parathyr: parathyroid gland; petit:
peritoneum; pituit: pituitary; skel. mus: skeletal muscle.
LIST OF SELECTED SEQUENCES
[0038] SEQ ID NO: 1
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8,
wherein X.sub.1-X.sub.8 are amino acids positions in a target
peptide of a length of 8 amino acids and X in each case is any
amino acid; [0039] SEQ ID NO: 2
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-
, wherein X.sub.1-X.sub.9 are amino acids positions in a target
peptide of a length of 9 amino acids and X in each case is any
amino acid; [0040] SEQ ID NO: 3
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10, wherein X.sub.1-X.sub.10 are amino acids positions in a
target peptide of a length of 10 amino acids and X in each case is
any amino acid; [0041] SEQ ID NO: 4
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11, wherein X.sub.1-X.sub.11 are amino acids
positions in a target peptide of a length of 11 amino acids and X
in each case is any amino acid; [0042] SEQ ID NO: 5
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12, wherein X.sub.1-X.sub.12 are amino acids
positions in a target peptide of a length of 12 amino acids and X
in each case is any amino acid.
DETAILED DESCRIPTION OF THE INVENTION
[0043] Before the present invention is described in detail below,
it is to be understood that this invention is not limited to the
particular methodology, protocols and reagents described herein as
these may vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only and is not intended to limit the scope of the present
invention which will be limited only by the appended claims. Unless
defined otherwise, all technical and scientific terms used herein
have the same meanings as commonly understood by one of ordinary
skill in the art.
[0044] Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including all
patents, patent applications, scientific publications,
manufacturer's specifications, instructions etc.), whether supra or
infra, is hereby incorporated by reference in its entirety. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior invention.
Some of the documents cited herein are characterized as being
"incorporated by reference". In the event of a conflict between the
definitions or teachings of such incorporated references and
definitions or teachings recited in the present specification, the
text of the present specification takes precedence.
[0045] In the following, the elements of the present invention will
be described. These elements are listed with specific embodiments
however, it should be understood that they may be combined in any
manner and in any number to create additional embodiments. The
variously described examples and preferred embodiments should not
be construed to limit the present invention to only the explicitly
described embodiments. This description should be understood to
support and encompass embodiments which combine the explicitly
described embodiments with any number of the disclosed and/or
preferred elements. Furthermore, any permutations and combinations
of all described elements in this application should be considered
disclosed by the description of the present application unless the
context indicates otherwise.
Definitions
[0046] To practice the present invention, unless otherwise
indicated, conventional methods of chemistry, biochemistry, and
recombinant DNA techniques are employed which are explained in the
literature in the field (cf., e.g., Molecular Cloning: A Laboratory
Manual, 2.sup.nd Edition, J. Sambrook et al. eds., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor 1989).
[0047] In the following, some definitions of terms frequently used
in this specification to characterize the invention are provided.
These terms will, in each instance of its use, in the remainder of
the specification have the respectively defined meaning and
preferred meanings.
[0048] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural referents,
unless the content clearly dictates otherwise.
[0049] The term "amino acid" refers in the context of this
invention to any monomer unit that comprises a substituted or
unsubstituted amino group, a substituted or unsubstituted carboxy
group, and one or more side chains or groups, or analog of any of
these groups. Exemplary side chains include, e.g., thiol, seleno,
sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine,
cyano, halo, hydrazide, alkenyl, alkynl, ether, borate, boronate,
phospho, phosphono, phosphine, heterocyclic, enone, imine,
aldehyde, ester, thioacid, hydroxylamine, or any combination of
these groups. Other representative amino acids include, but are not
limited to, amino acids comprising photoactivatable cross-linkers,
metal binding amino acids, spin-labelled amino acids, fluorescent
amino acids, metal-containing amino acids, amino acids with novel
functional groups, amino acids that covalently or noncovalently
interact with other molecules, photocaged and/or photoisomerizable
amino acids, radioactive amino acids, amino acids comprising biotin
or a biotin analog, glycosylated amino acids, other carbohydrate
modified amino acids, amino acids comprising polyethylene glycol or
polyether, heavy atom substituted amino acids, chemically cleavable
and/or photocleavable amino acids, carbon-linked sugar-containing
amino acids, redox-active amino acids, amino thioacid containing
amino acids, and amino acids comprising one or more toxic moieties.
As used herein, the term "amino acid" includes the following twenty
natural or genetically encoded alpha-amino acids: alanine (Ala or
A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp
or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid
(Glu or E), glycine (Gly or G), histidine (His or H), isoleucine
(Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met
or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or
S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or
Y), and valine (Val or V). In cases where "X" residues are
undefined, these are to be interpreted as "any amino acid." The
structures of these twenty natural amino acids are shown in, e.g.,
Stryer et al., Biochemistry, 5th ed., Freeman and Company (2002).
Additional amino acids, such as selenocysteine and pyrrolysine, can
also be genetically coded for (Stadtman (1996) "Selenocysteine,"
Annu Rev Biochem. 65:83-100 and Ibba et al. (2002) "Genetic code:
introducing pyrrolysine," Curr Biol. 12(13):R464-R466). The term
"amino acid" also includes unnatural amino acids, modified amino
acids (e.g., having modified side chains and/or backbones), and
amino acid analogs. See, e.g., Zhang et al. (2004) "Selective
incorporation of 5-hydroxytryptophan into proteins in mammalian
cells," Proc. Natl. Acad. Sci. U.S.A. 101(24):8882-8887, Anderson
et al. (2004) "An expanded genetic code with a functional
quadruplet codon" Proc. Natl. Acad. Sci. U.S.A. 101(20):7566-7571,
Ikeda et al. (2003) "Synthesis of a novel histidine analogue and
its efficient incorporation into a protein in vivo," Protein Eng.
Des. Sel. 16(9):699-706, Chin et al. (2003) "An Expanded Eukaryotic
Genetic Code," Science 301(5635):964-967, James et al. (2001)
"Kinetic characterization of ribonuclease S mutants containing
photoisomerizable phenylazophenylalanine residues," Protein Eng.
Des. Sel. 14(12):983-991, Kohrer et al. (2001) "Import of amber and
ochre suppressor tRNAs into mammalian cells: A general approach to
site-specific insertion of amino acid analogues into proteins,"
Proc. Natl. Acad. Sci. U.S.A. 98(25):14310-14315, Bacher et al.
(2001) "Selection and Characterization of Escherichia coli Variants
Capable of Growth on an Otherwise Toxic Tryptophan Analogue," J.
Bacteriol. 183(18):5414-5425, Hamano-Takaku et al. (2000) "A Mutant
Escherichia coli Tyrosyl-tRNA Synthetase Utilizes the Unnatural
Amino Acid Azatyrosine More Efficiently than Tyrosine," J. Biol.
Chem. 275(51):40324-40328, and Budisa et al. (2001) "Proteins with
{beta}-(thienopyrrolyl) alanines as alternative chromophores and
pharmaceutically active amino acids," Protein Sci. 10(7):1281-1292.
Amino acids can be merged into peptides, polypeptides, or proteins.
As used in this specification the term "peptide" refers to a short
polymer of amino acids linked by peptide bonds. It has the same
chemical (peptide) bonds as proteins but is commonly shorter in
length. The shortest peptide is a dipeptide, consisting of two
amino acids joined by a single peptide bond. There can also be a
tripeptide, tetrapeptide, pentapeptide, etc. Typically, a peptide
has a length of up to 8, 10, 12, 15, 18 or 20 amino acids. A
peptide has an amino end and a carboxyl end, unless it is a cyclic
peptide.
[0050] The term "virus" refers in the context of the present
invention to small obligate intracellular parasites, which by
definition contain either a RNA or DNA genome surrounded by a
protective protein coat, i.e. a capsid. The genome of a virus may
consist of DNA or RNA, which may be single stranded (ss) or double
stranded (ds), linear or circular. The entire genome may occupy
either one nucleic acid molecule (monopartite genome) or several
nucleic acid segments (multipartite genome). The virus can be a
double-stranded DNA virus, preferably Myoviridae, Siphoviridae,
Podoviridae, Herpesviridae, Adenoviridae, Baculoviridae,
Papillomaviridae, Polydnaviridae, Polyomaviridae, Poxviridae; a
single-stranded DNA virus, preferably Anelloviridae, Inoviridae,
Parvoviridae; double-stranded RNA virus, preferably Reoviridae; a
single-stranded RNA virus, preferably Coronaviridae,
Picornaviridae, Caliciviridae, Togaviridae, Flaviviridae,
Astroviridae, Arteriviridae, Hepeviridae; negative-sense
single-stranded RNA virus, preferably Arenaviridae, Filoviridae,
Paramyxoviridae, Rhabdoviridae, Bunyaviridae, Orthomyxoviridae,
Bornaviridae; a single-stranded RNA reverse transcribing virus,
preferably Retroviridae; or a double-stranded RNA reverse
transcribing virus, preferably Caulimoviridae, Hepadnaviridae.
[0051] The term "bacteriophage" (or "phage") refers in the context
of the present invention to a virus that infects and replicates in
bacteria and archaea. Bacteriophages are dependent on a host
organism, typically bacteria, to replicate in and inject their
genome, which is either comprised of proteins that encapsulate
desoxyribonucleic acid (DNA) or ribonucleic acid (RNA), into the
host organims's cytoplasm. Prominent examples of bacteriophages
used in biotechnology are bacteriophage T4 lambda (T4.lamda.)
phage, T7 phage, fd filamentous phage, in particular filamentous
M13 phage of which all have certain benefits and drawbacks.
[0052] The term "virus population" refers in the context of the
present invention to a high number of viruses which differ in the
genetic information encoding the antigen binding protein expressed
on their surface. The viral population can thus, express a library
of heterologous antigen binding proteins.
[0053] The term "phage display" or "phage library" refers in the
context of the present invention to a system that is used for
high-throughput screening of protein interactions. Briefly, a gene
encoding a protein of interest is inserted into a bacteriophage
coat protein gene which causes the bacteriophage to "display", i.e.
to show, the protein on its surface while keeping the gene encoding
the protein of interest in its DNA or RNA. This results in a
connection of genotype and phenotype. The proteins which are
displayed on the bacteriophage's surface can subsequently be
screened against other proteins, peptides or DNA sequences to study
their interaction between the displayed molecule and the molecules
to be screened. Such a molecule can be an antibody or a fragment
thereof, a TCR or a fragment thereof, a BCR or a fragment thereof
or a CAR or a fragment thereof. Fragments of TCRs may comprise the
alpha variable domain and the beta variable domain. Fragments in
the context of the present inventions are also defined in detail
below.
[0054] The term "cell" refers in the context of the present
invention to eukaryotic cells which contain a nucleus and cell
organelles and can be found in protozoa, fungi, plants and animals.
Animals can comprise mammalian cells. Mammalian cells comprise
inter alia human cells, rhodent cells, such as mouse, or rat cells,
monkey cells, pig cells or dog cells. Fungi cells inter alia
comprise yeast cells. Typical yeast cells used in biotechnology,
for example in a yeast surface display, are Saccharomyces
cerevisiae cells.
[0055] The term "yeast surface display" or "yeast display" or
"yeast library" refers in the context of the present invention to a
protein engineering technique using yeast cells that express
recombinant proteins of interest and incorporate these proteins
into their cell wall. This allows for isolation and engineering of
proteins, in particular antibodies or fragments thereof, TCRs or
fragments thereof, BCRs or fragments thereof or CARs or fragments
thereof. In detail, in the yeast surface display, the unit of
selection is a yeast cell that is decorated with tens of thousands
of copies of the protein of interest and that carries the plasmid
encoding that protein. The plasmid can be shuttled between
Saccharomyces cerevisiae, for display and sorting, and E. coli, for
DNA preparation and molecular biology. In the form of yeast display
pioneered by the Wittrup group (Chao et al., 2006), each .sup.10Fn3
variant is expressed as a genetic fusion with a native yeast
protein found in the cell wall, Aga2p. Aga2p is a domain of the
native yeast, an agglutinin mating factor; typically, it is cloned
upstream of the sequence encoding the .sup.10Fn3 variant. In
addition, an epitope tag, such as c-myc and V5, is engineered
immediately downstream from the sequence encoding the .sup.10Fn3
variant. Upon induction, the mating-factor secretory signal peptide
directs the fusion protein to be exported from the cell; it is
captured on the surface of the yeast cell wall by its binding
partner, Aga1p, to which it forms two disulfide bonds. The result
is a culture where each yeast cell displays between 10,000 and
100,000 copies of a single .sup.10Fn3 variant. On average, the more
thermostable the variant, the larger the number of its molecules on
the yeast surface (Hackel et al., 2010).
[0056] The term "immune cell" refers in the context of this
invention to a cell of the immune system. The immune system
comprises different cell types such as precursor cells comprising
lymphoid stem cells, which ultimately differentiate into B and T
lymphocytes and natural killer (NK) cells, and myeloblasts, which
ultimately differentiate into granulocytes and monocytes as well as
fully differentiated leukocytes. Differentiated leukocytes are
thymus-, spleen-, bone marrow or lymph node-derived cells and can
be categorized into the main groups of granulocytes, B-lymphocytes,
T-lymphocytes and monocytes, macrophages, and mast cells and
dendritic cells. Granulocytes are further divided into neutrophil,
eosinophil and basophil granulocytes, which phagocytose bacteria,
virus or fungi in the blood circulation. B-lymphocytes are
precursors of plasma cells and B-memory cells. The group of T-cells
comprises regulatory T-cells, memory T-cells, T helper cells and
cytotoxic T-cells. While T helper cells activate plasma cells and
natural killer cells, regulatory T-cells inhibit the function of B
and other T-cells and thus, slow down the immune response. T memory
cells are long-living and possess a memory for specific antigens,
and cytotoxic T-cells recognize and kill tumor cells or cells
attacked by viruses by interacting with tumor antigens or antigens
of the attacked cells. Examples of T-cells and their surface
phenotype described by the specific surface markers of the
respective T-cells are given in below Table 1 (according to Dong
and Martinez, Nature Reviews Immunology, 2010):
TABLE-US-00001 TABLE 1 Common T-cell surface markers
(non-exhaustive enumeration). Cell: Surface marker: Cytotoxic
T-cells: .alpha..beta. TCR, CD3, CD8 Regulatory T-cells:
.alpha..beta. TCR, CD3, CD4 Regulatory T-cells .alpha..beta. TCR,
CD3, CD4, CD25, CTLA4, GITR (natural and inducible): Natural Killer
cells: NK1.1, SLAMF1, SLAMF6, TGF.beta., V.alpha.24, J.alpha.18 T
helper cells: TH1 cells .alpha..beta. TCR, CD3, CD4, IL-12R,
IFN.gamma.R, CXCR3; TH2 cells .alpha..beta. TCR, CD3, CD4,
IL-4R,IL33R, CCR4, IL-17RB, CRTH2; TH9 cells .alpha..beta. TCR,
CD3, CD4; TH17 cells .alpha..beta. TCR, CD3, CD4, IL-23R, CCR6,
IL-1R, CD161; TH22 cells .alpha..beta. TCR, CD3, CD4, CCR10; TILs
Tumor-infiltrating lymphocytes T memory cells CCR7 hi, CD44,
CD62Lhi, TCR, CD3, IL-7R (CD127), IL-15R
[0057] The term "tumor-infiltrating lymphocytes" (TILs) refers in
the context of the present invention to T-cells and B-cells that
have migrated towards a tumor and can often be found in the tumor
stroma or the tumor itself. TILs typically comprise a cell
population of white blood cells that may be used in ACT or
autologous cell therapy. Such therapies have already shown
promising results, for example in patients with metastatic melanoma
in a variety of clinical trials (Guo et al.; "Recent updates on
cancer immunotherapy"; Precision Clinical Medicine, 1(2),
2018-65-74). In the context of ACT, TILs are expanded ex vivo from
surgically resected tumors or single cell suspensions isolated from
tumor fragments. TILs are expanded with a high doses of cytokines,
for example IL-2. Selected TIL lines that presented best tumor
reactivity are then further expanded in a "rapid expansion
protocol" (REP), which uses anti-CD3 activation for a typical
period of two weeks. The final post-REP TIL is infused back into
the patient. The process can also involve a preliminary
chemotherapy regimen to deplete endogenous lymphocytes in order to
provide the adoptively transferred TILs with enough access to
surround the tumor sites.
[0058] The term "immune cell enriched fraction" refers in the
context of this invention to a cell population, which is derived
from a naturally occurring cell population, e.g. blood, in which
the relative abundance of the immune cells has been increased in
comparison to their abundance in the naturally occurring cell
mixture. One ml of blood of a healthy human subject comprises, e.g.
4.7 to 6.1 million (male), 4.2 to 5.4 million (female)
erythrocytes, 4,000-11,000 leukocytes and 200,000-500,000
thrombocytes. Thus, in blood immune cells only constitute 0.06% to
0.25% of the total number of blood cells. An immune cell enriched
fraction of blood thus may comprise more than 0.25%, more
preferably more than 10%, even more preferably more than 50%, even
more preferably more than 80% and most preferably more than 90%
immune cells. The immune cell enriched fraction may be enriched for
one or more subtypes of immune cells. For example, the immune cell
enriched fraction may be enriched for lymphoid stem cells, T-cells,
B-cells, plasma cells or combinations. Usually, immune cells in
immune cell enriched fractions are selected by using one or more
fluorescently labelled antibodies that specifically bind to a
surface marker of the immune cells of interest. Suitable surface
markers to select T-cells or sub-fractions within the group of
T-cells are indicated in table 1 above. Cytotoxic T-cells can be
selected, e.g. by using an antibody that specifically binds to CD8
or by using antibodies that specifically bind to CD8 and CD3.
[0059] The term "cell population" refers in the context of this
invention to a plurality of cells which may be homogenous or
heterogenous, i.e. a mixture of cells of different characteristic.
Blood is an example of a cell population which is a mixture of
different cells. Homogenous cell populations can be obtained by
selection of a particular subtype or by clonal expansion.
[0060] The term "antigen binding protein" refers in the context of
this invention to one polypeptide or a complex of two or more
polypeptides that comprise a paratope (alternatively referred to as
"antigen binding site") that specifically binds to an antigen.
Examples of antigen binding proteins are single chain antibodies,
single chain TCRs, chimeric antigen receptor (CAR) and examples of
antigen binding complexes are antibodies, B cell receptors (BCRs)
or TCRs.
[0061] The term "chimeric antigen receptor" (CAR; also known as
chimeric immunoreceptor, chimeric T cell receptor, artificial T
cell receptor) in the context of the present invention refers to
engineered receptors, which graft an arbitrary specificity onto an
immune effector cell, preferably a T cell. Cells are genetically
equipped with a CAR, which is a composite membrane receptor
molecule and provides both targeting specificity and T cell
activation. The most common form of CARs are fusions of single
chain variable fragment (scFv) derived from monoclonal antibodies,
fused to CD3 transmembrane- and endodomain. The CAR targets the T
cell to a desired cellular target through an antibody-derived
binding domain in the extracellular moiety, and T cell activation
occurs via the intracellular moiety signalling domains when the
target is encountered. The transfer of the coding sequence of these
receptors into suitable cells, in particular T cells, is commonly
facilitated by retro- or lentiviral vectors. The receptors are
called chimeric because they are composed of parts from different
sources.
[0062] The term "epitope" refers in the context of this invention
to the functional epitope of an antigen. The functional epitope
comprises those residues, typically amino acids or polysaccharides
that contribute to the non-covalent interaction between the
paratope of the antigen binding protein and the antigen. The
non-covalent interaction comprises electrostatic forces, van der
Walls forces, hydrogen bonds, and hydrophobic interaction. The
functional epitope is a subgroup of the residues that constitute
the structural epitope of an antigen binding protein. The
structural epitope comprises all residues that are covered by an
antigen binding protein, i.e. the footprint of an antigen binding
protein. Typically, the functional epitope of an antigen bound by
an antibody comprises 4 to 10 amino acids. Similarly, the
functional epitope of a peptide that is MHC presented typically
comprises 4 to 8 amino acids.
[0063] The term "expression" refers in the context of this
invention to the presence of a protein or peptide, in particular a
PAI or a SPA in human tissue. The term expression of a protein or
peptide means that it is translated from its nucleic acid sequence
into its amino acid sequence during the process of protein
biosynthesis in the ribosomal machinery of the cell. The expressed
protein can be located intracellularly or extracellularly, e.g. on
the surface of cell. The human tissue wherein the protein is
expressed may be healthy or diseased tissue.
[0064] The term "protein antigen of interest" (PAI) refers in the
context of this invention to a protein or a portion of a protein or
a protein complex that comprises an epitope that is specifically
bound by the paratope of an antigen-binding protein. A PAI is
typically a naturally occurring protein and can be of any length.
It is preferred that the PAI comprises at least 25 amino acids. If
that the PAI is specifically bound by a TCR accordingly, it is
preferred that the length of the PAI is 8 to 12 amino acids. The
PAI may be a tumor associated target antigen (TAA), a viral protein
or a bacterial protein. The PAI is typically a tumor associated
antigen (TAA), which is to be specifically targeted in, e.g. a
tumor therapy.
[0065] The term "humanized mice" refers in the context of the
present invention to genetically modified mice which carry human
genes, cells, tissues and/or organs that exert their biological
function, e.g. are intact regarding their biological function.
Typically, immunodeficient mice are used as recipients for human
cells or tissues, because they can relatively easily accept
heterologous cells or tissues due to lack of host immunity.
Examples of humanized mice are the nude mouse, the severe combined
immunodeficiency (SCID) mouse, the NCG mouse, the NOG
(NOD/Shi-scid/IL-2R.gamma..sup.null) mouse or the NSG (NOD scid
gamma) mouse. Mice that accept human version of genes into their
respective mouse loci are called "knock-in" mice. B-cells and
T-cells can be isolated from humanized mice and be used in the
methods of the present invention.
[0066] The term "T-cell receptor libraries" refers in the context
of the present invention to a library that contains a high number
of different T cell receptor (TCR) proteins or fragments thereof,
wherein each TCR protein or fragment thereof is different.
[0067] A "viral antigenic peptide" in the context of the present
invention is shorter fragment of a viral protein that is presented
by a major histocompatibility complex (MHC) molecule on the surface
of an antigen presenting cell, which is typically a diseased cell.
The viral antigenic peptide is of a viral origin, i.e. the cell is
typically infected by said virus. The viral antigenic peptide in
the context of the present invention may be an antigenic peptide
selected from the group consisting of human immune deficiency virus
(HIV) antigenic peptides, human cytomegalovirus (HCMV) antigenic
peptides, cytomegalovirus (CMV) antigenic peptides, human
papillomavirus (HPV) antigenic peptides, hepatitis B virus (HBV)
antigenic peptides; hepatitis C virus (HCV) antigenic peptides;
Epstein-Barr virus (EBV) antigenic peptides, Influenza antigenic
peptides, preferably HIV, HBV, Influenza and HCMV antigenic
peptides. Viral antigenic peptides can be used in the method and
the embodiments described herein include, for example, viral
antigenic peptides as described in table 2 below. In one aspect,
viral antigenic peptides that are used in the method and embodiment
described herein include at least one viral antigenic peptide
comprising or consisting of an amino acid sequence selected from
the amino acid sequences of SEQ ID NO: 6 to SEQ ID NO: 8.
