U.S. patent application number 14/371910 was filed with the patent office on 2015-03-19 for dual antigen-induced bipartite functional complementation.
The applicant listed for this patent is JULUS-MAXIMILIANS-UNIVERSITAT WURZBURG. Invention is credited to Gernot Stuhler.
Application Number | 20150079093 14/371910 |
Document ID | / |
Family ID | 47715984 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150079093 |
Kind Code |
A1 |
Stuhler; Gernot |
March 19, 2015 |
DUAL ANTIGEN-INDUCED BIPARTITE FUNCTIONAL COMPLEMENTATION
Abstract
The present invention relates to a set of polypeptides and its
uses. In particular, the present invention relates to a set of
polypeptides whereby this set comprises two polypeptides each of
which comprises a targeting moiety "T" binding to an antigen "A"
and a fragment of "F" of a functional domain, wherein said two
polypeptides are not associated with each other in absence of a
substrate that has "A" at (on) its surface and wherein, upon
dimerization of "F", the resulting dimer becomes functional.
Furthermore, medical and diagnostic uses of said set are described.
Moreover, the present invention relates to nucleic acid molecule(s)
encoding said set of polypeptides. The present invention also
relates to a vector comprising the nucleotide sequence of nucleic
acid molecule(s) encoding said set of polypeptides. Furthermore,
the present invention relates to pharmaceutical compositions
comprising said set of polypeptides. Moreover, the present
invention relates to a kit comprising said set of polypeptides.
Inventors: |
Stuhler; Gernot; (Tubingen,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JULUS-MAXIMILIANS-UNIVERSITAT WURZBURG |
Wurzburg |
|
DE |
|
|
Family ID: |
47715984 |
Appl. No.: |
14/371910 |
Filed: |
January 14, 2013 |
PCT Filed: |
January 14, 2013 |
PCT NO: |
PCT/EP2013/050603 |
371 Date: |
July 11, 2014 |
Current U.S.
Class: |
424/136.1 ;
435/7.23; 536/23.53 |
Current CPC
Class: |
A61P 25/00 20180101;
C07K 16/2809 20130101; A61P 1/04 20180101; A61P 7/04 20180101; C07K
16/32 20130101; C07K 2317/73 20130101; C07K 16/289 20130101; C07K
16/30 20130101; A61P 9/00 20180101; C07K 16/3007 20130101; G01N
33/574 20130101; C07K 2317/34 20130101; A61P 31/00 20180101; A61P
35/00 20180101; C07K 2317/622 20130101; A61P 37/02 20180101; G01N
33/57484 20130101; C07K 16/2896 20130101; A61P 19/02 20180101; C07K
16/468 20130101; A61P 17/06 20180101; A61P 43/00 20180101; A61P
37/06 20180101; C07K 16/2863 20130101; A61P 37/08 20180101; C07K
2317/56 20130101; C07K 16/2833 20130101; G01N 2800/245 20130101;
A61P 29/00 20180101; C07K 16/2827 20130101; C07K 16/3061 20130101;
A61P 3/10 20180101; C07K 2317/31 20130101; A61K 2039/505
20130101 |
Class at
Publication: |
424/136.1 ;
435/7.23; 536/23.53 |
International
Class: |
C07K 16/30 20060101
C07K016/30; G01N 33/574 20060101 G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 13, 2012 |
EP |
12151125.7 |
Claims
1. A set of polypeptides comprising: a first polypeptide P1
comprising (i) a targeting moiety Ti, wherein said targeting moiety
T1 specifically binds to an antigen A1, and (ii) a fragment F1 of a
functional domain F, wherein neither said fragment F1 by itself nor
said polypeptide P1 by itself is functional with respect to the
function of said domain F, and a second polypeptide P2 comprising
(i) a targeting moiety T2, wherein said targeting moiety T2
specifically binds to an antigen A2, and (ii) a fragment F2 of said
functional domain F, wherein neither said fragment F2 by itself nor
said polypeptide P2 by itself is functional with respect to the
function of said domain F, wherein said antigen A1 is different
from said antigen A2, wherein said polypeptide P1 and said
polypeptide P2 are not associated with each other in the absence of
a substrate that has both antigens A1 and A2 at its surface, and
wherein, upon dimerization of said fragment F1 of said polypeptide
P1 with said fragment F2 of said polypeptide P2, the resulting
dimer is functional with respect to the function of said domain
F.
2. The set of polypeptides according to claim 1, wherein said
polypeptide P1 and said polypeptide P2 are not associated with each
other in the absence of a cell that carries both antigens A1 and A2
at its cell surface.
3. The set of polypeptides according to claim 1, wherein a cell
carrying both antigens A1 and A2 at its cell surface induces
dimerization of the fragment F1 of said polypeptide P1 with the
fragment F2 of said polypeptide P2, whereas a cell which does not
carry both antigens A1 and A2 at its cell surface does not induce
dimerization of the fragment F1 of said polypeptide P1 with the
fragment F2 of said polypeptide P2.
4. The set of polypeptides according to claim 1, wherein said
polypeptides P1 and P2 have, in the absence of said substrate or
cell, with each other a dissociation constant K.sub.D in the range
of 10.sup.-8 M to 10.sup.-2 M, in the range of 10.sup.-7 M to
10.sup.-3 M or in the range of 10.sup.-6 M to 10.sup.-3 M; and/or
said polypeptides P1 and P2 have, in the presence of said substrate
or cell, with each other a dissociation constant K.sub.D below
10.sup.-6 M, below 10.sup.-7 M below 10.sup.-8 M or below 10.sup.-9
M.
5. The set of polypeptides according to claim 1, wherein said
antigen A1 and/or said antigen A2 is an antigen expressed on the
surface of cells of a tumour or on the surface of
progenitor/precursor cells of a tumour.
6. (canceled)
7. The set of polypeptides according to claim 1, wherein the
combination of antigen A1 and antigen A2 is only found on cancerous
cells, and not on cells that are not cancerous.
8. The set of polypeptides according to claim 7, wherein the
combination of antigen A1 and antigen A2 is specific for cancerous
cells of a certain type of cancer.
9. The set of polypeptides according to claim 1, wherein said
antigen A1 is an MHC antigen being an allelic variant selected from
the group consisting of: HLA-A2, HLA-Cw6, HLA-A1, HLA-A3, HLA-A25,
HLA-B7, HLA-B8, HLA-B35, HLA-B44, HLA-Cw3, HLA-Cw4, and HLA-Cw7;
and/or said antigen A2 is an antigen that is specific for a certain
cell type or cell lineage selected from the group consisting of:
CD45; CD34; CD33; CD138; CD15; CD1a; CD2; CD3; CD4; CD5; CD8; CD20;
CD23; CD31; CD43; CD56; CD57; CD68; CD79a; CD146; surfactant
proteins; synaptophysin; CD56; CD57; nicotinic acetylcholine
receptor; muscle-specific kinase MUSK; voltage-gated calcium
channel (P/Q-type); voltage-gated potassium channel (VGKC);
N-methyl-D-aspartate receptor (NMDA); TSH; amphiphysin; HepPar-1;
ganglioside GQ1B, GD3 or GM 1; and glycophorin-A.
10.-13. (canceled)
14. The set of polypeptides according to claim 1, wherein any of
said antigens A1 and A2 is selected from the group consisting of:
HLA-A2; HLA-Cw6; EpCAM; CD20; CD33; CD38; CD45; Her2; EGFR; CD138;
CEA; CD19; PSMA; E-cadherin; Ca-125; Her-2/neu; gross cystic
disease fluid protein; BCA-225; CA 19-9; CD117; CD30; Epithelial
antigen BER-EP4, Epithelial membrane antigen and Epithelial Related
Antigen MOC-31; Epidermal growth factor receptor HER1; Platelet
derived growth factor receptor PDGFR alpha; Melanoma associated
marker/Mart 1/Melan-A; CD133; TAG 72; aquaporin-2 and a clonotypic
antibody on the surface of a B cell.
15. The set of polypeptides according to claim 1, wherein (i) one
of said antigens A1 and A2 is EpCAM and the other one is EGFR,
HER2/neu, CD 10, VEGF-R or MDR; (ii) one of said antigens A1 and A2
is MCSP and the other one is melanoferrin or EpCAM; (iii) one of
said antigens A1 and A2 is CA125 and the other one CD227; (iv) one
of said antigens A1 and A2 is CD56 and the other one is CD140b or
GD3 ganglioside; (v) one of said antigens A1 and A2 is EGFR and the
other one is HER2; (vi) one of said antigens A1 and A2 is PSMA and
the other one is HER2; (vii) one of said antigens A1 and A2 is
Sialyl Lewis and the other one is EGFR; (viii) one of said antigens
A1 and A2 is CD44 and the other one is ESA, CD24, CD133, MDR or
CD117; (ix) one of said antigens A1 and A2 is CD34 and the other
one is CD19, CD79a, CD2, CD7, HLA-DR, CD 13, CD 117, CD33 or CD 15;
(x) one of said antigens A1 and A2 is CD33 and the other one is
CD19, CD79a, CD2, CD7, HLA-DR, CD13, CD117 or CD15; (xi) one of
said antigens A1 and A2 is MUC1 and the other one is CD10, CEA or
CD57; (xii) one of said antigens A1 and A2 is CD38 and the other
one is CD138; (xiii) one of said antigens A1 and A2 is CD 24 and
the other one is CD29 or CD49f; (xiv) one of said antigens A1 and
A2 is carbonic anhydrase IX and the other one is aquaporin-2; (xv)
one of said antigens A1 and A2 is HLA-A2 and the other one is
EpCAM; (xvi) one of said antigens A1 and A2 is HLA-A2 and the other
one is CD45; (xvii) one of said antigens A1 and A2 is HLA-A2 and
the other one is EGFR; (xviii) one of said antigens A1 and A2 is
HLA-A2 and the other one is Her2; (xix) one of said antigens A1 and
A2 is HLA-A2 and the other one is CEA; (xx) one of said antigens A1
and A2 is EpCAM and the other one is CEA; (xxi) one of said
antigens A1 and A2 is CD45 and the other one is CD138; (xxii) one
of said antigens A1 and A2 is EGFR and the other one is CEA;
(xxiii) one of said antigens A1 and A2 is Her2 and the other one is
CEA; or (xxiv) one of said antigens A1 and A2 is CD19 and the other
one is a clonotypic antibody on the surface of a B cell.
16. The set of polypeptides according to claim 1, wherein said
targeting moiety T1 and/or T2 comprises an immunoglobulin module;
or wherein said targeting moiety T1 and/or T2 comprises an aptamer
or a natural ligand of said antigen A1 or antigen A2,
respectively.
17. The set of polypeptides according to claim 16, wherein said
targeting moiety T1 comprises an immunoglobulin module I1
comprising a V.sub.L domain linked to a V.sub.H domain or
comprising a variable domain V.sub.HH of a llama antibody, camel
antibody or shark antibody; and/or said targeting moiety T2
comprises an immunoglobulin module I2 comprising a V.sub.L domain
linked to a V.sub.H domain or comprising a variable domain V.sub.HH
of a llama antibody, camel antibody or shark antibody.
18. The set of polypeptides according to claim 17, wherein said
immunoglobulin module I1 comprises a scFv (single-chain variant
fragment), a Fab or a F(ab').sub.2 of an antibody or a complete
antibody; and/or said immunoglobulin module I2 comprises a scFv
(single-chain variant fragment), a Fab or a F(ab').sub.2 of an
antibody or a complete antibody.
19.-21. (canceled)
22. The set of polypeptides according to claim 1, wherein any one
of said targeting moiety T1 and T2 comprises an allergen or
substrate which binds to a clonotypic antibody on the surface of a
B cell.
23. The set of polypeptides according to claim 1, wherein said
functional domain F is or comprises an immunoglobulin module, or a
fluorescent molecule, or a molecule capable of mediating
bioluminescence.
24. The set of polypeptides according to claim 23, wherein said
functional domain F is a Fv (variant fragment) or a scFv
(single-chain variant fragment) of an antibody.
25.-28. (canceled)
29. The set of polypeptides according to claim 1, wherein said
fragment F1 comprises a V.sub.L domain of an antibody and said
fragment F2 comprises a V.sub.H domain of the same antibody; or
wherein said fragment F1 comprises a V.sub.H domain of an antibody
and said fragment F2 comprises a V.sub.L domain of the same
antibody.
30. The set of polypeptides according to claim 1, wherein said
fragment F1 comprises a V.sub.L domain of an anti-CD3, anti-His or
anti-DIG antibody and said fragment F2 comprises a V.sub.H domain
of the same antibody, or wherein said fragment F1 comprises a
V.sub.H domain of an anti-CD3 anti-His or anti-DIG antibody and
said fragment F2 comprises a V.sub.L domain of the same
antibody.
31. (canceled)
32. The set of polypeptides according to claim 23, wherein said
immunoglobulin module comprises a V domain selected from the group
consisting of: (i) a V domain of an anti-CD3 antibody comprising a
V.sub.L domain comprising SEQ ID NO: 2 and/or a V.sub.H domain
comprising SEQ ID NO: 1; (ii) a V domain of an anti-CD3 antibody
comprising a V.sub.L domain comprising SEQ ID NO: 4 and/or a
V.sub.H domain comprising SEQ ID NO: 3; (iii) a V domain of an
anti-CD3 antibody comprising a V.sub.L domain comprising SEQ ID NO:
6 and/or a V.sub.H domain comprising SEQ ID NO: 5; (iv) a V domain
of an anti-CD3 antibody comprising a V.sub.L domain comprising SEQ
ID NO: 8 and/or a V.sub.H domain comprising SEQ ID NO: 7; (v) a V
domain of an anti-CD3 antibody comprising a V.sub.L domain
comprising SEQ ID NO: 10 and/or a V.sub.H domain comprising SEQ ID
NO: 9; (vi) a V domain of an anti-His antibody comprising a V.sub.L
domain comprising SEQ ID NO: 12 and/or a V.sub.H domain comprising
SEQ ID NO: 11; and (vii) a V domain of an anti-DIG antibody
comprising a V.sub.L domain comprising SEQ ID NO: 14 and/or a
V.sub.H domain comprising SEQ ID NO: 30.
33. The set of polypeptides according to claim 1, wherein any of
the polypeptides P1 and P2 is or comprises an amino acid sequence
selected from the group consisting of SEQ ID NOS: 114-129 and
197.
34. A method of treating a patient who is suffering from cancer
and/or a tumour or method of diagnosing a patient who is suffering
from cancer and/or a tumour, said method comprising the step of
administering to said patient an effective amount of the set of
polypeptides according to claim 1.
35.-41. (canceled)
42. A nucleic acid molecule or a set of nucleic acid molecules
encoding the set of polypeptides or one of the polypeptides of the
set of polypeptides according to claim 1.
43. The nucleic acid molecule or set of nucleic acid molecules
according to claim 42 comprising a nucleotide sequence as depicted
in any one of SEQ ID NOS: 135-150 and 196.
44.-45. (canceled)
46. A kit comprising the set of polypeptides according to claim
1.
47. A kit comprising the nucleic acid molecule or the set of
nucleic acid molecules according to claim 42.
Description
[0001] The present invention relates to a set of polypeptides and
its uses. In particular, the present invention relates to a set of
polypeptides whereby this set comprises two polypeptides each of
which comprises a targeting moiety "T" binding to an antigen "A"
and a fragment of "F" of a functional domain, wherein said two
polypeptides are not associated with each other in absence of a
substrate that has "A" at (on) its surface and wherein, upon
dimerization of "F", the resulting dimer becomes functional.
Furthermore, medical and diagnostic uses of said set are described.
Moreover, the present invention relates to nucleic acid molecule(s)
encoding said set of polypeptides. The present invention also
relates to a vector comprising the nucleotide sequence of nucleic
acid molecule(s) encoding said set of polypeptides. Furthermore,
the present invention relates to pharmaceutical compositions
comprising said set of polypeptides. Moreover, the present
invention relates to a kit comprising said set of polypeptides.
[0002] The last years have seen a number of landmark papers
reporting outstanding efficacy of bispecific antibody constructs
for immune therapy of tumours in vitro and in pre-clinical and
early clinical trials. Today, a substantial number of different
bispecific constructs are available that differ in size,
composition, pharmacokinetics and ability to directly eliminate
neoplastic cells or to engage immune effector cells for tumour cell
lysis.
[0003] Antibody-based cancer immune strategies are highly promising
therapeutic options due to their excellent sensitivity and
specificity towards target structures.
[0004] The modular structural and functional organisation of
antibodies allows extensive manipulation by genetic engineering.
Different immunoglobulin-like domains can be separated and/or
joined without losing specific domain-associated functional
features. Moreover, they can be combined and linked with
heterologous protein domains but also with non-peptidic moieties.
It is therefore possible to develop fusion constructs in a rational
way devoid of the natural limitations of conventional
antibodies.
[0005] Antibody-based fusion proteins can be generated with novel
biological and/or pharmaceutical properties. There are promising
efforts to modify the capability of the Fc domain to elicit ADCC
(antibody dependent cell mediated cytotoxicity) and CDC
(complement-dependent cytotoxicity) by mutagenesis, dependent on
the intended application, either to reduce side effects (inhibitory
mutations) or to enhance therapeutic efficacy (activating
mutations). New applications that become possible by genetic
engineering are even more variate when the antigen binding domain
of antibodies is considered.
[0006] The antigen recognizing variable domains of the heavy
(V.sub.H) and light chain (V.sub.L) of an antibody can be joined by
a peptide linker via genetic engineering while preserving the
antigen binding capability. Such antigen binding single chain
variable fragments (scFvs) can be used as small antibody surrogates
with high tissue penetrating capability and low serum retention
time for clinical imaging procedures and radiotherapy and other
applications. Importantly, these scFv moieties can be easily
employed as antigen specific modules in the development of novel
recombinant therapeutics.
[0007] Recent reports indicate a tremendous potential of
recombinant bispecific antibodies in anti-tumour therapy. Such
bispecific antibodies recognise two antigens, one of which is
expressed by the tumour, whereas the other is usually found on an
immune cell. Most bispecific antibodies in anti-tumour therapy
target a tumour-associated lineage marker on the one hand and
CD3.epsilon., an invariant molecule of the T-cell receptor/CD3
complex on the other hand, thus recruiting T cells to destroy the
tumour [Muller and Kontermann, Bispecific antibodies for cancer
immunotherapy: Current perspectives. BioDrugs 2010,
24(2):89-98].
[0008] Despite the extensive options for manipulating antibody
structure and function, the therapeutic efficacy of such
antibody-based reagents is limited by the nature of the addressed
antigen, the accessibility of the antigen in tumour and
tumour-associated tissues and the aptitude of the antibody to
elicit or mediate the desired cell death inducing function.
[0009] For example, when patients are treated with bispecific
constructs directed against antigens also expressed on tissues with
vital functions, severe side effects are observed. This is a severe
problem, since, with the exception of an unknown number of
individually mutated cell surface molecules and the monoclonal B-
or T-cell receptor in case of lymphomas, tumour specific antigens
that discriminate a transformed cell from its healthy progenitor
are not available.
[0010] Since therapeutic concepts based on the use of bispecific
antibodies usually rely on the recruitment of effector cells, it
appears that the more effective the tool (bispecific construct),
the more likely side effects do occur, and even minute expression
of antigen on non-transformed tissue can cause uncontrollable
off-target effects.
[0011] In 2008, SCIENCE published the first report on the clinical
efficacy of the single-chain bispecific T cell engaging (BiTE)
antibody MT103/blinatumomab; it induces remissions in about 80% of
lymphoma patients relapsed or refractory to standard
immune-chemotherapy at serum levels about 5 orders of magnitude
lower than serum levels reported for the monoclonal antibody
rituximab (Bargou, R. et al Science 321, 974-977, 2008). This
publication and subsequent reports on confirmatory phase II trials
in acute lymphatic leukemia (ALL) ushered in a new era of
bispecific antibodies, until then in grave demise for almost two
decades due to systemic toxicity and little or no therapeutic
activity. Mainly in the wake of that SCIENCE paper, bispecific
antibodies became a burgeoning field again in which more than 35
different formats were counted (Reichert, Drug Discov Today. 17
(2012) 954-963). These formats differ in size and are optimized for
affinity to the antigen, stability, ability to recruit effector
cells (mostly T cells) and pharmacokinetics. Affinity or avidity of
the constructs are manipulated by affinity maturation using diverse
techniques or simply by joining multiple scFv domains in line in
order to create a multivalent construct. Even trispecific
antibodies are reported that are designed to display enhance
binding capabilities by addressing two instead of one target
molecule. Stability of the formats can be optimized by adding
immunoglobulin-like domains in order to mimic naturally occurring
antibodies and to simultaneously enhance pharmacokinetic properties
like prolonged half life in serum and protection from proteolytic
digestion by proteases. Moreover, stability of the formats can be
enhanced by optimizing the production. Since linker sequences which
are utilized to covalently join scFv domains often leads to
aggregates, production lines have been established that first
produce two or three polypeptides that can be easily reassembled in
order to generate a functional drug. Such techniques utilize
directed disulphid-bridges or crosslinking reagents to covalently
join two different polypeptides. Other techniques make use of
hetero- or homo-dimerization domains like leucine-zipper domains,
Fc-domains and others like knob into hole technologies (see, for
example, WO 2007/062466). Moreover, V.sub.H and V.sub.L
interactions, which can be stabilized by the binding of the
antigen, have been used in so called open-sandwich immunoassays for
the detection of the antigen (Ueda, Nature Biotechnology 14 (1996),
1714-1718; Ohmuro-Matsuyama (2012) Detection of Protein
Phosphorylation by
Open-Sandwich Immunoassay, Integrative Proteomics, Dr. Hon-Chiu
Leung (Ed.), ISBN: 978-953-51-0070-6; WO 2004/016782/EP-A1
1536005.)