TABLE-US-00002 TABLE 2 List of viral antigenic peptides SEQ ID
Amino acid NO: sequence Virus MHC 6 SLYNTVATL HIV HLA-A*02:01 7
GILGFVFTL Influenza A HLA-A*02:01 8 NLVPMVATV HCMV HLA-A*02:01
[0068] A "bacterial antigenic peptide" in the context of the
present invention is shorter fragment of a bacterial protein that
is presented by an MHC molecule on the surface of an antigen
presenting cell, which is typically a diseased cell. The bacterial
antigenic peptide is of a bacterial origin, i.e. the cell is
typically infected by a bacterium. Such bacterial antigenic
peptides have been discovered in the context of infections from,
for example, Mycobacterium tuberculosis. Accordingly, the bacterial
antigenic peptide in the context of the present invention may be a
Mycobacterium tuberculosis antigenic peptide.
[0069] The term "tumor associated antigen" (TAA) refers in the
context of this invention to autologous cellular antigens derived
from all protein classes, such as enzymes, receptors, transcription
factors, etc. that are preferentially or exclusively expressed by
tumor cells. TAA can be broadly categorized into aberrantly
expressed self-antigens, mutated self-antigens, and tumor-specific
antigens. TAAs that are preferentially expressed by tumor cells,
are also found in normal tissues. However, their expression differs
from that of normal tissues by their degree of expression in the
tumor, by alterations in their protein structure in comparison with
their normal counterparts, or by their aberrant subcellular
localization within tumor cells. The TAA peptides that can be used
in the methods and embodiments described herein include, for
example, TAA peptides described in U.S. Publication 20160187351,
U.S. Publication 20170165335, U.S. Publication 20170035807, U.S.
Publication 20160280759, U.S. Publication 20160287687, U.S.
Publication 20160346371, U.S. Publication 20160368965, U.S.
Publication 20170022251, U.S. Publication 20170002055, U.S.
Publication 20170029486, U.S. Publication 20170037089, U.S.
Publication 20170136108, U.S. Publication 20170101473, U.S.
Publication 20170096461, U.S. Publication 20170165337, U.S.
Publication 20170189505, U.S. Publication 20170173132, U.S.
Publication 20170296640, U.S. Publication 20170253633, U.S.
Publication 20170260249, U.S. Publication 20180051080, and U.S.
Publication No. 20180164315, the contents of each of these
publications and sequence listings described therein, which are
herein incorporated by reference in their entirety. Furthermore,
the TAA in the context of the present invention is a specific
ligand of MHC-class-I-molecules or MHC-class-II-molecules,
preferably MHC-class-I-molecules.
[0070] In an aspect, the antigen binding protein selected by the
method of the present invention selectively recognize cells which
present a TAA peptide described in one of more of the patents and
publications listed above. In another aspect, TAA peptides that may
be used in the methods and embodiments described herein include at
least one TAA consisting of an amino acid sequence selected from
the amino acid sequences of SEQ ID NO: 9 to 164. In an aspect, the
antigen binding protein selected by the method of the present
invention selectively binds cells which present a TAA peptide/MHC
complex, wherein the TAA peptide comprises or consists of an amino
acid sequence of SEQ ID NO: 1 to 164. Further examples of TAAs are
listed in table 3.
TABLE-US-00003 TABLE 3 List of TAAs SEQ ID Amino Acid NO: Sequence
9 YLYDSETKNA 10 HLMDQPLSV 11 GLLKKINSV 12 FLVDGSSAL 13 FLFDGSANLV
14 FLYKIIDEL 15 FILDSAETTTL 16 SVDVSPPKV 17 VADKIHSV 18 IVDDLTINL
19 GLLEELVTV 20 TLDGAAVNQV 21 SVLEKEIYSI 22 LLDPKTIFL 23 YLMDDFSSL
24 KVWSDVTPL 25 LLWGHPRVALA 26 KIWEELSVLEV 27 LLIPFTIFM 28
FLIENLLAA 29 LLWGHPRVALA 30 FLLEREQLL 31 SLAETIFIV 32 TLLEGISRA 33
ILQDGQFLV 34 VIFEGEPMYL 35 SLFESLEYL 36 SLLNQPKAV 37 GLAEFQENV 38
KLLAVIHEL 39 TLHDQVHLL 40 TLYNPERTITV 41 KLQEKIQEL 42 SVLEKEIYSI 43
RVIDDSLVVGV 44 VLFGELPAL 45 GLVDIMVHL 46 FLNAIETAL 47 ALLQALMEL 48
ALSSSQAEV 49 SLITGQDLLSV 50 QLIEKNWLL 51 LLDPKTIFL 52 RLHDENILL 53
GLPSATTTV 54 GLLPSAESIKL 55 KTASINQNV 56 SLLQHLIGL 57 YLMDDFSSL 58
LMYPYIYHV 59 KVWSDVTPL 60 LLWGHPRVALA 61 VLDGKVAVV 62 GLLGKVTSV 63
KMISAIPTL 64 GLLETTGLLAT 65 TLNTLDINL 66 VIIKGLEEI 67 YLEDGFAYV 68
KIWEELSVLEV 69 LLIPFTIFM 70 ISLDEVAVSL 71 KISDFGLATV 72 KLIGNIHGNEV
73 ILLSVLHQL 74 LDSEALLTL 75 VLQENSSDYQSNL 76 HLLGEGAFAQV 77
SLVENIHVL 78 SLSEKSPEV 79 AMFPDTIPRV 80 FLIENLLAA 81 FTAEFLEKV 82
ALYGNVQQV 83 LFQSRIAGV 84 ILAEEPIYIRV 85 FLLEREQLL 86 LLLPLELSLA 87
SLAETIFIV 88 AILNVDEKNQV 89 RLFEEVLGV 90 YLDEVAFML 91 KLIDEDEPLFL
92 KLFEKSTGL 93 SLLEVNEASSV 94 GVYDGREHTV 95 GLYPVTLVGV 96
ALLSSVAEA 97 TLLEGISRA 98 SLIEESEEL 99 ALYVQAPTV 100 KLIYKDLVSV 101
ILQDGQFLV 102 SLLDYEVSI 103 LLGDSSFFL 104 VIFEGEPMYL 105 ALSYILPYL
106 FLFVDPELV 107 SEWGSPHAAVP 108 ALSELERVL 109 SLFESLEYL 110
KVLEYVIKV 111 VLLNEILEQV 112 SLLNQPKAV 113 KMSELQTYV 114
ALLEQTGDMSL 115 VIIKGLEEITV 116 KQFEGTVEI 117 KLQEEIPVL 118
GLAEFQENV 119 NVAEIVIHI 120 ALAGIVTNV 121 NLLIDDKGTIKL 122
VLMQDSRLYL 123 KVLEHVVRV 124 LLWGNLPEI 125 SLMEKNQSL 126 KLLAVIHEL
127 ALGDKFLLRV 128 FLMKNSDLYGA 129 KLIDHQGLYL 130 GPGIFPPPPPQP
131 ALNESLVEC 132 GLAALAVHL 133 LLLEAVWHL 134 SIIEYLPTL 135
TLHDQVHLL 136 SLLMWITQC 137 FLLDKPQDLSI 138 YLLDMPLWYL 139
GLLDCPIFL 140 VLIEYNFSI 141 TLYNPERTITV 142 AVPPPPSSV 143 KLQEELNKV
144 KLMDPGSLPPL 145 ALIVSLPYL 146 FLLDGSANV 147 ALDPSGNQLI 148
ILIKHLVKV 149 VLLDTILQL 150 HLIAEIHTA 151 SMNGGVFAV 152 MLAEKLLQA
153 YMLDIFHEV 154 ALWLPTDSATV 155 GLASRILDA 156 ALSVLRLAL 157
SYVKVLHHL 158 VYLPKIPSW 159 NYEDHFPLL 160 VYIAELEKI 161
VHFEDTGKTLLF 162 VLSPFILTL 163 HLLEGSVGV
[0071] Furthermore, the TAA antigenic peptide in the context of the
present invention is a specific ligand of MHC-class-I-molecules or
MHC-class-II-molecules, preferably MHC-class-I-molecules.
[0072] The term "tumor-specific antigen" refers in the context of
this invention to antigens that are exclusively expressed on tumor
cells. They include neo-antigens that arise due to mutations, e.g.
point mutations or frame-shift mutations, in the tumor cell.
Examples for tumor specific antigens are p53 or BCR-ABL.
[0073] The term "MHC" refers in the context of this invention to
the abbreviation for the phrase "major histocompatibility complex".
MHC's are a set of cell surface receptors that have an essential
role in establishing acquired immunity against altered natural or
foreign proteins in vertebrates, which in turn determines
histocompatibility within a tissue. The main function of MHC
molecules is to bind to antigens derived from altered proteins or
pathogens and display them on the cell surface for recognition by
appropriate T-cells. The human MHC is also called HLA (human
leukocyte antigen) complex or HLA. The MHC gene family is divided
into three subgroups: class I, class II, and class III. Complexes
of peptide and MHC class I are recognized by CD8-positive T-cells
bearing the appropriate TCR, whereas complexes of peptide and MHC
class II molecules are recognized by CD4-positive-helper-T-cells
bearing the appropriate TCR. Since both types of response, CD8 and
CD4 dependent, contribute jointly and synergistically to the
anti-tumor effect, the identification and characterization of
tumor-associated antigens and corresponding TCRs is important in
the development of cancer immunotherapies such as vaccines and cell
therapies.
[0074] The term "MHC-I" refers in the context of the present
invention to MHC class I molecules or MHC-I. The MHC I molecule
consists of an alpha chain, also referred to as MHC I heavy chain
and a beta chain, which constitutes a beta 2 microglobulin
molecule. The alpha chain, comprises three alpha domains, i.e.
alpha1 domain, alpha2 domain and alpha3 domain. Alpha1 and alpha2
domain mainly contribute to forming the peptide pocket to produce a
peptide ligand MHC (pMHC) complex. MHC-I typically bind peptides
that are derived from cytosolic antigenic proteins and which are
degraded by the proteasome after ubiquitylation and subsequently
transported through a specific transporter associated with antigen
processing (TAP) from the cytosol to the endoplasmatic reticulum
(ER). MCH I typically binds peptides of 8-12 amino acids in
length.
[0075] The term "MHC-H" refers in the context of the present
invention to MHC class II molecules or MHC-II. The MHC-II molecule
consists of an alpha and a beta chain, wherein the alpha chain
comprises two alpha domains, alpha1 domain, alpha2 domain and the
beta chain comprises two beta domains, beta domain1 and beta
domain2 MHC II typically fold in the ER in complex with a protein
called invariant chain and are then transported to late endosomal
compartments where the invariant chain is cleaved by cathepsin
proteases and a short fragment remains bound to the peptide-binding
groove of MHC II, termed class II-associated invariant chain
peptide (CLIP). This placeholder peptide is then normally exchanged
against higher affinity peptides, which are derived from
proteolytically degraded proteins available in endocytic
compartments. MHC-II typically binds peptides of 10-30 amino acids
in length or peptides of 13-25 amino acids in length.
[0076] The term "HLA" refers in the context of the present
invention to molecules which differ between different human beings
in amino acid sequence. However, HLAs can be identified by an
internationally agreed nomenclature, the IMGT nomenclature, of HLA.
The HLA-A gene is located on the short arm of chromosome 6 and
encodes the larger, .alpha.-chain, constituent of HLA-A. Variation
of HLA-A .alpha.-chain is key to HLA function. This variation
promotes genetic diversity in the population. Since each HLA has a
different affinity for peptides of certain structures, greater
variety of HLAs means greater variety of antigens to be `presented`
on the cell surface. Each individual can express up to two types of
HLA-A, one from each of their parents. Some individuals will
inherit the same HLA-A from both parents, decreasing their
individual HLA diversity. However, the majority of individuals
receive two different copies of HLA-A. The same pattern follows for
all HLA groups. In other words, every single person can only
express either one or two of the 2432 known HLA-A alleles coding
for currently 1740 active proteins. HLA-A*02 signifies a specific
HLA allele, wherein the letter A signifies to which HLA gene the
allele belongs to and the prefix "*02 prefix" indicates the A2
serotype. In MHC class I dependent immune reactions, peptides not
only have to be able to bind to certain MHC class I molecules
expressed by tumor cells, they subsequently also have to be
recognized by T-cells bearing specific TCRs.
[0077] The term "target peptide" (TP) refers in the context of this
invention to a shorter peptide, part or fragment of the protein
antigen of interest (PAI). The amino acid sequence of a target
peptide comprises typically 8-12 amino acids in length, 8-11 amino
acids in length or 8-10 amino acids in length. Preferably, the
amino acid sequence of a target peptide comprises typically 8-11
amino acids in length. The target peptide may be bound to an MHC-I
molecule or an MHC-II molecule. Whether a target peptide binds to
an MHC-I or MHC-II molecule depends on the target peptide's natural
origin, i.e. whether it is synthesized in the cytoplasm and
processed in the proteasome or absorbed by endocytosis and
subsequently processed. Moreover, it depends on the length of the
target peptide whether it will bind to the binding groove of an
MHC-I or an MHC-II molecule. In one example a target peptide of a
length of 8-12, 8-11 or 8-10 amino acids is typically bound to a
MHC-I. In another example, the amino acid sequence of a target
peptide may comprise 13-23 amino acids in length, preferably 13-18
amino acids in length. A target peptide of a length of 13-25 or
13-18 amino acids is typically bound to an MHC-II.
[0078] The term "antigen complex" (AC) refers in the context of
this invention to a complex comprising an antigen that is directly
or indirectly, e.g. through an MHC or peptide binding part thereof,
attached to the surface of a carrier or a soluble multimerized MHC
or peptide binding part thereof. Such a carrier can be a cell or
synthetic material. If the antigen is attached to a cell, the cell
may be an antigen-presenting cells (APCs), preferably a human APC.
Synthetic materials for carriers can be beads or particles,
preferably microbeads, microparticles or nanoparticles. Such beads
can be magnetic or paramagnetic beads. Beads or microparticles are
usually made of polymers and can be covalently or non-covalently
coated with a first member of a pair of coupling residues. The
second member of the pair of coupling residues is covalently or
non-covalently coupled to the MHC or peptide binding part thereof.
A preferred pair of first and second coupling residues comprises
streptavidin and biotin member. The skilled person is aware of
other pairs of coupling residues. Accordingly, in a preferred
embodiment the carrier may be coated with streptavidin which will
allow the immobilization of MHC molecules or peptide binding parts
thereof that comprise a biotin moiety. Conversely, a carrier coated
with biotin allows the immobilization of MHC molecules or peptide
binding parts thereof that comprise a streptavidin moiety. A
soluble multimerized MHC or peptide binding part thereof may
comprise two or more MHCs, wherein each is covalently or
non-covalently, preferably covalently coupled to a third member of
a pair of coupling residues and a fourth member of a pair or
coupling residues, wherein the fourth member has at least two
binding sites for the third member, preferably 3, 4, 5, 6, 7, or 8
binding sites and particularly preferred 4 binding sites. Biotin is
a preferred third member of a pair of coupling residues and
streptavidin is a preferred fourth member of a pair of coupling
residues. Streptavidin has four binding sites for biotin. Thus, if
MHC peptide complexes comprising biotin are contacted with
streptavidin a soluble tetramer will form in which four peptide
loaded MHCs (or peptide binding fragments thereof) are
non-covalently bound to streptavidin. Thus, in a preferred
embodiment the soluble multimerized MHC or peptide binding fragment
thereof is a complex comprising four MHC peptide complexes, wherein
each of the MHC peptide complex is attached covalently to one
biotin, which are in turn bound non-covalently to streptavidin.
[0079] The term "pair of coupling residues" refers to two entities
that specifically and non-covalently bind to each other with high
affinity. Preferably, the K.sub.d is less than 10.sup.-10 mol/L,
more preferably less than 10.sup.-11 mol/L, more preferably less
than 10-12 mol/L and even more preferably less than 10.sup.-13
mol/L. Preferably, at least one of the members of a binding pair
has a molecular weight below 500 g/mol/. Such a molecule can be
attached covalently to one chain of the MHC or peptide binding
fragment thereof without interfering with the ability of the MHC to
interact with a TCR. Preferred pairs of coupling residues are
biotin-streptavidin, and biotin-avidin. Alternatively, one member
of a pair of coupling residues can be a protein that is fused to
one chain of an MHC. Examples include chitin binding protein (CBP),
maltose binding protein (MBP), Strep-tag glutathione-S-transferase
(GST), poly(His) tag, V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag and
NE-tag. The other member of the pair is determined by the
respective protein tag, i.e. chitin, maltose, biotin, glutathione,
metal matrix, e.g. Ni-matrix, or an antibody that specifically
binds to the V5-, Myc-, HA-, Spot-, T7- or NE-tag.
[0080] The term "similar protein antigen" (SPA) refers in the
context of this invention to a protein or a portion of a protein or
a protein complex that comprises an epitope bound by the paratope
of an antigen binding protein. The amino acid sequence of the SPA
is determined by the PAI. The amino acid sequence of the SPA
differs in at least one amino acid from the amino acid sequence of
the given PAI, i.e. the PAI of interest. It serves the purpose of
identifying antigen binding proteins that bind the PAI and at the
same time the SPA, i.e. that do not exhibit the desired specificity
and/or selectivity to the PAI. Such antigen binding proteins may
elicit off-tumor/off target toxicity. For a given PAI and antigen
binding protein combination, the SPA falls into one of three
categories:
(1) Similar Amino Acid Sequence, Identical Epitope:
[0081] If the amino acids of the SPA that differ with respect to
the PAI do not contribute to the epitope bound by a given
PAI-specific antigen binding protein then the antigen binding
protein will bind with the same affinity both to the PAI and the
SPA. An antigen binding protein with this property will be counter
selected by the methods of the present invention.
(2) Similar Amino Acid Sequence, Similar Epitope:
[0082] If at least one of the amino acids of the SPA that differ
with respect to the PAI contributes to the epitope bound by a given
PAI-specific antigen binding protein than the antigen binding
protein will bind with a different affinity to the PAI and the SPA.
An antigen binding protein that exhibits significantly lower
binding to the SPA than to the PAI may be selected by the methods
of the present invention. In this respect significantly lower
binding means that the difference between the binding to the PAI
and the SPA is at least 2-fold, at least 3-fold, at least 4-fold,
at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold,
at least 9-fold, at least 10-fold, at least 15-fold, at least
20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at
least 70-fold, at least 100-fold, at least 200-fold, preferably at
least 50-fold, more preferably at least 100-fold at identical
concentration of the PAI and the SPA.
(3) Similar Amino Acid Sequence, Different Epitope:
[0083] If the diverging amino acids are located at positions that
contributes to the epitope bound by a given PAI-specific antigen
binding protein then the antigen binding protein may not bind to
the SPA at all. An antigen binding protein with this property will
be selected by the methods of the present invention.
[0084] The amino acid sequences of the SPAs are generally based on
the amino acid sequences of naturally occurring proteins, since
such proteins may be expressed on healthy tissue of a tumor
patient. Preferably, the SPA is a naturally occurring protein or a
fragment thereof. In particular the SPA is present in the same
species as the PAI. Thus, it is desired that the SPAs included in
the method of the invention have amino acid sequences that allow
identification and counterselection of antigen binding proteins in
category (1) and (2). The SPA is only likely to allow the
counterselection of unsuitable antigen binding proteins if its
amino acid sequence is closely related to the amino acid sequence
of the PAI. It is, thus, preferred that the amino acid sequence of
the SPA used in the method of the present invention has a
similarity to the amino acid sequence of the PAI of at least 50%,
at least 60%, at least 70%, at least 80%, of at least 90% or at
least 95%. Thus, in a preferred embodiment the SPA differs by 1-20,
more preferably by 2-10 amino acids from the amino acid sequence of
the PAI. It is preferred that the SPA, in particular the target
similar peptide (TSP) used in the method of the invention is
expressed on healthy tissue, preferably with more than 10 copies
per cell, preferably more than 20 copies per cell, preferably more
than 50 copies per cell and preferably more than 100 copies per
cell. The relative strength of expression can be determined by a
variety of art known methods including FACS analysis of healthy and
diseased cells with fluorescently labeled antigen binding proteins
or mass spectrometry. Gene expression analysis can also be
performed using RNA sequencing approaches. Another criteria for the
selection of a SPA to be used in the method of the invention is its
frequency of presentation on primary normal tissues. The frequency
describes how often a SPA is presented on normal, i.e. a healthy
tissue--in contrast to the copy number which defines the number of
SPAs of a given healthy tissue, for example a cell. Together with
the copy number the frequency is an important criterion to select a
SPA for a given PAI. The higher the similarity to the PAI and the
higher the presentation frequency and the copy number per cell
(CpC) on normal tissues, the higher the relevance of a SPA.
[0085] The term "target similar peptide" (TSP) refers in the
context of this invention to a shorter peptide, part or fragment of
the SPA. The amino acid sequence of a TSP comprises typically 8 to
16 amino acids in length. The TSP is typically MHC presented.
Similarly to the SPA, the TSP has a similarity to the amino acid
sequence of the TP of at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%. The TSPs typically have a length of 8 to
16 amino acids, such as 8-12, preferably of 8 to 11 amino acids. In
particular, TSPs have a length of 8 to 11 or 8-12 amino acids when
they bind to MHC-I. In another example, TSPs typically have a
length of 13 to 25 amino acids when they bind to MHC-II. TSPs may
differ in one or more amino acids from the TP in as long as they
meet the similarity scores outlined above and which may be
determined as explained below. Thus, if the amino acid sequence of
a TSP of 8 amino acids length is aligned to a given TP it may
comprise between 1 to 8 amino acids that are similar to the
corresponding amino acids of the TP. The other amino acids may be
identical or dissimilar to the TP. Accordingly, a TSP of 9 amino
acids length may comprise between 1 to 9 amino acids that are
similar to the corresponding amino acids of the TP; a TSP of 10
amino acids length may comprise between 1 to 10 amino acids that
are similar to the corresponding amino acids of the TP; a TSP of 11
amino acids length may comprise between 1 to 11 amino acids that
are similar to the corresponding amino acids of the TP, a TSP of 12
amino acids length may comprise between 1 to 12 amino acids that
are similar to the corresponding amino acids of the TP. If the TSP
is bound to MHC II it typically has a length of 13 to 25 amino
acids and accordingly, a TSP of 13 amino acids length may comprise
between 1 to 13 amino acids that are similar to the corresponding
amino acids of the TP; a TSP of 14 amino acids length may comprise
between 1 to 14 amino acids that are similar to the corresponding
amino acids of the TP; a TSP of 15 amino acids length may comprise
between 1 to 15 amino acids that are similar to the corresponding
amino acids of the TP, a TSP of 16 amino acids length may comprise
between 1 to 16 amino acids that are similar to the corresponding
amino acids of the TP, a TSP of 17 amino acids length may comprise
between 1 to 17 amino acids that are similar to the corresponding
amino acids of the TP; a TSP of 18 amino acids length may comprise
between 1 to 18 amino acids that are similar to the corresponding
amino acids of the TP; a TSP of 19 amino acids length may comprise
between 1 to 19 amino acids that are similar to the corresponding
amino acids of the TP, a TSP of 20 amino acids length may comprise
between 1 to 20 amino acids that are similar to the corresponding
amino acids of the TP. Another criteria for the selection of a TSP
to be used in the method of the present invention is its frequency
of presentation on primary normal tissues. The frequency of
presentation describes how often a TSP is presented on normal, i.e.
a healthy tissue--in contrast to the copy number which defines the
number of TSPs on different samples of a given healthy tissue, e.g.
if a certain TSP is detected on 6 out of 12 samples of adipose
tissue is has a frequency of presentation of 50%. The frequency of
presentation of a given TSP can be determined by art known methods
including MS analysis as used in Example 4 (see FIGS. 6 to 8, which
indicate frequency of presentation for three different TSPs). Thus,
it is preferred that a TSP is used in the method of the invention,
which has a frequency of presentation of at least 10% in at least
one healthy tissue, preferably, at least 20% in at least one
healthy tissue, more preferably at least 30% in at least one
healthy tissue. Preferably, the selected TSP is presented in at
least one, preferably at least two, more preferably at least three
healthy tissues. These tissues are preferably selected from those
that were analyzed regarding their presentation of TSP1, and TSP2,
respectively, in FIGS. 6 and 7.
[0086] Together with the copy number the frequency is an important
criterion to select a TSP for a given TP. The higher the similarity
to the TP and the higher the presentation frequency and CpC on
normal tissues, the higher the relevance of a TSP.
[0087] The term "irrelevant antigen complex" (IAC) refers in the
context of this invention to an AC comprising an irrelevant protein
antigen (IPA). Such an AC can be, e.g. an APC, or a multimerized
MHC-peptide complex that is soluble or MHC-peptide complexes
immobilized on a carrier. The irrelevant protein antigen is defined
in the following.
[0088] The term "irrelevant protein antigen" (IPA) refers in the
context of this invention to a protein antigen which is not bound
by a selected TCR. TCRs are screened for binding their respective
target peptides. Upon binding of a TCR to its target peptide a
desired immune reaction or T-cell mediated immune response is
triggered. Such a desired immune response will not be triggered by
an irrelevant peptide because an irrelevant peptide will not be
bound by a TCR in the screening. The IPA may be a protein encoded
by a housekeeping gene. Typically, the IPA has a similarity to the
amino acid sequence of the TP of at least less than 50%, at least
less than 40%, at least less than 30%, at least less than 20%, at
least less than 10%.
[0089] The term "irrelevant peptide" (IP) refers in the context of
this invention to a shorter peptide, part or fragment of the IPA.
The amino acid sequence of an IP comprises typically 8-16 amino
acids in length. Such an IP is typically bound to MHC-I. In some
examples, when an IP is bound to a MHC-II, an IP may comprise 13-25
amino acids in length. An IP may also comprise 13-18 amino acids in
length when bound to a MHC-II. The IP may be encoded by a
housekeeping gene.
[0090] "Housekeeping genes" in the context of this invention are
typically constitutive genes that are required for the maintenance
of basic cellular function and are expressed in all cells of an
organism under normal and pathophysiological conditions. Although
some housekeeping genes are expressed at relatively constant rates
in most non-pathological situations, the expression of other
housekeeping genes may vary depending on experimental conditions.
Housekeeping genes account for majority of the active genes in the
genome, and their expression is obviously vital to survival. The
housekeeping gene expression levels are fine-tuned to meet the
metabolic requirements in various tissues. Examples for
housekeeping genes are listed (non-exhaustive) as follows:
Transcription factor, translation factors, repressor molecules, RNA
splicing molecules, RNA binding proteins, ribosomal proteins,
mitochondrial ribosomal proteins, RNA polymerases, protein
processing genes, heat shock proteins, histone, cell cycle,
apoptosis, oncogenes, DNA repair, DNA replication, metabolism
involved genes, e.g. genes involved in carbohydrate metabolism,
citrate cycle, lipid metabolism, amino acid metabolism, NADH
dehydrogenase, cytochrome C oxidase, ATPase, lysosome, proteasome,
ribonuclease, thioreductases, receptors, channels, transporters,
HLA/immunoglobulin/cell recognition, kinases, cytoskeletal, growth
factors, tumor necrosis factor .alpha.. Similarly to the IPA, the
IP has a similarity to the amino acid sequence of the TP of at
least less than 50%, at least less than 40%, at least less than
30%, at least less than 20%, at least less than 10%, preferably at
least less than 30%, at least less than 20%, at least less than
10%.
[0091] The term "amino acid sequence identity" refers in the
context of this invention to the percentage of sequence identity
and is determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the sequence in the
comparison window can comprise additions or deletions (i.e. gaps)
as compared to the reference sequence (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 base or amino acid residue
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 window of comparison and multiplying the result by
100 to yield the percentage of sequence identity.
[0092] The term "identical" refers in the context of two or more
polypeptide or nucleic acid sequences, refers to two or more
sequences or subsequences that are the same, i.e. comprise the same
sequence of amino acids or nucleic acids. Sequences are
"substantially identical" to each other if they have a specified
percentage of amino acid residues that are the same (e.g., at least
70%, at least 75%, at least 80, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% identity over a specified
region), when compared and aligned for maximum correspondence over
a comparison window, or designated region as measured using one of
the following sequence comparison algorithms or by manual alignment
and visual inspection. These definitions also refer to the
complement of a test sequence. Accordingly, the term "at least 80%
sequence identity" is used throughout the specification with regard
to polypeptide and polynucleotide sequence comparisons. This
expression preferably refers to a sequence identity of at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% to the respective reference polypeptide or to the
respective reference polynucleotide.
[0093] The term "sequence comparison" refers in the context of this
invention to the process wherein one sequence acts as a reference
sequence, to which test sequences are compared. When using a
sequence comparison algorithm, test and reference sequences are
inputted into a computer, if necessary subsequence coordinates are
designated, and sequence algorithm program parameters are
designated. Default program parameters are commonly used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities or
similarities for the test sequences relative to the reference
sequence, based on the program parameters. In case where two
sequences are compared and the reference sequence is not specified
in comparison to which the sequence identity percentage is to be
calculated, the sequence identity is to be calculated with
reference to the longer of the two sequences to be compared, if not
specifically indicated otherwise. If the reference sequence is
indicated, the sequence identity is determined on the basis of the
full length of the reference sequence indicated by SEQ ID, if not
specifically indicated otherwise.
[0094] In a sequence alignment, the term "comparison window" refers
to those stretches of contiguous positions of a sequence which are
compared to a reference stretch of contiguous positions of a
sequence having the same number of positions. It is preferred that
the entire length of the PAI, preferably the TP is used as
comparison window in the alignment with the SPA and TSP,
respectively. If the TP is, e.g. a 10 amino acid long MHC 1
presented peptide the similarity is determined in a comparison
window of 10 amino acids. In this case a 9 amino acids long SPA
with one amino acid mismatch has an identity of 80% to the given
TP.
[0095] Methods of alignment of sequences for comparison are well
known in the art. Optimal alignment of sequences for comparison can
be conducted, for example, by the local homology algorithm of Smith
and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology
alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443,
1970), by the search for similarity method of Pearson and Lipman
(Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerized
implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Ausubel et al., Current
Protocols in Molecular Biology (1995 supplement)). Algorithms
suitable for determining percent sequence identity and sequence
similarity are the BLAST and BLAST 2.0 algorithms, which are
described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977),
and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively.
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information
(http://www.ncbi.nlm nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
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). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. 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, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, 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.
[0096] The term "similarity" of two amino acid sequences takes into
consideration the relatedness of two amino acids at a given
position (see, for example below Table 4). The similarity of two
amino acid sequences, e.g. in a TP and a TSP, can be determined
using the BLAST algorithm, which performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993). Preferred
settings for such an alignment are: wordlength of 3, and
expectation (E) of 10, and the use of the BLOSUM62 or PMBEC scoring
matrix (Kim et al. 2009 BMC Bioinformatics), preferably the PMBEC
scoring matrix is used in the determination of the similarity.
These matrices quantify amino acid similarity based for example on
evolutionary or functional similarity between amino acids, which
correlate well with the similarity according to physicochemical
parameters. For each substitution of an amino acid in a given TP
sequence a score (decimal value) can be calculated by using these
matrices, which indicates the similarity of the amino acid in the
TP sequence with the substituted amino acid in the TSP sequence.
Multiple substitutions can be considered by summing up the effect
(scores) of individual substitutions in the TP sequence. By
definition, the maximum score which can be achieved for a TSP is
provided by the unsubstituted TP sequence, whereas any substitution
leading to a TSP will be penalized in the scoring matrix and
ultimately lead to a lower score of a TSP. This maximum score is
however dependent on the length and amino acid sequence of the TP
(i.e. different TP sequences will have different maximum scores).
Typically, a longer amino acid sequences results in a higher score.
However, the score of a TP depends on the score allotted to the
amino acids it consists of. In order to be able to calculate and
compare the similarity of a TSP in reference to a TP without
considering the difference of maximum scores of distinct TP
sequences the respective decimal values are converted, which are
the result of calculating the similarity of a TSP in reference to a
TP, into a percentage score wherein the maximum score of a TP
sequence will therefore, always be 100%.
[0097] Another measure of similarity provided by the BLAST
algorithm is the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between amino acid
sequences would occur by chance. For example, an amino acid is
considered similar to a reference sequence if the smallest sum
probability in a comparison of the test amino acid to the reference
amino acid is less than about 0.2, typically less than about 0.01,
and more typically less than about 0.001. Semi-conservative and
especially conservative amino acid substitutions, wherein an amino
acid is substituted with a chemically related amino acid are
preferred. Typical substitutions are among the aliphatic amino
acids, among the amino acids having aliphatic hydroxyl side chain,
among the amino acids having acidic residues, among the amide
derivatives, among the amino acids with basic residues, or the
amino acids having aromatic residues. Typical semi-conservative and
conservative substitutions are indicated in below Table 4.
TABLE-US-00004 TABLE 4 Amino acids and conservative and semi-
conservative substitutions, respectively. Semi-conservative Amino
acid Conservative substitution substitution A G; S; TN; V; C C A;
V; L M; I; F; G D E; N; Q A; S; T; K; R; H E D; Q; N A; S; T; K; R;
F W; Y; L; M; H I; V; A G A S; N; T; D; E; N; Q H Y; F; K; R L; M;
A I V; L; M; A F; Y; W; G K R; H D; E; N; Q; S; T; A L M; I; V; A
F; Y; W; H; C M L; I; V; A F; Y; W; C; N Q D; E; S; T; A; G; K; R P
V; I L; A; M; W; Y; S; T; C; F Q N D; E; A; S; T; L; M; K; R R K; H
N; Q; S; T; D; E; A S A; T; G; N D; E; R; K T A; S; G; N; V D; E;
R; K; I V A; L; I M; T; C; N W F; Y; H L; M; I; V; C Y F; W; H L;
M; I; V; C
[0098] Changing from A, F, H, I, L, M, P, V, W or Y to C is
semi-conservative if the new cysteine remains as a free thiol.
Furthermore, the skilled person will appreciate that glycines at
sterically demanding positions should not be substituted and that P
should not be introduced into parts of the protein which have an
alpha-helical or a beta-sheet structure.
[0099] The term "detectable label" refers in the context of this
invention to a molecule which labels a different molecule or a cell
by allowing this different molecule to be selected due to a
property or specific characteristics the label exerts. Molecules
that are eligible for labelling are proteins, DNA or RNA or
synthetic materials such as beads or other suitable materials.
Regarding proteins, labeling strategies result in the covalent
attachment of different molecules, including biotin, reporter
enzymes, fluorophores, magnetic labels and radioactive isotopes, to
the target protein or peptide or nucleotide sequence. Single-cell
can be labeled using short DNA or RNA barcode `tags` to identify
reads that originate from the same cell in a sequencing experiment.
Labels may be a fluorescent label, e.g. xanthens, acridines,
oxazines, cynines, styryl dyes, coumarines, porphines,
metal-ligand-complexes, fluorescent proteins, nanocrystals,
perylenes and phtalocyanines, phycoerythrin (SA-PE),
streptavidin-allophycocyanin (SA-APC) or
streptavidin-brilliant-violet 421 (SA-BV421); RNA-barcodes or
DNA-barcodes or a radioactive label. A radioactive label is
typically a molecule wherein one or more atoms are replaced by the
radioactive counterparts, i.e. radio isotopes. Proteins, peptides,
DNA or RNA may be labeled radioactively. Magnetic labels may
comprise magnetic beads or magnetic nanoparticles whjch can be
coated with e.g. antibodies against a particular surface antigen.
Magnetic labels may be used in magnetic-activated cell sorting
(MACS).
[0100] The term "detectably different" refers in the context of
this invention to a scenario wherein two labels are present but may
only be different in the signal they are emitting. For example, two
cells may be labelled with a fluorescent label and are thus, not
distinguishable by the characteristics of the label as such, i.e.
the fluorescence. However, the fluorescence label attached to one
cell may signal in red wherein the fluorescence signal attached to
the second cell may signal in green. The two labels of the
exemplified cells are thus, detectably different.
[0101] The term "flow cytometry analysis" refers in the context of
this invention to a sorting technique comprising the measurement of
chemical and physical properties of a specific cell populations or
cell subpopulations in a sample. The sample usually is a suspension
and is adjusted to result in a flow of one cell at a time through a
detection unit, typically a laser beam that excites a fluorophore
and a light detector. The detected signal, e.g. light scattered by
the flow through of the cell, is characteristic to the cell, i.e.
its components. Multiple cells can be analyzed by this technique in
a short period of time. Routine applications of flow cytometry are
cell counting, cell sorting, determination of cell characteristic
and functions, diagnosis of diseases, e.g. cancer, detection of
biomarkers or detection of microorganisms. A popular flow cytometry
technique is fluorescence activated cell sorting (FACS). The FACS
technique harnesses the ability to label a target cell/cells with
fluorescent dye tags or labels which allows for the cell sorting
based on the individual labeling profile of a particular cell
population.
[0102] The term "magnetic-activated cell sorting" (MACS) refers to
a sorting technique that harnesses functional micro- or
nanoparticles that are conjugated with antibodies corresponding to
particular cell surface antigens. Under application of a magnetic
field gradient, the magnetically targeted cells can be separated in
either a positive or negative fashion with the respect to the
antigen employed. A skilled person is well aware of the different
kind of sorting analyses.
[0103] The term "specifically binding" refers in the context of
this invention to the binding of an antigen binding protein or
fragments thereof, e.g. an antibody or fragments thereof or a TCR
or fragments thereof, to a specific binding site of its target when
the target comprises specific and non-specific binding sites.
However, sometimes binding of a protein to closely related proteins
is unavoidable, then the actual binding to the target may be
specific but the protein is deemed to be non-specific in relation
to the intended target binding. An antigen binding protein or a
fragment thereof of the present invention is considered to bind
specifically to a given antigen, if it binds to the antigen with a
K.sub.d of 10.sup.-5 M or less when measured by surface plasmon
resonance (SPR) at RT. The dissociation constant (K.sub.d) for the
target to which the binding moiety specifically binds is at least
2-fold, at least 5-fold, at least 7-fold, 10-fold, preferably at
least 20-fold, more preferably at least 50-fold, even more
preferably at least 100-fold, 200-fold, 500-fold or 1000-fold lower
than the dissociation constant (K.sub.d) for the target to which
the binding moiety of the antigen binding protein or fragment
thereof does not bind specifically, for example the similar protein
antigen (SPA), preferably the target similar peptide (TSP).
[0104] Typically, if the antigen binding protein of the present
invention that specifically binds to a given TP is a TCR or a
fragment thereof it has a K.sub.d in the range of 3.times.10.sup.-5
to 1.times.10.sup.-7, 2.times.10.sup.-5 to 5.times.10.sup.-7,
1.times.10.sup.-5 to 1.times.10.sup.-60r 5.times.10.sup.-6 to
1.times.10.sup.-6. In this situation, it is preferred that the
antigen binding protein at the same time has a K.sub.d for the TP,
that is at least 2-fold lower, at least 5-fold, at least 7-fold,
10-fold, preferably at least 20-fold, more preferably at least
50-fold, even more preferably at least 100-fold, 200-fold, 500-fold
or 1000-fold lower than the K.sub.d for the target to which the
binding moiety of the antigen binding protein does not bind
specifically, for example the TSP. Thus, for example a selected TCR
may bind to the TP with a K.sub.D of 1.times.10.sup.-6 and to the
TSP with a K.sub.D of 1.times.10.sup.-5.
[0105] Typically, if the antigen binding protein of the present
invention that specifically binds to a given TP is an affinity
maturated TCR or a fragment thereof or a soluble molecule in a
bispecific format or fragment thereof, such as a TCER.RTM. molecule
or a fragment thereof, the K.sub.D in the range of
9.times.10.sup.-9 to 1.times.10.sup.-12, 8.times.10.sup.-9 to
5.times.10.sup.-12, 7.times.10.sup.-9 to 1.times.10.sup.-11,
6.times.10.sup.-9 to 2.times.10.sup.-11, 5.times.10.sup.-9 to
5.times.10.sup.-11, 4.times.10.sup.-9 to 8.times.10.sup.-11,
3.times.10.sup.-9 to 1.times.10.sup.-10. In this situation, it is
preferred that the antigen binding protein at the same time has a
K.sub.d for the TP, that is at least 2-fold lower, at least 5-fold,
at least 7-fold, 10-fold, preferably at least 20-fold, more
preferably at least 50-fold, even more preferably at least
100-fold, 200-fold, 500-fold or 1000-fold lower than the K.sub.d
for the target to which the binding moiety of the antigen binding
protein does not bind specifically, for example the TSP. Molecules
in the bispecific format referred to herein as "TCER.RTM."
molecules or "TCER.RTM." typically comprise a first polypeptide
that specifically binds to a surface molecule on a T cell and a
second polypeptide that specifically binds to a MHC-peptide
complex.
[0106] Typically, if the antigen binding protein of the present
invention is an antibody or a fragment thereof or a B-cell that
specifically binds to a given PAI, the K.sub.D is in the range of
9.times.10.sup.-9 to 1.times.10.sup.-12, 8.times.10.sup.-9 to
5.times.10.sup.-12, 7.times.10.sup.-9 to 1.times.10.sup.-11,
6.times.10.sup.-9 to 2.times.10.sup.-11, 5.times.10.sup.-9 to
5.times.10.sup.-11, 4.times.10.sup.-9 to 8.times.10.sup.-11,
3.times.10.sup.-9 to 1.times.10.sup.-1.degree.. In this situation,
it is preferred that the antigen binding protein at the same time
has a K.sub.d for the PAI, that is at least 2-fold lower, at least
5-fold, at least 7-fold, 10-fold, preferably at least 20-fold, more
preferably at least 50-fold, even more preferably at least
100-fold, 200-fold, 500-fold or 1000-fold lower than the K.sub.d
for the target to which the binding moiety of the antigen binding
protein does not bind specifically, for example the SPA.
[0107] In some instances, in particular in context of TCRs, if the
antigen binding protein of the present invention, in particular in
context of a TCR, specifically binds to a given TP might be
determined by using a functional assay, for example, in a TCR
activation assay, such as an IFN.gamma.-release assay. Accordingly,
a specific binding may be characterized by a response, such as
signal that is detected for a TP is more than 30%, more than 40%,
more than 50%, more than 60%, more than 70%, more than 80%, more
than 90%, more than 100% of the response, i.e. the signal, obtained
for the TSP in such an assay.
[0108] The term "selectively binding" refers in the context of this
invention to the characteristic of an antigen binding protein, such
as a TCR or antibody, to selectively recognize or bind to
preferably only one specific epitope and preferably shows no or
substantially no binding (no cross-reactivity) to another epitope,
peptide or protein. Assessing the threshold of epitope binding by
flow cytometry can be assessed by using non-tetramer stained
controls. The gates can be set according to the non-tetramer
stained control in a way that <0.01% cells appear in this gate.
This gate can be applied to a sample from the same donor that has
been stained with tetramer of interest. Cells which appear in this
gate are considered to bind selectively to the epitope of
interest.
[0109] The term "T-cell receptor" (TCR) refers in the context of
this invention to a heterodimeric cell surface protein of the
immunoglobulin super-family, which is associated with invariant
proteins of the CD3 complex involved in mediating signal
transduction. TCRs exist in .alpha..beta. and .gamma..delta. forms,
which are structurally similar but have quite distinct anatomical
locations and probably functions. The extracellular portion of
native heterodimeric .alpha..beta. TCR and .gamma..beta. TCR each
contain two polypeptides, each of which has a membrane-proximal
constant domain, and a membrane-distal variable domain. Each of the
constant and variable domains include an intra-chain disulfide
bond. The variable domains contain the highly polymorphic loops
analogous to the complementarity determining regions (CDRs) of
antibodies. The use of TCR gene therapy overcomes a number of
current hurdles. It allows equipping the subjects' (patients') own
T-cells with desired specificities and generation of sufficient
numbers of T-cells in a short period of time, avoiding their
exhaustion. The TCR will be transduced into potent T-cells (e.g.
central memory T-cells or T-cells with stem cell characteristics),
which may ensure better persistence, preservation and function upon
transfer. TCR-engineered T-cells will be infused into cancer
patients rendered lymphopenic by chemotherapy or irradiation,
allowing efficient engraftment but inhibiting immune suppression.
Native alpha-beta heterodimeric TCRs have an alpha chain and a beta
chain. Each alpha chain comprises variable, joining and constant
regions, and the beta chain also usually contains a short diversity
region between the variable and joining regions, but this diversity
region is often considered as part of the joining region. The
constant, or C, regions of TCR alpha and beta chains are referred
to as TRAC and TRBC respectively (Lefranc, (2001), Curr Protoc
Immunol Appendix 1: Appendix 10). Each variable region, herein
referred to as alpha variable domain and beta variable domain,
comprises three "complementarity determining regions" (CDRs)
embedded in a framework sequence, one being the hypervariable
region named CDR3. The alpha variable domain CDRs are referred to
as CDRa1, CDRa2, CDRa3, and the beta variable domain CDRs are
referred to as CDRb1, CDRb2, CDRb3. There are several types of
alpha chain variable (Valpha) regions and several types of beta
chain variable (Vbeta) regions distinguished by their framework,
CDR1 and CDR2 sequences, and by a partly defined CDR3 sequence. The
Valpha types are referred to in IMGT nomenclature by a unique TRAY
number, Vbeta types are referred in IMGT nomenclature to by a
unique TRBV number (Folch and Lefranc, (2000), Exp Clin Immunogenet
17(1): 42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet
17(2): 83-96; LeFranc and LeFranc, (2001), "T-cell Receptor
Factsbook", Academic Press). For more information on immunoglobulin
antibody and TCR genes see the international ImMunoGeneTics
information System.RTM., Lefranc M-P et al (Nucleic Acids Res. 2015
January; 43 (Database issue): D413-22; and http://www.imgt.org/). A
conventional TCR antigen-binding site usually includes six CDRs,
comprising the CDR set from each of an alpha and a beta chain
variable region, wherein CDR1 and CDR3 sequences are relevant to
the recognition and binding of the peptide antigen that is bound to
the MHC protein and the CDR2 sequences are relevant to the
recognition and binding of the MHC protein. Analogous to
antibodies, TCRs comprise framework regions which are amino acid
sequences interposed between CDRs, i.e. to those portions of TCR
alpha and beta chain variable regions that are relatively conserved
among different TCRs. The alpha and beta chains of a TCR each have
four FRs, herein designated FR1-a, FR2-a, FR3-a, FR4-a, and FR1-b,
FK3 b, FR4-b, respectively. Accordingly, the alpha chain variable
domain may thus be designated as
(FR1-a)-(CDRa1)-(FR2-1)-(CDRa2)-(1-R3-a)-(CDRa3)-(FR4-a) and the
beta chain variable domain may thus be designated as
(FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b).