[0012] However, bi/tri-specific and bi- or multivalent constructs
described in the art have disadvantages. First, the absence of
truly specific tumor antigens that can be addressed as target
molecule. In fact, the more potent the bispecific antibody format,
the more severe are collateral damages, because the target antigens
addressed so far are differentiation antigens shared by tumours and
non-maligant cells. In consequence, bi- or tri-specific formats of
the prior art cannot discriminate malignant from non-malignant
cells. In this respect, tri-specific constructs, developed for high
avidity binding to target cells, may turn out to confer a high
degree of off-target effects because binding of one target molecule
in general suffice to recruit immune cells for destruction of a
cell which express either target molecule. Thus, tri-specific
construct enhance avidity on the cost of specificity. Recent
multi-parameter analyses indicate that tumor cells can be
distinguished from their respective non-transformed tissues of
origin because of the expression of aberrant antigen signatures.
Today, these findings constitute an integral part of the World
Health Organization (WHO) classification system of hematopoietic
neoplasms, and also hold true for cancer and cancer stem or cancer
initiating cells of other provenance. Thus, it would be
advantageous to target cells that simultaneously express a
combination of antigens that together signify a malignant state.
None of the antibodies disclosed by prior art is able to
discriminate between cells that express a combination of target
antigens from single antigen positive cells. Second, a major
problem of bi-specific antibody technologies using, for example,
complete CD3 modules (e.g. a anti Cd3 scFv) is the inherent ability
of these proteins to stimulate or pre-stimulate T cells
irrespective of binding to the target antigen on target cells and
many side effects observed so far appear to be associated with
errant T cell function.
[0013] Thus, there is a need in the art for more specific treatment
options in cancer treatment, in particular there is a need for
improved ways to identify and/or eliminate cancer cells with higher
specificity and reduce side-effects.
[0014] Similar needs exist in the field of allogeneic stem cell
transplantation, i.e. the transplantation of stem cells obtained
from another person to a patient. A patient suffering from relapsed
or refractory leukaemia or another haematological disease may be
treated by chemotherapy/irradiation (to eliminate the malignant
haematopoietic cells) in combination with a transplantation of
healthy haematopoietic cells from a donor. If elimination of
malignant cells is incomplete, the tumour may grow back from the
surviving malignant recipient cells despite the presence of healthy
cells provided by the transplantation. As a result, survival rates
among patients undergoing tumour treatment and allogeneic
transplantation are significantly reduced.
[0015] However, it is difficult to eliminate (and, similarly, to
identify) the surviving malignant cells with high specificity, and
thus despite various attempts, good solutions to this problem have
not been found. Accordingly, there exists a need in the art to
provide improved ways to specifically identify and/or eliminate
such malignant recipient cells with minimal side effects on other
cells.
[0016] The graft (allogenic stem cells), given shortly after the
conditioning therapy (radiation/chemotherapy) can replace and
reconstitute hematopoiesis. The graft is harvested from either bone
marrow or from stimulated peripheral blood cells and contains about
one percent of hematopoetic stem cells which are the source of
newly built blood cells. In addition, the graft normally contains a
huge number of immune cells, especially T lymphocytes, that are
part of the adoptive immune system and that can be very beneficial
in cases where these T cells mount an immune attack against
leukemic cells. This situation is well described and known as graft
versus leukemia effect. On the other side, an errant immune
response which directs T cells against the patient, known as graft
versus host disease, is also frequently observed.
[0017] To minimize graft versus host disease, grafts are usually
selected on the basis of HLA (human leukocyte antigen) or MHC
(major histocompatibility complex). The closer the antigens between
donor and recipient match the lower is the probability of severe
graft versus host disease. However, for many patients, a full
matched graft cannot be found. In these cases, a bone marrow or
peripheral blood stem cells are utilized that differ in one or even
more HLA molecules. These clinical situation requires a strict
immunosuppressive regimen after transplantation to keep the T cell
system strictly under control.
[0018] It is therefore one object of the present invention to
provide for improved ways to specifically identify and/or eliminate
specific kinds of cells. Moreover, it is an object of the present
invention to provide for improved ways to specifically identify
and/or eliminate cells that have a specific combination of two
specific antigens at their cell surface. Furthermore, it is an
object of the present invention to provide for improved ways to
specifically identify and/or eliminate cancerous cells.
Furthermore, it is an object of the present invention to provide
for improved ways to specifically identify and/or eliminate cells
that (1) are of a certain origin (such as, in the situation of a
tissue or cell transplantation, cells originating from the
recipient or from the donor) and that (2) belong to a specific cell
type or cell lineage (such as haematopoietic cells).
[0019] The objects of the present invention are solved by a set of
polypeptides comprising: [0020] a first polypeptide P1 comprising
[0021] (i) a targeting moiety T1, [0022] wherein said targeting
moiety T1 specifically binds to an antigen A1, and [0023] (ii) a
fragment F1 of a functional domain F, [0024] wherein neither said
fragment F1 by itself nor said polypeptide P1 by itself is
functional with respect to the function of said domain F, [0025]
and [0026] a second polypeptide P2 comprising [0027] (i) a
targeting moiety T2, [0028] wherein said targeting moiety T2
specifically binds to an antigen A2, and [0029] (ii) a fragment F2
of said functional domain F, [0030] wherein neither said fragment
F2 by itself nor said polypeptide P2 by itself is functional with
respect to the function of said domain F, [0031] wherein said
antigen A1 is different from said antigen A2, [0032] wherein said
polypeptide P1 and said polypeptide P2 are not associated with each
other in the absence of a substrate that has both antigens A1 and
A2 at or on its surface, more specifically a cell that carries both
antigens A1 and A2 at or on its cell surface, and [0033] wherein,
upon dimerization of said fragment F1 of said polypeptide P1 with
said fragment F2 of said polypeptide P2, the resulting dimer is
functional with respect to the function of said domain F.
[0034] The objects of the present invention are also solved by a
set of polypeptides comprising: [0035] a first polypeptide P1
comprising [0036] (i) a targeting moiety T1, [0037] wherein said
targeting moiety T1 specifically binds to an antigen A1, and [0038]
(ii) a fragment F1 of a functional domain F, [0039] wherein neither
said fragment F1 by itself nor said polypeptide P1 by itself is
functional with respect to the function of said domain F, [0040]
and [0041] a second polypeptide P2 comprising [0042] (i) a
targeting moiety T2, [0043] wherein said targeting moiety T2
specifically binds to an antigen A2, and [0044] (ii) a fragment F2
of said functional domain F, [0045] wherein neither said fragment
F2 by itself nor said polypeptide P2 by itself is functional with
respect to the function of said domain F, [0046] wherein said
antigen A1 is different from said antigen A2, [0047] wherein [0048]
(a) said fragment F1 comprises a V.sub.L domain of an antibody and
said fragment F2 comprises a V.sub.H domain of the same antibody;
or wherein said fragment F1 comprises a V.sub.H domain of an
antibody and said fragment F2 comprises a V.sub.L domain of the
same antibody; or [0049] (b) said fragment F1 comprises a V.sub.L
domain of an antibody and said fragment F2 comprises a V.sub.H
domain of the same antibody; or wherein said fragment F1 comprises
a V.sub.H domain of an antibody and said fragment F2 comprises a
V.sub.L domain of the same antibody; and [0050] wherein said
polypeptide P1 and said polypeptide P2 are not associated with each
other in the absence of a substrate that has both antigens A1 and
A2 at or on its surface, more specifically a cell that carries both
antigens A1 and A2 at or on its cell surface, and [0051] wherein,
upon dimerization of said fragment F1 of said polypeptide P1 with
said fragment F2 of said polypeptide P2, the resulting dimer is
functional with respect to the function of said domain F.
[0052] The objects of the present invention are also solved by a
set of polypeptides comprising: [0053] a first polypeptide P1
comprising [0054] (i) a targeting moiety T1, [0055] wherein said
targeting moiety T1 specifically binds to an antigen A1, and [0056]
(ii) a fragment F1 of a functional domain F, [0057] wherein neither
said fragment F1 by itself nor said polypeptide P1 by itself is
functional with respect to the function of said domain F, [0058]
and [0059] a second polypeptide P2 comprising [0060] (i) a
targeting moiety T2, [0061] wherein said targeting moiety T2
specifically binds to an antigen A2, and [0062] (ii) a fragment F2
of said functional domain F, [0063] wherein neither said fragment
F2 by itself nor said polypeptide P2 by itself is functional with
respect to the function of said domain F, [0064] wherein said
antigen A1 is different from said antigen A2, [0065] wherein [0066]
(c) said fragment F1 comprises a V.sub.L domain of an antibody and
said fragment F2 comprises a V.sub.H domain of the same antibody;
or wherein said fragment F1 comprises a V.sub.H domain of an
antibody and said fragment F2 comprises a V.sub.L domain of the
same antibody; [0067] (d) said polypeptide P1 and said polypeptide
P2 are not associated with each other in the absence of a substrate
that has both antigens A1 and A2 at its surface, more specifically
a cell that carries both antigens A1 and A2 at its cell surface; or
[0068] (e) said fragment F1 comprises a V.sub.L domain of an
antibody and said fragment F2 comprises a V.sub.H domain of the
same antibody; or wherein said fragment F1 comprises a V.sub.H
domain of an antibody and said fragment F2 comprises a V.sub.L
domain of the same antibody; and [0069] wherein said polypeptide P1
and said polypeptide P2 are not associated with each other in the
absence of a substrate that has both antigens A1 and A2 at its
surface, more specifically a cell that carries both antigens A1 and
A2 at its cell surface, and [0070] wherein, upon dimerization of
said fragment F1 of said polypeptide P1 with said fragment F2 of
said polypeptide P2, the resulting dimer is functional with respect
to the function of said domain F, and wherein said polypeptides P1
and P2, in particular said fragments F1 and F2, have, in the
absence of a substrate or cell, with each other a dissociation
constant K.sub.D in the range of 10.sup.-8 M to 10.sup.-2 M.
[0071] The present invention further refers to the following items:
[0072] 1. A set of polypeptides comprising: [0073] a first
polypeptide P1 comprising [0074] (i) a targeting moiety T1, [0075]
wherein said targeting moiety T1 specifically binds to an antigen
A1, and [0076] (ii) a fragment F1 of a functional domain F, [0077]
wherein neither said fragment F1 by itself nor said polypeptide P1
by itself is functional with respect to the function of said domain
F, [0078] and [0079] a second polypeptide P2 comprising [0080] (i)
a targeting moiety T2, [0081] wherein said targeting moiety T2
specifically binds to an antigen A2, and [0082] (ii) a fragment F2
of said functional domain F, [0083] wherein neither said fragment
F2 by itself nor said polypeptide P2 by itself is functional with
respect to the function of said domain F, [0084] wherein said
antigen A1 is different from said antigen A2, [0085] wherein said
polypeptide P1 and said polypeptide P2 are not associated with each
other in the absence of a substrate that has both antigens A1 and
A2 at its surface, more specifically a cell that carries both
antigens A1 and A2 at its cell surface, and [0086] wherein, upon
dimerization of said fragment F1 of said polypeptide P1 with said
fragment F2 of said polypeptide P2, the resulting dimer is
functional with respect to the function of said domain F. [0087] 2.
The set of polypeptides according to item 1, wherein a cell
carrying both antigens A1 and A2 at its cell surface induces
dimerization of the fragment F1 of said polypeptide P1 with the
fragment F2 of said polypeptide P2, whereas a cell which does not
carry both antigens A1 and A2 at its cell surface does not induce
dimerization of the fragment F1 of said polypeptide P1 with the
fragment F2 of said polypeptide P2. [0088] 3. The set of
polypeptides according to item 1 or 2, wherein said targeting
moiety T1 comprises an immunoglobulin module, preferably an
immunoglobulin module I1 comprising a V.sub.L domain linked to a
V.sub.H domain, more preferably an immunoglobulin module I1 that
comprises a scFv (single-chain variant fragment) of an antibody, or
an immunoglobulin module comprising a variable domain V.sub.HH of a
llama antibody, camel antibody or shark antibody, [0089] and/or
said targeting moiety T2 comprises an immunoglobulin module,
preferably an immunoglobulin module I2 comprising a V.sub.L domain
linked to a V.sub.H domain, more preferably an immunoglobulin
module I2 that comprises a scFv (single-chain variant fragment) of
an antibody, or an immunoglobulin module comprising a variable
domain V.sub.HH of a llama antibody, camel antibody or shark
antibody, [0090] or wherein said targeting moiety T1 and/or said
targeting moiety T2 comprises an aptamer or a natural ligand of
said antigen A1 or antigen A2, respectively [0091] 4. The set of
polypeptides according to any of the preceding items, wherein said
antigen A1 and/or said antigen A2 is an antigen expressed on the
surface of cells of a tumour or on the surface of
progenitor/precursor cells of a tumour, preferably an antigen
expressed on the surface of cells of a haematologic tumour or an
antigen expressed on the surface of cells of a non-haematologic
tumour. [0092] 5. The set of polypeptides according to any of the
preceding items, wherein the combination of antigen A1 and antigen
A2 is only found on cancerous cells, and not on cells that are not
cancerous, and wherein, preferably, the combination of antigen A1
and antigen A2 is specific for cancerous cells of a certain type of
cancer. [0093] 6. The set of polypeptides according to any of the
preceding items, wherein said antigen A1 is an MHC antigen,
preferably an allelic variant of any of HLA-A, HLA-B, HLA-C,
HLA-DQ, HLA-DR, or HLA-DM, more preferably an allelic variant of an
MHC class I molecule, more preferably an allelic variant selected
from the group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A25,
HLA-B7, HLA-B8, HLA-B35, HLA-B44, HLA-Cw3, HLA-Cw4, and HLA-Cw7,
and/or said antigen A2 is an antigen that is specific for a certain
cell type or cell lineage. [0094] 7. The set of polypeptides
according to any of the preceding items, wherein said functional
domain F is an immunoglobulin module, preferably a scFv
(single-chain variant fragment) of an antibody, or a fluorescent
molecule, preferably GFP or a GFP variant, or a molecule capable of
mediating bioluminescence, preferably Gaussia luciferase. [0095] 8.
The set of polypeptides according to any of the preceding items,
wherein said functional domain F is a domain that specifically
binds to a carrier molecule, preferably a carrier molecule that is
a peptide or a carbohydrate molecule, or an affinity tag,
preferably an affinity tag selected from the group consisting of a
FLAG-tag, a myc-tag, a glutathione-S-transferase(GST)-tag, a
hemagglutinin(HA)-tag, a polyhistidine(His)-tag and a maltose
binding protein(MBP)-tag. [0096] 9. The set of polypeptides
according to any of the preceding items, wherein said functional
domain F is a domain that specifically binds to a radioactive
compound, a domain that specifically binds to a toxin molecule that
by itself is not capable of penetrating through the cell membrane
of a human cell and that is internalized into a human cell upon
association with the cell membrane of said cell, a domain that
specifically binds to a fluorescent molecule, or a domain that
specifically binds to a molecule capable of mediating
bioluminescence. [0097] 10. The set of polypeptides according to
any of the preceding items, wherein said fragment F1 comprises a
V.sub.L domain of an antibody and said fragment F2 comprises a
V.sub.H domain of the same antibody, wherein, preferably, said
antibody is an anti-CD3 antibody, or wherein said fragment F1
comprises a V.sub.H domain of an antibody and said fragment F2
comprises a V.sub.L domain of the same antibody, wherein,
preferably, said antibody is an anti-CD3 antibody. [0098] 11. The
set of polypeptides according to any of the preceding items for use
in the treatment of a patient who is suffering from a tumour or for
diagnostic use in a patient who is suffering from a tumour,
preferably for use in the treatment of a patient who is suffering
from a tumour and undergoing allogeneic tissue or cell
transplantation or meant to undergo such transplantation or for
diagnostic use in a patient who is suffering from a tumour and
undergoing or meant to undergo allogeneic tissue or cell
transplantation, wherein, preferably, said set of polypeptides is
administered to said patient. [0099] 12. A nucleic acid molecule or
a set of nucleic acid molecules encoding the set of polypeptides or
one of the polypeptides of the set of polypeptides according to any
of the preceding items. [0100] 13. A vector comprising the
nucleotide sequence of the nucleic acid molecule according to item
12 or the sequence of one of the nucleic acid molecules of the set
of nucleic acid molecules according to item 12. [0101] 14. A
pharmaceutical composition comprising either the set of
polypeptides according to any of items 1 to 11 or the nucleic acid
molecule/set of nucleic acid molecules according to item 12 or the
vector according to item 13, wherein, preferably, said
pharmaceutical composition further comprises a pharmaceutically
acceptable carrier. [0102] 15. A kit comprising the set of
polypeptides according to any of items 1-11.
[0103] Preferably, said antigen A1 is a cell surface molecule.
Preferably, said antigen A2 is a cell surface molecule. Preferably,
said antigen A1 is specific for the malignant state of a cell.
Preferably, said antigen A2 is specific for a certain cell type or
cell lineage or for the malignant state of a cell. Preferably, said
antigen A1 is specific for a malignant cell type. Preferably, said
antigen A2 is specific for a malignant cell type.
[0104] In one aspect, the present invention relates to the set of
polypeptides as defined and described herein, wherein, however, the
antigen A1 is the same as the antigen A2. Hence, in such a set of
polypeptides P1 and P2, the F1 fragment may be linked to the
targeting moiety T1 and the F2 fragment may be linked to the
targeting moiety T2, whereas both T1 and T2 specifically bind to
the same antigen. In this context, the epitope on antigen A1, to
which the targeting moiety T1 binds, may be the same or a different
epitope as the epitope on the antigen A2, to which the targeting
moiety T2 binds. In case the epitope on antigen A1 is the same as
the epitope on the antigen A2, polypeptide P1 may comprise a
targeting moiety which is identical to the targeting moiety
comprised in P2. Also this aspect of the invention is based on the
advantage that the set of polypeptides P1 and P2 with the disrupted
F domain displays no off target effects (for example no
pre-activation of CD3-displaying T cells and, hence, less toxic
properties and/or side effects, for example as compared to
conventional bispecific antibodies).
[0105] In one embodiment, said fragment F1 and said fragment F2
together are said functional domain F.
[0106] In one embodiment, said polypeptide P1 and said polypeptide
P2 are not covalently linked to each other in the absence of a
substrate that has both antigens A1 and A2 at its surface, more
specifically a cell that carries both antigens A1 and A2 at its
cell surface.
[0107] In one embodiment, said polypeptide P1 and said polypeptide
P2 are not covalently linked to each other.
[0108] Said polypeptide P1 and polypeptide P2 and/or, in
particular, said fragment F1 and fragment F2 as comprised therein,
more particular the V.sub.H and V.sub.L which may be comprised
therein, are not associated with each other, in particular when
administered to a subject in need of medical intervention. i.e. in
need of therapy and/or diagnosis. Accordingly, the pharmaceutical
or diagnostic means provided herein comprise the two polypeptides
P1 and P2 as comprised in the herein defined "set of polypeptides"
in non-associated form. The association of said two polypeptides
take place in vivo under the presence of said substrate or cell.
Under the presence of said substrate or cell, the association of
said two polypeptides may be (further) stabilized by a stabilizing
agent (for example an antigen, like, for example, CD3, HIS or DIG
as described herein). Preferably, they are not associated with each
other in the absence of said substrate or cell and/or do not
dimerizise in the absence of said substrate or cell. More
preferably, they are not associated with each other in the absence
of said substrate or cell and/or do not dimerizise in the absence
of said substrate or cell even if an agent is present which
stabilizes association and/or dimerization of polypeptide P1 and
polypeptide P2 and/or, in particular, fragment F1 and fragment F2,
i.e. even if said polypeptide P1 and polypeptide P2 and/or, in
particular, said fragment F1 and fragment F2 is present in an
stabilizing agent/P1(F1)/P2(F2)-trimeric complex (for example in an
antigen/VH/VL-trimeric complex).