[0110] A "disease caused by a virus or bacteria" may also be
referred to as a viral or bacterial infection. In the context of
the present invention, the virus causing the disease may be
selected from the group constituted of for example, human
immunodeficiency viruses (HIV), Humane Cytomegalievirus (HCMV),
cytomegalovirus (CMV), human papillomavirus (HPV), Hepatitis B
virus (HBV), Hepatitis C virus (HCV), human papillomavirus
infection (HPV), Epstein-Barr virus (EBV), Influenza virus,
preferably human immunodeficiency viruses (HIV). In the context of
the present invention, the bacteria causing the disease may be
Mycobacterium tuberculosis. The disease caused by this bacterium
is, thus, tuberculosis. It will be understood by the skilled in the
art, that when the antigen binding protein targets a viral
antigenic peptide, for instance, a HIV peptide, the antigen binding
protein may be for use in the treatment of HIV. Accordingly, an
antigen binding protein targeting the viral or bacterial antigenic
peptide TA-C is thus, suitable for use in the treatment of virus or
bacteria from which said antigenic viral or bacterial antigenic
peptide, is derived.
[0111] The term "immune disease" refers in the context of this
invention to a disease triggered by the immune system. The term
"disease" refers to an abnormal condition, especially an abnormal
medical condition such as an illness or injury, wherein a tissue,
an organ or an individual is not able to efficiently fulfil its
function anymore. In contrast, healthy tissue, organs or
individuals are referred to herein if no abnormal conditions are
present and the tissue, organ or individual is without pathological
finding. In healthy tissues random migration of cells is absent,
cells adhere to each other in structures characterizing the tissue
and assist in its function. No metastasis is present in healthy
tissue. Typically, but not necessarily, a disease is associated
with specific symptoms or signs indicating the presence of such
disease. The presence of such symptoms or signs may thus, be
indicative for a tissue, an organ or an individual suffering from a
disease. An alteration of these symptoms or signs may be indicative
for the progression of such a disease. A progression of a disease
is typically characterized by an increase or decrease of such
symptoms or signs which may indicate a "worsening" or "bettering"
of the disease. The "worsening" of a disease is characterized by a
decreasing ability of a tissue, organ or organism to fulfil its
function efficiently, whereas the "bettering" of a disease is
typically characterized by an increase in the ability of a tissue,
an organ or an individual to fulfil its function efficiently. A
tissue, an organ or an individual being at "risk of developing" a
disease is in a healthy state but shows potential of a disease
emerging. Typically, the risk of developing a disease is associated
with early or weak signs or symptoms of such disease. In such case,
the onset of the disease may still be prevented by treatment.
Examples of a disease include but are not limited to infectious
diseases, traumatic diseases, inflammatory diseases, cutaneous
conditions, endocrine diseases, intestinal diseases, neurological
disorders, joint diseases, genetic disorders, autoimmune diseases,
and various types of cancer. Healthy tissue as defined herein
usually comprises or consists of healthy cells.
[0112] The term "neoplastic disease" refers in the context of this
invention to diseases characterized by an abnormal growth of cells,
also known as a tumor. Neoplastic diseases are conditions that
cause tumor growth. Malignant tumors are cancerous and can grow
slowly or quickly and carry the risk of metastasis or spreading to
multiple tissues and organs. By "tumor" is meant an abnormal group
of cells or tissue that grows by a rapid, uncontrolled cellular
proliferation and continues to grow after the stimuli that
initiated the new growth cease. Tumors show partial or complete
lack of structural organization and functional coordination with
the normal tissue, and usually form a distinct mass of tissue,
which may be either benign or malignant. A neoplastic disease may
result in cancer, wherein exemplified cancer type diseases include
but are not limited to Basal cell carcinoma, Bladder cancer, Bone
cancer, Brain tumor, Breast cancer, Burkitt lymphoma, Cervical
cancer, Colon Cancer, Cutaneous T-cell lymphoma, Esophageal cancer,
Retinoblastoma, Gastric (Stomach) cancer, Gastrointestinal stromal
tumor, Glioma, Hodgkin lymphoma, Kaposi sarcoma, Leukemias,
Lymphomas, Melanoma, Oropharyngeal cancer, Ovarian cancer,
Pancreatic cancer, Pleuropulmonary blastoma, Prostate cancer,
Throat cancer, Thyroid cancer, and Urethral cancer.
[0113] The term "treating" or "treatment" refers in the context of
the present invention to a therapeutic use, e.g. for a subject in
need thereof, i.e. suffering a disease or disorder) and means
reversing, alleviating, inhibiting the progress of one or more
symptoms of such a disease, disorder or condition. Therefore,
treatment does not only refer to a treatment that leads to a
complete cure of the disease, but also to treatments that slow down
the progression of the disease and/or prolong the survival of the
subject.
[0114] The term "immune cell specific surface marker" refers in the
context of this invention to cell surface antigens, which serve as
monograms to help identify and classify immune cells. Examples of
such markers that characterize different T-cell subtypes are
indicated in Table 1 above. The majority of immune cell specific
surface markers are molecules or antigens within cell's plasma
membrane. These molecules serve not only as markers but they also
have key functional roles.
[0115] The term "growth factor" or "differentiation factor" is used
in the context of this invention interchangeably and refers to
molecules that are capable of stimulation cellular growth, cell
proliferation and cellular differentiation and regulate multiple
cellular processes. Growth factors are usually proteins or steroid
hormones. Examples of prevailing molecules are listed in the
following (non-exhaustive enumeration): Growth factors, such as
colony stimulating factor (CSF), Macrophage colony-stimulating
factor (M-CSF), Granulocyte colony-stimulating factor (G-CSF) and
Granulocyte macrophage colony-stimulating factor (GM-CSF);
epidermal growth factor (EGF); erythropoietin (EPO); fibroblast
growth factor (FGF); foetal bovine somatotropin (FBS); hepatocyte
growth factor (HGF); insulin; insulin like growth factor (IGF);
interleukins; neuregulins; neutrotrophins; T-cell growth factor
(TCGF); transforming growth factor (TGF); tumor necrosis factor
alpha (TNF.alpha.); vascular endothelial growth factor (VEGF).
[0116] The term "nucleic acid" refers in the context of this
invention to single or double-stranded oligo- or polymers of
deoxyribonucleotide or ribonucleotide bases or both. Nucleotide
monomers are composed of a nucleobase, a five-carbon sugar (such as
but not limited to ribose or 2'-deoxyribose), and one to three
phosphate groups. Typically, a nucleic acid is formed through
phosphodiester bonds between the individual nucleotide monomers. In
the context of the present invention, the term nucleic acid
includes but is not limited to ribonucleic acid (RNA) and
deoxyribonucleic acid (DNA) molecules but also includes synthetic
forms of nucleic acids comprising other linkages (e.g., peptide
nucleic acids as described in Nielsen et al. (Science
254:1497-1500, 1991). Typically, nucleic acids are single- or
double-stranded molecules and are composed of naturally occurring
nucleotides. The depiction of a single strand of a nucleic acid
also defines (at least partially) the sequence of the complementary
strand. The nucleic acid may be single or double stranded or may
contain portions of both double and single stranded sequences.
Exemplified, double-stranded nucleic acid molecules can have 3' or
5' overhangs and as such are not required or assumed to be
completely double-stranded over their entire length. The nucleic
acid may be obtained by biological, biochemical or chemical
synthesis methods or any of the methods known in the art, including
but not limited to methods of amplification, and reverse
transcription of RNA. The term nucleic acid comprises chromosomes
or chromosomal segments, vectors (e.g., expression vectors),
expression cassettes, naked DNA or RNA polymer, primers, probes,
cDNA, genomic DNA, recombinant DNA, cRNA, mRNA, tRNA, microRNA
(miRNA) or small interfering RNA (siRNA). A nucleic acid can be,
e.g., single-stranded, double-stranded, or triple-stranded and is
not limited to any particular length. Unless otherwise indicated, a
particular nucleic acid sequence comprises or encodes complementary
sequences, in addition to any sequence explicitly indicated.
[0117] The term "vector" refers in the context of this invention to
a polynucleotide that encodes a protein of interest or a mixture
comprising polypeptide(s) and a polynucleotide that encodes a
protein of interest, which is capable of being introduced or of
introducing proteins and/or nucleic acids comprised therein into a
cell. Examples of vectors include but are not limited to plasmids,
cosmids, phages, viruses or artificial chromosomes. A vector is
used to introduce a gene product of interest, such as e.g. foreign
or heterologous DNA into a host cell. Vectors may contain
"replicon" polynucleotide sequences that facilitate the autonomous
replication of the vector in a host cell. Foreign DNA is defined as
heterologous DNA, which is DNA not naturally found in the host
cell, which, for example, replicates the vector molecule, encodes a
selectable or screenable marker, or encodes a transgene. Once in
the host cell, the vector can replicate independently of or
coincidental with the host chromosomal DNA, and several copies of
the vector and its inserted DNA can be generated. In addition, the
vector can also contain the necessary elements that permit
transcription of the inserted DNA into an mRNA molecule or
otherwise cause replication of the inserted DNA into multiple
copies of RNA. Vectors may further encompass "expression control
sequences" that regulate the expression of the gene of interest.
Typically, expression control sequences are polypeptides or
polynucleotides such as promoters, enhancers, silencers,
insulators, or repressors. In a vector comprising more than one
polynucleotide encoding for one or more gene products of interest,
the expression may be controlled together or separately by one or
more expression control sequences. More specifically, each
polynucleotide comprised on the vector may be control by a separate
expression control sequence or all polynucleotides comprised on the
vector may be controlled by a single expression control sequence.
Polynucleotides comprised on a single vector controlled by a single
expression control sequence may form an open reading frame. Some
expression vectors additionally contain sequence elements adjacent
to the inserted DNA that increase the half-life of the expressed
mRNA and/or allow translation of the mRNA into a protein molecule.
Many molecules of mRNA and polypeptide encoded by the inserted DNA
can thus be rapidly synthesized. Such vectors may comprise
regulatory elements, such as a promoter, enhancer, terminator and
the like, to cause or direct expression of said polypeptide upon
administration to a subject. Examples of promoters and enhancers
used in the expression vector for animal cell include early
promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR
promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et
al. 1987), promoter (Mason J O et al. 1985) and enhancer (Gillies S
D et al. 1983) of immunoglobulin H chain and the like. Any
expression vector for animal cell can be used, as long as a gene
encoding the human antibody C region can be inserted and expressed.
Examples of suitable vectors include pAGE107 (Miyaji H et al.
1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al.
1984), pKCR (O'Hare K et al. 1981), pSG1 beta d2-4-(Miyaji H et al.
1990) and the like. Other examples of plasmids include replicating
plasmids comprising an origin of replication, or integrative
plasmids, e.g. pUC, pcDNA, pBR.
[0118] The term "antigen binding part" or "or antigen binding
fragment" in the context of the present invention refers to
molecules, in particular amino acid chains, that are shorter in
length but which retain the binding specificity and/or selectivity
of the parent protein because it still comprises the essential
amino acid sequence or sequences which are responsible for the
binding specificity and/or selectivity of the parent protein. An
"antigen binding part" or "or antigen binding fragment" is
considered to have retained the binding specificity, if it's
K.sub.d to the target of the parent protein measured as outlined
below is at least 10-fold higher or less, 5-fold higher or less,
3-fold higher or less 2-fold higher or less or identical to the
K.sub.d of the parent protein. Antigen binding fragments of TCRs
are, e.g. the variable domains of the alpha and beta chain, and
antigen binding parts of antibodies are the variable light and
heavy chain. Antigen binding parts of a TCR, BCR or an antibody are
the CDRs that are positioned in the respective variable regions of
the alpha and beta or light and heavy chain. Thus, if the
Assessment of binding and/or specificity of an antigen binding
protein, e.g., an antibody, TCR, BCR or immunologically functional
part or fragment thereof, can be conducted by binding affinity
measurements of e.g. a TCR to its target peptide or an antibody to
its antigen. The term "fragment" used herein refers to naturally
occurring fragments (e.g. splice variants or peptide fragments) as
well as artificially constructed fragments, in particular to those
obtained by gene-technological means.
[0119] The term "K.sub.d" (measured in "mol/L", sometimes
abbreviated as "M") in the context of the present invention refers
to the dissociation equilibrium constant of the particular
interaction between a binding moiety (e.g. an antibody or fragment
thereof) and a target molecule (e.g. an antigen or epitope
thereof). Affinity can be measured by common methods known in the
art, including but not limited to surface plasmon resonance (SPR)
based assay (such as the BIAcore assay); biolayer interferometry
(BLI), enzyme-linked immunoabsorbent assay (ELISA); and competition
assays (e.g. radio immuno assays (RIA)). Low-affinity antibodies
generally bind antigen slowly and tend to dissociate readily,
whereas high-affinity antibodies generally bind antigen faster and
tend to remain bound longer. A variety of methods of measuring
binding affinity are known in the art, any of which can be used for
the purposes of the present invention. The IQ's indicated for
various antigen binding proteins throughout this disclosure are
measured at room temperature, i.e. 20.degree. C., by SPR.
[0120] The term "B-cell receptor" (BCR) refers to a receptor with
an antigen like structure present on the surface of B cells. A
B-cell is activated by its first encounter with an antigen that
binds to its receptor (its "cognate antigen"), the cell
proliferates and differentiates to generate a population of
antibody-secreting plasma B-cells and memory B-cells. The BCR
controls B-cell activation by biochemical signaling and by physical
acquisition of antigens from immune synapses with
antigen-presenting cells and has two crucial functions upon
interaction with the antigen. One function is signal transduction,
involving changes in receptor oligomerization. The second function
is to mediate internalization for subsequent processing of the
antigen and presentation of peptides to helper T-cells. The portion
of the BCR that recognizes antigens is made up of three disparate
genetic regions, termed V, D, and J, that are spliced and
recombined at the genetic level in a combinatorial process unique
to the immune system. The immunoglobulin molecules that form a type
1 transmembrane receptor protein are usually located on the outer
surface of a B-lymphocyte. Structurally, the BCR comprises a
membrane-bound immunoglobulin molecule of one isotype (IgD, IgM,
IgA, IgG, or IgE) and a signal transduction moiety: A
Ig-.alpha./Ig-.beta. (CD79) heterodimer, linked by disulfide
bridges. Each member of the dimer spans the plasma membrane and has
a cytoplasmic tail bearing an immunoreceptor tyrosine-based
activation motif (ITAM).
[0121] The term "antibody" in the context of the present invention
refers to secreted immunoglobulins which lack the transmembrane
region and can thus, be released into the bloodstream and body
cavities. Human antibodies are grouped into different isotypes
based on the heavy chain they possess. There are five types of
human Ig heavy chains denoted by the Greek letters: .alpha.,
.gamma., .delta., .epsilon., and .mu.. The type of heavy chain
present defines the class of antibody, i.e. these chains are found
in IgA, IgD, IgE, IgG, and IgM antibodies, respectively, each
performing different roles, and directing the appropriate immune
response against different types of antigens. Distinct heavy chains
differ in size and composition; and may comprise approximately 450
amino acids (Janeway et al. (2001) Immunobiology, Garland Science).
IgA is found in mucosal areas, such as the gut, respiratory tract
and urogenital tract, as well as in saliva, tears, and breast milk
and prevents colonization by pathogens (Underdown & Schiff
(1986) Annu. Rev. Immunol. 4:389-417). IgD mainly functions as an
antigen receptor on B-cells that have not been exposed to antigens
and is involved in activating basophils and masT-cells to produce
antimicrobial factors (Geisberger et al. (2006) Immunology
118:429-437; Chen et al. (2009) Nat. Immunol. 10:889-898). IgE is
involved in allergic reactions via its binding to allergens
triggering the release of histamine from masT-cells and basophils.
IgE is also involved in protecting against parasitic worms (Pier et
al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG
provides the majority of antibody-based immunity against invading
pathogens and is the only antibody isotype capable of crossing the
placenta to give passive immunity to fetus (Pier et al. (2004)
Immunology, Infection, and Immunity, ASM Press). In humans there
are four different IgG subclasses (IgG1, 2, 3, and 4), named in
order of their abundance in serum with IgG1 being the most abundant
(66%), followed by IgG2 (23%), IgG3 (.about.7%) and IgG
(.about.4%). The biological profile of the different IgG classes is
determined by the structure of the respective hinge region. IgM is
expressed on the surface of B-cells in a monomeric form and in a
secreted pentameric form with very high avidity. IgM is involved in
eliminating pathogens in the early stages of B-cell mediated
(humoral) immunity before sufficient IgG is produced (Geisberger et
al. (2006) Immunology 118:429-437). Antibodies are not only found
as monomers but are also known to form dimers of two Ig units (e.g.
IgA), tetramers of four Ig units (e.g. IgM of teleost fish), or
pentamers of five Ig units (e.g. mammalian IgM). Antibodies are
typically made of four polypeptide chains comprising two identical
heavy chains and identical two light chains which are connected via
disulfide bonds and resemble a "Y"-shaped macro-molecule. Each of
the chains comprises a number of immunoglobulin domains out of
which some are constant domains and others are variable domains
Immunoglobulin domains consist of a 2-layer sandwich of between 7
and 9 antiparallel .about.-strands arranged in two .about.-sheets.
Typically, the heavy chain of an antibody comprises four Ig domains
with three of them being constant (CH domains: CHI. CH2. CH3)
domains and one of the being a variable domain (VH). The light
chain typically comprises one constant Ig domain (CL) and one
variable Ig domain (V L). The VH and VL regions can be further
subdivided into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR). Each VH and VL is
composed of three CDRs and four 1-Rs, arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. The
constant regions of the antibodies may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (e.g., effector cells) and the first component
(Clq) of the classical complement system. Term antibody as used
herein also encompasses a chimeric antibody, a humanized antibody
or a human antibody.
[0122] The term "antigen-binding fragment" of an antibody, TCR, or
BCR or CAR (or "binding portion" or "fragment"), as used herein,
refers to one or more fragments of an antibody TCR, BCR or CAR that
retain the ability to specifically bind to an antigen. It has been
shown that the antigen-binding function of an antibody, of a TCR,
of a BCR or CAR can be performed by fragments of a full-length
antibody, TCR, BCR or CAR. Examples of binding fragments of
antibodies encompassed within the term "antigen-binding portion of
an antibody, BCR or CAR "include (i) Fab fragments, monovalent
fragments consisting of the VL, VH, CL and CH domains; (ii)
F(ab').sub.2 fragments, bivalent fragments comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii)
Fd fragments consisting of the VH and CH domains; (iv) Fv fragments
consisting of the VL and VH domains of a single arm of an antibody,
(v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which
consist of a VH domain; (vi) isolated complementarity determining
regions (CDR), and (vii) combinations of two or more isolated CDRs
which may optionally be joined by a synthetic linker. Furthermore,
although the two domains of the Fv fragment, VL and VH, are coded
for by separate genes, they can be joined, using recombinant
methods, by a synthetic linker that enables them to be made as a
single protein chain in which the VL and VH regions pair to form
monovalent molecules (known as single chain Fv (scFv); see e.g.,
Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain
antibodies are also intended to be encompassed within the term
"antigen-binding fragment" of an antibody, BCR or CAR. The term
"antigen binding portion of a TCR" comprises at least CDR1 and CDR3
of the alpha and beta chain of a TCR, preferably CDR1, CDR2 and
CDR3 of the alpha and beta chain. While these CDRs are preferably
comprised in the context of their natural framework regions, they
may also be comprised in another protein--a so called protein
scaffold--that positions them to each other in a similar way as
they are positioned in an alpha and/or beta chain. The antigen
binding portion of a TCR comprises preferably the variable domain
of the alpha and beta chain. The antigen binding fragments of
antibodies, TCRs, BCRs or CARs can be included in a monomeric,
dimeric, trimeric, tetrameric or multimeric protein complex to
provide such complex with one or more different antigen binding
specificities. Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding fragment of an antibody". Further
formats in which antigen binding fragments of an antibody are used
to create monovalent, bivalent or multivalent binding molecules are
known in the art and are termed diabody, a tetrabody, and a
nanobody. Similarly, to scFV's single chain TCRs comprise the
variable domains of alpha and beta chain on one protein chain
linked by a linker.
[0123] As used in this specification the term "subject" relates to
an "individual", "subject", or "patient" which are used
interchangeably herein and refer to any mammal that may benefit
from the present invention. In particular, the "individual" is a
human being. The subject can be a healthy subject.
[0124] The term "subject in need thereof" in the context of this
invention refers to a subject that suffers or is at risk of
suffering a disease, for example a proliferative disease or
disorder, a disease caused by a virus or a disease caused by
bacteria. Such a proliferative disease or disorder, for example
cancer, involve the unregulated and/or inappropriate proliferation
of cells. The proliferative disorder or disease may be, for
example, a tumor disease characterized by the expression of the
TAA, more particular of the TAA, in a cancer or tumor cell of said
tumor disease.
[0125] Accordingly, a particularly preferred cancer is a TA
positive cancer, in particular a TAA positive cancer.
[0126] Abbreviations of frequently used terms throughout the claims
and specification of the present invention:
TABLE-US-00005 TABLE 5 Abbreviations of frequently used terms.
Antigen complex AC B-cell receptor BCR Chimeric antigen receptor
CAR Complementary determining regions CDR Fluorescence activated
cell sorting FACS Human leukocyte antigen HLA Irrelevant antigen
complex IAC Irrelevant peptide IP Irrelevant protein antigen IPA
Magnetic activated cell sorting MACS Major histocompatibility
complex I/II MHC I/II Protein antigen of interest PAI Similar
protein antigen SPA T-cell receptor TCR Target peptide TP Target
similar peptide TSP Tumor-associated antigen TAA
Embodiments
[0127] In the following different aspects of the invention are
defined in more detail. Each aspect so defined may be combined with
any other aspect or aspects unless clearly indicated to the
contrary. In particular, any feature indicated as being preferred
or advantageous may be combined with any other feature or features
indicated as being preferred or advantageous.
[0128] Immunotherapy constitutes an exciting and rapidly evolving
field, and the demonstration that genetically modified T-cell
receptors (TCRs) can be used to produce T-lymphocyte populations of
desired specificity offers new opportunities for antigen-specific
T-cell therapy.
[0129] Overall, TCR-modified T-cells have the ability to target a
wide variety of self and non-self-targets through the normal
biology of a T-cell. However, "off-tumor/on-target" or
"off-tumor/off-target" effects can lead to tremendously undesired
effects of immune related toxicity. By including similar protein
antigens (similar peptides) already at the stage of identification
of TCRs, the inventors are able to exclude a proportion of
cross-reactive T-cells before the characterization process and by
that enhancing the efficiency of the whole TCR discovery procedure.