[0109] In one specific embodiment, said polypeptide P1 and
polypeptide P2 and/or, in particular, said fragment F1 and fragment
F2 as comprised therein, more particular the V.sub.H and V.sub.L
which may be comprised therein, are associated with each other
and/or dimerizise into a three-part-complex-formation, preferably
by an interaction mediated by an agent which stabilizes association
and/or dimerization of polypeptide P1 and polypeptide P2 and/or, in
particular, fragment F1 and fragment F2 (for example by an
antigen-mediated interaction). Most preferably, however, this
association and/or dimerization only occurs in the presence of said
substrate or cell.
[0110] The affinity strength with which, for example,
leucine-zippers and/or constant domains, like immunoglobulin CH3 or
Fc fragments, hetero- and homodimerize is estimated to be at a
dissociation constant K.sub.D in the range of .about.10.sup.-8 to
10.sup.-11 M (see, for example, Zhu (1997) Protein Sci. 6, 781-8;
Pluckthun (1997) Immunotech. 3, 83-105). This K.sub.D range is
clearly below the K.sub.D with which, in the absence of said
substrate or cell, association and/or dimerization of said
polypeptides P1 and P2, in particular of said fragments F1 and F2,
of this invention might occur. Hence, in one embodiment,
polypeptide P1 and polypeptide P2 and/or, in particular, fragment
F1 and fragment F2 as comprised therein, more particular the
V.sub.H and V.sub.L which may be comprised therein, associate with
each other and/or dimerizise in the absence of said substrate or
cell only with a K.sub.D which is above the K.sub.D of, for
example, hetero- and homodimerization of leucine-zippers and/or
constant domains, like immunoglobulin CH3 or Fc fragments. In the
presence of said substrate or cell, it is envisaged that
polypeptide P1 and polypeptide P2 and/or, in particular, fragment
F1 and fragment F2 as comprised therein, more particular the
V.sub.H and V.sub.L which may be comprised therein, associate with
each other and/or dimerizise with a K.sub.D which is in the range
of the K.sub.D of, for example, hetero- and homodimerization of
leucine-zippers and/or constant domains, like immunoglobulin CH3 or
Fc fragments, or even below this range.
[0111] The interaction strength of, for example, isolated VH and VL
domains in general is of low affinity. Using calorimetric,
fluorometric or ultraviolet difference spectroscopy and/or circular
dichroisma techniques, dissociation constants K.sub.D of 10.sup.-9
to 10.sup.-6 M have been determined (see, for example, Worn J M B
(2001) 305, 989-1010; Pluckthun (1992) Immunological Reviews No
130). Using surface plasmon resonance techniques (SPR biosensor
BIAcore or BIAcore 2000, Pharmacia) and an anti HEL-Antibody system
(anti-hen egg lysozyme antibody HyHEL-10), Ueda (loc. cit.) and
Ohmuro-Matsuyama (loc. cit.) found that isolated VH and VL domains
do not dimerize at all (K.sub.a<10.sup.5/M, below detection
limit). However, association of the VH and VL peptides was
significantly enhanced in the presence of cognate antigens
(Ka.about.10.sup.9/M) with a remarkable reduction of the
dissociation rate of the antigenNHNL-trimeric complex with a
calculated
K.sub.d.about.2.73.times.10.sup.-5.+-.1.43.times.10.sup.-6/s at 1.4
.mu.M of the antigen. Hence, it is particularly envisaged in the
context of this invention that the K.sub.D with which, in the
absence of said substrate or cell, association and/or dimerization
of said polypeptides P1 and P2, in particular of said fragments F1
and F2, of this invention might occur is only at, or even above,
the K.sub.D or range of K.sub.D of isolated VH and VL domains, for
example as has been estimated in the context of Worn (loc. cit.),
Pluckthun (1992; loc. cit.), Ueda (loc. cit.) and Ohmuro-Matsuyama
(loc. cit.), in particular above the K.sub.D or range of K.sub.D of
the antigen/VH/VL-trimeric complex as has been estimated in the
context of Worn (loc. cit.), Pluckthun (1992; loc. cit.), Ueda
(loc. cit.) and Ohmuro-Matsuyama (loc. cit.). In the presence of
said substrate or cell, it is envisaged that polypeptide P1 and
polypeptide P2 and/or, in particular, fragment F1 and fragment F2
as comprised therein, more particular the V.sub.H and V.sub.L which
may be comprised therein, associate with each other and/or
dimerizise with a K.sub.D which is (far) below the K.sub.D or range
of K.sub.D of isolated VH and VL domains, for example as has been
estimated in the context of Worn (loc. cit.), Pluckthun (1992; loc.
cit.), Ueda (loc. cit.) and Ohmuro-Matsuyama (loc. cit.),
preferably at, or even below, the K.sub.D or range of K.sub.D of
the antigen/VH/VL-trimeric complex as has been estimated in the
context of Pluckthun (loc. cit.), Ueda (loc. cit.) and
Ohmuro-Matsuyama (loc. cit.)
[0112] In one aspect, polypeptide P1 and polypeptide P2 and/or, in
particular, fragment F1 and fragment F2 as comprised therein, more
particular the V.sub.H and V.sub.L which may be comprised therein,
are not associated in the absence of said substrate or cell and/or
do not dimerizise in the absence of said substrate or cell. If at
all, they associate with each other and/or dimerizise in the
absence of said substrate or cell only with a K.sub.D above
10.sup.-8 M, preferably above 10.sup.-6 M, more preferably above
10.sup.-5 M and more preferably above 10.sup.-4M. In another
aspect, if at all, they associate with each other and/or dimerizise
in the absence of said substrate or cell only with a K.sub.D in the
range of 10.sup.-8 M to 10.sup.-2 M, preferably 10.sup.-7 M to
10.sup.-3 M, more preferably 10.sup.-6 M to 10.sup.-3 M and even
more preferably 10.sup.-5 M to 10.sup.-3 M. I another aspect,
polypeptide P1 and polypeptide P2 and/or, in particular, fragment
F1 and fragment F2 as comprised therein, more particular the
V.sub.H and V.sub.L which may be comprised therein, are associated
in the presence of said substrate or cell and/or dimerizise in the
presence of said substrate or cell. In particular, they associate
with each other and/or dimerizise in the presence of said substrate
or cell with a K.sub.D below 10.sup.-6 M, preferably below
10.sup.-7 M, more preferably below 10.sup.-8 M and more preferably
below 10.sup.-9M. They may also associate with each other and/or
may dimerizise in the presence of said substrate or cell with a
K.sub.D in the range of 10.sup.-11 M to 10.sup.-6 M, more
preferably 10.sup.-11 M to 10.sup.-7 M and even more preferably
10.sup.-11 M to 10.sup.-8 M.
[0113] In a preferred embodiment, the above even applies in case an
agent is present which stabilizes association and/or dimerization
of polypeptide P1 and polypeptide P2 and/or, in particular,
fragment F1 and fragment F2. For example, such an stabilizing agent
in accordance with this invention may be an antigen, like, for
example, CD3, HIS or DIG as described herein, capable to bind to
the domain F which, for example, may comprise a V.sub.H and a
V.sub.L of an antibody (F1 and F2, respectively, or F2 and F3,
respectively).
[0114] Being "present", in the context of this invention and, in
particular, in the context of the above (i.e. with respect to said
agent and/or said substrate or cell and/or said antigens A1 and
A2), particularly means being present at a concentration in a range
of 0.01 .mu.M to 1 mM, in a range of 0.1 to 500 .mu.M, in a range
of 0.1 to 300 in a range of 0.1 to 100 .mu.M, in a range of 1 to
500 in a range of 10 to 500 .mu.M. Being "absent", in the context
of this invention and, in particular, in the context of above (i.e.
with respect to said agent and/or said substrate or cell and/or
said antigens A1 and A2), particularly means being present at a
concentration below the above ranges or below 1 mM, 500 .mu.M, 300
.mu.M, 100 .mu.M, 10 .mu.M, 1 .mu.M, 0.1 .mu.M, 0.01 .mu.M, 0.001
.mu.M or 1 nM wherein the lower values are preferred.
[0115] The person skilled in the art is readily in the position to
measure the K.sub.D of dimerization, in particular, of P1 and P2,
more particular of F1 and F2 as comprised therein, more particular
of the V.sub.H and V.sub.L which may be comprised therein. Examples
of respective measuring methods are x-ray crystallography; nuclear
magnet resonance (NMR); isothermal calorimetry (ITC); cryo-electro
microscopy (CEM); mass spectrometry (MS); surface Plasmon resonance
(SPR). Such methods are, for example, described in Protein Surface
Recognition: Approaches for Drug Discovery: Approaches for the
Inhibition of Protein-Protein Interactions for Drug Discovery (Eds:
Ernest Giralt, Mark Peczuh, Xavier Salvatella John Wiley &
Sons; 12 Nov. 2010). Further examples of respective measuring
methods are circular Dichroism Analysis; small Zone Gel Filtration
Chromatography; Fluorescence Gel Retardation; Sedimentation
Equilibrium; Fluorescence Polarization Assay; Blot Overlay or Far
Western Blot Analysis; Affinity Capillary Electrophoresis Analysis;
Fluorescence Resonance Energy Transfer (FRET); such methods are,
for example described in Protein'Protein Interactions: Methods and
Applications: 261 (Methods in Molecular Biology); Haian Fu
(Editor); Humana Press; 1 (23. Marz 2004). A preferred method to
measure the K.sub.D in accordance with this invention is
Fluorescence Correlation Spectroscopy (FCS). This method is, for
example, described in Douglas Magde (Physical Review Letters 29,
11, 1972, S. 705-708).
[0116] In one particular aspect, the K.sub.DS referred to herein
(i) apply to, (ii) are at or (iii) are to be measured at a
temperature of 4 to 38.degree. C., preferably 4 to 20.degree. C.
(for example 10.degree. C.) or 20 to 38.degree. C. (for example
30.degree. C.), and/or a pH of 4.5 to 8 (for example a pH of 7),
"Not associated" in the context of the present invention
particularly means not functionally associated with respect of the
function of the domain F, i.e. not allowing F1 and F2 to form a
functional F. Hence, in one aspect of the invention, P1 and P2 may
be bound to each other (for example covalently) as far as no
functional domain F is formed by F1 and F2. It is, however,
preferred that P1 and P2 are separated.
[0117] In one embodiment, said antigen A1 and/or said antigen A2 is
a molecule.
[0118] In one embodiment, said antigen A1 and/or said antigen A2 is
proteinaceous.
[0119] In one embodiment, said antigen A1 and/or said antigen A2 is
non-proteinaceous.
[0120] In one embodiment, said targeting moiety T1 binds
non-covalently to said antigen A1.
[0121] In one embodiment, said targeting moiety T2 binds
non-covalently to said antigen A2.
[0122] In one embodiment, a substrate having both antigens A1 and
A2 at its surface induces dimerization of the fragment F1 of said
polypeptide P1 with the fragment F2 of said polypeptide P2, whereas
a substrate which does not have both antigens A1 and A2 at its cell
surface does not induce dimerization of the fragment F1 of said
polypeptide P1 with the fragment F2 of said polypeptide P2.
[0123] In one embodiment, a cell carrying both antigens A1 and A2
at its cell surface induces dimerization of the fragment F1 of said
polypeptide P1 with the fragment F2 of said polypeptide P2, whereas
a cell which does not carry both antigens A1 and A2 at its cell
surface does not induce dimerization of the fragment F1 of said
polypeptide P1 with the fragment F2 of said polypeptide P2. In this
context "induces dimerization" particularly means "allows
juxtaposition and subsequent dimerization".
[0124] In one embodiment, said targeting moiety T1 comprises an
immunoglobulin module and/or said targeting moiety T2 comprises an
immunoglobulin module.
[0125] In one embodiment, said targeting moiety T1 comprises an
immunoglobulin module I1 which comprises a V.sub.L domain linked to
a V.sub.H domain, preferably an immunoglobulin module I1 that
comprises a scFv (single-chain variant fragment) of an antibody, a
Fab or a F(ab').sub.2 (for example with additional parts of, for
example, an Fc domain) of an antibody or a complete antibody.
and/or said targeting moiety T2 comprises an immunoglobulin module
I2 which comprises a V.sub.L domain linked to a V.sub.H domain,
preferably an immunoglobulin module I2 that comprises a scFv
(single-chain variant fragment) of an antibody a Fab or a
F(ab').sub.2 (for example with additional parts of, for example, an
Fc domain) of an antibody or a complete antibody.
[0126] In one embodiment, said targeting moiety T1 and/or said
targeting moiety T2 comprises an immunoglobulin module which
comprises a variable domain V.sub.HH of a llama antibody, a camel
antibody, or a shark antibody.
[0127] In one embodiment, said targeting moiety T1 and/or said
targeting moiety T2 is an aptamer, or a natural ligand of said
antigen A1 or antigen A2, respectively.
[0128] In one embodiment, said targeting moiety T1 and/or said
targeting moiety T2 comprises a Fv or scFv ((single-chain) variant
fragment) of an antibody.
[0129] In one embodiment, the immunoglobulin module comprised in
the targeting moiety T1 and T2 comprises a V domain selected from
the group consisting of: [0130] (i) a V domain of an anti-HLA-A2
antibody comprising a V.sub.L domain comprising SEQ ID NOS: 78 and
79 (CDRs 1 and 3) and DAS (CDR 2) and/or a V.sub.H domain
comprising SEQ ID NOS: 75-77 (CDRs 1-3); [0131] (ii) a V domain of
an anti-HLA-Cw6 antibody comprising a V.sub.L domain comprising SEQ
ID NOS: 83 and 84 (CDRs 1 and 3) and DDS (CDR 2) and/or a V.sub.H
domain comprising SEQ ID NOS: 80-82 (CDRs 1-3); [0132] (iii) a V
domain of an anti-EpCAM antibody comprising a V.sub.L domain
comprising SEQ ID NOS: 88 and 89 (CDRs 1 and 3) and WAS (CDR 2)
and/or a V.sub.H domain comprising SEQ ID NOS: 85-87 (CDRs 1-3);
[0133] (iv) a V domain of an anti-Her2 antibody comprising a
V.sub.L domain comprising SEQ ID NOS: 93 and 94 (CDRs 1 and 3) and
SAS (CDR 2) and/or a V.sub.H domain comprising SEQ ID NOS: 90-92
(CDRs 1-3); [0134] (v) a V domain of an anti-EGFR1 antibody
comprising a V.sub.L domain comprising SEQ ID NOS: 98 and 99 (CDRs
1 and 3) and DAS (CDR 2) and/or a V.sub.H domain comprising SEQ ID
NOS: 95-97 (CDRs 1-3); [0135] (vi) a V domain of an anti-CEA
antibody comprising a V.sub.L domain comprising SEQ ID NOS: 103 and
104 (CDRs 1 and 3) and SAS (CDR 2) and/or a V.sub.H domain
comprising SEQ ID NOS:100-102 (CDRs 1-3); [0136] (vii) a V domain
of an anti-CD45 antibody comprising a V.sub.L domain comprising SEQ
ID NOS: 107 and 108 (CDRs 1 and 3) and LAS (CDR 2) and/or a V.sub.H
domain comprising SEQ ID NOS: 105 and 106 (CDRs 1 and 2) and CDR3
or SEQ ID NOS:132-134 (CDRs 1-3); [0137] (viii) a V domain of an
anti-CD138 antibody comprising a V.sub.L domain comprising SEQ ID
NOS: 112 and 113 (CDRs and 1 and 3) and YTS (CDR 2) and/or a
V.sub.H domain comprising SEQ ID NOS: 109-111 (CDRs 1-3); and
[0138] (ix) a V domain of an anti-CD19 antibody comprising a
V.sub.L domain comprising SEQ ID NOS: 158 and 159 (CDRs 1 and 3)
and DAS (CDR 2) and/or a V.sub.H domain comprising SEQ ID NOS:
155-157 (CDRs 1-3).
[0139] In a further, preferred, embodiment, the immunoglobulin
module comprised in the targeting moiety T1 and/or T2 comprises a V
domain selected from the group consisting of: [0140] (i) a V domain
of an anti-HLA-A2 antibody comprising a V.sub.L domain comprising
SEQ ID NO: 52 and/or a V.sub.H domain comprising SEQ ID NO: 51;
[0141] (ii) a V domain of an anti-HLA-Cw6 antibody comprising a
V.sub.L domain comprising SEQ ID NO: 54 and/or a V.sub.H domain
comprising SEQ ID NO: 53; [0142] (iii) a V domain of an anti-EpCAM
antibody comprising a V.sub.L domain comprising SEQ ID NO: 56
and/or a V.sub.H domain comprising SEQ ID NO: 55; [0143] (iv) a V
domain of an anti-Her2 antibody comprising a V.sub.L domain
comprising SEQ ID NO: 58 and/or a V.sub.H domain comprising SEQ ID
NO: 57; [0144] (v) a V domain of an anti-EGFR1 antibody comprising
a V.sub.L domain comprising SEQ ID NO: 60 and/or a V.sub.H domain
comprising SEQ ID NO: 59; [0145] (vi) a V domain of an anti-CEA
antibody comprising a V.sub.L domain comprising SEQ ID NO: 62
and/or a V.sub.H domain comprising SEQ ID NO: 61; [0146] (vii) a V
domain of an anti-CD45 antibody comprising a V.sub.L domain
comprising SEQ ID NO: 64 and/or a V.sub.H domain comprising SEQ ID
NO: 63; and [0147] (viii) a V domain of an anti-CD138 antibody
comprising a V.sub.L domain comprising SEQ ID NO: 66 and/or a
V.sub.H domain comprising SEQ ID NOS: 65; [0148] (ix) a V domain of
an anti-CD19 antibody comprising a V.sub.L domain comprising SEQ ID
NO: 153 and/or a V.sub.H domain comprising SEQ ID NO: 152.
[0149] In a further, preferred, embodiment, the immunoglobulin
module comprised in the targeting moiety T1 and/or T2 comprises a V
domain comprising any one of SEQ ID NOS: 67-74 and 154.
[0150] In one embodiment, polypeptide P1 has the general structure
F1-T1 and/or polypeptide P2 has the general structure F2-T2. The F
fragment and T moieties may be separated by a linker (e.g.
F1-linker-T1 and/or F2-linker-T2) and/or flanked by (an) additional
amino acid stretche(s) 1 and/or 2 (stretch-F1-(linker)-T1-stretch2
and/or stretch1-F2-(linker)-T2-stretch2). It is preferred that the
above general structure is from the N terminus to the C terminus of
the polypeptides, i.e. N-F1-T1-C and/or N-F2-T2-C, N-F1-linker-T1-C
and/or N-F2-linker-T2-C and N-stretch1-F1-(linker)-T1-stretch2-C
and/or N-stretch1-F2-(linker)-T2-stretch2-C. In case the targeting
moiety is or comprises an immunoglobulin module I, like an Fv or
scFv, polypeptide P1 may have the general structure F1-VH1-VL1
and/or polypeptide P2 may have the general structure F2-VH2-VL2 or
polypeptide P1 may have the general structure F1-VL1-VH1 and/or
polypeptide P2 may have the general structure F2-VL2-VH2. Also in
these cases the F fragment and T moieties may be separated by a
linker (e.g. F1-linker-VH/VL1-VL/VH1 and/or
F2-linker-VH/VL2-VL/VH2) and/or flanked by (an) additional amino
acid stretche(s) 1 and/or 2
(stretch1-F1-(linker)-VH/VL1-VL/VH1-stretch2 and/or
stretch1-F2-(linker)-VH/VL2-VL/VH2-stretch2). Also in this case, it
is preferred that the above general structure is from the N
terminus to the C terminus of the polypeptides, i.e.
N-F1-VH/VL1-VL/VH1-C and/or N-F2-VH/VL2-VL/VH2-C,
N-F1-linker-VH/VL1-VL/VH1-C and/or N-F2-linker-VH/VL2-VL/VH2-C and
N-stretch1-F1-(linker)-VH/VL1-VL/VH1-stretch2-C and/or
N-stretch1-F2-(linker)-VH/VL2-VL/VH2-stretch2-C. There may also a
linker be present between VH and VL or VL and VH.
[0151] The above described linker, in particular the between the V
domains, may comprise 1 to 25 amino acids, preferably 12 to 20
amino acids, preferably 12 to 16 or 15 to 20 amino acids. The above
described linker may comprise one or more (G.sub.3S) and/or
(G.sub.4S) motives, in particular 1, 2, 3, 4, 5 or 6 (G.sub.3S)
and/or (G.sub.4S) motives, preferably 3 or 4 (G.sub.3S) and/or
(G.sub.4S) motives, more preferably 3 or 4 (G.sub.4S) motives.