For that purpose, 1D-labeled or 2D-labeled similar-peptide
multimers are included to the staining panel. An in-house database
allowed the inventors to identify highly relevant, target-sequence
similar peptides found on normal human tissue. Such target-sequence
similar peptides pose a safety risk if recognized by a TCR that is
supposed to be developed towards clinical use. Therefore, the
inventors have developed an in-house search algorithm that combines
public and in house genomic database searches for target-similar
peptides with results of actual MS-detected peptides on healthy
tissue from the in-house database. The inventors use a set of
target-similar peptides early during TCR identification, enabling
early de-selection of cross-reactive TCRs. For this purpose,
fluorochrome (streptavidin) labelled peptide major
histocompatibility complex (pMHC) tetramers are generated both for
the target peptide as well as for target-similar peptides,
distinguishable by at least one different fluorochrome. Cells
positive for both the target as well as the similar peptide are
excluded from T-cell sorting for downstream TCR identification.
[0130] This surprising finding provides inter alia the following
advantages over the art: (i) reduction of cross-reactivity of
selected TCRs with similar peptides on healthy tissues (ii)
increased safety profile of selected TCRs; (iii) efficient and fast
identification and characterization of TCRs due to early stage
selection of target specific TCRs; (v) specific TCR selection by
exclusion of similar peptide binding during sorting (vi) TCRs
exerting reduced off target and off-tumor cytotoxicity; and (vii)
the provision of improved specific, selective and safe TCRs.
[0131] A first aspect of the invention relates to a method for
selecting a cell or a virus expressing on its surface an
antigen-binding protein specifically and/or selectively binding to
a protein antigen of interest (PAI) comprising the following steps:
[0132] (i) providing a cell population or a virus population;
[0133] (ii) contacting the cell population or the virus population
of step (i) with a first antigen complex (1.sup.st AC) comprising
the PAI and a detectable label A or with the PAI comprising a
detectable label A; [0134] (iii) contacting the cell population or
the virus population of step (i) with at least a second antigen
complex (2.sup.nd AC) comprising a similar protein antigen (SPA),
wherein the amino acid sequence of the SPA differs by at least 1
amino acid from the amino acid sequence of the PAI and wherein the
2.sup.nd AC comprises a detectable label B; or with the SPA and a
detectable label B; and [0135] (iv) selecting at least cell or a
virus that specifically and/or selectively binds to the 1.sup.st
AC, wherein the detectable label A and the detectable label B are
detectably different from each other.
[0136] In one embodiment of the first aspect of the invention a
cell is selected based on the principle of counterselection:
MHC-presented short peptides of tumor antigens (TP) that are
preferably expressed on diseased tissue are labeled with a
detectable label and peptides with a similar sequence (TSP) that
are expressed on healthy tissues are labeled with a detectable
label. The labels used in this approach are detectably different.
Upon contacting a cell, preferably a T-cell, with the TP and the
TSP, the cell binds to either the TP, the TSP or to both the TP and
the TSP or none and is either selected based on a positive
selection criterion or on a negative selection criterion. In a
conventional sorting approach, the following cells with their
respective detectable cell signal can be identified: One cell can
be detected by detecting the signal of the TP's label, i.e. the
peptide of interest in case a cell is bound to the MHC presented
peptide. Another cell can signal by the detection of the TP's
label, i.e. the peptide of interest in case an immune cell is bound
to the MHC presented peptide and by the detection of the TSP's
label. A third cell can solely signal by the detection of the TSP's
label. Positively selected are only those cells which are
detectable by the TP's label because, in case the cell is a T-cell,
this is the cell with a TCR of interest capable of binding to the
MHC-presented peptides. The counterselection (or negative
selection) can be described as follows: Cells detected by two
labels, i.e. the label of TP and the TSP are negatively selected
because these cells bind to the TSP beside binding to the TP. Cells
only detected by the TSP's label are also negatively selected as
they do only bind the TSP which is expressed on healthy tissue and
generally, binding to TSP should be avoided in order to decrease
off target effects. Often, the binding of a given cell to a given
PAI, preferably a TP and one or more SPAs, preferably TSPs, is not
all or nothing. Thus, the selection may also be based on relative
differences of the binding of the PAI, preferably a TP, and a SPA,
preferably a TSP. Cells are considered to specifically bind to a
PAI, preferably a TP, if their binding is at least 2-fold, at least
3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least
7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at
least 15-fold, at least 20-fold, at least 30-fold, at least
40-fold, at least 50-fold, at least 70-fold, at least 100-fold, at
least 200-fold stronger at identical concentration of the PAI and
the SPA. Preferably, the binding is at least 10-fold stronger to
the PAI, preferably the TP, than to any of the one or more SPAs,
preferably TSPs used in the method of the invention. It is even
more preferably, that the binding is at least 20-fold, more
preferably at least 50-fold stronger. The strength of the binding
or affinity of a given cell, for example a T-cell or its TCR, or a
B-cell can be determined by a variety of assays and is commonly
indicated as the dissociation constant (K.sub.d) of the TCR.
However, for the purpose of the method of the present invention an
exact determination of the K.sub.d of the cell to a given PAI,
preferably a TP and one or more SPAs, preferably TSPs, is not
required.
[0137] It is sufficient to determine relative binding strength,
which can be, for example, determined by FACS analysis of cells in
which the fluorescence signal of a given TP with the fluorescence
signal of one or more TSPs is compared. In such a determination the
relative molar amounts of the PAI, preferably the TP, and of the
one or more SPA, preferably TSPs, has to be taken into
consideration. If TP and TSPs are added to the cells at the same
molar amounts and differences in fluorescent intensity of the
respective labels used are accounted for, a cell specifically binds
to a PAI, preferably TP, if an equimolar basis of the SPA,
preferably TSP, shows at least 10-fold stronger fluorescence
attributable to the PAI, preferably to the TP, than to the SPA,
preferably TSP. The skilled person in the art understands that a
change of the molar ratios of PAI, preferably TP, and the SPAs,
preferably TSPs, which are contacted with the cell population in
steps (ii) and (iii) can be accounted for when selecting the cells
by adapting the gating accordingly.
[0138] In one embodiment of the first aspect of the present
invention the selected cell is a mammalian cell that expresses a
heterologous antigen binding protein or a yeast cell that expresses
a heterologous antigen binding protein. The mammalian cell can be
any mammalian cell, such as a human cell, a mouse cell, preferably
a humanized mouse cell, a rat cell, a pig cell, a monkey cell or a
dog cell. Typically, the mammalian cell can be any antigen
presenting cell (APC). Preferably, the mammalian cell is a human
cell. In particular, the mammalian cell is engineered to express a
heterologous antigen binding protein, such as a TCR or fragments
thereof, or a BCR or fragments thereof or a CAR or fragments
thereof or an antibody or fragments thereof. If the selected cell
is a yeast cell expressing a heterologous antigen binding protein,
it is preferred that such a yeast cell is a Saccharomyces
cerevisiae yeast cell. In particular, the yeast cell is engineered
to express a heterologous antigen binding protein, such as a TCR or
fragments thereof, or a BCR or fragments thereof or a CAR or
fragments thereof or an antibody or fragments thereof.
[0139] In another embodiment of the first aspect of the present
invention the method selects a virus. The selected virus can be any
virus selected from the group consisting of a double-stranded DNA
virus, preferably Myoviridae, Siphoviridae, Podoviridae,
Herpesviridae, Adenoviridae, Baculoviridae, Papillomaviridae,
Polydnaviridae, Polyomaviridae, Poxviridae; a single-stranded DNA
virus, preferably Anelloviridae, Inoviridae, Parvoviridae;
double-stranded RNA virus, preferably Reoviridae; a single-stranded
RNA virus, preferably Coronaviridae, Picornaviridae, Caliciviridae,
Togaviridae, Flaviviridae, Astroviridae, Arteriviridae,
Hepeviridae; negative-sense single-stranded RNA virus, preferably
Arenaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae,
Bunyaviridae, Orthomyxoviridae, Bornaviridae; a single-stranded RNA
reverse transcribing virus, preferably Retroviridae; or a
double-stranded RNA reverse transcribing virus, preferably
Caulimoviridae, Hepadnaviridae. More preferably, the selected virus
is a bacteriophage. The bacteriophage is preferably selected from
the group consisting of bacteriophage T4 lambda (T4.lamda.) phage,
T7 phage, fd filamentous phage, preferably filamentous phage M13. A
selected virus, for example a phage, can be bound to beads, for
example magnetic beads which are suitable for sequential magnetic
sorting. In this embodiment, it is preferred that labels, such as
label A and label B are barcode labels, preferably RNA-barcodes or
DNA-barcodes as described herein above.
[0140] In another embodiment of the first aspect of the present
invention the method for selecting a cell comprises in step (i)
providing a cell population. Preferably, the cell population
comprises eukaryotic cells. More preferably eukaryotic cells are
mammalian cells expressing a library of heterologous antigen
binding proteins or yeast cells expressing a library of
heterologous antigen binding proteins. The method of the first
aspect of the present invention can, thus, be used for example in a
yeast display.
[0141] In another embodiment of the first aspect of the invention
the method for selecting a virus comprises in step (i) providing a
virus population. Preferably, the virus population comprises
viruses expressing a library of heterologous antigen binding
proteins. In a preferred embodiment the virus population comprises
bacteriophages and thus, the method of the first aspect of the
present invention can be used, for example, in a phage display.
[0142] Another embodiment of the first aspect the present invention
relates to a method for selecting an immune cell expressing on its
surface an antigen-binding protein specifically and/or selectively
binding to a protein antigen of interest (PAI) comprising the
following steps: [0143] (i) providing a cell population comprising
immune cells; [0144] (ii) contacting the cell population of step
(i) with a first antigen complex (1.sup.st AC) comprising the PAI
and a detectable label A or with the PAI comprising a detectable
label A; [0145] (iii) contacting the cell population of step (i)
with at least a second antigen complex (2.sup.nd AC) comprising a
similar protein antigen (SPA), wherein the amino acid sequence of
the SPA differs by at least 1 amino acid from the amino acid
sequence of the PAI and wherein the 2.sup.nd AC comprises a
detectable label B; or with the SPA and a detectable label B; and
[0146] (iv) selecting at least one immune cell that specifically
and/or selectively binds to the 1.sup.st AC, wherein the detectable
label A and the detectable label B are detectably different from
each other.
[0147] In one embodiment of the first aspect of the invention an
immune cell is selected based on the principle of counterselection:
MHC-presented short peptides of tumor antigens (TP) that are
preferably expressed on diseased tissue are labeled with a
detectable label and peptides with a similar sequence (TSP) that
are expressed on healthy tissues are labeled with a detectable
label. The labels used in this approach are detectably different.
Upon contacting an immune cell, preferably a T-cell, with the TP
and the TSP, the immune cell binds to either the TP, the TSP or to
both the TP and the TSP or none and is either selected based on a
positive selection criterion or on a negative selection criterion.
In a conventional sorting approach, the following cells with their
respective detectable cell signal can be identified: One cell can
be detected by detecting the signal of the TP's label, i.e. the
peptide of interest in case an immune cell is bound to the MHC
presented peptide. Another cell can signal by the detection of the
TP's label, i.e. the peptide of interest in case an immune cell is
bound to the MHC presented peptide and by the detection of the
TSP's label. A third cell can solely signal by the detection of the
TSP's label. Positively selected are only those cells which are
detectable by the TP's label because, in case the immune cell is a
T-cell, this is the cell with a TCR of interest capable of binding
to the MHC-presented peptides. The counterselection (or negative
selection) can be described as follows: Cells detected by two
labels, i.e. the label of TP and the TSP are negatively selected
because these cells bind to the TSP beside binding to the TP. Cells
only detected by the TSP's label are also negatively selected as
they do only bind the TSP which is expressed on healthy tissue and
generally, binding to TSP should be avoided in order to decrease
off target effects. Often, the binding of a given immune cell to a
given PAI, preferably a TP and one or more SPAs, preferably TSPs,
is not all or nothing. Thus, the selection may also be based on
relative differences of the binding of the PAI, preferably a TP,
and a SPA, preferably a TSP Immune cells are considered to
specifically bind to a PAI, preferably a TP, if their binding is at
least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at
least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at
least 10-fold, at least 15-fold, at least 20-fold, at least
30-fold, at least 40-fold, at least 50-fold, at least 70-fold, at
least 100-fold, at least 200-fold stronger at identical
concentration of the PAI and the SPA. Preferably, the binding is at
least 10-fold stronger to the PAI, preferably the TP, than to any
of the one or more SPAs, preferably TSPs used in the method of the
invention. It is even more preferably, that the binding is at least
20-fold, more preferably at least 50-fold stronger. The strength of
the binding or affinity of a given immune cell, e.g. a T-cell or
its TCR can be determined by a variety of assays and is commonly
indicated as the dissociation constant (K.sub.d) of the TCR.
However, for the purpose of the method of the present invention an
exact determination of the K.sub.d of the immune cell to a given
PAI, preferably a TP and one or more SPAs, preferably TSPs, is not
required. It is sufficient to determine relative binding strength,
which can be, for example, determined by FACS analysis of immune
cells in which the fluorescence signal of a given TP with the
fluorescence signal of one or more TSPs is compared. In such a
determination the relative molar amounts of the PAI, preferably the
TP, and of the one or more SPA, preferably TSPs, has to be taken
into consideration. If TP and TSPs are added to the immune cells at
the same molar amounts and differences in fluorescent intensity of
the respective labels used are accounted for, an immune cell
specifically binds to a PAI, preferably TP, if an equimolar basis
of the SPA, preferably TSP, shows at least 10-fold stronger
fluorescence attributable to the PAI, preferably to the TP, than to
the SPA, preferably TSP. The skilled person in the art understands
that a change of the molar ratios of PAI, preferably TP, and the
SPAs, preferably TSPs, which are contacted with the cell population
in steps (ii) and (iii) can be accounted for when selecting the
cells by adapting the gating accordingly.
[0148] In one embodiment the cell population of step (i) is
contacted with at least a second antigen complex (2.sup.nd AC)
comprising a SPA, preferably a TSP, at least a third antigen
complex (3.sup.rd AC) comprising a SPA, preferably a TSP, at least
a fourth antigen complex (4.sup.th AC) comprising a SPA, preferably
a TSP, at least a fifth antigen complex (5.sup.th AC) comprising a
SPA, preferably a TSP, at least a sixth antigen complex (6.sup.th
AC) comprising a SPA, preferably a TSP, at least a seventh antigen
complex (7.sup.th AC) comprising a SPA, preferably a TSP, at least
a eighth antigen complex (8.sup.th AC) comprising a SPA, preferably
a TSP, at least a ninth antigen complex (9.sup.th AC) comprising a
SPA, preferably a TSP, at least a fifth antigen complex (10.sup.th
AC) comprising a SPA, preferably a TSP. Accordingly, it is
preferred that not more than ten SPAs, preferably TSPs, are used,
not more than nine SPAs, preferably TSPs are used, not more than
eight SPAs, preferably TSPs, are used, not more than seven SPAs,
preferably TSPs are used, not more than six SPAs, preferably TSPs
are used, not more than five SPAs, preferably TSPs are used, not
more than four SPAs, preferably TSPs, are used, not more than three
SPAs, preferably TSPs are used, not more than two SPAs, preferably
TSPs, are used or not more than one SPA, preferably one TSP is
used. Accordingly, it is preferred that in the method of the
present invention between 1-10 different TSPs are used, preferably
2-8 different TSPs and more preferably 3-5 different TSPs. In an
even more preferred embodiment, three TSPs are used.
[0149] In one embodiment the method of the first aspect of the
invention selects an immune cell. Preferably this immune cell is a
T-cell or a B-cell. More preferably, the T-cell is a CD4 T-cell. In
an even more preferred embodiment the T-cell is a CD8 T-cell. The
signaling domain preferably comprises CD3. In another embodiment of
the first aspect of the invention the immune cell expresses on its
surface an antigen binding protein. It is preferred that the
antigen binding protein is a TCR or an antigen binding fragment
thereof if the immune cell to be selected is a T-cell. It is
preferred that the antigen binding protein is a BCR or an antigen
binding fragment thereof if the immune cell to be selected is a
B-cell. Such an antigen recognition or antigen binding site is
preferably a single chain variable fragment (scFv) and preferably
targets a PAI that is a TAA. The costimulatory domain preferably
comprises CD28 or 4-1 BB. The signaling domain preferably comprises
CD3. It is also preferred that the antigen binding protein is a CAR
or an antigen binding fragment thereof if the immune cell to be
selected is a T-cell.
[0150] In another embodiment of the first aspect of the invention
the method for selecting an immune cell comprises in step (i)
providing a cell population comprising immune cells. The cell
population comprising immune cells is derived from peripheral blood
of healthy subjects. In another embodiment, the cell population
comprising immune cells is derived from peripheral blood from
diseased subjects. Preferably the cell population comprising immune
cells is derived from an immune cell enriched fraction of the
peripheral blood of a healthy or diseased subject. Preferably, the
immune cell enriched fraction is enriched in stem cells, T-cells,
B-cells or plasma cells. It is even more preferred that the immune
enriched fraction is enriched in CD4 T-cells and/or CD8 T-cells. In
another embodiment the cell population comprising immune cells can
be derived from tumor-infiltrating lymphocytes (TILs) or TCR
libraries. Preferably, the TCR library contains a high number of
different T cell receptor (TCR) proteins or fragments thereof,
wherein each TCR protein or fragment thereof is different.
[0151] In another embodiment of the first aspect of the invention
the immune cell or cells in the immune cell enriched fraction are
selected by detectably labeling one or more immune cell specific
surface markers. It is preferred that the immune cell surface
markers are selected from the group consisting of CD3, CD8, CD4 and
CD19.
[0152] In another embodiment of the first aspect of the invention,
the cell population of step (i) a can be incubated in the presence
of growth factors and/or cytokines in a further step. Preferably,
cytokines are interleukins. More preferably, interleukins are
selected from the group consisting of IL-1, IL-2, IL-7, IL-10,
IL-12, Il-15, IL-17, IL-21 and IL-23. Most preferably, the cell
population of step (i) is incubated with IL-2, IL-7, IL-15 and/or
IL-21.
[0153] In another embodiment the protein antigen of interest (PAI)
is a tumor associated antigen (TAA), a viral protein or a bacterial
protein. It is preferred that if the PAI is a target peptide, i.e.
a shorter fragment of the PAI, the target peptide is a viral
antigenic peptide or a bacterial antigenic peptide. In another
embodiment of the first aspect of the invention the diseased
subject suffers from a disease selected from the group consisting
of an immune disease, a neoplastic disease, a disease cause by a
virus or a disease caused by bacteria. Preferably the neoplastic
disease is cancer. Preferably, diseases caused by a virus is a
viral infection; and a disease caused by bacteria is a bacterial
infection. Preferably, the viral infection is caused by a virus
selected from the group consisting of HIV, HCMV, CMV, HPV, HBV,
HCV, HPV, EBV, Influenza virus. More preferably the viral infection
is caused by HIV. Preferably, the bacterial infection is caused by
Mycobacterium tuberculosis. Such a disease is tuberculosis.
[0154] In another preferred embodiment of the first aspect the
method comprises in step (ii) contacting the cell population of
step (i) with a first antigen complex (1.sup.st AC) comprising the
PAI and a detectable label A or with the PAI and a detectable label
A. The 1.sup.st AC comprising the PAI and the label A is preferably
an antigen presenting cell. In another preferred embodiment the
1.sup.st AC is a complex comprising a particle, the PAI and the
detectable label A. More preferably, the particle is a nano- or a
microbead. It is also preferred that an MHC molecule is linked to a
nano- or microbead. In another preferred embodiment the 1.sup.st AC
consists of the PAI and the detectable label A. In another
embodiment the 1.sup.st AC is a complex comprising a particle, the
SPA and the detectable label B. More preferably, the particle is a
nano- or a microbead. It is also preferred that an MHC molecule is
linked to a nano- or microbead. In another preferred embodiment the
1.sup.st AC consists of the SPA and the detectable label B.
[0155] Alternatively, the PAI may comprising a detectable label.
This is a preferred embodiment, if the PAI is an amino acid chain
and the label is covalently linked to this amino acid chain.
Examples are fluorescent labels or fluorescent proteins as GFP or
EGFP. In the latter case it is preferred that the fluorescent
proteins are linked to the PAI by a peptide bond.
[0156] In another preferred embodiment the method of the first
aspect of the invention further comprises one or more of the steps
of contacting the cell population with a further AC comprising a
further PAI and a further detectable label and a further AC
comprising a further SPA and a further detectable label. Using a
third AC comprising a further PAI and a further detectable label C
and a fourth AC comprising a further SPA and a further detectable
label D mirrors the so called 2D Multimer multiplexing (2DMM)
approach which allows specific rare cell detection with high
sensitivity (0.0001%) in a highly cell saving manner Two multimers
labeled with different fluorochromes for each specificity (peptide
MHC) enable the identification of up to 36 different specificities
in one sample by using 9 different fluorochromes. In one preferred
embodiment the cell population of step (i) is contacted with a
third antigen complex (3.sup.rd AC) comprising the PAI and a
detectable label C that is detectably different from one or more or
all of the other detectable labels of the other ACs contacted with
the cell population. Preferably the label is detectably different
from at least the detectable label A, preferably from at least the
detectable label A and a detectable label D, if a detectable label
D is present. A detectable label D is present if the cell
population of step (i) is contacted with a fourth antigen complex
(4.sup.th AC) comprising the PAI and a detectable label D that is
detectably different from one or more or all of the other
detectable labels of the other ACs contacted with the cell
population. The detectable label D is preferably detectably
different from at least the detectable label A. It is also
preferred that the detectable label D is detectably different from
at least the detectable label A and the detectable label C. In one
embodiment the cell population of step (i) is contacted with a
fifth antigen complex (5.sup.th AC) comprising the SPA and a
detectable label E that is detectably different from one or more or
all of the other detectable labels of the other ACs contacted with
the cell population. It is preferred that the label E is detectably
different from at least the detectable label B. It is also
preferred that the detectable label E is detectably different from
at least the detectable label B and a detectable label F, (6) if a
detectable label F is present. A detectable label F is present if
the cell population of step (i) is further contacted with a sixth
antigen complex (6.sup.th AC) comprising the SPA and a detectable
label F that is detectably different from one or more or all of the
other detectable labels of the other ACs contacted with the cell
population. It is preferred that the label F is detectably
different from at least the detectable label B. It is also
preferred that the detectable label F is detectably different from
at least the detectable label B and the detectable label E.
[0157] In another embodiment the cell population of step (i) is
contacted with a first antigen complex (1.sup.st AC) comprising the
PAI, preferably a TP, and a detectable label A; and with at least a
second antigen complex (2.sup.nd AC) comprising a similar protein
antigen (SPA), preferably a TSP, wherein the amino acid sequence of
the SPA, preferably the TSP, differs by at least 1 amino acid from
the amino acid sequence of the PAI, preferably the TP, and wherein
the 2.sup.nd AC comprises a detectable label B which is detectably
different to label A; and with one to ten, i.e. one, two, three,
four, five, six, seven, eight, nine or ten, preferably two to four,
most preferably two further antigen complexes (ACs), wherein each
comprises a different SPA, preferably a different TSP, that differs
in at least one amino acid sequence from the amino acid sequence of
the SPA of the 2.sup.nd AC, and wherein each further AC comprises
one or more labels, wherein the one or more label is detectably
different to the one or more labels of the 2.sup.nd AC. It is
preferred that the 1.sup.st AC comprises a further, second label
which is detectably different to the one or more labels of the
2.sup.nd AC and to the one or more labels of the further ACs. It is
also preferred that the cell population is a T-cell population. It
is further preferred that the selected immune cell that
specifically and/or selectively binds to the 1.sup.st AC is a
T-cell. It is also preferred that the antigen-binding protein on
the surface of the selected T-cell which is specifically and/or
selectively binding to the PAI is a TCR.
[0158] In another embodiment the cell population of step (i) is
contacted with a first antigen complex (1.sup.st AC) comprising the
PAI, preferably a TP, and a detectable label A; and with at least a
second antigen complex (2.sup.nd AC) comprising a similar protein
antigen (SPA), preferably a TSP, wherein the amino acid sequence of
the SPA, preferably the TSP, differs by at least 1 amino acid from
the amino acid sequence of the PAI, preferably the TP, and wherein
the 2.sup.nd AC comprises a detectable label B; and with one to
ten, i.e. one, two, three, four, five, six, seven, eight, nine or
ten, preferably two to four, most preferably two further antigen
complexes (AC) wherein each comprises a different SPA, preferably a
different TSP, that differs in at least one amino acid sequence
from the amino acid sequence of the SPA of the 2.sup.nd AC, and
wherein each further AC comprises one or more labels, wherein the
one or more labels is the same as the one or more labels of the
2.sup.nd AC. It is also preferred that the cell population is a
T-cell population. It is further preferred that the selected immune
cell that specifically and/or selectively binds to the 1.sup.st AC
is a T-cell. It is also preferred that the antigen-binding protein
on the surface of the selected T-cell which is specifically and/or
selectively binding to the PAI is a TCR.
[0159] In another embodiment the cell population of step (i) is
contacted with a first antigen complex (1.sup.st AC) comprising the
PAI, preferably a TP, and a detectable label A; and with at least a
second antigen complex (2.sup.nd AC) comprising a similar protein
antigen (SPA), preferably a TSP, wherein the amino acid sequence of
the SPA, preferably the TSP, differs by at least 1 amino acid from
the amino acid sequence of the PAI, preferably the TP, and wherein
the 2.sup.nd AC comprises a detectable label B which is detectably
different to label A and the 1.sup.st AC comprises at least one
further detectable label and the 2.sup.nd AC comprises at least one
further detectable label, which are the same. It is preferred that
the cell population is a T-cell population. It is further preferred
that the selected immune cell that specifically and/or selectively
binds to the 1.sup.st AC is a T-cell. It is also preferred that the
antigen-binding protein on the surface of the selected T-cell which
is specifically and/or selectively binding to the PAI is a TCR.
[0160] In another embodiment the cell population of step (i) is
contacted with a first antigen complex (1.sup.st AC) comprising the
PAI, preferably a TP, and a detectable label A; and with at least a
second antigen complex (2.sup.nd AC) comprising SPA, preferably a
TSP, wherein the amino acid sequence of the SPA, preferably the
TSP, differs by at least 1 amino acid from the amino acid sequence
of the PAI, preferably the TP, and wherein the 2.sup.nd AC
comprises a detectable label B which is detectably different to
label A and the 1.sup.st AC comprises at least one further
detectable label and the 2.sup.nd AC comprises at least one further
detectable label, which are different. It is preferred that the
cell population is a T-cell population. It is further preferred
that the selected immune cell that specifically and/or selectively
binds to the 1.sup.st AC is a T-cell. It is also preferred that the
antigen-binding protein on the surface of the selected T-cell which
is specifically and/or selectively binding to the PAI is a TCR.
[0161] In another embodiment the cell population of step (i) is
contacted with a first antigen complex (1.sup.st AC) comprising the
PAI, preferably a TP, and a detectable label A; and with at least a
second antigen complex (2.sup.nd AC) comprising a similar protein
antigen (SPA), preferably a TSP, wherein the amino acid sequence of
the SPA, preferably the TSP, differs by at least 1 amino acid from
the amino acid sequence of the PAI, preferably the TP, and wherein
the 2.sup.st AC comprises a detectable label B which is detectably
different to label A and the 1.sup.st AC comprises at least one
further detectable label and the 2.sup.nd AC comprises at least one
further detectable label, which are the same and the cell
population of step (i) is contacted with one or more further
antigen complexes (ACs) wherein each comprises a SPA that differs
in at least one amino acid sequence from the amino acid sequence of
the SPA of the 2.sup.nd AC the one or more further AC comprises at
least one further detectable label; wherein the at least one
further label is selected in such that it allows to distinguish the
1.sup.st AC from the 2.sup.nd AC and the one or more further ACs.
It is preferred that the cell population is a T-cell population. It
is further preferred that the selected immune cell that
specifically and/or selectively binds to the 1.sup.st AC is a
T-cell. It is also preferred that the antigen-binding protein on
the surface of the selected T-cell which is specifically and/or
selectively binding to the PAI is a TCR.
[0162] In another embodiment the cell population of step (i) is
contacted with a first antigen complex (1.sup.st AC) comprising the
PAI, preferably a TP, and a detectable label A; and with at least a
second antigen complex (2.sup.nd AC) comprising a similar protein
antigen (SPA), preferably a TSP, wherein the amino acid sequence of
the SPA, preferably the TSP, differs by at least 1 amino acid from
the amino acid sequence of the PAI, preferably the TP, and wherein
the 2.sup.nd AC comprises a detectable label B which is detectably
different to label A and the 1.sup.st AC comprises at least one
further detectable label and the 2.sup.nd AC comprises at least one
further detectable label, which are different and the cell
population of step (i) is contacted with one or more further
antigen complexes (ACs) wherein each comprises a SPA that differs
in at least one amino acid sequence from the amino acid sequence of
the SPA of the 2.sup.nd AC and, wherein the one or more further AC
comprises at least one further detectable label; wherein the at
least one further label is selected in such that it allows to
distinguish the 1.sup.st AC from the 2.sup.nd AC and the one or
more further ACs. It is preferred that the cell population is a
T-cell population. It is further preferred that the selected immune
cell that specifically and/or selectively binds to the 1.sup.st AC
is a T-cell. It is also preferred that the antigen-binding protein
on the surface of the selected T-cell which is specifically and/or
selectively binding to the PAI is a TCR.
[0163] In another embodiment the cell population of step (i) is
contacted with a first antigen complex (1.sup.st AC) comprising the
PAI, preferably a TP, and a detectable label A; and with at least a
second antigen complex (2.sup.nd AC) comprising a similar protein
antigen (SPA), preferably a TSP, wherein the amino acid sequence of
the SPA, preferably the TSP, differs by at least 1 amino acid from
the amino acid sequence of the PAI, preferably the TP, and wherein
the 2.sup.st AC comprises a detectable label B which is detectably
different to label A and the 1.sup.st AC comprises at least one
further detectable label and the 2.sup.nd AC comprises at least one
further detectable label, which are the same and the cell
population of step (i) is contacted with one to ten, i.e. one, two,
three, four, five, six, seven, eight, nine or ten, preferably two
to four, more preferably two further antigen complexes (ACs)
wherein each comprises a different SPA, preferably a different that
differs in at least one amino acid sequence from the amino acid
sequence of the SPA of the 2.sup.nd AC and wherein the one or more
further AC comprises at least one further detectable label; wherein
the at least one further label is selected in such that it allows
to distinguish the 1.sup.st AC from the 2.sup.nd AC and the one or
more further ACs. It is preferred that the cell population is a
T-cell population. It is further preferred that the selected immune
cell that specifically and/or selectively binds to the 1.sup.st AC
is a T-cell. It is also preferred that the antigen-binding protein
on the surface of the selected T-cell which is specifically and/or
selectively binding to the PAI is a TCR.
[0164] In each of the above embodiments it is preferred that each
of the SPAs, in particular each of the TSPs has a similarity to the
amino acid sequence of the PAI, in particular to the amino acid
sequence of the TP of at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%.
[0165] In another embodiment the detectable labels are provided.
The skilled person in the art is well aware of how to label a
protein antigen of interest, target peptide, similar protein
antigen or target similar peptide of interest. Detectable labels as
specified above with A-F, are independently selected from the group
consisting of magnetic labels, fluorescent label, RNA-barcodes; DNA
barcodes; or radioactive labels. Preferably, magnetic labels may
comprise magnetic beads or magnetic nanoparticles which can be
coated with e.g. antibodies against a particular surface antigen.
Magnetic labels may be used in magnetic-activated cell sorting
(MACS). Preferably, the detectable label is a fluorescent label
selected from the group consisting of xanthens, acridines,
oxazines, cyanines, styryl dyes, coumarins, porphines,
metal-ligand-complexes, fluorescent proteins, nanocrystals,
perylenes and phtalocyanines. Also preferred is the use of
phycoerythrin (SA-PE), streptavidin-allophycocyanin (SA-APC) or
streptavidin-brilliant-violet 421 (SA-BV421) as fluorescent labels
for the detectable labels A-F. In another preferred embodiment the
1.sup.st AC is a complex of a MHC-I or MHC-II and the PAI, and the
PAI is a target peptide (TP). It is preferred that the TP is TAA.
Additionally or alternatively, the 2nd AC is a complex of a MHC-I
or MHC-II and the SPA, and the SPA is a target similar peptide
(TSP). In a further preferred embodiment the 1.sup.st AC and the
2.sup.nd AC is a soluble multimerized MHC-peptide complex.
Functional Differences and Similarities of PAI and SPA:
[0166] As noted above the PAI is preferably expressed on diseased
tissues, contrary to the SPA which is preferably expressed on
healthy human tissues and thus, is selected based on the expression
on healthy tissues. The inventors developed an in-house
high-throughput technology platform (XPRESIDENT) including a large
immunopeptidome database (comprising peptides which have been
previously found to be presented on healthy tissues. SPAs are
preferably from MHC, preferably HLA typed source, i.e. the SPAs are
capable of binding to the respective MHC, preferably HLA molecule.
This is required in the case the immune cell is a T-cell and the
SPA is presented to the T-cell bound to an HLA molecule in order to
allow the T-cell to recognize HLA presented SPAs. It is thus,
preferred to select a SPA that is known to be presented on the same
HLA allotype as the PAI. Preferably, SPAs are used in the method of
the invention that are expressed on cells of healthy tissue with
more than 10 copies per cell, preferably more than 20 copies per
cell, preferably more than 50 copies per cell and even more
preferably more than 100 copies per cell. The counterselection of
T-cells that are capable of binding to such relatively abundant
SPAs and at the same time to the PAI is desired to avoid
off-target/off-tumor toxicity.
Number of TSP:
[0167] TCRs of T-cells recognize a subgroup of amino acids within a
given TP, i.e. the epitope of the TCR. Thus, if too many different
TSP are used, it is likely that there will be no TCR that
predominantly or exclusively binds to the TP but not to the TSP.
Accordingly, it is preferred that not more than 10 TSPs, not more
than 9 TSPs are used, not more than TSPs are used, not more than
seven TSPs are used, not more than six TSPs are used, not more than
five TSPs are used, not more than four TSPs are used, not more than
three TSPs are used, not more than 2 TSPs are used or not more than
1 TSP is used. Accordingly, it is preferred that in the method of
the present invention between 1-10 different TSPs, between 2-8
different TSPs, between 3-5 different TSPs or between one to three
different TSPs are used. In a preferred embodiment three TSPs are
used. The TSPs are fragments of SPAs and are selected on the basis
of the same criteria as outlined for the SPA above. Similarly, TSPs
are selected that are strongly expressed in healthy tissue.
Accordingly, the TSPs to be included in the method of the invention
are those, with high sequence similarity as defined above, i.e. it
is preferred that each of the SPAs, in particular each of the TSPs
has a similarity to the amino acid sequence of the PAI, in
particular to the amino acid sequence of the TP of at least 50%, at
least 60%, at least 70%, at least 80%, or at least 90%, and that
show the highest expression on healthy tissue.
Length of TP and TSP:
[0168] In one embodiment, the TP comprises 8-11 amino acids in
length. The TP may also comprise 12 amino acids. In one embodiment,
the TP comprises 13-25 amino acids in length. In another
embodiment, the TP comprises 13-18 amino acids in length.
Typically, the TSPs are chosen to have the same length as the given
TP. However, alternatively, the length of the TSP may be longer or
shorter by one to three amino acids as the TP. In the embodiments
in which the TP is MHC presented, the length of the one or more
TSPs are chosen in such that they can also be MHC presented. For
example, if the TP comprises 8 amino acids in length, it is
preferred that the TSP either has a length of 7 or less amino acids
or a length of 8 or more amino acids. More preferably, a mixture of
TSPs comprising sequences of different amino acids in length are
used. In an embodiment wherein the TP is bound to MHC-I, the TP
comprises 8-12 amino acids in length. In another embodiment,
wherein the TP is bound to MHC-I, the TP comprises 8-11 amino acids
in length. In an embodiment wherein the TP is bound to MHC-I, the
TP comprises 8-10 amino acids in length. In an embodiment wherein
the TP is bound to MHC-II, the TP comprises 13-23 amino acids in
length. In a preferred embodiment wherein the TP is bound to
MHC-II, the TP comprises 13-18 amino acids in length.
Structural Difference/Similarity of TP and TSP:
[0169] In another embodiment the TSP is selected from the
XPRESIDENT database of healthy tissue-presented HLA bound peptides
based on high sequence similarity (similarity BLAST search) to the
TP. The XPRESIDENT database comprises peptides presented by
different HLA allotypes on healthy or diseased tissues. It is
preferred that the TSP and the TP are presented by the same HLA
allotype. HLA allotypes presenting TSP and TPs can be selected from
the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G,
HLA-H, HLA-J, HLA-K, HLA-L. Preferably the HLA-A protein is
selected from the group consisting of HLA-A1, HLA-A2, HLA-A3, and
HLA-A11. Preferred HLA-A alleles are HLA-A*02:01; HLA-A*01:01,
HLA-A*03:01 or HLA-A*24:02. Preferred HLA-B alleles are
HLA-B*07:02; HLA-B*08:01, HLA-B*15:01, HLA-B*35:01 or
HLA-B*44:05.
[0170] Generally, for most of the HLA allotypes listed above, the
second amino acid (when counting from the N-terminus) and the
C-terminal amino acid of a given MHC presented peptide are not
comprised in the epitope of that peptide recognized by a TCR that
specifically binds to that peptide.
[0171] In another embodiment the amino acid sequence of the TSP has
a length of 8 to 16 amino acids and the TP has a length of 8 amino
acids and wherein the amino acid sequence of the TSP differs from
the amino acid sequence of the TP as follows:
TABLE-US-00006 (SEQ ID NO: 1)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8
[0172] (i) at position X.sub.1, X.sub.2 and X.sub.3, and wherein
position X.sub.4 to X.sub.8 are identical or similar, preferably
identical to the TP; or [0173] (ii) at position X.sub.4, X.sub.5
and X.sub.6, and wherein positions X.sub.1 to X.sub.3 and X.sub.7
and X.sub.8 are identical or similar, preferably identical to the
TP; or [0174] (iii) at position X.sub.7 and X.sub.8, and wherein
position X.sub.1 to X.sub.6 are identical or similar, preferably
identical to the TP.
[0175] In a preferred embodiment the positions X.sub.1, X.sub.2 and
X.sub.3 are mutated in the TSP wherein the position X.sub.4 to
X.sub.8 are identical compared to the TP. In another preferred
embodiment positions X.sub.4, X.sub.5 and X.sub.6 are mutated in
the TSP and positions X.sub.1 to X.sub.3 and X.sub.7 and X.sub.8
are identical to the TP. In another preferred embodiment position
X.sub.7 and X.sub.8 in the TSP are mutated and position X.sub.1 to
X.sub.6 are identical to the TP. In another preferred embodiment
position X.sub.7 and X.sub.8 in the TSP are mutated and position
X.sub.1 to X.sub.6 are identical to the TP.
[0176] In another preferred embodiment a mixture of the TSP
described above in (i) to (iii), i.e. TSP with different mutation
patterns are used in the method of the first aspect of the
invention. In another preferred embodiments a mixture of the TSP
described above in (i) to (iii), i.e. TSP with different mutation
patterns and also different amino acid sequences in length are
used. The use of such a mixture of TSP allows the fast and
efficient positive selection of immune cells binding to the TP and
negative selection of immune cells binding to one or more TSPs.
[0177] In another preferred embodiment the amino acid sequence of
the TSP has a length of 8 to 16 amino acids and the TP has a length
of 9 amino acids and the amino acid sequence of the TSP differs
from the amino acid sequence of the TP
TABLE-US-00007 (SEQ ID NO: 2)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9
[0178] (i) at position X.sub.1, X.sub.2 and X.sub.3, and wherein
position X.sub.4 to X.sub.9 are identical or similar, preferably
identical to the TP; [0179] (ii) at position X.sub.4, X.sub.5 and
X.sub.6, and wherein position X.sub.1 to X.sub.3 and positions
X.sub.7 to X.sub.9 are identical or similar, preferably identical
to the TP; or [0180] (iii) at position X.sub.4, X.sub.5, X.sub.6
and X.sub.7, and wherein position X.sub.1 to X.sub.3 and positions
X.sub.8 to X.sub.9 are identical to the TP; or [0181] (iv) at
position X.sub.7, X.sub.8 and X.sub.9, and wherein position X.sub.1
to X.sub.6 are identical or similar, preferably identical to the
TP.
[0182] In a preferred embodiment the positions X.sub.1, X.sub.2 and
X.sub.3 are mutated in the TSP wherein the position X.sub.4 to
X.sub.9 are identical compared to the TP. In another preferred
embodiment positions X.sub.4, X.sub.5 and X.sub.6 and X.sub.7 are
mutated in the TSP and positions X.sub.1 to X.sub.3 and positions
X.sub.8 and X.sub.9 are identical to the TP. In another preferred
embodiment positions X.sub.7 to X.sub.9 are mutated in the TSP and
positions X.sub.1 to X.sub.6 are identical to the TP. In another
preferred embodiment position X.sub.7 to X.sub.9 in the TSP are
mutated and position X.sub.1 to X.sub.6 are identical to the TP. In
another preferred embodiment a mixture of the TSP described above
in (i) to (iv), i.e. TSP with different mutation patterns are used
in the method of the first aspect of the invention. In another
preferred embodiments a mixture of the TSP described above in (i)
to (iv), i.e. TSP with different mutation patterns and also
different amino acid sequences in length are used.
[0183] In another preferred embodiment the amino acid sequence of
the TSP has a length of 8 to 16 amino acids, the TP has a length of
10 amino acids and the amino acid sequence of the TSP differs from
the amino acid sequence of the TP
TABLE-US-00008 (SEQ ID NO: 3)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10
[0184] (i) at position X.sub.1, X.sub.2 and X.sub.3, wherein
position X.sub.4 to X.sub.10 are identical or similar, preferably
identical to the TP; [0185] (ii) at position X.sub.4, X.sub.5,
X.sub.6 and X.sub.7, wherein position X.sub.1 to X.sub.3 and
positions X.sub.8 to X.sub.10 are identical or similar, preferably
identical to the TP; or [0186] (iii) at position X.sub.4, X.sub.5
and X.sub.6, and wherein position X.sub.1 to X.sub.3 and positions
X.sub.7 to X.sub.10 are identical or similar, preferably identical
to the TP; or [0187] (iv) at position X.sub.8, X.sub.9 and
X.sub.10, wherein position X.sub.1 to X.sub.7 are identical or
similar, preferably identical to the TP.
[0188] In a preferred embodiment the positions X.sub.1, X.sub.2 and
X.sub.3 are mutated in the TSP wherein the position X.sub.4 to
X.sub.10 are identical compared to the TP. In another preferred
embodiment positions X.sub.4 to X.sub.7 are mutated in the TSP and
positions X.sub.1 to X.sub.3 and positions X.sub.8 and X.sub.10 are
identical to the TP. In another preferred embodiment position
X.sub.4 to X.sub.6 are mutated in the TSP and position X.sub.1 to
X.sub.3 and positions X.sub.7 to X.sub.10 are identical to the TP.
In another preferred embodiment positions X.sub.8 to X.sub.10 are
mutated in the TSP and positions X.sub.1 to X.sub.7 are identical
to the TP. In another preferred embodiment a mixture of the TSP
described above in (i) to (iv), i.e. TSP with different mutation
patterns are used in the method of the first aspect of the
invention. In another preferred embodiments a mixture of the TSP
described above in (i) to (iv), i.e. TSP with different mutation
patterns and also different amino acid sequences in length are
used.
[0189] In another preferred embodiment the amino acid sequence of
the TSP has a length of 8 to 16 amino acids, the TP has a length of
11 amino acids and the amino acid sequence of the TSP differs from
the amino acid sequence of the TP
TABLE-US-00009 (SEQ ID NO: 4)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.s-X.sub.9-X-
.sub.10X.sub.11
[0190] (i) at position X.sub.1, X.sub.2 and X.sub.3, wherein
position X.sub.4 to X.sub.11 are identical or similar, preferably
identical to the TP; [0191] (ii) at position X.sub.4, X.sub.5,
X.sub.6 and X.sub.7, wherein position X.sub.1 to X.sub.3 and
positions X.sub.8 to X.sub.11 are identical or similar, preferably
identical to the TP; or [0192] (iii) at position X.sub.4, X.sub.5
and X.sub.6, and wherein position X.sub.1 to X.sub.3 and positions
X.sub.7 to X.sub.11 are identical or similar, preferably identical
to the TP; or [0193] (iv) at position X.sub.8, X.sub.9, X.sub.10
and X.sub.11, wherein position X.sub.1 to X.sub.7 are identical or
similar, preferably identical to the TP; or [0194] (v) at position
X.sub.9, X.sub.10 and X.sub.11, wherein position X.sub.1 to X.sub.8
are identical or similar, preferably identical to the TP.
[0195] In a preferred embodiment the positions X.sub.1, X.sub.2 and
X.sub.3 are mutated in the TSP wherein the position X.sub.4 to
X.sub.11 are identical compared to the TP. In another preferred
embodiment positions X.sub.4 to X.sub.7 are mutated in the TSP and
positions X.sub.1 to X.sub.3 and positions X.sub.8 to X.sub.11 are
identical to the TP. In another preferred embodiment position
X.sub.4 to X.sub.6 are mutated in the TSP and position X.sub.1 to
X.sub.3 and positions X.sub.7 to X.sub.11 are identical to the TP.
In another preferred embodiment positions X.sub.5 to X.sub.11 are
mutated in the TSP and positions X.sub.1 to X.sub.7 are identical
to the TP. In another preferred embodiment positions X.sub.9 to
X.sub.11 are mutated in the TSP and positions X.sub.1 to X.sub.8
are identical to the TP.
[0196] In another preferred embodiment a mixture of the TSP
described above in (i) to (iv), i.e. TSP with different mutation
patterns are used in the method of the first aspect of the
invention. In another preferred embodiments a mixture of the TSP
described above in (i) to (iv), i.e. TSP with different mutation
patterns and also different amino acid sequences in length are
used.