[0152] In one embodiment, said immunoglobulin module I1 and said
fragment F1 are separated by a linker comprising 1 to 12,
preferably 3 to 12, amino acids, and/or said immunoglobulin module
I2 and said fragment F2 are separated by a linker comprising 1 to
12, preferably 3 to 12, amino acids.
[0153] In one embodiment, the V.sub.L domain of I1 is linked to the
V.sub.H domain of I1 by a linker comprising 12 to 25 amino acids,
preferably a linker with the sequence (G.sub.3S).sub.3 or
(G.sub.3S).sub.4 or (G.sub.4S).sub.3 or (G.sub.4S).sub.4 and/or the
V.sub.L domain of I2 is linked to the V.sub.H domain of I2 by a
linker comprising 12 to 25 amino acids, preferably a linker with
the sequence (G.sub.3S).sub.3 or (G.sub.3S).sub.4 or
(G.sub.4S).sub.3 or (G.sub.4S).sub.4.
[0154] As mentioned, the linker as describe above may comprise
(G.sub.3S) and/or (G.sub.4S) motives. Alternative linkers may
consist of or comprise the GEGTSTGSGGSGGSGGAD motive. The person
skilled in the art can without further ado find and use further
(peptide) linker known in the art.
[0155] The said additional amino acid stretches 1 and/or 2 may
consist of or comprise 1 to 200, 1 to 100, 1 to 70, 1 to 65, 1 to
50, 1 to 25 or 1 to 20 amino acids.
[0156] In one embodiment, said antigen A1 and/or said antigen A2 is
an antigen expressed on the surface of cells of a tumour or on the
surface of progenitor/precursor cells of a tumour, preferably an
antigen expressed on the surface of cells of a haematologic tumour,
more preferably an antigen expressed on the surface of cells
selected from the group consisting of acute myeloic leukemia cells,
chronic myeloic leukemia cells, acute lymphatic leukemia cells,
chronic lymphatic leukemia cells, lymphoma cells,
myeloproliferative syndrome cells, myelodysplastic cells, more
preferably myeloma cells, or said antigen A1 and/or said antigen A2
is an antigen expressed on the surface of cells of a
non-haematologic tumour, preferably a cell selected from the group
consisting of renal cell carcinoma cells, bladder cancer cells,
lung cancer cells, mesothelioma cells, prostate cancer cells, brain
cancer cells, bone cancer cells, sarcoma cells, soft tissue cancer
cells, ovarian cancer cells, cervix cancer cells, breast cancer
cells, endometrial cancer cells, uterine cancer cells, germ cell
tumour cells, anal cancer cells, rectal carcinoma cells, colon
carcinoma cells, small intestine carcinoma cells, gastric carcinoma
cells, gastrointestinal stroma tumour cells, liver carcinoma cells,
pancreas carcinoma cells, bile duct carcinoma cells, gall bladder
carcinoma cells, head and neck cancer cells, hypopharyngeal cancer
cells, laryngeal cancer cells, cells of a cancer of the esophagus,
skin cancer cells, preferably melanoma cells, cells of a childhood
cancer, cells of an endocrine tumour, cells of a carcinoid tumour,
thymoma cells, thyroid cancer cells, cells of an islet cell tumour,
cells of an adrenal cell tumour, cells of a neuroendocrine tumour
and cells of a cancer of unknown primary (cancer of unknown primary
origin). Detailed information on such cancers can be found in the
relevant literature, such as "Cancer Medicine", JF Holland, E Frei
(editors), Mcgraw-Hill Professional, 8th edition (2010) and
references cited therein.
[0157] In one embodiment, the combination of antigen A1 and antigen
A2 is only found on blood cells or precursor cells of blood cells,
preferably on only one type of blood cells.
[0158] In one embodiment, the combination of antigen A1 and antigen
A2 is only found on target, in particular, cancerous cells, and not
(or only to a negligible extent) on cells that are not target
cells, in particular, that are not cancerous. In a preferred
embodiment, the combination of antigen A1 and antigen A2 is
specific for cancerous cells of a certain type of cancer.
[0159] In one embodiment, the combination of antigen A1 and antigen
A2 distinguishes a certain kind of cells, preferably a certain type
of cancer cells, from any other cells.
[0160] "Certain type of cancer" in this context may mean type of
cancer characterized by the same organ in which the cancer is
formed or, preferred, type cancer characterized by the same pair of
(aberrant) antigens A1 and A2.
[0161] In one embodiment, the combination of antigen A1 and antigen
A2 is found on progenitor/precursor cells that are
progenitor/precursor cells of a tumour and not on
progenitor/precursor cells that are not progenitor/precursor cells
of a tumour.
[0162] In one embodiment, said antigen A1 is an antigen that is
specific for the malignant state of a cell and said antigen A2 is
an antigen that is specific for the cell type or cell lineage of
said cell.
[0163] In one embodiment, [0164] a) antigen A1 is EpCAM (epithelial
cell adhesion molecule) and antigen A2 is CD 10 (cluster of
differentiation 10), HER2/neu (human epidermal growth factor
receptor 2), VEGF-R (vascular endothelial growth factor receptor),
EGFR (epidermal growth factor receptor; also called HER1 (human
epidermal growth factor receptor 1) or ErbB1) or MDR (multidrug
resistance protein), or [0165] b) antigen A1 is MCSP
(melanoma-associated chondroitin sulfate proteoglycan) and antigen
A2 is melanoferrin or EpCAM, or [0166] c) antigen A1 is CA125
(cancer antigen 125/carbohydrate antigen 125) and antigen A2 is
CD227 (PEM (polymorphic epithelial mucin) or MUC1 (mucin-1)), or
[0167] d) antigen A1 is CD56 and antigen A2 is CD140b (PDGFR.beta.
(platelet-derived growth factor receptor beta)) or GD3 ganglioside,
or [0168] e) antigen A1 is EGFR and antigen 2 is HER2, or [0169] f)
antigen A1 is PSMA (prostate-specific membrane antigen) and antigen
2 is HER2, or [0170] g) antigen 1 is Sialyl Lewis and antigen 2 is
EGFR, or [0171] h) antigen 1 is CD44 and antigen 2 is ESA
(epithelial surface antigen) (CD326, EpCAM), CD24, CD133, MDR
(multidrug resistance protein) or CD117, or [0172] i) antigen 1 is
CD34 and antigen 2 is CD19, CD79a, CD2, CD7, HLA-DR (human
leukocyte antigen DR), CD13, CD117, CD33 or CD15, or [0173] j)
antigen 1 is CD33 and antigen 2 is CD19, CD79a, CD2, CD7, HLA-DR
(human leukocyte antigen DR), CD13, CD117 or CD15, or [0174] k)
antigen 1 is MUC1 and antigen 2 is CD10, CEA or CD57, or [0175] l)
antigen 1 is CD38 and antigen 2 is CD138, or [0176] m) antigen 1 is
CD 24 and antigen 2 is CD29 or CD49f, or [0177] n) antigen 1 is
carbonic anhydrase IX and antigen 2 is aquaporin, preferably
aquaporin-2.
[0178] In one embodiment, said antigen A1 and/or said antigen A2 is
selected from the group consisting of HLA-A (HLA-A major
histocompatibility complex, class I, A [Homo sapiens]; Gene ID:
3105 updated on 13 Jan. 2013; DAQB-90C11.16-002; Chromosome: 6;
NC.sub.--000006.11 (29910247.29913661); for HLA-A2: 1. mRNA=LOCUS
NM.sub.--001242758=Version NM.sub.--001242758.1
GI:337752169=GenBank: AY191309.1 PRI 13 Jan. 2013; 2.
Protein=P79495 [UniParc]. Last modified May 1, 1997. Version 1.;
for HLA-Cw6: mRNA=LOCUS HUMMHCCW6A=GenBank: VERSION M28160.1
GI:531197PRI (18 Aug. 1994); Protein=Q29963 [UniParc]. Last
modified Aug. 22, 2003. Version 2.); EpCAM (EPCAM epithelial cell
adhesion molecule [Homo sapiens]; also known as ESA; KSA; M4S1;
MK-1; DIAR5; EGP-2; EGP40; KS1/4; MIC18; TROP1; EGP314; HNPCC8;
TACSTD1.; Gene ID: 4072, updated on 6 Jan. 2013; mRNA=VERSION
NM.sub.--002354.2 GI:218505669PRI 6 Jan. 2013; Protein=P16422
[UniParc]. last modified Nov. 13, 2007. Version 2.); CD45 (PTPRC
protein tyrosine phosphatase, receptor type, C [Homo sapiens]; also
known asLCA; LY5; B220; CD45; L-CA; T200; CD45R; GP180; Gene ID:
5788, updated on 13 Jan. 2013; mRNA=VERSION NM.sub.--002838.4
GI:392307006 PRI 13 Jan. 2013; Protein=P08575-1=Isoform 1, Last
modified Jul. 19, 2003. Version 2.; Protein=P08575-2=Isoform 2);
Her2 (ERBB2 v-erb-b2 erythroblastic leukemia viral oncogene homolog
2, neuro/glioblastoma derived oncogene homolog (avian) [Homo
sapiens]; also known asNEU; NGL; HER2; TKR1; CD340; HER-2; MLN 19;
HER-2/neu; gene ID: 2064, updated on 13 Jan. 2013; mRNA transcript
variant 1=VERSION NM.sub.--004448.2 GI:54792095, PRI 6 Jan. 2013;
mRNA transcript variant 2=VERSION NM.sub.--001005862.1 GI:54792097,
PRI 6 Jan. 2013; Protein=P04626-1=Isoform 1, Last modified Aug. 13,
1987. Version 1.; Protein=P04626-2=Isoform 2;
Protein=P04626-3=Isoform 3; Protein=P04626-4=Isoform 4); EGFR (EGFR
epidermal growth factor receptor [Homo sapiens]; also known asERBB;
HER1; mENA; ERBB1; PIG61; Gene ID: 1956, updated on 13 Jan. 2013;
mRNA transcript variant 1=VERSION NM.sub.--005228.3 GI:41327737,
PRI 13 Jan. 2013; mRNA transcript variant 2=VERSION
NM.sub.--201282.1 GI:41327731, PRI 13 Jan. 2013; mRNA transcript
variant 3=VERSION NM.sub.--201283.1 GI:41327733, PRI 13 Jan. 2013;
mRNA transcript variant 4=VERSION NM.sub.--201284.1 GI:41327735,
PRI 13 Jan. 2013; Protein=P00533-1=Isoform 1, Last modified Nov. 1,
1997. Version 2.; Protein=P00533-2=Isoform 2;
Protein=P00533-3=Isoform 3; Protein=P00533-4=Isoform 4); CD138
(SDC1 syndecan 1 [Homo sapiens]; Gene ID: 6382, updated on 6 Jan.
2013; mRNA transcript variant 1=VERSION NM.sub.--001006946.1
GI:55749479, PRI 6 Jan. 2013; mRNA transcript variant 2=VERSION
NM.sub.--002997.4 GI:55925657, PRI 6 Jan. 2013; Protein=P18827
[UniParc]. Last modified May 5, 2009. Version 3.); CEA (CEACAM5
carcinoembryonic antigen-related cell adhesion molecule 5 [Homo
sapiens]; also known asCEA; CD66e; Gene ID: 1048, updated on 13
Jan. 2013; mRNA=VERSION NM.sub.--004363.2 GI:98986444, PRI 13 Jan.
2013; P06731, Last modified Jan. 11, 2011. Version 3.); and CD19
(CD19 CD19 molecule [Homo sapiens]; also known asB4; CVID3; Gene
ID: 930, updated on 5 Jan. 2013; mRNA transcript 1=VERSION
NM.sub.--001178098.1 GI:296010920, PRI 6 Jan. 2013; mRNA transcript
2=VERSION NM.sub.--001770.5 GI:296010919, PRI 6 Jan. 2013;
Protein=P15391 [UniParc]. Last modified Nov. 13, 2007. Version
6).
[0179] In one embodiment, said antigen A1 and/or said antigen A2 is
an MHC antigen, preferably an allelic variant of any of HLA-A,
HLA-B, HLA-C, HLA-DQ, HLA-DR, or HLA-DM, more preferably an allelic
variant of an MHC class I molecule, more preferably an allelic
variant selected from the group consisting of HLA-A1, HLA-A2,
HLA-A3, HLA-A25, HLA-B7, HLA-B8, HLA-B35, HLA-B44, HLA-Cw3,
HLA-Cw4, HLA-Cw6, and HLA-Cw7.
[0180] In one embodiment, said antigen A1 is HLA-A2.
[0181] In one embodiment, said antigen A1 and/or said antigen A2 is
selected from the group consisting of CD45, aquaporin, preferably
aquaporin-2, scavenger receptor class B member 1 (SCARB1), CD34,
CD33, CD138, CD15, CD1a, CD2, CD3, CD4, CD5, CD8, CD20, CD23, CD31,
CD43, CD56, CD57, CD68, CD79a, CD146, synaptophysin, CD56, CD57,
nicotinic acetylcholine receptor, muscle-specific kinase (MUSK),
voltage-gated calcium channel (P/Q-type), voltage-gated potassium
channel (VGKC), N-methyl-D-aspartate receptor (NMDA), TSH (thyroid
stimulating hormone) receptor, amphiphysin, HepPar-1, ganglioside
GQ1B, ganglioside GD3, ganglioside GM1 and glycophorin-A.
[0182] In a preferred embodiment, said antigen A1 is an MHC antigen
and said antigen A2 is an antigen that is specific for a certain
cell type or cell lineage.
[0183] In one embodiment, said functional domain F is an
immunoglobulin module, preferably a scFv (single-chain variant
fragment) of an antibody more preferably a Fv (variant fragment) of
an antibody, or a fluorescent molecule, preferably a bimolecular
fluorescence complementation molecule, more preferably GFP or a GFP
variant, or a molecule capable of mediating bioluminescence,
preferably a luciferase molecule, more preferably Gaussia
luciferase.
[0184] In one embodiment, said functional domain F is a Fv (variant
fragment) of an antibody.
[0185] In one embodiment, said functional domain F specifically
binds or is capable of specifically binding to an antigen. In a
specific aspect, said antigen may be an antigen that is present on
cells of the human immune system. In a preferred embodiment, said
binding activates said cells of the human immune system.
[0186] In one embodiment, said functional domain F is a T cell
engaging domain, preferably a T cell engaging domain specifically
binding to CD2, CD3, CD5, T cell receptor or CD28, more preferably
a T cell engaging domain specifically binding to CD3.epsilon., an
NK cell (natural killer cell) engaging domain, preferably a NK cell
engaging domain specifically binding to CD1a, CD16a or CD56, a
domain engaging macrophage cells, preferably a domain engaging
macrophage cells specifically binding to CD16a, CD32a, CD32b, CD89
or CD64, a monocyte engaging domain, preferably a monocyte engaging
domain specifically binding to CD32a, CD32b, CD64 or CD89, a
granulocyte engaging domain, preferably a granulocyte engaging
domain specifically binding to CD16b, CD32a, CD32b, CD64, or CD89,
a domain engaging neutrophil granulocytes, preferably a domain
engaging neutrophil granulocytes that specifically binds to CD89
(Fc.alpha.RI), or a domain engaging activated neutrophil
granulocytes, monocytes and/or macrophages, preferably a domain
engaging activated neutrophil granulocytes, monocytes and/or
macrophages that specifically binds to CD64 (Fc.gamma.RI).
[0187] In one embodiment, said functional domain F is a domain that
specifically binds to an antigen linked to a diagnostic or
therapeutic compound.
[0188] In one embodiment, said functional domain F is a domain that
specifically binds to a carrier molecule or an affinity tag.
Preferably, said carrier molecule is linked to a diagnostic or
therapeutic compound. Preferably, said affinity tag is linked to a
diagnostic or therapeutic compound.
[0189] Preferably, said affinity tag is selected from the group
consisting of a FLAG-tag, a myc-tag, a
glutathione-S-transferase(GST)-tag, a hemagglutinin(HA)-tag, a
polyhistidine(His)-tag, a digoxigenin (DIG)-tag and a maltose
binding protein(MBP)-tag.
[0190] Preferably, said carrier molecule is a peptide or a
carbohydrate molecule. In a preferred embodiment, said functional
domain F is a domain that specifically binds to a carrier molecule,
preferably a carrier molecule linked to a diagnostic or therapeutic
compound, wherein said carrier molecule is selected from the group
consisting of gelatine, inulin, dextrane and hydroxyethyl
starch.
[0191] In one embodiment, said therapeutic compound is a
radioactive compound, preferably a radioactive compound comprising
.sup.90Y, .sup.177Lu, .sup.131I, .sup.32P, .sup.10B, or .sup.213Bi.
In one embodiment, said therapeutic compound is a toxin.
Preferably, said toxin is selected from the group consisting of B.
anthracis edema factor, B. anthracis lethal factor, C. perfringens
iota toxin, C. botulinum C2 toxin, C. difficile
ADP-ribosyltransferase, C. diphtheriae diphteria toxin fragment A,
Burgholderia sp. shiga toxin (subunit A), Clostridium perfringens
str. 13 toxin pfoA perfringolysin O, Ricin A chain, plant RIP
bouganin, Human RNASE3 ribonuclease (RNase A family, 3) and anthrax
lethal factor endopeptidase. A further non-limiting example of a
toxin in accordance with this invention is a toxin being or
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS 160 to 168.
[0192] In one embodiment, said diagnostic compound is a radioactive
compound, preferably a radioactive compound comprising .sup.99mTc,
.sup.111In, .sup.82Rb or .sup.201Tl. In one embodiment, said
diagnostic compound is a fluorescent compound, preferably GFP, a
GFP variant, or a fluorescent small-molecule compound such as FITC
(fluorescein isothiocyanate), PE (phycoerythrin), an alexa fluor
dye (such as AlexaFluor488 or related dyes) or a cyanine dye (such
as Cy3 (Indocarbocyanine) or Cy5 (Indodicarbocyanine) or related
dyes), In one embodiment, said diagnostic compound is a molecule
capable of mediating bioluminescence, preferably a luciferase
molecule, more preferably Gaussia luciferase.
[0193] In one embodiment, said fragment F1 comprises a V.sub.L
domain of an antibody and said fragment F2 comprises a V.sub.H
domain of the same antibody, wherein, preferably, said antibody is
an anti-CD3 antibody, more preferably an anti-CD3.epsilon.
antibody, or an anti-His or anti-DIG antibody or said fragment F1
comprises a V.sub.H domain of an antibody and said fragment F2
comprises a V.sub.L domain of the same antibody, wherein,
preferably, said antibody is an anti-CD3 antibody, more preferably
an anti-CD3.epsilon. antibody, or an anti-His or anti-DIG
antibody.
[0194] In another embodiment, the V.sub.L and V.sub.H domains as
comprised in the F1 and F2 fragment, respectively, or in the F2 and
F1 fragment, respectively may also of two different antibodies,
either specific for the same Antigen (and for the same or a
different epitope) or for different Antigen. This is, for example,
envisaged to be employed where new specifications are to be created
(for example in phage-display approaches).
[0195] In another embodiment, the immunoglobulin module comprised
in the F domain comprises a V domain selected from the group
consisting of: [0196] (i) a V domain of an anti-CD3 antibody
comprising a V.sub.L domain comprising SEQ ID NOS: 18-20 (CDRs 1-3)
and/or a V.sub.H domain comprising SEQ ID NOS: 15-17 (CDRs 1-3);
[0197] (ii) a V domain of an anti-CD3 antibody comprising a V.sub.L
domain comprising SEQ ID NOS: 24-26 (CDRs 1-3) and/or a V.sub.H
domain comprising SEQ ID NOS: 21-23 (CDRs 1-3); [0198] (iii) a V
domain of an anti-CD3 antibody comprising a V.sub.L domain
comprising SEQ ID NOS: 30-32 (CDRs 1-3) and/or a V.sub.H domain
comprising SEQ ID NOS: 27-29 (CDRs 1-3); [0199] (iv) a V domain of
an anti-CD3 antibody comprising a V.sub.L domain comprising SEQ ID
NOS: 36 and 37 (CDRs 1 and 3) and DTS (CDR 2) and/or a V.sub.H
domain comprising SEQ ID NOS: 33-35 (CDRs 1-3); [0200] (v) a V
domain of an anti-CD3 antibody comprising a V.sub.L domain
comprising SEQ ID NOS: 41 and 42 (CDRs 1 and 3) and YTN (CDR 2)
and/or a V.sub.H domain comprising SEQ ID NOS: 38-40 (CDRs1-3); and
[0201] (vi) a V domain of an anti-His antibody comprising a V.sub.L
domain comprising SEQ ID NOS: 46 and 47 (CDRs 1 and 3) and KVS (CDR
2) and/or a V.sub.H domain comprising SEQ ID NOS: 43-45 (CDRs 1-3);
[0202] (vii) a V domain of an anti-DIG antibody comprising a
V.sub.L domain comprising SEQ ID NOS: 50 and 131 (CDRs 1 and 3) and
YSS (CDR 2) and/or a V.sub.H domain comprising SEQ ID NOS: 48 and
49 (CDRs 1 and 2) and A (CDR 3).
[0203] In another, preferred embodiment, the immunoglobulin module
comprised in the F domain comprises a V domain selected from the
group consisting of: [0204] (i) a V domain of an anti-CD3 antibody
comprising a V.sub.L domain comprising SEQ ID NO: 2 and/or a
V.sub.H domain comprising SEQ ID NO: 1; [0205] (ii) a V domain of
an anti-CD3 antibody comprising a V.sub.L domain comprising SEQ ID
NO: 4 and/or a V.sub.H domain comprising SEQ ID NO: 3; [0206] (iii)
a V domain of an anti-CD3 antibody comprising a V.sub.L domain
comprising SEQ ID NO: 6 and/or a V.sub.H domain comprising SEQ ID
NO: 5; [0207] (iv) a V domain of an anti-CD3 antibody comprising a
V.sub.L domain comprising SEQ ID NO: 8 and/or a V.sub.H domain
comprising SEQ ID NO: 7; [0208] (v) a V domain of an anti-CD3
antibody comprising a V.sub.L domain comprising SEQ ID NO: 10
and/or a V.sub.H domain comprising SEQ ID NO: 9; and [0209] (vi) a
V domain of an anti-His antibody comprising a V.sub.L domain
comprising SEQ ID NO: 12 and/or a V.sub.H domain comprising SEQ ID
NO: 11; [0210] (vii) a V domain of an anti-DIG antibody comprising
a V.sub.L domain comprising SEQ ID NO: 14 and/or a V.sub.H domain
comprising SEQ ID NO: 30.
[0211] In one embodiment, said functional domain F is a domain that
specifically binds to a toxin molecule, preferably a toxin molecule
that by itself is not capable of penetrating through the cell
membrane of a human cell and that, preferably, is internalized into
a human cell upon association with the cell membrane of said cell,
wherein, preferably, said association with the cell membrane of
said cell is mediated by specifically binding to a heterodimer
formed from two molecules, preferably two molecules associated with
said cell membrane, wherein, preferably, said two molecules are the
polypeptides P1 and P2 as described herein. In one embodiment, said
functional domain F is a domain that specifically binds to the
A-component (active component) of a bacterial two-component A-B
toxin. In one embodiment said functional domain F is a domain that
specifically binds to a toxin selected from the group consisting of
B. anthracis edema factor, B. anthracis lethal factor, C.
perfringens iota toxin, C. botulinum C2 toxin, C. difficile
ADP-ribosyltransferase, C. diphtheriae diphteria toxin fragment A,
Burgholderia sp. shiga toxin (subunit A), Clostridium perfringens
str. 13 toxin pfoA perfringolysin O, Ricin A chain, plant RIP
bouganin, Human RNASE3 ribonuclease (RNase A family, 3) and anthrax
lethal factor endopeptidase. A further non-limiting example of a
toxin in accordance with this invention is a toxin being or
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS 160 to 168.
[0212] In one embodiment, said functional domain F is a domain that
specifically binds to a fluorescent molecule, preferably a
fluorescent molecule that by itself is not capable of penetrating
through the cell membrane of a human cell. Preferably, said
fluorescent molecule is GFP or a GFP variant or a molecule that is
or comprises a fluorescent small-molecule compound such as FITC
(fluorescein isothiocyanate), PE (phycoerythrin), an alexa fluor
dye (such as AlexaFluor488 or related dyes) or a cyanine dye (such
as Cy3 (Indocarbocyanine) or Cy5 (Indodicarbocyanine) or related
dyes).
[0213] In one embodiment, said functional domain F is a domain that
specifically binds to a molecule capable of mediating
bioluminescence, preferably to a luciferase molecule, more
preferably to Gaussia luciferase.
[0214] In one embodiment, said functional domain F is a fluorescent
molecule, preferably a bimolecular fluorescence complementation
molecule, more preferably GFP or a GFP variant, such as YFP, CFP,
Venus, or Cerulean.
[0215] Examples of particular polypeptides P1 or P2 comprised in
the set of polypeptides according to this invention are
polypeptides comprising an amino acid sequence selected from the
group consisting of SEQ ID NOS: 114-129 and 197.
[0216] In general, the present invention relates to the treatment
or elimination of any undesired cell population and the treatment
or prevention of any disorder or disease which comes along with
this undesired cell population. For this purpose, the set of
polypeptides of this invention is to be used.
[0217] In one embodiment, said set of polypeptides is a set of
polypeptides for use in the treatment of a patient who is suffering
from a tumour or cancer or for diagnostic use in a patient who is
suffering from a tumour or cancer, preferably for use in the
treatment of a patient who is suffering from a tumour or cancer and
undergoing allogeneic tissue or cell transplantation or meant to
undergo such transplantation, or for diagnostic use in a patient
who is suffering from a tumour or cancer and undergoing or meant to
undergo allogeneic tissue or cell transplantation, wherein,
preferably, said set of polypeptides is administered to said
patient.
[0218] Examples of tumours to be treated or diagnosed are those for
which the tumour or cancer cells are described herein above with
respect to the antigens A1 and/or A2.
[0219] In one embodiment, said treatment involves the elimination
of recipient tissue/cells of a certain cell type, preferably a
cancerous cell type, or recipient precursor cells giving rise to a
certain cell type, preferably to a cancerous cell type, optionally
after or in parallel to transplantation to the recipient of donor
tissue/cells of said same cell type or donor precursor cells giving
rise to said same cell type.
[0220] In one embodiment, the set of polypeptides of the invention
is for use in an allogeneic transplantation setting for
haematopoietic neoplasias, for example, with mismatched HLA
antigens, in particular for use in therapeutically exploiting this
mismatch situation. In this exemplary situation, the dual
information of recipient HLA haplotype (HLA.sub.patient) and
haematopoietic lineage origin (CD45) is displayed exclusively on
leukemic blasts and other haematopoietic cells of the patient. All
other cells of recipient origine express the recipient haplotype
but not the hematopoietic lineage antigen CD45 (e.g. recipient
non-hematopoietic cells are positive for HLA-A2 but negative for
CD45). Likewise, all donor hematopoietic cells express donor HLA
haplotype molecules that means that they are CD45 positive but
HLA-A2 negative in the situation a mismatch transplantation where
the patient but not the donor is positive for HLA-A2. Consequently,
the present invention also relates to bimolecular and complementing
single-chain antibody constructs directed against HLA-A2, in cases
where the patient but not the donor is HLA-A2 positive, and a
second construct specific for the haematopoietic lineage marker
CD45 to specifically target all hematopoietic cells of the patient
including all hematologic neoplasms. Hence, the first polypeptide
P1 may comprise a single-chain variable fragment antibody construct
directed against the HLA of the patient (targeting moiety T1) fused
to the V.sub.L fragment of F1 antiCD3 (for example, fragment F1).
The second polypeptide P2 may comprise a single-chain variable
fragment construct specific for a haematopoietic lineage marker
(for example, CD45; targeting moiety T2), fused to the V.sub.H
split-fragment of F2 anti CD3-Fv (fragment F2).
[0221] In one embodiment, said elimination involves the destroying
of said recipient tissue/cells or said recipient precursor cells by
cells of the immune system, by a toxin or by a radioactive
compound.
[0222] In one embodiment, said set of polypeptides is a set of
polypeptides for diagnostic use in a patient undergoing allogeneic
tissue or cell transplantation, wherein, preferably, said patient
is a patient suffering from a tumour.
[0223] In one embodiment, said diagnostic use involves the specific
detection of recipient cells of a certain cell type or cell lineage
among recipient cells of different cell type or cell lineage and
donor cells of the same or different type or cell lineage.
[0224] In one embodiment, said diagnostic use involves the specific
detection of recipient cells that are malignant cells among
recipient cells that are not malignant and among donor cells. In
one embodiment, said set of polypeptides is administered to a
patient.
[0225] Preferably, said patient is a mammal, more preferably a
human being.
[0226] In one embodiment, said administration occurs by bolus
administration or by continuous administration.
[0227] In one embodiment, the polypeptides P1 and P2 of said set of
polypeptides are administered in parallel. In another embodiment,
the polypeptides P1 and P2 of said set of polypeptides are
administered sequentially.
[0228] In one embodiment, one of the polypeptides P1 or P2 of said
set of polypeptides is administered by bolus administration,
whereas the other one is administered by continuous
administration.
[0229] In one embodiment, the amount of polypeptide administered is
in the range of from 0.5 .mu.g/m.sup.2 per day to 500 .mu.g/m.sup.2
per day for the polypeptide P1 or for the polypeptide P2 or for
each of the polypeptides P1 and P2, preferably in the range of from
5 .mu.g/m.sup.2 per day to 200 .mu.g/m.sup.2 per day for the
polypeptide P1 or for the polypeptide P2 or for each of the
polypeptides P1 and P2, more preferably in the range of from 10
.mu.g/m.sup.2 per day to 80 .mu.g/m.sup.2 per day for the
polypeptide P1 or for the polypeptide P2 or for each of the
polypeptides P1 and P2.
[0230] In one embodiment, the amount of polypeptide administered is
in the range of from 0.05 .mu.g/m.sup.2 per day to 0.5
.mu.g/m.sup.2 per day for the polypeptide P1 or for the polypeptide
P2 or for each of the polypeptides P1 and P2.
[0231] In one embodiment, the amount of polypeptide P1 administered
is different from the amount of polypeptide P2 administered.
[0232] In one embodiment, the amount of polypeptide administered is
in the range of from 0.5 .mu.g/m.sup.2 per day to 50 .mu.g/m.sup.2
per day for the polypeptide P1 or for the polypeptide P2 or for
each of the polypeptides P1 and P2. In one embodiment, the amount
of polypeptide administered is in the range of from 50
.mu.g/m.sup.2 per day to 100 .mu.g/m.sup.2 per day for the
polypeptide P1 or for the polypeptide P2 or for each of the
polypeptides P1 and P2. In one embodiment, the amount of
polypeptide administered is in the range of from 100 .mu.g/m.sup.2
per day to 200 .mu.g/m.sup.2 per day for the polypeptide P1 or for
the polypeptide P2 or for each of the polypeptides P1 and P2. In
one embodiment, the amount of polypeptide administered is in the
range of from 200 .mu.g/m.sup.2 per day to 300 .mu.g/m.sup.2 per
day for the polypeptide P1 or for the polypeptide P2 or for each of
the polypeptides P1 and P2. In one embodiment, the amount of
polypeptide administered is in the range of from 300 .mu.g/m.sup.2
per day to 400 .mu.g/m.sup.2 per day for the polypeptide P1 or for
the polypeptide P2 or for each of the polypeptides P1 and P2. In
one embodiment, the amount of polypeptide administered is in the
range of from 400 .mu.g/m.sup.2 per day to 500 .mu.g/m.sup.2 per
day for the polypeptide P1 or for the polypeptide P2 or for each of
the polypeptides P1 and P2. In one embodiment, the amount of
polypeptide administered is in the range of from 500 .mu.g/m.sup.2
per day to 1 mg/m.sup.2 per day for the polypeptide P1 or for the
polypeptide P2 or for each of the polypeptides P1 and P2.
[0233] Further reference points for deriving the amounts of the
polypeptides P1 and P2 to be administered can also be obtained by
consulting studies carried out with bispecific antibody constructs
(e.g. Bargou R et al., Tumor regression in cancer patients by very
low doses of a T cell-engaging antibody. Science. 2008;
321(5891):974-7; and Topp M S et al. Targeted therapy with the
T-cell-engaging antibody blinatumomab of chemotherapy-refractory
minimal residual disease in B-lineage acute lymphoblastic leukemia
patients results in high response rate and prolonged leukemia-free
survival. J Clin Oncol. 2011, 29:2493-8).
[0234] In one embodiment, said administration occurs continuously
for at least 12 hours or for at least 1 day or for at least 2 days
or for at least 3 days or for at least 4 days or for at least 5
days or for at least 6 days or for at least 7 days or for at least
8 days or for at least 9 days or for at least 10 days or for at
least 11 days or for at least 12 days or for at least 13 days or
for at least 14 days or for at least 15 days or for at least 16
days or for at least 17 days or for at least 18 days or for at
least 19 days or for at least 20 days or for at least 21 days or
for at least 22 days or for at least 23 days or for at least 24
days or for at least 25 days or for at least 26 days or for at
least 27 days or for at least 28 days or for at least 29 days or
for at least 30 days or for at least 5 weeks or for at least 6
weeks.
[0235] In one embodiment, said administration of said set of
polypeptides or of one of the polypeptides of said set of
polypeptides occurs intravenously, preferably by intravenous
injection.
[0236] In one embodiment, said administration of said set of
polypeptides or of one of the polypeptides of said set of
polypeptides occurs subcutaneously, preferably by subcutaneous
injection.
[0237] In one embodiment, said set of polypeptides is administered
in combination with one or more drugs selected from the group
consisting of an immunomodulatory drug, and/or a steroid,
preferably prednisolone or prednisone.
[0238] In one embodiment, said set of polypeptides is administered
in combination with a radioactive compound, preferably a
radioactive compound linked to an antigen, a carrier molecule or an
affinity tag, wherein said radioactive compound, said antigen, said
carrier molecule or said affinity tag is specifically bound by said
functional domain F.
[0239] In one embodiment, said set of polypeptides is administered
in combination with a toxin, preferably a toxin linked to an
antigen, a carrier molecule or an affinity tag, wherein said toxin,
said antigen, said carrier molecule or said affinity tag is
specifically bound by said functional domain F.
[0240] In one embodiment, said set of polypeptides is administered
in combination with a fluorescent molecule, preferably a
fluorescent molecule linked to an antigen, a carrier molecule or an
affinity tag, wherein said fluorophore, said antigen, said carrier
molecule or said affinity tag is specifically bound by said
functional domain F.
[0241] In one embodiment, said functional domain F is a domain that
specifically binds to an antigen which is not recognized as foreign
by the immune system of said patient to whom said set of
polypeptides is administered.
[0242] In one embodiment two sets of polypeptides as described
above (a first set of polypeptides and a second set of
polypeptides) are administered simultaneously or sequentially. In
one preferred embodiment, said first set of polypeptides has
different fragments F1 and F2 than said second set of polypeptides.
In one preferred embodiment, said first set of polypeptides has the
same fragments F1 and F2 as said second set of polypeptides. In one
preferred embodiment, the targeting moieties T1 and T2 of said
first set of polypeptides bind to the same antigens as the
targeting moieties T1 and T2, respectively, of said second set of
polypeptides. In one preferred embodiment, the targeting moieties
T1 and T2 of said first set of polypeptides bind to different
antigens than the targeting moieties T1 and T2 of said second set
of polypeptides.
[0243] In one embodiment, said patient has undergone cancer
treatment before treatment with said set of polypeptides, said
cancer treatment preferably being chemotherapy, radiation therapy
or operative removal of the tumour, or undergoes cancer treatment
parallel to treatment with said set of polypeptides, said cancer
treatment preferably being chemotherapy, radiation therapy or
operative removal of the tumour.
[0244] In one embodiment, said set of polypeptides or one of the
polypeptides of said set of polypeptides has been produced by means
of a prokaryotic or eukaryotic expression system or by de novo
peptide synthesis.
[0245] In one embodiment, said set of polypeptides or one of the
polypeptides of said set of polypeptides is generated inside said
patient by protein expression from a nucleic acid introduced into
said patient.
[0246] Many patients suffer from allergic or auto-immune diseases.
In many of these cases, a clonal B cell population produce an
errant antibody that reacts with antigens expressed by the
patients' tissues or complex with an allergen, causing anaphylactic
reactions. In both cases, it is desirable to specifically eliminate
the errant B cell clone.
[0247] To this end, one may modify the combinatorial system in a
way so that one arm (P1 or P2, in particular T1 or T2) recognizes a
B cell associated antigen (e.g. CD19, CD20, CD38 or CD138) and the
other arm (P2 or P1, in particular T2 or T1, respectively) is the
allergen or the substrate bound by the antibody that causes the
autoimmune disease. When these two constructs bind to a B cell that
is CD19 (CD20, CD38 or CD138) positive and simultaneously displays
the clonotypic antibody on the surface, the attached anti-CD3 VH
and VL can interact and reconstitute the CD3 binding site exactly
on the B cell. This allergen-specific or antigen-specific assembly
will ultimately result in the clonal depletion of the Target B
cells.
[0248] Hence, in accordance with this invention, any of said
antigens A1 and A2 may also be a clonotypic antibody on the surface
of a B cell, in particular a B cell that causes an autoimmune
disorder.
[0249] In this context, for example, one of said antigens A1 and A2
may be CD19 and the other one may be a clonotypic antibody on the
surface of a B cell, in particular a B cell that causes an
autoimmune disorder.
[0250] In accordance with this aspect of the invention, any one of
said targeting moiety T1 and T2 may comprise an allergen or
substrate which binds to the clonotypic antibody on the surface of
the B cell and/or which is, upon binding to the clonotypic
antibody, capable to cause an autoimmune disorder. Non-limiting
examples of an allergen comprised in any one of said targeting
moiety T1 and T2 are hair allergens, like, for example, dog-hair,
cat-hair (e.g. Fel d 1, Feld d1A, Feld d1B) or guinea-pig-hair
allergens, or pollen allergens, like, for example, birch, grass,
pollen allergens. Further non-limiting examples are mite allergens
(for example Tyr p 2, Der P1, Der f 2), cat allergens (for example
Fel d 1, Feld d1A, Feld d1B), peanut allergens (for example
Conglutin-7), rot fungus allergens (for example Alt a 1), dog
allergens (for example Can f 1), sprue wheat allergens (for example
Alpha/beta-gliadin), german cockroach allergens (for example Bla g
1.02 variant allergen), birch tree or (major) pollen allergens (for
example Cyn d 1, Pha a 1, Dac g 3, Ph1 p 2, Ph1 p 1, Profilin, Bet
v 1-L, Bet v 1-A), major apple allergens (for example Mal d 1),
cow's milk allergens (for example alpha-lactalbumin,
alpha-S1-casein), chicken egg allergens (for example lysozyme C,
ovalbumin) and Horse allergens (for example latherin, Equ c 1), and
the like. A further non-limiting and preferred example of an
allergen comprised in any one of said targeting moiety T1 and T2 is
the antigen for human myeloma cell line U266 antibody IgE-ND. A
further non-limiting and preferred example of an allergen comprised
in any one of said targeting moiety T1 and T2 is an allergen being
or comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS 169 to 195.
[0251] In this context, the inventions also relates to the set of
polypeptides as described herein, and, in particular in the above
aspect, for use in treating or preventing a disorder selected from
the group consisting of [0252] (i) an autoimmune disorder; and
[0253] (ii) a hypersensitivity disorder.
[0254] Non-limiting examples of an autoimmune disorder to be
treated or prevented in accordance with this invention are selected
from the group consisting of [0255] (i) allergic disorders; [0256]
(ii) Multiple Sclerosis; [0257] (iii) Psoriasis; [0258] (iv)
Systemic Lupus Erythematosus; [0259] (v) Sjogren's syndrome; [0260]
(vi) Rheumatoid Arthritis; [0261] (vii) Idiopathic Thrombocytopenic
Purpura; [0262] (viii) Diabetes; [0263] (xi) Vasculitis; [0264] (x)
Crohn's disease; and [0265] (xi) Amyloidosis.
[0266] Non-limiting examples of a hypersensitivity disorder to be
treated or prevented in accordance with this invention are selected
from the group consisting of allergies (type I hypersensitivity
reaction according to Coombs and Gell classification), an antibody
dependent cytotoxic reaction (type II hypersensitivity reaction), a
immune complex disease (type III hypersensitivity reaction),
delayed type hypersensitivity (type IV hypersensitivity reaction)
and a receptor mediated autoimmune disease (type V hypersensitivity
reaction).
[0267] In a preferred embodiment, said autoimmune or
hypersensitivity disorder comes along with or is triggered by
allogenic stem cell transplantation (i.e. any of type I to type V
hypersensitivity disorder according to the Coombs and Gell
classification).
[0268] Many cells which are infected by a pathogen (for example a
virus, like, for example, HIV, EBV, CMV) express pathogen-encoded
proteins on their cell surface. Hence, in accordance with this
invention, any of said antigens A1 and A2 may also be such a
pathogen-encoded protein, like, for example, a HIV, EBV or CMV
protein on the surface of a cell. In this context, the inventions
also relates to the set of polypeptides as described herein for use
in treating or preventing an infectious disease, for example a
viral infectious disease. Particular examples of pathogen-encoded
proteins can be derived from http://www.uniprot.org/uniprot/and are
HIV gp120 (Q78706); EBV LMP-2 (P13285); CMV gB (P06473); HBV HBS
(Q9JG36); HCV E1 (C4B751); HCV E2 (Q6TRB1); Human adenovirus C
serotype 2 HAdV-2 (P03276).