[0197] In another preferred embodiment the amino acid sequence of
the TSP has a length of 8-16 amino acids, the TP has a length of 12
amino acids and the amino acid sequence of the TSP differs from the
amino acid sequence of the TP
TABLE-US-00010 (SEQ ID NO: 5)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10X.sub.11X.sub.12
[0198] (i) at position X.sub.1, X.sub.2 and X.sub.3, wherein
position X.sub.4 to X.sub.12 are identical or similar to the TP;
[0199] (ii) at position X.sub.4, X.sub.5, X.sub.6 and X.sub.7,
wherein position X.sub.1 to X.sub.3 and positions X.sub.5 to
X.sub.12 are identical or similar to the TP; or [0200] (iii) at
position X.sub.4, X.sub.5 and X.sub.6, and wherein position X.sub.1
to X.sub.3 and positions X.sub.7 to X.sub.12 are identical or
similar to the TP; or [0201] (iv) at position X.sub.8, X.sub.9,
X.sub.10, X.sub.11 and X.sub.12, wherein position X.sub.1 to
X.sub.7 are identical or similar to the TP; or [0202] (v) at
position X.sub.9, X.sub.10, X.sub.11 and X.sub.12, wherein position
X.sub.1 to X.sub.8 are identical or similar to the TP.
[0203] In a preferred embodiment the positions X.sub.1, X.sub.2 and
X.sub.3 are mutated in the TSP wherein the position X.sub.4 to
X.sub.12 are identical compared to the TP. In another preferred
embodiment positions X.sub.4 to X.sub.7 are mutated in the TSP and
positions X.sub.1 to X.sub.3 and positions X.sub.8 to X.sub.12 are
identical to the TP. In another preferred embodiment position
X.sub.4 to X.sub.6 are mutated in the TSP and position X.sub.1 to
X.sub.3 and positions X.sub.7 to X.sub.12 are identical to the TP.
In another preferred embodiment positions X.sub.8 to X.sub.12 are
mutated in the TSP and positions X.sub.1 to X.sub.7 are identical
to the TP. In another preferred embodiment positions X.sub.9 to
X.sub.12 are mutated in the TSP and positions X.sub.1 to X.sub.8
are identical to the TP.
[0204] In another preferred embodiment a mixture of the TSP
described above in (i) to (iv), i.e. TSP with different mutation
patterns are used in the method of the first aspect of the
invention. In another preferred embodiments a mixture of the TSP
described above in (i) to (iv), i.e. TSP with different mutation
patterns and also different amino acid sequences in length are
used.
[0205] In another embodiment of the first aspect of the invention
the amino acid sequence of the SPA or of at least one protein or
peptide comprised in the SPA has a similarity to the amino acid
sequence of the PAI of at least 50%, at least 60%, at least 70%, at
least 80%, or of at least 90% or of at least 95%. In another
embodiment the amino acid sequence of the SPA or of at least one
protein or peptide comprised in the SPA has less than or 90%, less
than or 89%, less than or 88%, less than or 87% or less than or 86%
amino acid identity to the PAI. In another embodiment the amino
acid sequence of the SPA or of at least one protein or peptide
comprised in the SPA has less than or 85%, less than or 84%, less
than or 83%, less than or 82%, less than or 81% or less than or 80%
amino acid identity to the PAI.
[0206] In another embodiment of the first aspect of the invention
the amino acid sequence of the TSP has less than or 96%, less than
or 95%, less than or 94%, less than or 93%, less than or 92% or
less than or 91% amino acid identity to the TP. In another
embodiment the amino acid sequence of the TSP has less than 90%,
less than or 89%, less than or 88%, less than or 87% or less than
or 86% amino acid identity to the TP. In another embodiment the
amino acid sequence of the TSP has less than or 85%, less than or
84%, less than or 83%, less than or 82%, less than or 81% or less
than or 80% amino acid identity to the TP.
[0207] In another embodiment the absolute expression of a TSP on
healthy tissue is correlated with the lowest sequence identity of
TSPs included in the method of the invention. If a TSP is highly
expressed on healthy tissue, TCRs which only bind with low affinity
to the TSP may nevertheless bind to the TSP expressed in healthy
tissue due to avidity effects. Thus, if a given TSP has a low copy
number on healthy tissue, it is included in the method of the
present invention only, if it shows a high similarity to the TP.
Correspondingly, if a given TSP has a high copy number on healthy
tissue, it is included in the method of the present invention
although it may have a low similarity to the TP. For example, if
TSPs have a copy number below 10 per healthy cell than it is
included, if it has at least a 90% sequence similarity to the TP.
If the copy number of TSPs are between 1 to 25 such TSPs are
included, if they have at least 85% sequence similarity with the
TP. If the copy number of TSPs are between 25 to 100/cell such TSPs
are included, if they have at least 80% sequence similarity with
the TP. If the copy number of TSPs are between 100 to 250/cell such
TSPs are included, if they have at least 75% sequence similarity
with the TP. If the copy number of TSPs are above 250/cell such
TSPs are included, if they have at least 50% sequence similarity
with the TP.
[0208] In another embodiment of the method of the first aspect of
the present invention the steps (ii) and (iii) of the method are
carried out consecutively or concomitantly. In another embodiment
of the first aspect of the invention steps (a), (b), (c) and (d) as
outline above are carried out consecutively or concomitantly.
Whether steps (a), (b), (c) and (d) are combined depends on the use
of the number of ACs labeled with a detectable label.
[0209] In another embodiment of the method of the first aspect of
the present invention step (iv) comprises positively selecting
(selecting) cells bound to the 1.sup.st AC, 1.sup.st and 3.sup.rd
or 1.sup.st, 3.sup.rd and 4.sup.th AC. In another embodiment step
(iv) comprises negatively selecting (excluding) cells bound to the
2.sup.nd AC, the 2.sup.nd and 5.sup.th or the 2.sup.nd, 5.sup.th
and 6.sup.th AC. In another preferred embodiment the step (iv)
comprises selecting cells bound to the 1.sup.st AC, 1.sup.st and
3.sup.rd or 1.sup.st, 3.sup.rd and 4.sup.th AC and excluding cells
bound to the 2.sup.nd AC, the 2.sup.nd and 5.sup.th or the
2.sup.nd, 5.sup.th and 6.sup.th AC.
[0210] In another embodiment of the method of the first aspect of
the present invention the detectable label is detected by flow
cytometry analysis. In a preferred embodiment the detectable label
is detected by FACS analysis. In another preferred embodiment the
detectable label is detected by preparative sorting analysis.
[0211] In another embodiment of the method of the first aspect of
the present invention the cells comprised in the population of step
i) of the method of the first aspect of the invention are T-cells
and are phenotyped. In another embodiment the cells comprised in
the population of step i) are B-cells and are phenotyped.
[0212] In another embodiment of the method of the first aspect of
the present invention the phenotyping of T-cells comprises the
determination of one or more T-cell marker. T-cell marker are
preferably selected from the group consisting of CD3, CD4, CD8,
CD11a, CD14, CD19, CD25, CD27, CD28, CD44, CD45RA, CD45RO, CD57,
CD62L, CD69, CD122, CD127, CD137 CD197 (CCR7), IFN.gamma., IL-2,
TNF.alpha., IL7R and telomer length. In another preferred
embodiment T-cell markers are CD45RA, CD45RO, CD197, CD25, CD27,
CD57, CD95, CD127 and CD62L It is particularly preferred that CD69
and CD137 are used for the phenotyping of T-cells. In another
preferred embodiment the phenotyping of B-cells comprises the
determination of one or more B-cell marker. B-cell marker are
preferably selected from the group consisting of CD19, CD27, CD45R,
CD21, CD40, CD20, CD38, and CD83.
[0213] In another embodiment of the method of the first aspect of
the present invention the method further comprises the step of
contacting the cell population of step (i) with an irrelevant
antigen complex (IAC) comprising an irrelevant protein antigen
(IPA), wherein the amino acid sequence of the IPA when aligned with
the amino acid sequence of the PAI is identical to the PAI at two
amino acid positions or less and wherein the IAC comprises a
detectable label G that is detectably different from the detectable
label A. Preferably, an irrelevant protein antigen is the gene
product of a housekeeping gene. The housekeeping gene product is
expressed in all cells of an organism under normal and
pathophysiological conditions which make it suitable to function as
a reference gene because it is usually not up or down regulated
under different or varying cell conditions. Generally, a natural
immune cell population should not comprise any immune cells binding
to housekeeping genes or peptides derived therefrom. Thus, the
inclusion of an IPC in the method of the invention allows the
identification of T-cells that nonspecifically bind to AC, which
are also undesirable.
[0214] In another embodiment the amino acid sequence of at least
one IP is selected by one or more of the following criteria:
presentation of the IP on healthy tissue; the IP is derived from a
HLA typed source; or the binding to the respective HLA. It is
preferred that the amino acid sequence of the IP or of at least one
protein or peptide comprised in the IPA has less than 50%, less
than 40%, less than 30%, less than 29%, less than 28%, less than
27%, less than 26%, less than 25%, less than 24%, less than 23%,
less than 22%, less than 21%, less than 20%, less than 19%, less
than 18%, less than 17%, less than 16%, less than 15%, less than
10%, less than 5% amino acid identity to the PAI. In another
embodiment the IAC is a complex of a MHC-I or MHC-II and an IP. It
is preferred that the amino acid sequence of the IP when aligned
with the amino acid sequence of the TP is identical to the TP at
one or none amino acid positions. Preferably, the IP is encoded by
a housekeeping gene.
[0215] A second aspect of the invention further relates to a method
for determining the sequence of a nucleic acid encoding an
antigen-binding protein or an antigen-binding part thereof
comprising the steps of: [0216] (i) isolating the nucleic acid
encoding the antigen-binding protein or the antigen-binding part
thereof from the cell selected in the method of the first aspect of
the invention; and [0217] (ii) determining the sequence of the
nucleic acid. In a preferred embodiment the nucleic acid is
isolated from the selected immune cell by methods well known in the
art, e.g. organic extraction, solid phase extraction, e.g. using a
resin comprising a styrene-divinylbenzene co-polymer containing
iminodiacetic acid groups. In another embodiment the isolated
nucleic acid is either DNA or RNA. In another embodiment it is
preferred to amplify the nucleic acid after isolation. Preferably,
amplification is conducted by polymerase chain reaction (PCR). More
preferably the nucleic acid is amplified in a rapid amplification
of cDNA-ends with PCR (RACE PCR). In another embodiment the
synthesis of DNA from an RNA template, via reverse transcription,
produces complementary DNA (cDNA). Reverse transcriptases (RTs) use
an RNA template and a short primer complementary to the 3' end of
the RNA to direct the synthesis of the first strand cDNA, which can
be used directly as a template for the PCR. In another embodiment
the sequence of the isolated nucleic acid can be determined by
known methods in the art, for example next generation sequencing,
e.g. Illumina (Solexa) sequencing by simultaneously identifying DNA
bases, as each base emits a unique fluorescent signal, and adding
them to a nucleic acid chain, Roche 454 sequencing based on
pyrosequencing, a technique which detects pyrophosphate release,
again using fluorescence, after nucleotides are incorporated by
polymerase to a new strand of DNA, or ion torrent: Proton/PGM
sequencing measuring the direct release of protons from the
incorporation of individual bases by DNA polymerases.
[0218] A third aspect of the invention relates to a method for
producing a cell expressing a nucleic acid encoding an
antigen-binding protein or an antigen-binding part thereof
comprising the steps of: [0219] (i) providing the nucleic acid
sequence encoding the antigen-binding protein or an antigen-binding
part thereof from the cell selected in the method of the first
aspect of the invention; [0220] (ii) producing a nucleic acid
vector comprising the nucleic acid sequence provided in step (i)
optionally under the control of an expression control element; and
[0221] (iii) introducing the nucleic acid vector of step (ii) into
a host cell. In one embodiment the antigen-binding protein or an
antigen binding part thereof is cloned into a vector.
[0222] In one embodiment the antigen-binding protein is a TCR or an
antigen binding fragment thereof; a BCR or an antigen binding
fragment thereof or an antibody or an antigen binding fragment
thereof. In another embodiment, the antigen binding protein is a
TCR or the part thereof comprise at least the variable domains of
the alpha and beta chain. Preferably, the sequence of the TCR or
antigen binding part thereof is inserted into a suitable vector. In
another embodiment the amino acid sequence of the TCR, BCR or
antibody comprises six CDRs. In another embodiment two or three
CDRs of the variable alpha and/or beta domain of an identified TCR
are inserted into the framework or another TCR or antibody.
Preferably, the gene sequence of one, two or three CDRs of the
variable alpha domain of a TCR are cloned into a suitable vector
comprising framework regions. The expression vector may either
comprise nucleic acids encoding both the light or heavy chain or
alpha and beta chain (or the variable domains thereof)--soc-called
"tandem type"--or they may be encoded by nucleic acids comprised in
separate vectors. It is preferred that humanized antibody
expression vectors of the tandem type are used (shitara K et al. J
Immunol Methods. 1994 Jan. 3; 167(1-2):271-8). Examples of tandem
type humanized antibody expression vector include e.g. pKANTEX93
(WO 97/10354), and pEE18.
[0223] In another embodiment the vector of step (ii) is introduced
into a host cell. In one embodiment such recombinant host cells can
be used for the production of at least one antigen binding protein
of the invention or part thereof. Preferably, the host cell is
transformed, transduced or transfected with a nucleic acid and/or a
vector encoding the antigen binding protein or antigen binding part
thereof. Transduction or transfection of host cells with nucleic
acid encoding the antigen binding protein or part of the antigen
binding protein is conducted using methods well known in the art,
for example methods described in US20190216852. In another
embodiment the host cells comprising the antigen binding protein or
antigen binding part thereof can be a eukaryotic cell, e.g., plant,
animal, fungi, or algae, or can be a prokaryotic cell, e.g.,
bacteria or protozoa. The host cell can be a cultured cell or a
primary cell, i.e., isolated directly from an organism, e.g., a
human. The host cell can be an adherent T-cell or a suspended cell,
i.e., a cell that grows in suspension. For purposes of producing an
antigen binding protein or part of the antigen binding protein,
such as a recombinant TCR or fragment thereof, the host cell is
preferably a mammalian cell. Most preferably, the host cell is a
human cell. While the host cell can be of any cell type, can
originate from any type of tissue, and can be of any developmental
stage, the host cell preferably is a peripheral blood leukocyte
(PBL) or a peripheral blood mononuclear cell (PBMC) or a B-cell.
More preferably, the host cell is a T-cell. The T-cell can be any
T-cell, such as a cultured T-cell, e.g., a primary T-cell, or a
T-cell from a cultured T-cell line, e.g., Jurkat, SupT1, etc., or a
T-cell obtained from a mammal, preferably a T-cell or T-cell
precursor from a human patient. If obtained from a mammal, the
T-cell can be obtained from numerous sources, including but not
limited to blood, bone marrow, lymph node, the thymus, or other
tissues or fluids. Preferably, the T-cell is a human T-cell. More
preferably, the T-cell is a T-cell isolated from a human. The
T-cell can be any type of T-cell and can be of any developmental
stage, including but not limited to, CD4-positive and/or
CD8-positive, CD4-positive helper T-cells, e.g., Th1 and Th2 cells,
CD8-positive T-cells (e.g., cytotoxic T-cells), tumor infiltrating
cells (TILs), memory T-cells, naive T-cells. Preferably, the T-cell
is a CD8-positive T-cell or a CD4-positive T-cell. In another
embodiment the host cell may be any cell for recombinant
expression. Preferably, the host cell is a Chinese hamster ovary
(CHO) cell.
[0224] A fourth aspect of the invention relates to a method for
treating a subject in need thereof comprising the steps of: [0225]
(i) providing a cell population of the subject comprising immune
cells; [0226] (ii) contacting the cell population of step (i) with
a first antigen complex (1.sup.st AC) comprising a PAI and a
detectable label A or with the PAI comprising a detectable label A;
[0227] (iii) contacting the cell population of step (i) with at
least a second antigen complex (2.sup.nd AC) comprising a SPA,
wherein the amino acid sequence of the SPA differs by at least 1
amino acid from the amino acid sequence of the PAI and wherein the
2.sup.nd AC comprises a detectable label B; and [0228] (iv)
selecting at least one cell that specifically binds to the 1.sup.st
AC, [0229] wherein the detectable label A and the detectable label
B are detectably different from each other [0230] (v) increasing
the number of the at least one selected cell by cultivation; and
[0231] (vi) reintroducing the cultivated cells into the
subject.
[0232] This approach is an ACT approach. Preferably, the selected
cells are transferred into the subject after being genetically
engineered and functionally improved. Preferably, the cells are
originated from the subject to which they are transferred to after
being genetically engineered, i.e. donor of the cells and receptor
of engineered cells is identical. The subject is a subject in need
thereof as defined herein above. Preferably, the subject in need
thereof suffers or is at risk of suffering from a disease selected
from the group consisting of immune diseases or neoplastic
diseases, a disease caused by a virus or a disease caused by
bacteria. It is preferred that the neoplastic disease is cancer. It
is preferred that the disease caused by a virus is HIV. It is
preferred that the disease cause by a bacterium is
tuberculosis.
[0233] A fifth aspect of the invention relates to a method for
selecting an immune cell expressing on its surface an
antigen-binding protein specifically binding to a protein antigen
of interest (PAI) comprising the following steps: [0234] (i)
providing a cell population comprising immune cells; [0235] (ii)
contacting the cell population of step (i) with a first antigen
complex (1.sup.st AC) comprising the PAI and a detectable label A
or with the PAI comprising a detectable label A; [0236] (iii)
contacting the cell population of step (i) with at least a second
antigen complex (2.sup.nd AC) comprising an irrelevant protein
antigen (IPA), wherein the amino acid sequence of the IPA when
aligned with the amino acid sequence of the PAI is identical to the
PAI at two amino acids positions or less and wherein the IAC
comprises a detectable label G; or with the IPA and a detectable
label G; and [0237] (iv) selecting at least one cell that
specifically binds to the 1.sup.st AC, wherein the detectable label
A and the detectable label G are detectably different from each
other. The selection process according to the fifth aspect of the
invention is carried out as outlined above for the first aspect of
the invention.
EXAMPLES
Example 1: Direct Sorting of Target-Peptide Specific T Cells with
and without Prior Target-Specific Expansion
[0238] FIGS. 2 and 3 show a comparison of two different approaches
which lead to sorting of target-specific T cells while sparing
cross-reactive T cells which recognize target and similar peptides.
Method 1 (FIG. 2) does not require prior amplification of
target-specific T cells. PBMCs are isolated and enriched for T cell
populations by magnetic bead separation. The T cell population is
stained with fluorochrome-conjugated target-peptide and
similar-peptide tetramers. Subsequently, those cells can be further
enriched for target-specific T cells by using magnetic bead
separation targeting one of the fluorochrome-conjugates of the
target-tetramers. The target peptide-specific T cell population is
then stained for surface markers such as CD4 and CD8 as well as
viability markers to exclude dead cells. By using flow cytometric
sorting approaches the target-specific T cells can be sorted for
desired surface marker expression while sparing target+similar
peptide-specific T cells as shown in FIG. 2. Method 2 (FIG. 3)
utilizes stimulation with target-peptide HLA-coated artificial
antigen-presenting cells to amplify low frequency target-specific T
cells. Here, enriched CD8 T cells are cultivated in individual
vessels to allow for the growth of mono- or oligoclonal
target-peptide specific T cell populations. After repeated
stimulation with artificial antigen-presenting cells the individual
mono- or oligoclonal populations are stained with surface markers
as well as target- and similar-peptide tetramers (target and
similar peptide tetramers are labelled with 2 distinct
fluorochromes each in a 2D staining approach) which allows for
distinction of target-specific and cross-reactive mono- or
oligoclonal T cell populations as shown in FIG. 3.
Example 2: Functional Assessment of T Cell Receptors Derived from T
Cells Sorted with Target-Peptide Multimers Only
[0239] To assess functionality and specificity of TCRs identified
by sorting with target multimers, T cell receptor mRNA is generated
by in vitro transcription and subsequently used to transfect CD8
positive T cells of healthy donors by electroporation. Eighteen
hours after electroporation 20,000 transfected T cells are then
co-incubated with T2 cells loaded either with target peptide,
different target-sequence similar peptides, an irrelevant peptide
or unloaded T2 cells at a 1:1 ratio. Supernatants are harvested 24
h after start of co-culture and analyzed for secreted IFN-.gamma.
by ELISA-technique. Cytokine secretion demonstrates antigen
recognition and activity of the respective T cells as illustrated
in FIG. 4. Whereas all TCRs in FIGS. 4A, B and C recognize the
target (positive control), TCRs in FIG. 4A and FIG. 4B are also
cross-reactive towards target-sequence similar peptides expressed
on normal tissue and are thus excluded from further analysis, only
"clean" TCRs (FIG. 4C) are worth to be selected for further
characterization. ("Target"=target peptide; TP; "SIM 1-10"=target
similar peptides; TSPs 1-10).
Peptides Used in this Example: TP and SIM 1-SIM 10 are all 9mers.
[0240] TP and SIM 1 differ in amino acid position 2, 5, 8 and 9
wherein SIM 1 has an isoleucine residue at position 2, a threonine
residue at position 5, a leucine residue at position 8 and a valine
residue at position 9. [0241] TP and SIM 2 differ in position 3, 4
and 7, wherein SIM 2 has an isoleucine residue at position 3, a
glutamic acid residue at position 4 and a glutamine residue at
position 7. [0242] TP and SIM 3 differ in position 2, 7, 8 and 9,
wherein SIM 3 has an isoleucine residue at position 3, a glutamic
acid residue at position 7 and 8 and an isoleucine residue at
position 9. [0243] TP and SIM 4 differ in position 4, 5 and 8,
wherein SIM 4 has a lysine residue at position 4, an asparagine
residue at position 5 and a tyrosine residue at position 8. [0244]
TP and SIM 5 differ in position 4, 7 and 8, wherein SIM 5 has an
asparagine residue at position 4, a proline residue at position 7
and a tyrosine residue at position 8. [0245] TP and SIM 6 differ in
position 6, 7 and 8, wherein SIM 6 has valine residue at position 6
and a leucine residue at position 7 and 8. [0246] TP and SIM 7
differ in position 5, 6 and 8, wherein SIM 7 has lysine residue at
position 5, a glutamine residue at position 6 and a methionine
residue at position 8. [0247] TP and SIM 8 differ in position 3, 5,
7 and 9, wherein SIM 8 has serine residue at position 3, a glutamic
acid residue at position 5 and a valine residue at position 7 and
9. [0248] TP and SIM 9 differ in position 2, 4, 5 and 9, wherein
SIM 9 has valine residue at position 2, a glycine residue at
position 4, an alanine residue at position 5 and a valine residue
at position 9. [0249] TP and SIM 10 differ in position 1, 4 and 6,
wherein SIM 10 has valine residue at position 1, a histidine
residue at position 4 and a glutamine residue at position 9. [0250]
TP and control peptide differ in positions 4-9.
Example 3: Functional Assessment of T Cell Receptors Derived from
Target-Peptide as Well as Target- and Similar-Peptide Specific T
Cells
[0251] To this end, T cell receptor mRNA is generated using in
vitro transcription and subsequently used to transfect
NFAT-luciferase Jurkat cells by electroporation. The transfected
Jurkat cells start to express the newly introduced TCRs transiently
on their surface. Eighteen hours after electroporation 50,000
Jurkat cells are then co-incubated with T2 cells at a 1:1 ratio
loaded either with a target peptide or the similar peptides which
are used for sorting, as well as a control peptide or no peptide.