[0269] The objects of the present invention are also solved by a
nucleic acid molecule or a set of nucleic acid molecules encoding
the set of polypeptides or one of the polypeptides of the set of
polypeptides as defined in the embodiments above, wherein,
preferably, said nucleic acid molecule or the nucleic acid
molecules of said set of nucleic acid molecules comprises an export
signal that mediates secretion of the encoded polypeptide(s) by a
bacterial or eukaryotic cell.
[0270] A non-limiting example of the nucleic acid molecule or set
of nucleic acid molecules according to this invention comprises one
or more of the nucleotide sequences as depicted in any one of SEQ
ID NOS: 135-150 and 196.
[0271] The objects of the present invention are also solved by a
vector comprising the nucleotide sequence of the nucleic acid
molecule as defined above or the sequence of one of the nucleic
acid molecules of the set of nucleic acid molecules as defined
above.
[0272] The objects of the present invention are also solved by a
cell comprising said nucleic acid/set of nucleic acids or said
vector.
[0273] The objects of the present invention are also solved by a
pharmaceutical composition comprising either the set of
polypeptides as defined above or the nucleic acid molecule/set of
nucleic acid molecules as defined above or the vector as defined
above, wherein, preferably, said pharmaceutical composition further
comprises a pharmaceutically acceptable carrier.
[0274] The objects of the present invention are also solved by a
kit comprising the set of polypeptides as defined above and/or the
nucleic acid molecule or the set of nucleic acid molecules
according the invention and/or the vector according the
invention.
[0275] In one embodiment, the polypeptides of said set of
polypeptides comprised by said kit are contained in a single
vial.
[0276] In one preferred embodiment, the polypeptides of said set of
polypeptides comprised by said kit are contained in separate
vials.
[0277] In one embodiment, one or more of the polypeptides of said
set of polypeptides comprised by said kit are freeze-dried.
[0278] In one embodiment, one or more of the polypeptides of said
set of polypeptides comprised by said kit are in solution.
[0279] The objects of the present invention are also solved by a
method for treatment of a patient who is suffering from a [0280]
(i) tumour or cancer and/or who is undergoing allogeneic cell or
tissue transplantation; [0281] (ii) an autoimmune disorder; or
[0282] (iii) a hypersensitivity disorder. [0283] Said method may
comprise the steps: [0284] obtaining a set of polypeptides, said
set of polypeptides comprising [0285] a first polypeptide P1
comprising [0286] (i) a targeting moiety T1, wherein said targeting
moiety T1 specifically binds to an antigen A1, and [0287] (ii) a
fragment F1 of a functional domain F, [0288] wherein neither said
fragment F1 by itself nor said polypeptide P1 by itself is
functional with respect to the function of said domain F, [0289]
and [0290] a second polypeptide P2 comprising [0291] (i) a
targeting moiety T2, wherein said targeting moiety T2 specifically
binds to an antigen A2, said antigen A2 being a cell surface
molecule that is specific for a certain cell type or cell lineage,
and [0292] (ii) a fragment F2 of said functional domain F, [0293]
wherein neither said fragment F2 by itself nor said polypeptide P2
by itself is functional with respect to the function of said domain
F, [0294] wherein said antigen A1 is different from said antigen
A2, [0295] wherein said polypeptide P1 and said polypeptide P2 are
not associated with each other in the absence of a substrate that
has both antigens A1 and A2 at its surface, more specifically a
cell that carries both antigens A1 and A2 at its cell surface, and
[0296] wherein, upon dimerization of said fragment F1 of said
polypeptide P1 with said fragment F2 of said polypeptide P2, the
resulting dimer is functional with respect to the function of said
domain F, [0297] administering said set of polypeptides to said
patient.
[0298] In such method of treatment, said set of polypeptides is as
defined in the embodiments above.
[0299] The objects of the present invention are also solved by a
method of using the set of polypeptides as described above for
treatment of a patient undergoing cell or tissue
transplantation.
[0300] The objects of the present invention are also solved by the
use of a set of proteins as defined in the embodiments above for
the manufacture of a medicament for the treatment of a patient
suffering from the above defined and described diseases a disorder
or, for example, a patient suffering from cancer and/or undergoing
cell or tissue transplantation.
[0301] As used herein, the term "polypeptide" refers to a linear
molecular chain of amino acids containing more than 30 amino acids.
Optionally, a polypeptide may include one or more disulfide bonds
or be chemically modified. Moreover, optionally a non-proteinaceous
element (such as a fluorophore, RNA-aptamer, DNA-aptamer, or small
molecule) may be attached to said linear molecular chain of amino
acids. Such polypeptides can be produced by any known method. The
polypeptide can for example be generated by expression from a
nucleic acid coding for said polypeptide, or can be synthesized by
solid phase synthesis methods, or be produced by conjugation or
linkage of existing molecules, e.g., by chemical linkage.
[0302] The term "polypeptide P1" is used to refer to a polypeptide
comprising (i) a targeting moiety, wherein said targeting moiety
specifically binds to an antigen, and (ii) a fragment of a
functional domain, wherein neither said fragment by itself nor said
polypeptide P1 by itself is functional with respect to the function
of said functional domain. The term "polypeptide P2" is used to
refer to a polypeptide comprising (i) a targeting moiety, wherein
said targeting moiety specifically binds to an antigen, and (ii) a
fragment of a functional domain, wherein neither said fragment by
itself nor said polypeptide P2 by itself is functional with respect
to the function of said functional domain.
[0303] The term "domain", as used herein, refers to a linear
molecular chain of amino acids that includes the amino acid
sequence of an entire polypeptide or a portion of a polypeptide.
Optionally, a domain may include one or more disulfide bonds or be
chemically modified. Moreover, optionally a domain may comprise a
non-proteinaceous element (such as a fluorophore). In one
embodiment, however, the term "domain" does not comprise compounds
that are chemically modified or comprise non-proteinaceous
element(s).
[0304] A "functional domain", as used herein, is a domain that is
capable of fulfilling a certain function, such as specific binding
to a certain binding partner or antigen, specific activation of a
certain receptor, mediation of toxic effects, or fluorescence upon
excitation with light of an appropriate wavelength.
[0305] The term "functional domain F" is preferably meant to also
include compounds that are non-proteinaceous. In one embodiment,
however, it refers to a proteinaceous compound or a functional part
thereof.
[0306] The term "a fragment of a domain", as used herein, refers to
a linear molecular chain of amino acids that corresponds to a part
of a domain, but not the entire domain. Optionally, a fragment of a
domain may include one or more disulfide bonds or be chemically
modified. Moreover, optionally a domain may comprise a
non-proteinaceous element or part of such a non-proteinaceous
element.
[0307] The term "fragment F1" is used to refer to a fragment of a
functional domain. The term "fragment F2" is used to refer to a
fragment of a functional domain.
[0308] The pairwise abbreviations P1, P2; T1, T2; F1, F2; A1, A2;
and I1, I2, as used herein, are meant to designate different
polypeptides, targeting moieties, fragments, antigens, and
immunoglobulin modules, respectively. They are synonymous to first
polypeptide, second polypeptide; first targeting moiety, second
targeting moiety; first fragment, second fragment; first antigen,
second antigen; and first immunoglobulin module, second
immunoglobulin module, respectively.
[0309] The term "moiety", as used herein, refers to a linear
molecular chain of amino acids that includes the amino acid
sequence of an entire polypeptide or a portion of a polypeptide.
Optionally, a moiety may include one or more disulfide bonds or be
chemically modified. Moreover, optionally a moiety may comprise a
non-proteinaceous element (such as an oligonucleotide). In one
embodiment, however, the term "moiety" does not comprise compounds
that are chemically modified or comprise non-proteinaceous
element(s). The term "targeting moiety T1" is used to refer to a
moiety that specifically binds to an antigen, for example antigen
A1. The term "targeting moiety T2" is used to refer to a moiety
that specifically binds to an antigen, for example antigen A2.
[0310] As used herein, a "linker" is a sequence of amino acids
within a polypeptide that connects two parts of said polypeptide or
two domains comprised by said polypeptide.
[0311] The term "nucleic acid molecule", as used by the present
invention, defines a linear molecular chain consisting of more than
30 nucleotides. The term includes DNA, such as cDNA or genomic DNA,
and RNA.
[0312] The term "construct", as used herein, refers to a nucleic
acid molecule comprising one or more recombinant nucleotide
sequences. The term also includes polypeptides that are expressed
from a recombinant nucleotide sequence or that are artificially
made or recombinant molecules that comprise two or more amino acid
sequences that are not naturally found within the same protein.
[0313] The term "specifically binds to" or "specifically binds", as
used by the present invention in the context of a molecule or
domain that specifically binds to an interaction partner or antigen
or that specifically binds an interaction partner or antigen, means
that a molecule or domain binds to said interaction partner or
antigen, preferably by non-covalent binding, or is capable of
binding said interaction partner or antigen, preferably by
non-covalent binding, and does not or essentially not cross-react
with any other interaction partner or antigen with a structure
similar to that of the interaction partner or antigen.
[0314] In the context of a targeting moiety (such as targeting
moiety T1 or T2) specifically binding to an antigen (such as
antigen A1 or A2), the term "specifically binds to" is meant to
refer to a situation where either said targeting moiety is capable
of specifically binding to said antigen, or where it actually binds
thereto.
[0315] In the context of a T cell engaging domain, an NK cell
engaging domain, domain engaging macrophage cells, a monocyte
engaging domain, a granulocyte engaging domain, a domain engaging
neutrophil granulocytes, or a domain engaging activated neutrophil
granulocytes, monocytes and/or macrophages, the term "specifically
binding to" an antigen or molecule or "specifically binds to" an
antigen or molecule is meant to refer to a situation where either
the respective domain is capable of specifically binding to said
antigen or molecule, or where it actually binds thereto.
[0316] In the context of a functional domain being a domain that
"specifically binds to" an antigen, a molecule, a compound, a
carrier molecule or an affinity tag, the term "specifically binds
to" is meant to refer to a situation where either said functional
domain is capable of specifically binding to said antigen,
molecule, compound, carrier molecule or affinity tag, or where it
actually binds thereto.
[0317] In the context of a toxin, fluorophore, antigen, carrier
molecule or affinity tag being "specifically bound by" a functional
domain, this is meant to refer to a situation where either said
functional domain is capable of specifically binding to said toxin,
fluorophore, antigen, carrier molecule or affinity tag, or where it
actually binds thereto.
[0318] As used in the present application, a molecule or antigen is
"specific for a certain cell type or cell lineage" if it is
expressed by said cell type/cells of said cell lineage, but not (or
only to a negligible extent) by other cell types or cells of other
cell lineage. In some embodiments, a molecule or antigen is
"specific for a certain cell type or cell lineage" if it is
expressed by said cell type/cells of said cell lineage, and not
more than a few other cell types or cells of other cell lineage
besides said cell type/cells of said cell lineage express said
antigen as well, while most other cell types or cells of other cell
lineage besides said cell type/cells of said cell lineage do not
express said antigen (or only to a negligible extent). The term
"specific for a certain cell type or cell lineage" may also mean
that said molecule or antigen is expressed by said cell type/cells
of said cell lineage at a higher rate or at a higher proportion or
amount than by other cell types/cells of other cell lineages, in
the sense that there may be a small but detectable expression of
said molecule also in other cell types/cells of other cell
lineages. The term "marker", as used herein in the context of a
marker for a certain cell type or cell lineage, can refer to a
molecule or antigen that is specific for a cell type or cells of a
cell lineage, respectively, as described above.
[0319] As used herein, the term "aptamer" refers to a small
compound composed of oligonucleic acid (such as RNA or DNA) or
peptidic or non-peptidic molecule that binds to a specific target
molecule with high affinity.
[0320] As used herein, the term "carrier molecule" refers to a
molecule or part of a molecule that is not recognized as foreign by
the immune system of a patient to whom the set of polypeptides
according to the invention is administered or that causes no or
only a weak immune reaction by a patient to whom the set of
polypeptides according to the invention is administered.
Preferably, such a "carrier molecule" is being bound by or capable
of being bound by another molecule, such as an antibody. In some
embodiments, a "carrier molecule" is a molecule or part of a
molecule that In certain embodiments, the carrier molecule is
attached covalently or non-covalently to a second molecule or part
of a second molecule, for example a fluorophore or toxin.
[0321] The term "MHC" refers to the Major Histocompatibility
Complex, which is a set of genes encoding a group of molecules
comprising cell-surface molecules that are required for antigen
presentation to T-cells and that are also responsible for rapid
graft rejections. In humans, the MHC includes the genes HLA-A,
HLA-B, HLA-C, HLA-DP, HLA-HQ, and HLA-DR. In the present
application, the term is used to refer to the genes of the Major
Histocompatibility Complex as well to the gene products encoded by
these genes. The term "HLA" refers to Human Leukocyte Antigens. As
used herein, "HLA" is the human form of "MHC".
[0322] The term "allelic variant", as used herein, denotes any of
two or more alternative forms of a gene occupying the same
chromosomal locus. For example, HLA-A1, HLA-A2, and HLA-A3 are
three of the allelic variants of HLA-A. The term allelic variant is
also used herein to denote a protein encoded by an allelic variant
of a gene.
[0323] The term "antigen", as used herein, refers to a molecule
known to be specifically bound by or capable of being specifically
bound by an antibody or the antigen-binding part of an antibody. In
its broadest meaning, "antigen A1" refers to an antigen as defined
above. In its broadest meaning "antigen A2" refers to an antigen as
defined above. The designations "antigen A1" and "antigen A2" have
been chosen in order to allow for distinction between "antigen A1"
and "antigen A2". An "MHC antigen" is an antigen that is also a
molecule belonging to the major histocompatibility complex. MHC
antigens include MHC class I antigens (in humans, the antigens
HLA-A, -B, and -C) and MHC class II antigens (in humans, the
antigens HLA-DP, -DQ, and -DR). The phrase that a cell "carries an
antigen" or "carries an antigen at its cell surface" is meant to
refer to a situation where a cell expresses an antigen that is
present at the cell surface of said cell and accessible for an
antibody from outside said cell. The phrase that a substrate "has
an antigen at its surface" is meant to refer to a situation where
said antigen is present at the surface of said substrate and
accessible for an antibody applied to said substrate.
[0324] The term "an antigen that is specific for the malignant
state of a cell", as used herein, refers to an antigen that a
malignant cell of a certain cell type (such as a malignant B-cell
tumour cell) carries at its cell surface, but that a cell of the
same cell type that is not malignant (such as a non malignant
B-cell) does not (or only to a negligible extent) carry at its cell
surface.
[0325] The term "an antigen/molecule that is specific for a
malignant cell type", as used herein, refers to an antigen/molecule
that a malignant cell of a certain cell type (such as malignant
B-cell tumour cell) carries at its cell surface, but that a cell of
the same cell type that is not malignant (such as a non malignant
B-cell) or cells of other cell types (such as T-cells or
hepatocytes) do not (or only to a negligible extent) carry at their
cell surface. In some embodiments, the term "an antigen/molecule
that is specific for a malignant cell type" refers to an
antigen/molecule that a malignant cell of a certain cell type (such
as malignant B-cell tumour cell) carries at its cell surface, but
that a cell of the same cell type that is not malignant (such as a
non malignant B-cell) does not (or only to a negligible extent)
carry at its cell surface, and that only cells of a few other cell
types besides that certain cell type carry at their cell surface,
while cells of most other cell types do not (or only to a
negligible extent). The term "an antigen/molecule that is specific
for a malignant cell type" may also mean that said antigen/molecule
is expressed by said malignant cell of a certain cell type at a
higher rate or at a higher proportion or amount than by a cell of
the same cell type that is not malignant, in the sense that there
may be a small but detectable expression of said molecule also in a
cell of the same cell type that is not malignant. The term
"marker", as used herein in the context of a marker for the
malignant state of a certain cell or for a malignant cell type, can
refer to a molecule or antigen that is specific for the malignant
state of a certain cell or for a malignant cell type, respectively,
as described above.
[0326] The term "immunoglobulin domain", as used herein, refers to
a domain that essentially consists of a globular region of an
antibody chain. Immunoglobulin domains are characterized in that
they retain the immunoglobulin fold characteristic of antibody
molecules. Immunoglobulins, such as IgG, IgE, or IgM, are composed
of a varying number of heavy and light chains. Each heavy and light
chain contains a constant region and a variable region. Each light
chain variable region (V.sub.L) and each heavy chain variable
region (V.sub.H) contains three hypervariable regions, also called
"complementarity-determining regions" or "CDRs". The CDRs are
primarily responsible for binding of the immunoglobulin to an
antigen.
[0327] The terms "V.sub.H" or "V.sub.H domain" are used
interchangeably and refer to the variable region of an
immunoglobulin heavy chain of an antibody. The terms "V.sub.L" or
"V.sub.L domain" are used interchangeably and refer to the variable
region of an immunoglobulin light chain of an antibody.
[0328] The term "immunoglobulin module", as used herein, refers to
a molecule, part of a molecule or molecular assembly which
comprises one or more, preferably two or more, immunoglobulin
domains and which is capable of binding to an antigen. Preferably,
an "immunoglobulin module" comprises a linear molecular chain of
amino acids that includes the amino acid sequence of one or more,
preferably two or more, immunoglobulin domains. Optionally, an
"immunoglobulin module" comprises one ore more, preferably two or
more, disulfide bonds. Included in the term "immunoglobulin module"
are molecules or parts of a molecule that comprise or consist of a
"single-chain variant fragment" of an antibody. Included in the
term "immunoglobulin module" are also molecules or parts of a
molecule that comprise or consist of a V.sub.HH domain of a llama
antibody, a camel antibody, or a shark antibody.
[0329] The term "immunoglobulin module I1" is used to refer to an
immunoglobulin module comprising a V.sub.L domain linked to a
V.sub.H domain. Preferably, said V.sub.L domain and said V.sub.H
domain of said immunoglobulin module I1 are derived from the same
antibody. Preferably, said V.sub.L domain and said V.sub.H domain
of said immunoglobulin module I1 form a dimer. Preferably, said
dimer is capable of specifically binding to an antigen. Said
antigen may be, for example, the antigen A1. In one embodiment,
said "immunoglobulin module I1" comprises a "single-chain variant
fragment" of an antibody that is capable of specifically binding to
an antigen, for example the antigen A1.
[0330] The term "immunoglobulin module I2" is used to refer to an
immunoglobulin module comprising a V.sub.L domain linked to a
V.sub.H domain. Preferably, said V.sub.L domain and said V.sub.H
domain of said immunoglobulin module I2 are derived from the same
antibody. Preferably, said V.sub.L domain and said V.sub.H domain
of said immunoglobulin module I2 form a dimer. Preferably, said
dimer is capable of specifically binding to an antigen. Said
antigen may be, for example, the antigen A2. In one embodiment,
said "immunoglobulin module I2" comprises a "single-chain variant
fragment" of an antibody that is capable of specifically binding to
an antigen, for example the antigen A2.
[0331] Within a construct of an immunoglobulin module comprising a
V.sub.L domain linked to a V.sub.H domain, the V.sub.L domain may
be positioned N- or C-terminally of the corresponding V.sub.H
domain. The skilled person is able to determine which arrangement
of the V.sub.H and V.sub.L domains is more suitable for a specific
single-chain variant fragment domain.
[0332] The terms "Fv" and "variant fragment", as used herein,
refers to a fragment of an antibody that is the minimum antibody
fragment which contains a complete antigen recognition and binding
site. This region consists of a dimer of one heavy and one light
chain variable region in a tight, non-covalent association
(V.sub.H-V.sub.L dimer). In this configuration, the V.sub.H and
V.sub.L domain together define an antigen binding site with antigen
binding specificity on the surface of the V.sub.H-V.sub.L
dimer.
[0333] The terms "scFv", "single chain Fv", and "single-chain
variant fragment" are used interchangeably and are meant to
designate an antibody or portion of an antibody in which the
variable region of the heavy chain (V.sub.H) and the variable
region of the light chain (V.sub.L) of a traditional two chain
antibody have been joined to form one chain. Typically, a linker is
inserted between the two chains to allow for proper folding and
creation of an active binding site.
[0334] The term "llama antibody", as used herein, refers to an
antibody or part of an antibody derived from llama. The term "camel
antibody", as used herein, refers to an antibody or part of an
antibody derived from camel. The term "shark antibody", as used
herein, refers to an antibody or part of an antibody derived from
shark. Llama, camel and shark antibodies have an antigen binding
moiety that is built up by one single domain, V.sub.HH, (rather
than a V.sub.H and a V.sub.L chain)
[0335] The expression "T cell engaging domain", as used herein, is
meant to refer to a domain that specifically binds to an antigen
that is present on the cell surface of T cells. Preferably, binding
of said T cell engaging domain to said antigen activates said T
cell. Similarly, the expression "NK cell engaging domain" refers to
a domain that specifically binds to an antigen that is present on
the cell surface of Natural Killer cells. Preferably, binding of
said NK cell engaging domain to said antigen activates said Natural
Killer cells. The expression "domain engaging macrophage cells"
refers to a domain that specifically binds to an antigen that is
present on the cell surface of macrophage cells. Preferably,
binding of said domain engaging macrophage cells to said antigen
activates said macrophage cells. The expression "monocyte engaging
domain" refers to a domain that specifically binds to an antigen
that is present on the cell surface of monocytes. Preferably,
binding of said monocyte engaging domain to said antigen activates
said monocytes. The expression "granulocyte engaging domain" refers
to a domain that specifically binds to an antigen that is present
on the cell surface of granulocytes. Preferably, binding of said
granulocyte engaging domain to said antigen activates said
granuloctyes. The expression "domain engaging neutrophil
granulocytes" refers to a domain that specifically binds to an
antigen that is present on the cell surface of neutrophil
granulocytes. Preferably, binding of said domain engaging
neutrophil granulocytes to said antigen activates said neutrophil
granuloctyes. The expression "domain engaging activated neutrophil
granulocytes, monocytes and/or macrophages" refers to a domain that
specifically binds to an antigen that is present on the cell
surface of activated neutrophil granulocytes, monocytes and/or
macrophages. Preferably, binding of said domain engaging activated
neutrophil granulocytes, monocytes and/or macrophages to said
antigen activates said monocytes and/or macrophages.
[0336] The term "molecule capable of mediating bioluminescence", as
used herein, refers to a molecule (or functional part of a
molecule) that has an enzymatic activity which in the presence of
the appropriate substrate(s) catalyzes a reaction that causes
bioluminescence. The term includes luciferases, such as the
luciferases of firefly or Gaussia.
[0337] The term "GFP variant", as used herein, refers to a molecule
that has an amino acid sequence derived from the amino acid
sequence of green fluorescent protein from Aequorea victoria by
introducing alterations resulting in greater fluorescence or
fluoresce in different colors. The term is meant to include, among
others, YFP (yellow fluorescent protein), CFP (cyan fluorescent
protein), Venus (Nagai T et al., A variant of yellow fluorescent
protein with fast and efficient maturation for cell-biological
applications. Nat Biotechnol. 2002 January; 20(1):87-90), Cerulean
(Enhanced CFP with S72A, Y145A and H148D substitutions).
[0338] "Enhanced GFP" (and, analogously, "enhanced YFP", "enhanced
CFP") refers to a GFP (YFP, CFP) which has been "humanized", as
reported in Kain et al. (1995) Biotechniques 19(4):650-55.
"Humanized" refers to changes made to the GFP (YFP, CFP) nucleic
acid sequence to optimize the codons for expression of the protein
in human cells.
[0339] The term "bimolecular fluorescence complementation
molecule", as used herein, refers to a fluorescent molecule that
can be provided as two fragments which by themselves are not
fluorescent, but which upon heterodimerization between the two
fragments form a dimer that is capable of fluorescence.
[0340] The term "therapeutic compound", as used herein, refers to a
compound suited for preventing, treating, alleviating or curing a
disease or disease state. Preferably, a "therapeutic compound" is a
compound that, upon entry into a cell, is capable of causing the
death of that cell. In some embodiments, a therapeutic compound can
be a chemical or radioactive compound that damages vital cellular
structures or interrupts vital cellular processes.
[0341] The term "diagnostic compound", as used herein, refers to a
compound that can be detected by common detection methods, such as
methods used in the clinic or in biochemical or medical diagnostic
laboratories, for example a fluorescent compound, a radioactive
compound, or a molecule mediating bioluminescence.
[0342] The term "progenitor/precursor cells" is meant to refer to
immature, undifferentiated or partially differentiated cells that
are typically found in post-natal animals/humans and have the
potential to differentiate into a specific cell type or into
specific cell types. The term "progenitor/precursor cells of a
tumour" designates progenitor/precursor cells with altered
properties (e.g. regarding their proliferation behaviour or gene
expression pattern) that give rise to tumour cells. Examples for
such progenitor/precursor cells of a tumour are e.g. leukemic
precursor or progenitor cells.
[0343] The term "cancer", as used herein, refers to a malignant
cell, group of cells, or malignant neoplasia. The term is meant to
comprise carcinomas, sarcomas, lymphomas, leukemias, germ cell
tumours, and blastomas. A "cancerous cell" is a cell that is part
of or derived from a cancer. The term "tumour" is used
interchangeably with the term "cancer".
[0344] As used herein, the term "haematologic tumour" refers to a
cancer of the blood or blood building system (such as bone marrow
cells, blood-building cells, and precursor cells of mature blood
cells). In some embodiments, the term "haematologic tumour" refers
to a haematologic neoplasia. As used herein, the term
"non-haematologic tumour" refers to a tumour that is not a
haematologic tumour.
[0345] The term "a patient who is undergoing allogeneic tissue or
cell transplantation", as used herein, refers to a situation where
a patient receives or has received transplanted cells or
transplanted tissue that has/have been obtained from another
person. A preferred situation as to this aspect is the situation
with mismatched HLA antigens. The unit ".mu.g/m.sup.2", as used
herein in the context of an amount of a polypeptide administered,
refers to a certain amount of polypeptide per square meter of body
surface of the patient to whom said polypeptide is administered
(the peptide may be administered by any adequate route of
administration, such as by intravenous or subcutaneous injection).
For example, the expression "The amount of polypeptide administered
is 50 .mu.g/m.sup.2 per day for the polypeptide P1." is meant to
refer to a situation where the amount of polypeptide P1
administered per day is 50 .mu.g per square meter of body surface
of the patient to whom the polypeptide P1 is administered. In the
case of a patient having a body surface of 2 m.sup.2 this would
mean that 100 .mu.g of polypeptide P1 are administered per day.
[0346] The present inventors have surprisingly found that with a
set of polypeptides according to the invention the above-indicated
problems of the prior art can be overcome and the above-described
objects can be accomplished. Moreover, the present inventors have
surprisingly found that with a set of polypeptides according to the
invention, cells with a specific combination of two antigens can be
identified and/or eliminated with high specificity and reduced
side-effects.
[0347] It is one advantage of the combinatorial strategy of the
invention that no preformed F units (for example anti-CD3 units)
are used. The F1 and CD3 V.sub.H and V.sub.L) do not heterodimerize
per se, not even in the presence of an agent which stabilizes their
dimerization (for example an antigen capable to bind to the domain
F, like for example, CD3, HIS or DIG), and thus do not result in a
functional F domain (for example do not stimulate T cells).
Exclusively in situations where both complementary constructs P1
and P2 simultaneously bind on the surface of a given cell, the two
components F1 and F2 reconstitute the F domain (for example, the
CD3 binding site). Thus, function of the F domain (for example T
cell activation) takes place precisely where needed but not
systemically. Hence, it can be assumed that the combinatorial
strategy of the invention has less toxic effects, for example as
compared to normal bispecific antibody strategies. This is also
evidenced by the appended examples, in particular by the in vivo
model for allogeneic transplantation, where HLA-A2 positive mice
did not suffer any clinical effects after infusion of HLA-A2
reactive constructs.
[0348] In particular, to tag cells that express a predefined
antigen signature, two single-chain polypeptides were designed as
parts of the final bipartite (bi-molecular) construct
(bi-moleculer/trispecific antibody construct), each composed of an
antigen-binding single-chain variable fragment (scFv) and either
the variable light (VL) or variable heavy chain (VH) domain of an
antibody. When these two hybrid fragments bind their respective
antigens on the surface of a single cell, the VL and VH domains
interact with each other to reconstitute the original antigen
binding site and thus fulfill the desired requirements.
[0349] As mentioned, it is one advantage of the set of polypeptides
of the invention that binding of both target antigens on the cell
surface is requisite for functional heterodimerization.
Self-assembly of the two complementary parts and subsequent T cell
stimulation after binding of only one arm to its antigen can be
ruled out, thus corroborating published data showing that V.sub.H
or V.sub.L binding by itself is of low affinity and that
V.sub.H/V.sub.L heterodimers tend to dissociate rapidly in the
absence of antigen (Colman, 1987, Nature 326, 358-363; Amit, 1986,
Science 233, 747-753; Law, 2002, Int Immunol 14, 389-400; Ueda,
1996, Nat Biotechnol 14, 1714-1718).
[0350] In contrast to the homo- or hetero-dimerization domains well
known in the art (leucine-zipper, Fc-domains, knob in the hole
etc), VH and HL interactions are of low affinity. However, it has
been shown that VH/VL interaction can be stabilized after binding
to the specific antigen. Without being bound by theory, VH/VL
interaction in accordance with this invention takes place only in
situations after both fragments have previously bound to their
cognate target antigens, for example on the surface of a target
cell. Also without being bound by theory, after simultaneous
on-target binding, the constructs are brought into close proximity
so that they can form a trimeric complex with the antigen. The thus
on-target complemented trispecific heterodimer of the invention is
functional with respect to the function of the domain F, for
example, engages and stimulates T cells for tumor cell destruction
if anti CD3 is reconstituted.
[0351] Beside one advantage of the constructs of the invention P1
and P2, e.g. the combinatorial nature of the immune response
elicited, it was surprisingly found in the context of this
invention that the bi-molecular construct with the disrupted F
domain, for example scFv-anti CD3, displays no off target
effects.
[0352] The set of polypeptides according to this invention, in
particular the polypeptides P1 and P2 comprised therein, have the
further advantage to be more stable and/or have an improved shelf
life (in particular at 4.degree. C.) as compared to conventional
bispecific constructs like BiTE constructs. These conventional
bispecific constructs tend to aggregate (in particular at 4.degree.
C.).
[0353] It is envisaged that the polypeptides of this invention P1
and P2, more particular of F1 and F2 as comprised therein, more
particular of the V.sub.H and V.sub.L which may be comprised
therein, due to their hydrophobic interface, are capable to bind
albumin. This leads to an improved retention time; i.e. longer
bioavailability in vivo but also in vitro, like, for example, in
serum or blood samples.
[0354] The set of polypeptides according to the present invention
comprises a first polypeptide P1 and a second polypeptide P2. The
first polypeptide P1 comprises a first targeting moiety T1 (which
is capable of specifically binding to an antigen A1) fused to a
first fragment F1 of a functional domain F (see FIG. 1A, top). The
second polypeptide P2 comprises a second targeting moiety T2 (which
is capable of specifically binding to an antigen A2) linked to a
second fragment F2 of the functional domain F (see FIG. 1A,
bottom). Importantly, the fragments F1 resp. F2 of the functional
domain are non-toxic by their own and unable to exert any
biological function unless there is partnering between the two
polypeptides P1 and P2. When both polypeptides P1 and P2
simultaneously bind to their antigens on the surface of a single
cell that expresses both antigens A1 and A2, the fragments F1 and
F2 of the functional domain F are brought together in close
proximity, they hetero-dimerize and thus complement the desired
biological function (see FIG. 1B). On the other hand, a cell that
expresses either only antigen A1 (FIG. 1C) or only antigen A2 (FIG.
1D) or none of the antigens does not cause complementation of the
biological function. Thus, the biological function is achieved with
high specificity only in the presence of cells having both antigens
A1 and A2 at their cell surface upon simultaneous binding of both
polypeptides P1 and P2 to such a cell. Depending on the nature of
the functional domain F, different objects, such as specific
identification/detection or elimination of cells that express both
antigens A1 and A2, can be accomplished.
[0355] In one exemplary embodiment, this inventive principle is
applied for the specific elimination of tumour cells:
[0356] Novel histopathological and flow cytometry analyses have
revealed that tumour cells can be detected and distinguished from
their non-transformed counterparts not by single surface markers
but by the expression of aberrant antigen combinations/profiles, as
is known for haematopoietic neoplasias and cancer and cancer stem
cells of various other provenience. Thus, while a single antigen
may not be sufficient to specifically identify a certain tumour
cell, a specific combination of two antigens may allow
discriminating the tumour cell from any other type of cell.
[0357] For example, the set of polypeptides according to the
invention may be used to specifically eliminate cancer cells
characterized by the simultaneous expression of the antigens CD33
and CD19 at their cell surface. This combination of antigens is
found on certain types of acute leukemia cells and distinguishes
these cells from any other cells (such as non-malignant cells),
which may carry either CD33 or CD19 at their cell surface, but do
not carry both CD33 and CD19 at their cell surface (Ossenkoppele et
al., Review of the relevance of aberrant antigen expression by flow
cytometry in myeloid neoplasms. Br J Haematol 2011,
153(4):421-36).
[0358] To specifically eliminate these leukemic cells carrying both
CD33 and CD19 at their cell surface, the first targeting moiety T1
of the first polypeptide P1 may be a single chain variable Fragment
(scFv) specific for CD33. As fragment F1 of the functional domain
F, the light chain variable domain V.sub.L of an anti CD3 antibody
may be chosen. The second targeting moiety T2 of the second
polypeptide P2 may be a scFv specific for CD19. As the fragment F2
of the functional domain F the heavy chain variable domain V.sub.H
of that anti CD3 antibody may be chosen. The light chain variable
domain V.sub.L and the heavy chain variable domain V.sub.H of the
anti CD3 antibody are each non-toxic by their own. They are also
unable to exert their biological function (i.e. to effectively bind
the CD3 antigen) unless there is partnering between the
polypeptides P1 and P2.
[0359] In the presence of a leukemic cell having both CD33 and CD19
at its cell surface, both polypeptides P1 and P2 simultaneously
bind to that cell. As a consequence, the fragments F1 and F2 of the
functional group F (i.e. the heavy and light chain of the Fv anti
CD3 variable domain of that anti-CD3 antibody) are brought together
in close proximity, they hetero-dimerize and thus complement the
desired biological function, enabling the dimer of P1 and P2 to
specifically bind to CD3.
[0360] CD3 is a cell surface molecule that is present on the
surface of T cells. The molecule is part of the T cell signaling
complex, and cross-linking of CD3 molecules on the surface of a T
cell after binding of a CD3-specific antibody leads to activation
of the T cell. By engaging CD3 antigens on the surface of T cells,
heterodimers of polypeptides P1 and P2 are capable of recruiting T
cells and activating them. As a result, typical effector mechanisms
of a cytotoxic T cell response are elicited, leading to cell lysis:
release of lytic granules containing the cytotoxic proteins
perforin, granzymes, and granulysin. Perform forms pores into the
membrane of the target cell through which the granzymes can enter
and induce apoptosis. These effects lead to specific destruction of
leukemic cells that carry both CD33 and the CD 19 antigen at their
cell surface.
[0361] Other cells than the leukemic cells do not have both the
CD33 and CD19 antigen at their cell surface. Therefore, they cannot
recruit both polypeptides P1 and P2, and no complementation of the
CD3 binding capability and engagement of CD3 positive T lymphocytes
is achieved. Consequently, other cells besides the leukemic cells
are unaffected, and destruction of the malignant cells with
exquisite specificity is achieved.
[0362] This is in stark contrast to conventional bispecific
antibodies. A conventional bispecific construct that engages T
cells and has specificity for cells expressing CD33 would mediate
the destruction of all CD33 positive cells. Since CD33 is myeloid
lineage marker which is expressed on many myeloid cells and myeloid
progenitor cells, the destruction of these cells would result in
long lasting aplasia and probably death of the patient. A
conventional bispecific construct that engages T cells and has
specificity for CD 19 positive cells would lead to the elimination
of all cells carrying the CD 19 antigen at their cell surface. CD
19 is expressed on a significant subset of B-lymphocytes.
Destruction of these cells would lead to a severe defect of the
immune system. Thus, besides eliminating leukemic cells that
simultaneously express CD33 and CD19 on the surface, the
application of conventional bispecific antibodies with specificity
for CD33 and CD19 would lead to elimination of myeloid cells and a
substantial subset of B-lymphocytes.
[0363] Thus, while conventional bispecific antibodies recognize
only one antigen on the cell to be eliminated, effector activation
according to the present invention requires the simultaneous
recognition of two specific antigens on the surface of the cell to
be identified/eliminated. In consequence, the present invention
achieves significantly improved specificity and reduced side
effects.
[0364] It is clear to a person of skill in the art that, within the
principle of the present invention, diverse variations to the
exemplary embodiment described above are possible.
[0365] For example, the approach described in the above exemplary
embodiment can easily be adapted for the identification/elimination
of other types of tumour cells besides CD33 and CD19 positive acute
leukemia cells simply by choosing appropriate targeting moieties T1
and T2 that specifically bind to antigens A1 and A2, respectively,
that are present simultaneously on the cells to be
identified/eliminated, but not present simultaneously on other cell
types. As quoted above, many if not all cancer cells (but also
progenitor/precursor cells of cancer cells) express a number of
cell surface molecules which per se are widely expressed on normal
tissues, but are indicative for the malignant phenotype if
expressed in a non-physiological combination. For example, CD34 is
a marker for haematopoietic stem cells and CD7 can be detected on a
subset of lymphoid cells. The combination of CD34 and CD7, however,
is strongly associated with malignancy, and aberrant co-expression
of the two antigens can be detected on a substantial proportion of
acute myelogenous leukemias (Ossenkoppele et al., Review of the
relevance of aberrant antigen expression by flow cytometry in
myeloid neoplasms. Br J Haematol 2011, 153(4):421-36.). Similarly,
aberrant co-expression of CD44 and CD 117 has been described for
ovarian cancer stem cells, CD44 and CD24 for pancreas cancer
initiating cells and the combination of EpCAM and CD44 in colon and
breast cancer stem cells (Natasha Y. Frank, Tobias Schatton, Markus
H. Frank; The therapeutic promise of the cancer stem cell concept.
J Clin Invest. 2010; 120:41-50). Expression of CD24 and CD29, as
well as CD24 and CD49f has been found to be specific for breast
carcinoma (Vassilopoulos A et al. Identification and
characterization of cancer initiating cells from BRCA1 related
mammary tumours using markers for normal mammary stem cells. Int J
Biol Sci 2008; 4:133-142). Moreover, combinations with highly
expressed antigen levels are indicative for a number of
malignancies, like CD38 and CD138 for myeloma.
[0366] In addition to the cancer-specific antigen combinations
listed above and those known from the scientific literature,
additional combinations of two antigens that are expressed
simultaneously on specific tumour cells but not on other cells can
be derived in a straight-forward manner by the person of skill in
the art.
[0367] Firstly, the skilled person may arrive at an antigen
combination that is specific for a certain cancer by combining an
antigen that is specific for the malignant state of the respective
cell type with an appropriate cell type marker or cell lineage
marker. For example, carbonic anhydrase IX is a marker strongly
associated with renal cell carcinoma and metastases of renal cell
carcinoma and thus represents a marker for the malignant state of
renal cells. This membrane located marker, however, is also
expressed on normal cells of the intestinal tract. By selecting as
second antigen a renal lineage marker like aquaporin, the resulting
combination of two antigens is specific for renal cell carcinoma
cells and cells resulting from metastasis of renal cell carcinoma,
while neither non-malignant kidney cells (which do not express
carbonic anhydrase IX) nor cells from the intestinal tract (which
do not express aquaporins) are characterized by the selected pair
of antigens.
[0368] Detailed information on markers for the malignant state of
various cell types and on markers for numerous cell types or cell
lineages is available from the literature and web-based resources
(see below for details) or can be obtained by straight-forward
experimentation (see below).
[0369] Examples for markers for the malignant state of a cell
include: E-cadherin for epithelial cells and ductal-type breast
carcinoma cells; Ca-125 for Epitheloid malignancies and ovary
cancer cells, adenocarcinoma cells and breast cancer cells;
Her-2/neu for breast cancer cells; gross cystic disease fluid
protein (BRST-2 protein) for breast cancer cells; BCA-225 (breast
carcinoma associated glycoprotein) for lung and breast cancer
cells; CA 19-9 (carbohydrate antigen 19-9) for pancreas, bile duct
and intestinal tract cancer cells; CEA for colorectal cancer cells;
CD117 (c-kit) for gist (gastrointestinal stromal tumour) cells (and
myeloid and mast cells); CD30 for Reed-Sternberg cells (and Ki-1
activated T-cells and B-cells); Epithelial antigen (BER-EP4),
Epithelial membrane antigen, and Epithelial Related Antigen
(MOC-31) for epithelial cancer cells; Epidermal growth factor
receptor (HER1) for cells of various cancers; Platelet derived
growth factor receptor (PDGFR) alpha for cells of various cancers;
Melanoma associated marker/Mart 1/Melan-A for melanoma cells; CD133
for cancer stem cell populations and others; TAG 72 (tumour
associated gp 72) for adenocarcinoma cells.