Upon specific binding of the TCR to its cognate peptide-HLA,
signaling leads to NFAT activation which in turn leads to
expression of luciferase. After overnight incubation luciferase
substrate is added and a luminescence signal can be detected when
the T cell is activated. FIG. 5 shows that TCRs derived from
target-tetramer binding T cells lead to functional activation when
stimulated with target-peptide loaded T2 cells. ("Target"=target
peptide; TP; "SIM 1-3"=target similar peptides; TSPs 1-3).
Peptides Used in this Example: TP and SIM 1-SIM 3 are all 9mers.
[0252] TP and SIM 1 differ in amino acid position 4, 6 and 7
wherein SIM 1 has a glutamic acid residue at position 4, a leucine
residue at position 6 and an isoleucine residue at position 7.
[0253] TP and SIM 2 differ in position 2, 7 and 8, wherein SIM 2
has a methionine residue at position 2, a glutamic acid residue at
position 7 and lysine residue at position 8. [0254] TP and SIM 3
differ in position 1, 5 and 6, wherein SIM 3 has a phenylalanine
residue at position 1, a glycine residue at position 5 and a serine
residue at position 6. [0255] TP and control peptide differ in
positions 1 and 4-9.
[0256] Overall SIM 1 has a similarity to TP of 77%, SIM 2 has a
similarity to TP of 77% and SIM 3 has a similarity to TP of 75%
using BLASTP, BLOSUM62 scoring matrix, a word length of 3, and
expectation (E) of 10.
Example 4: Relevant and Irrelevant Target Similar Peptides for a
Given Target Peptide
[0257] The relevance of a peptide as TSP to a given TP is
determined mainly by its similarity to the TP, and can additionally
be guided by its frequency of presentation as well as and
quantitative presentation level (copy numbers per cell (CpC)) on
primary normal tissues. The higher the similarity to the TP and the
higher the presentation frequency and CpC on normal tissues, the
higher the relevance of a TSP. Table 6 shows example sequences of a
TP, two corresponding TSPs as well as an IP. Per peptide, the
number of identical amino acids (aa) to the TP, the similarity
based on the pmbec positional scoring matrix in comparison to the
TP sequence and the CpC range on normal tissues is depicted. FIGS.
6, 7 and 8 additionally shows the peptide presentation profiles of
the two TSPs as well as the IP. TSP (TSP1) has 4 identical amino
acids to the TP but a higher overall similarity to the target as
compared to TSP2 which has 5 identical amino acids in comparison to
the target peptide. Both TSPs are considered relevant based on
their similarity to the TP and their presentation on normal tissues
(FIGS. 6 and 7). The depicted IP shows an even higher presentation
frequency on normal tissues (FIG. 8) and is in general also
presented at a higher copy number per cell. The sequence similarity
as well as the number of identical amino acids is however rather
low (17% similarity and 0 identical amino acids).
TABLE-US-00011 TABLE 6 Similarity Number of to TP CpC range Amino
acid identical (PMBEC normal equence aa to TP Score) tissue TP
VLLHHQIGL 9 100% n.a. (SEQ ID NO: 164) TSP1 ALMYHTITL 4 63% 5-60
(SEQ ID NO: 165) TSP2 LLLAHIIAL 5 55% 15-35 (SEQ ID NO: 166) IP
AIVDKVPSV 0 17% 55-600 (SEQ ID NO: 167)
Sequence CWU 1
1
16718PRTHomo sapiensMISC_FEATURE(1)..(8)X can be any amino acid
1Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 529PRTHomo
sapiensMISC_FEATURE(1)..(9)X can be any amino acid 2Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5310PRTHomo sapiensMISC_FEATURE(1)..(10)X can
be any amino acid 3Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
10411PRTHomo sapiensMISC_FEATURE(1)..(11)X can be any amino acid
4Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10512PRTHomo
sapiensMISC_FEATUREX can be any amino acidmisc_feature(1)..(12)Xaa
can be any naturally occurring amino acid 5Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa1 5 1069PRTHIV-005 6Ser Leu Tyr Asn Thr Val
Ala Thr Leu1 579PRTInfluenza A MP 7Gly Ile Leu Gly Phe Val Phe Thr
Leu1 589PRTHuman cytomegalovirus pp 65 8Asn Leu Val Pro Met Val Ala
Thr Val1 5910PRTArtificial SequenceTAA 9Tyr Leu Tyr Asp Ser Glu Thr
Lys Asn Ala1 5 10109PRTArtificial SequenceTAA 10His Leu Met Asp Gln
Pro Leu Ser Val1 5119PRTArtificial SequenceTAA 11Gly Leu Leu Lys
Lys Ile Asn Ser Val1 5129PRTArtificial SequenceTAA 12Phe Leu Val
Asp Gly Ser Ser Ala Leu1 51310PRTArtificial SequenceTAA 13Phe Leu
Phe Asp Gly Ser Ala Asn Leu Val1 5 10149PRTArtificial SequenceTAA
14Phe Leu Tyr Lys Ile Ile Asp Glu Leu1 51511PRTArtificial
SequenceTAA 15Phe Ile Leu Asp Ser Ala Glu Thr Thr Thr Leu1 5
10169PRTArtificial SequenceTAA 16Ser Val Asp Val Ser Pro Pro Lys
Val1 5178PRTArtificial SequenceTAA 17Val Ala Asp Lys Ile His Ser
Val1 5189PRTArtificial SequenceTAA 18Ile Val Asp Asp Leu Thr Ile
Asn Leu1 5199PRTArtificial SequenceTAA 19Gly Leu Leu Glu Glu Leu
Val Thr Val1 52010PRTArtificial SequenceTAA 20Thr Leu Asp Gly Ala
Ala Val Asn Gln Val1 5 102110PRTArtificial SequenceTAA 21Ser Val
Leu Glu Lys Glu Ile Tyr Ser Ile1 5 10229PRTArtificial SequenceTAA
22Leu Leu Asp Pro Lys Thr Ile Phe Leu1 5239PRTArtificial
SequenceTAA 23Tyr Leu Met Asp Asp Phe Ser Ser Leu1
5249PRTArtificial SequenceTAA 24Lys Val Trp Ser Asp Val Thr Pro
Leu1 52511PRTArtificial SequenceTAA 25Leu Leu Trp Gly His Pro Arg
Val Ala Leu Ala1 5 102611PRTArtificial SequenceTAA 26Lys Ile Trp
Glu Glu Leu Ser Val Leu Glu Val1 5 10279PRTArtificial SequenceTAA
27Leu Leu Ile Pro Phe Thr Ile Phe Met1 5289PRTArtificial
SequenceTAA 28Phe Leu Ile Glu Asn Leu Leu Ala Ala1
52911PRTArtificial SequenceTAA 29Leu Leu Trp Gly His Pro Arg Val
Ala Leu Ala1 5 10309PRTArtificial SequenceTAA 30Phe Leu Leu Glu Arg
Glu Gln Leu Leu1 5319PRTArtificial SequenceTAA 31Ser Leu Ala Glu
Thr Ile Phe Ile Val1 5329PRTArtificial SequenceTAA 32Thr Leu Leu
Glu Gly Ile Ser Arg Ala1 5339PRTArtificial SequenceTAA 33Ile Leu
Gln Asp Gly Gln Phe Leu Val1 53410PRTArtificial SequenceTAA 34Val
Ile Phe Glu Gly Glu Pro Met Tyr Leu1 5 10359PRTArtificial
SequenceTAA 35Ser Leu Phe Glu Ser Leu Glu Tyr Leu1
5369PRTArtificial SequenceTAA 36Ser Leu Leu Asn Gln Pro Lys Ala
Val1 5379PRTArtificial SequenceTAA 37Gly Leu Ala Glu Phe Gln Glu
Asn Val1 5389PRTArtificial SequenceTAA 38Lys Leu Leu Ala Val Ile
His Glu Leu1 5399PRTArtificial SequenceTAA 39Thr Leu His Asp Gln
Val His Leu Leu1 54011PRTArtificial SequenceTAA 40Thr Leu Tyr Asn
Pro Glu Arg Thr Ile Thr Val1 5 10419PRTArtificial SequenceTAA 41Lys
Leu Gln Glu Lys Ile Gln Glu Leu1 54210PRTArtificial SequenceTAA
42Ser Val Leu Glu Lys Glu Ile Tyr Ser Ile1 5 104311PRTArtificial
SequenceTAA 43Arg Val Ile Asp Asp Ser Leu Val Val Gly Val1 5
10449PRTArtificial SequenceTAA 44Val Leu Phe Gly Glu Leu Pro Ala
Leu1 5459PRTArtificial SequenceTAA 45Gly Leu Val Asp Ile Met Val
His Leu1 5469PRTArtificial SequenceTAA 46Phe Leu Asn Ala Ile Glu
Thr Ala Leu1 5479PRTArtificial SequenceTAA 47Ala Leu Leu Gln Ala
Leu Met Glu Leu1 5489PRTArtificial SequenceTAA 48Ala Leu Ser Ser
Ser Gln Ala Glu Val1 54911PRTArtificial SequenceTAA 49Ser Leu Ile
Thr Gly Gln Asp Leu Leu Ser Val1 5 10509PRTArtificial SequenceTAA
50Gln Leu Ile Glu Lys Asn Trp Leu Leu1 5519PRTArtificial
SequenceTAA 51Leu Leu Asp Pro Lys Thr Ile Phe Leu1
5529PRTArtificial SequenceTAA 52Arg Leu His Asp Glu Asn Ile Leu
Leu1 5539PRTArtificial SequenceTAA 53Gly Leu Pro Ser Ala Thr Thr
Thr Val1 55411PRTArtificial SequenceTAA 54Gly Leu Leu Pro Ser Ala
Glu Ser Ile Lys Leu1 5 10559PRTArtificial SequenceTAA 55Lys Thr Ala
Ser Ile Asn Gln Asn Val1 5569PRTArtificial SequencePRAME-004 56Ser
Leu Leu Gln His Leu Ile Gly Leu1 5579PRTArtificial SequenceTAA
57Ser Leu Leu Gln His Leu Ile Gly Leu1 5589PRTArtificial
SequenceTAA 58Ser Leu Leu Gln His Leu Ile Gly Leu1
5599PRTArtificial SequenceTAA 59Lys Val Trp Ser Asp Val Thr Pro
Leu1 56011PRTArtificial SequenceTAA 60Leu Leu Trp Gly His Pro Arg
Val Ala Leu Ala1 5 10619PRTArtificial SequenceTAA 61Val Leu Asp Gly
Lys Val Ala Val Val1 5629PRTArtificial SequenceTAA 62Gly Leu Leu
Gly Lys Val Thr Ser Val1 5639PRTArtificial SequenceTAA 63Lys Met
Ile Ser Ala Ile Pro Thr Leu1 56411PRTArtificial SequenceTAA 64Gly
Leu Leu Glu Thr Thr Gly Leu Leu Ala Thr1 5 10659PRTArtificial
SequenceTAA 65Thr Leu Asn Thr Leu Asp Ile Asn Leu1
5669PRTArtificial SequenceTAA 66Val Ile Ile Lys Gly Leu Glu Glu
Ile1 5679PRTArtificial SequenceTAA 67Tyr Leu Glu Asp Gly Phe Ala
Tyr Val1 56811PRTArtificial SequenceTAA 68Lys Ile Trp Glu Glu Leu
Ser Val Leu Glu Val1 5 10699PRTArtificial SequenceTAA 69Leu Leu Ile
Pro Phe Thr Ile Phe Met1 57010PRTArtificial SequenceTAA 70Ile Ser
Leu Asp Glu Val Ala Val Ser Leu1 5 107110PRTArtificial SequenceTAA
71Lys Ile Ser Asp Phe Gly Leu Ala Thr Val1 5 107211PRTArtificial
SequenceTAA 72Lys Leu Ile Gly Asn Ile His Gly Asn Glu Val1 5
10739PRTArtificial SequenceTAA 73Ile Leu Leu Ser Val Leu His Gln
Leu1 5749PRTArtificial SequenceTAA 74Leu Asp Ser Glu Ala Leu Leu
Thr Leu1 57513PRTArtificial SequenceTAA 75Val Leu Gln Glu Asn Ser
Ser Asp Tyr Gln Ser Asn Leu1 5 107611PRTArtificial SequenceTAA
76His Leu Leu Gly Glu Gly Ala Phe Ala Gln Val1 5 10779PRTArtificial
SequenceTAA 77Ser Leu Val Glu Asn Ile His Val Leu1
5789PRTArtificial SequenceTAA 78Ser Leu Ser Glu Lys Ser Pro Glu
Val1 57910PRTArtificial SequenceTAA 79Ala Met Phe Pro Asp Thr Ile
Pro Arg Val1 5 10809PRTArtificial SequenceTAA 80Phe Leu Ile Glu Asn
Leu Leu Ala Ala1 5819PRTArtificial SequenceTAA 81Phe Thr Ala Glu
Phe Leu Glu Lys Val1 5829PRTArtificial SequenceTAA 82Ala Leu Tyr
Gly Asn Val Gln Gln Val1 5839PRTArtificial SequenceTAA 83Leu Phe
Gln Ser Arg Ile Ala Gly Val1 58411PRTArtificial SequenceTAA 84Ile
Leu Ala Glu Glu Pro Ile Tyr Ile Arg Val1 5 10859PRTArtificial
SequenceTAA 85Phe Leu Leu Glu Arg Glu Gln Leu Leu1
58610PRTArtificial SequenceTAA 86Leu Leu Leu Pro Leu Glu Leu Ser
Leu Ala1 5 10879PRTArtificial SequenceTAA 87Ser Leu Ala Glu Thr Ile
Phe Ile Val1 58811PRTArtificial SequenceTAA 88Ala Ile Leu Asn Val
Asp Glu Lys Asn Gln Val1 5 10899PRTArtificial SequenceTAA 89Arg Leu
Phe Glu Glu Val Leu Gly Val1 5909PRTArtificial SequenceTAA 90Tyr
Leu Asp Glu Val Ala Phe Met Leu1 59111PRTArtificial SequenceTAA
91Lys Leu Ile Asp Glu Asp Glu Pro Leu Phe Leu1 5 10929PRTArtificial
SequenceTAA 92Lys Leu Phe Glu Lys Ser Thr Gly Leu1
59311PRTArtificial SequenceTAA 93Ser Leu Leu Glu Val Asn Glu Ala
Ser Ser Val1 5 109410PRTArtificial SequenceTAA 94Gly Val Tyr Asp
Gly Arg Glu His Thr Val1 5 109510PRTArtificial SequenceTAA 95Gly
Leu Tyr Pro Val Thr Leu Val Gly Val1 5 10969PRTArtificial
SequenceTAA 96Ala Leu Leu Ser Ser Val Ala Glu Ala1
5979PRTArtificial SequenceTAA 97Thr Leu Leu Glu Gly Ile Ser Arg
Ala1 5989PRTArtificial SequenceTAA 98Ser Leu Ile Glu Glu Ser Glu
Glu Leu1 5999PRTArtificial SequenceTAA 99Ala Leu Tyr Val Gln Ala
Pro Thr Val1 510010PRTArtificial SequenceTAA 100Lys Leu Ile Tyr Lys
Asp Leu Val Ser Val1 5 101019PRTArtificial SequenceTAA 101Ile Leu
Gln Asp Gly Gln Phe Leu Val1 51029PRTArtificial SequenceTAA 102Ser
Leu Leu Asp Tyr Glu Val Ser Ile1 51039PRTArtificial SequenceTAA
103Leu Leu Gly Asp Ser Ser Phe Phe Leu1 510410PRTArtificial
SequenceTAA 104Val Ile Phe Glu Gly Glu Pro Met Tyr Leu1 5
101059PRTArtificial SequenceTAA 105Ala Leu Ser Tyr Ile Leu Pro Tyr
Leu1 51069PRTArtificial SequenceTAA 106Phe Leu Phe Val Asp Pro Glu
Leu Val1 510711PRTArtificial SequenceTAA 107Ser Glu Trp Gly Ser Pro
His Ala Ala Val Pro1 5 101089PRTArtificial SequenceTAA 108Ala Leu
Ser Glu Leu Glu Arg Val Leu1 51099PRTArtificial SequenceTAA 109Ser
Leu Phe Glu Ser Leu Glu Tyr Leu1 51109PRTArtificial SequenceTAA
110Lys Val Leu Glu Tyr Val Ile Lys Val1 511110PRTArtificial
SequenceTAA 111Val Leu Leu Asn Glu Ile Leu Glu Gln Val1 5
101129PRTArtificial SequenceTAA 112Ser Leu Leu Asn Gln Pro Lys Ala
Val1 51139PRTArtificial SequenceTAA 113Lys Met Ser Glu Leu Gln Thr
Tyr Val1 511411PRTArtificial SequenceTAA 114Ala Leu Leu Glu Gln Thr
Gly Asp Met Ser Leu1 5 1011511PRTArtificial SequenceTAA 115Val Ile
Ile Lys Gly Leu Glu Glu Ile Thr Val1 5 101169PRTArtificial
SequenceTAA 116Lys Gln Phe Glu Gly Thr Val Glu Ile1
51179PRTArtificial SequenceTAA 117Lys Leu Gln Glu Glu Ile Pro Val
Leu1 51189PRTArtificial SequenceTAA 118Gly Leu Ala Glu Phe Gln Glu
Asn Val1 51199PRTArtificial SequenceTAA 119Asn Val Ala Glu Ile Val
Ile His Ile1 51209PRTArtificial SequenceTAA 120Ala Leu Ala Gly Ile
Val Thr Asn Val1 512112PRTArtificial SequenceTAA 121Asn Leu Leu Ile
Asp Asp Lys Gly Thr Ile Lys Leu1 5 1012210PRTArtificial SequenceTAA
122Val Leu Met Gln Asp Ser Arg Leu Tyr Leu1 5 101239PRTArtificial
SequenceMAG-003 123Lys Val Leu Glu His Val Val Arg Val1
51249PRTArtificial SequenceTAA 124Leu Leu Trp Gly Asn Leu Pro Glu
Ile1 51259PRTArtificial SequenceTAA 125Ser Leu Met Glu Lys Asn Gln
Ser Leu1 51269PRTArtificial SequenceTAA 126Lys Leu Leu Ala Val Ile
His Glu Leu1 512710PRTArtificial SequenceTAA 127Ala Leu Gly Asp Lys
Phe Leu Leu Arg Val1 5 1012811PRTArtificial SequenceTAA 128Phe Leu
Met Lys Asn Ser Asp Leu Tyr Gly Ala1 5 1012910PRTArtificial
SequenceTAA 129Lys Leu Ile Asp His Gln Gly Leu Tyr Leu1 5
1013012PRTArtificial SequenceTAA 130Gly Pro Gly Ile Phe Pro Pro Pro
Pro Pro Gln Pro1 5 101319PRTArtificial SequenceTAA 131Ala Leu Asn
Glu Ser Leu Val Glu Cys1 51329PRTArtificial SequenceTAA 132Gly Leu
Ala Ala Leu Ala Val His Leu1 51339PRTArtificial SequenceTAA 133Leu
Leu Leu Glu Ala Val Trp His Leu1 51349PRTArtificial SequenceTAA
134Ser Ile Ile Glu Tyr Leu Pro Thr Leu1 51359PRTArtificial
SequenceTAA 135Thr Leu His Asp Gln Val His Leu Leu1
51369PRTArtificial SequenceTAA 136Ser Leu Leu Met Trp Ile Thr Gln
Cys1 513711PRTArtificial SequenceTAA 137Phe Leu Leu Asp Lys Pro Gln
Asp Leu Ser Ile1 5 1013810PRTArtificial SequenceTAA 138Tyr Leu Leu
Asp Met Pro Leu Trp Tyr Leu1 5 101399PRTArtificial SequenceTAA
139Gly Leu Leu Asp Cys Pro Ile Phe Leu1 51409PRTArtificial
SequenceTAA 140Val Leu Ile Glu Tyr Asn Phe Ser Ile1
514111PRTArtificial SequenceTAA 141Thr Leu Tyr Asn Pro Glu Arg Thr
Ile Thr Val1 5 101429PRTArtificial SequenceTAA 142Ala Val Pro Pro
Pro Pro Ser Ser Val1 51439PRTArtificial SequenceTAA 143Lys Leu Gln
Glu Glu Leu Asn Lys Val1 514411PRTArtificial SequenceTAA 144Lys Leu
Met Asp Pro Gly Ser Leu Pro Pro Leu1 5 101459PRTArtificial
SequenceTAA 145Ala Leu Ile Val Ser Leu Pro Tyr Leu1
51469PRTArtificial SequenceTAA 146Phe Leu Leu Asp Gly Ser Ala Asn
Val1 514710PRTArtificial SequenceTAA 147Ala Leu Asp Pro Ser Gly Asn
Gln Leu Ile1 5 101489PRTArtificial SequenceTAA 148Ile Leu Ile Lys
His Leu Val Lys Val1 51499PRTArtificial SequenceTAA 149Val Leu Leu
Asp Thr Ile Leu Gln Leu1 51509PRTArtificial SequenceTAA 150His Leu
Ile Ala Glu Ile His Thr Ala1 51519PRTArtificial SequenceTAA 151Ser
Met Asn Gly Gly Val Phe Ala Val1 51529PRTArtificial SequenceTAA
152Met Leu Ala Glu Lys Leu Leu Gln Ala1 51539PRTArtificial
SequenceTAA 153Tyr Met Leu Asp Ile Phe His Glu Val1
515411PRTArtificial SequenceTAA 154Ala Leu Trp Leu Pro Thr Asp Ser
Ala Thr Val1 5 101559PRTArtificial SequenceTAA 155Gly Leu Ala Ser
Arg Ile Leu Asp Ala1 51569PRTArtificial SequenceTAA 156Ala Leu Ser
Val Leu Arg Leu Ala Leu1 51579PRTArtificial SequenceTAA 157Ser Tyr
Val Lys Val Leu His His Leu1 51589PRTArtificial SequenceTAA 158Val
Tyr Leu Pro Lys Ile Pro Ser Trp1 51599PRTArtificial SequenceTAA
159Asn Tyr Glu Asp His Phe Pro Leu Leu1 51609PRTArtificial
SequenceTAA 160Val Tyr Ile Ala Glu Leu Glu Lys Ile1
516112PRTArtificial SequenceTAA 161Val His Phe Glu Asp Thr Gly Lys
Thr Leu Leu Phe1 5 101629PRTArtificial SequenceTAA 162Val Leu Ser
Pro Phe Ile Leu Thr Leu1 51639PRTArtificial SequenceTAA 163His Leu
Leu Glu Gly Ser Val Gly Val1 51649PRTArtificial SequenceTarget
Peptide 164Val Leu Leu His His Gln Ile Gly Leu1 51659PRTArtificial
SequenceTarget Similar Peptide 1 165Ala Leu Met Tyr His Thr Ile Thr
Leu1 51669PRTArtificial SequenceTarget Similar Peptide 2 166Leu Leu
Leu Ala His Ile Ile Ala Leu1 51679PRTArtificial SequenceIrrelevant
Peptide 167Ala Ile Val Asp Lys Val Pro Ser Val1 5
* * * * *
References