[0370] Further examples for markers for a malignant state of a
cell/cells include: EpCAM, CD19, HER-2, HER-3, HER-4, PSMA, MUC-1
(mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, Lewis-Y, CD20,
CD33, CD44v6, Wue-1, Plasma Cell Antigen, (membrane-bound) IgE,
Melanoma Chondroitin Sulfate Proteoglycan (MCSP), STEAP,
mesothelin, Prostate Stem Cell Antigen (PSCA), sTn (sialylated Tn
antigen), FAP (fibroblast activation antigen), EGFRvIII, Ig.alpha.,
Ig.beta., MT-MMPs, Cora antigen, EphA2, L6 and CO-29, CCR5,
.beta.HCG, ganglioside GD3, 9-O-Acetyl-GD3, GM2, Globo H, fucosyl
GM1, Poly SA, GD2, Carboanhydrase IX (MN/CA IX), Sonic Hedgehog
(Shh), CCR8, TNF-alpha precursor, A33 Antigen, Ly-6, desmoglein 4,
E-cadherin neoepitope, Fetal Acetylcholine Receptor, CD25,
Muellerian inhibitor Substance (MIS) Receptor type II, endosialin,
SAS, CD63, TF-antigen, CD7, CD22, Ig.alpha.(CD79a), Ig.beta.
(CD79b), G250, gp100, F19-antigen and EphA2.
[0371] Examples for antigens that are specific for a certain cell
type/cell lineage or for a few cell types/cell lineages (cell type
markers/cell lineage markers) include: CD45 for hematopoietic
cells; CD34 for endothelial cells, stem cells, and stromal cells;
CD33 for myeloid cells; CD138 for plasma cells and a subset of
epithelial cells; CD15 for epithelial, myeloid, and Reed-Sternberg
cells; CD1a for cortical thymocyctes and Langerhans cells; CD2 for
thymic cells, T-cells, and Natural Killer (NK) cells; CD3 for
T-cells; CD4 for helper T-cells; CD5 for T-cells, a subset of
B-cells, and thymic carcinoma cells; CD8 for cytotoxic T-cells;
CD20 for B-cells; CD23 for activated B-cells; CD31 for endothelial
cells; CD43 for T-cells, myeloid cells, a subset of B-cells,
histiocytes, and plasma cells; CD56 for NK cells; CD57 for
neuroendocrine cells, and NK cells; CD68 for macrophages; CD79a for
B-cells and plasma cells; CD146 for the endothelial cell lineage;
surfactant proteins for lung cells; synaptophysin, CD56 or CD57 for
neuroendocrine cells; nicotinic acetylcholine receptor or
muscle-specific kinase (MUSK) for muscle cells; voltage-gated
calcium channel (P/Q-type) or voltage-gated potassium channel
(VGKC) or N-methyl-D-aspartate receptor (NMDA) for muscle cells and
neurons; TSH (thyroid stimulating hormone) receptor for thyreoid
gland; amphiphysin for muscle cells; HepPar-1 for hepatocytes;
ganglioside GQ1B, GD3 or GM1 for neuronal cells; and glycophorin-A
for cells of the erythropoietic cell lineage.
[0372] It should be noted that there are situations where it may be
advantageous to rely for the purposes of the present invention on
an antigen with a less than perfect specificity for the cell type
or cell lineage of interest. For example in situations where no
antigen is known that is found exclusively on the cell type/cell
lineage of interest and not on any other cell types/lineages or in
situations where it is not possible to confirm the exclusive
specificity of an antigen, also antigens that are present on one or
more other cell types/cell lineages besides the cell type/cell
lineage of interest may be considered. Similar consideration apply
for markers for the malignant state of a cell, or even for the
specificity of the combination of two antigens. Thus, there are for
example situations where for the purposes of the present invention
a combination of two antigens is selected that is specific not only
for the cells of interest, but also for one or more (a few) other
cell types/cell lineages/kinds of malignant cells.
[0373] Secondly, the skilled person may arrive at an antigen
combination that is specific for a certain cancer by
straightforward experimentation. This may comprise the steps of (1)
determining the surface antigens on the tumour cells to be
eliminated and (2) identifying among these tumour cell surface
antigens two antigens that are not present simultaneously on other
cell types (or, in some embodiments, present on only a few other
cell types).
[0374] Often, experimentation may not be necessary to determine the
surface antigens on tumour cells to be eliminated, because such
information may already be available for the respective type of
cancer from the printed literature (see, e.g. David J. Dabbs,
Diagnostic immunohistochemistry, Churchill Livingstone, 3rd edition
(2010); or F Lin and J Prichard, Handbook of Practical
Immunohistochemistry: Frequently Asked Questions, Springer, New
York, 1st edition (2011)). Even more extensive information is
available through web-based resources. For example, the Cancer
Genome Anatomy Project (CGAP) of the U.S. National Cancer Institute
(NCI) has systematically determined the gene expression profiles of
various normal, precancer, and cancer cells (Strausberg R L. The
Cancer Genome Anatomy Project: building a new information and
technology platform for cancer research. In: Molecular Pathology of
Early Cancer, 1999, (Srivastava, S., Henson, D. E., Gazdar, A.,
eds. IOS Press), pp. 365-370). The resources generated by the CGAP
initiative are freely available (http://cgap.nci.nih.gov/) and
include access to all CGAP data and the necessary analysis tools.
Similarly, the Cancer Genome Characterization Initiative (CGCI) of
the National Cancer Institute focuses on tools for characterizing
the genomic changes involved in different tumours, for example
genomic characterization methods including exome and transcriptome
analysis using second generation sequencing. The data generated by
CGCI is available through a publicly accessible database
(http://cgap.nci.nih.gov/cgci.html). Thus, in many cases
information about the presence or absence of various known cell
surface proteins on the tumour cells of interest can be derived by
simply checking these publicly accessible databases. If desired,
this information may then be verified in a second step by
immunocytochemical/immunohistochemical analysis of tumour
cells/tissue according to the methods described below.
[0375] If there is no information available on the proteins
expressed by the tumour cells/tissue of interest, the skilled
person can carry out a characterization of the antigens on the
tumour cells/tissue by immunocytochemical/immunohistochemical
methods with a panel of antibodies (see, e.g., "Handbook of
Practical Immunohistochemistry: Frequently Asked Questions" by F
Lin and J Prichard, Springer New York, 1st edition (2011); or
"Using Antibodies: A Laboratory Manual" by E Harlow and D Lane,
Cold Spring Harbor Laboratory Press (1998)). In brief, a
histological preparation or cells isolated from the tumour are
incubated with a first antibody directed at a potential surface
antigen and, after a washing step, incubation of a second antibody
directed against the Fc domain of the first antibody. This second
antibody is labelled with a fluorophore or an enzyme like HRP
(horse radish peroxidase), in order to visualize expression of the
targeted antigen. Panels of antibodies that can be used for high
throughput antigen profiling purposes of cell surface antigens are
commercially available from numerous manufacturers.
[0376] In addition, tools specifically dedicated to high throughput
proteomic cell characterization to identify and analyze cell
surface protein expression are commercially available, such as the
FACS (Fluorescence-activated cell sorting)-based high throughput
array technology BD FACS.TM. CAP (Combinational Antibody Profile)
of Becton, Dickinson & Company.
[0377] The immunocytochemical/immunohistochemical/proteomic
analysis described above may be preceded (or, in some cases,
replaced) by genome-wide gene expression profiling of tumour cells
or by mass spectrometric analysis of the proteins expressed by the
tumour cells/tissue of interest. For example, genome-wide gene
expression profiling of tumour cells can be carried out to check
for the expression of various cell surface molecules, and the
presence of such antigens on the cell surface of the tumour cells
may then be confirmed through antibody-based staining methods as
described above.
[0378] Further information about approaches to characterize the
surface antigens of (cancer) cells is available in the relevant
scientific literature (e.g. Zhou J, Belov L, Huang P Y, Shin J S,
Solomon M J, Chapuis P H, Bokey L, Chan C, Clarke C, Clarke S J,
Christopherson R I. Surface antigen profiling of colorectal cancer
using antibody microarrays with fluorescence multiplexing. J
Immunol Methods. 2010; 355:40-51; or Carter P, Smith L, Ryan M.
Identification and validation of cell surface antigens for antibody
targeting in oncology. Endocr Relat Cancer. 2004; 11:659-87).
[0379] In a next step, the skilled person may identify among the
cell surface antigens of the tumour cells a combination of two
antigens which is not expressed simultaneously on other cell
types.
[0380] Often, already the literature or publicly available
databases may provide detailed information about the presence or
absence of antigens from other cell types:
[0381] The expression of various cell surface molecules on diverse
cell types has been studied systematically by researchers in the
past decades by immunophenotyping and gene expression profiling of
almost any cell type of the body. For example, detailed information
on the expression of more than 360 "cluster of differentiation"
antigens (or CD antigens) is available in print (e.g. "Leukocyte
and Stromal Cell Molecules: The CD Markers" by Zola H, Swart B,
Nicholson I, and Voss E; John Wiley & Sons, 1st ed. (2007)) and
in online depositories (e.g.
www.hcdm.org/MoleculeInformation/tabid/54/Default.aspx), and
includes information on tissue distribution and expression levels
of antigens, as well as information about antigen reactive
antibodies and the epitopes these antibodies bind to.
[0382] Moreover, there are publicly available databases which
provide access to a large amount of genomic data generated by the
scientific community. For example, the Gene Expression Omnibus
(GEO) platform of the National Center for Biotechnology Information
(NCBI) of the United States (Barrett T et al., NCBI GEO: archive
for functional genomics data sets--10 years on. Nucleic Acids Res.
2011; 39(Database issue):D1005-10) archives and gives access to an
enormous collection of microarray, next-generation sequencing, and
other forms of high-throughput functional genomic data, and further
provides web-based interfaces and applications for easy access to
this information (http://www.ncbi.nlm.nih.gov/geo/).
[0383] Once a pair of two antigens has been identified through
these resources that appears to be absent from other cell types
besides the tumour cells of interest, a person skilled in the art
can easily validate the suitability of the antigen combination for
further development of P1 and P2-polypeptide constructs. Such
validation that the identified combination of two antigens is
indeed not expressed simultaneously on other cell types besides the
tumour cells can be carried out by
immunohistochemical/immunocytochemical analysis of a (optimally
large) collection of assorted cell types and/or tissues with
antibodies against the two antigens. Cells and tissues of any kind
can be obtained from ATCC (American Type Culture Collection), from
pathology departments and from tissue banks associated with
universities and research institutions. A suitable antigen
combination is defined as a pair of antibodies that stains
exclusively the tumour cells, but not healthy tissues or healthy
cells (i.e. both antibodies of the pair stain the tumour cells, but
no other tissues/cells are stained by both antibodies).
[0384] It should be noted that, while in many situations the
highest degree of specificity (preferably absolute specificity) is
of course desirable, there are situations where a lower degree of
specificity is acceptable. For example, if the set of polypeptides
is used for diagnostic purposes, some degree of crossreactivity
with other cell types or tissues may be acceptable (especially in
the case of solid tumours, since the additional positional
information helps to distinguish tumour cells from crossreacting
cells). Moreover, if the set of polypeptides is used for
therapeutic purposes, some degree of crossreactivity with other
cell types or tissues may also be acceptable, depending on the
severity the disease in a treated patient and on the cell
types/tissues affected by the crossreactivity. Other situations
where a lower degree of specificity may be acceptable may arise in
the context of a transplantation setting (see below).
[0385] In cases where no hint about a suitable antigen combination
can be derived from the literature or public databases, the
presence/absence of the cell surface antigens of the tumour cells
from other cell types can be checked by straightforward
experimentation. To this end, a variety of cell types and/or
tissues obtainable from the sources indicated above may be
subjected to proteomic cell characterization,
immunocytochemical/immunohistochemical analysis and/or gene
expression profiling. (It should be noted that such analysis of
non-tumour cells/tissues has to be carried out only once in order
to obtain data that can be used for the design of various
constructs according to the invention that may be adapted to
diverse different therapeutic or diagnostic situations.) Upon
comparison of the obtained results with the information about cell
surface antigens of the tumour cells of interest, a combination of
two antigens that is not present on any other cells besides the
tumour cells of interest can be easily identified.
[0386] A similar systematic approach to identify a pair of two
antigens that is specific for tumour cells is also described in a
recent publication by Balagurunathan, which relies on genome-wide
gene expression profiling followed by immunohistochemistry
(Yoganand Balagurunathan, Gene expression profiling-based
identification of cell-surface targets for developing multimeric
ligands in pancreatic cancer. Mol Cancer Ther 2008; 7. 3071-3080).
Using DNA microarrays, the authors of that manuscript generated
databases of mRNA gene expression profiles for a substantial number
of pancreatic cancer specimens and normal tissue samples. The
expression data for genes encoding cell-surface molecules were
analyzed by a multivariate rule-based computational approach in
order to identify gene combinations that are preferentially
expressed on tumour cells but not in normal tissues. Aberrant
co-expression of antigens constituting a tumour-specific antigen
combination was then confirmed using standard immunohistochemistry
techniques on pancreatic tumour tissue and normal tissue
microarrays.
[0387] Having identified and validated such a combination of
antigens that is specific for the tumour cells of interest, the
constructs of polypeptide P1 and polypeptide P2 can be engineered
by standard protein engineering techniques and methods of molecular
biology (see, e.g. G Howard and M Kaser, Making and Using
Antibodies: A Practical Handbook, CRC Press, 1st edition (2006);
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, New York (2001)).
[0388] For many cell surface molecules, specific monoclonal
antibodies are characterized and therefore readily available. Thus,
in many cases the skilled person may have access to hybridoma cells
of monoclonal antibodies that are specific for the antigens of the
identified combination of antigens. Having the option to choose
from a panel of antibodies specific for a given antigen, a person
skilled in the art may choose a reactive antibody which binds an
epitope close to the membrane, in order to minimize the distance of
the antigen expressing cell from the effector cell (Bluemel C,
Hausmann S, Fluhr P, Sriskandarajah M, Stallcup W B, Baeuerle P A,
Kufer P. Epitope distance to the target cell membrane and antigen
size determine the potency of T cell-mediated lysis by BiTE
antibodies specific for a large melanoma surface antigen. Cancer
Immunol Immunother. 2010 August; 59(8):1197-209). If no such
antibody is available against one or both antigens of the
identified combination of antigens, monoclonal antibodies against
the antigens can be generated by standard techniques (e.g. G Howard
and M Kaser, Making and Using Antibodies: A Practical Handbook, CRC
Press, 1st edition (2006)). Moreover, various companies offer full
services for the generation of custom-made monoclonal antibodies
and hybridoma cells.
[0389] DNA or mRNA coding for the variable domains of the
monoclonal antibodies of interest can be obtained from hybridomas
by PCR amplification or cloning (Orlandi R, Gussow P T, Jones:
Cloning immunoglobulin variable domains for expression by the
polymerase chain reaction. Proc Natl Acad Sci USA 1989,
86(10):3833-3837; Wang Z, Raifu M, Howard M, Smith L, Hansen D,
Goldsby R, Ratner D: Universal PCR amplification of mouse
immunoglobulin gene variable regions: the design of degenerate
primers and an assessment of the effect of DNA polymerase 3' to 5'
exonuclease activity. J Immunol Methods 2000, 233(1-2):167-177;
Essono S, Frobert Y, Grassi J, Cremino C, Boquet D: A general
method allowing the design of oligonucleotide primers to amplify
the variable regions from immunoglobulin cDNA. J Immunol Methods
2003, 279:251-266; G Howard and M Kaser, Making and Using
Antibodies: A Practical Handbook, CRC Press, 1st edition (2006)) or
from already established vectors comprising the DNA sequence of the
variable fragment of the respective antibody. Often, the sequence
can be extracted from public databases, where many sequences are
deposited, and then the construct may even be generated by gene
synthesis as it is offered by various commercial service providers
(e.g. Creative Biolabs, Shirley, USA).
[0390] To form the construct of polypeptide P1, the sequence coding
for the variable fragment Fv of an antibody specific for the first
antigen of the identified pair of antigens (or, optionally, the
sequence of a single chain variable fragment derived from that
sequence) is used for the first targeting moiety (T1) and linked
via a suitable linker (coding, e.g., for less than 12 aa) to a
sequence coding for the first fragment F1 of a functional domain
(e.g. the V.sub.L domain of an anti CD3 antibody). Likewise, to
form the construct of polypeptide P2 the sequence coding for the
variable fragment Fv of an antibody specific for the second antigen
of the identified pair of antigens (or, optionally, the sequence of
a single chain variable fragment derived from that sequence) is
used for the second targeting moiety (T2) and linked via a suitable
linker to a sequence coding for the second fragment F2 of that
functional domain (e.g. the V.sub.H domain of an anti CD3
antibody).
[0391] For any construct of a polypeptide P1 or P2 according to the
invention, modifications to the construct or to the sequences used
for forming the construct are considered in order to adapt the
construct to specific needs. For example, a construct can be
modified in a way that reduces or abolishes its immunogenicity in
humans. In case a sequence is derived from a non-human parent
antibody, such as a murine antibody, modifications to the sequence
can be carried out that result in a reduced immunogenicity in
humans while retaining or substantially retaining the
antigen-binding properties of the parent antibody (known to the
skilled person as "humanizing" an antibody/construct).
[0392] Various modifications of the above-described procedure and
adaptions in order to accommodate the embodiments and variations
described in this application are evident to the person of skill in
the art.
[0393] In addition to variations with respect to the antigens that
the targeting moieties T1 and T2 specifically bind to, various
other modifications are possible. For example, instead of single
chain variant fragments (scFv) as targeting moiety T1 and/or T2
other types of monovalent antibodies or antibody-like structures
can be employed. For example, an antibody/antibody-like structure
derived from a llama, camel or shark antibody can be used. Since
llama, camel and shark antibodies have an antigen binding moiety
that is built up by one single domain (rather than a V.sub.H and a
V.sub.L chain), the resulting polypeptide P1 or P2 is much smaller
and may thus better penetrate into tumour tissues.
[0394] Furthermore, since many tumour-relevant antigens are cell
surface-bound receptors, the single chain Fv of targeting moiety T1
and/or T2 can be replaced by the natural or artificial ligand of
such a cell surface-bound receptor. Like antibodies, these natural
or artificial ligands confer excellent specificity towards the
target receptor. Alternatively, the targeting moiety T1 and/or T2
can be an aptamer.
[0395] Moreover, in order to enhance binding affinity of a
targeting moiety towards the antigen, the targeting moiety can be
multimerized and/or altered by glycosylation or other types of
posttranslational or chemical modification or be optimized through
site directed mutagenesis or a phage display selection process.
[0396] Moreover, the fragments F1 and F2 (i.e. the V.sub.L and
V.sub.H fragments of anti CD3 Fv in the above-described exemplary
embodiment) can be replaced by fragments of a different functional
domain F, resulting in a different biological effect upon
complementation of the two fragments. By using fragments of anti
CD56, anti CD1a, or anti CD16a, natural killer cells can be
recruited and activated. By using fragments of anti CD 16, natural
killer cells, neutrophil polymorphonuclear leukocytes, monocytes
and macrophages can be recruited and activated. By using fragments
of anti CD32a, anti CD32b, anti CD89, anti CD16a, or anti CD64,
macrophages can be recruited and activated. By using fragments of
anti CD32a, anti CD32b, anti CD64, or anti CD89, monocytes can be
recruited and activated. By using fragments of anti CD16b, anti
CD89, anti CD32a, anti CD32b, or anti CD64, granulocytes can be
recruited and activated. Moreover, alternatively to anti CD3, T
cells can also be recruited and activated by using fragments of
anti CD2, anti CD5, anti CD28, or anti TCR (T cell receptor).
Further information or additional options regarding the recruitment
and activation of effector cells through antibody binding are
available from the published literature, e.g. "Bispecific
Antibodies" by Roland E. Kontermann (editor), Springer Berlin
Heidelberg; 1st Edition. (2011).
[0397] An additional option is to use a set of polypeptides P1 and
P2 with fragments F1 and F2 of a functional domain F that binds an
antigen on an effector cell upon complementation of the two
fragments, but wherein binding to this antigen of the effector cell
does not cause activation of said effector cell. This set of
polypeptides ("first set of polypeptides") is then used (e.g.
administered to a patient) in combination with a second set of
polypeptides with fragments of a functional domain F that upon
complementation binds to a second, different antigen on the same
effector cell, but wherein again binding to this antigen of the
effector cell does not cause activation of the effector cell. The
antigens to which the first and the second sets of polypeptides
bind are chosen in a way that, while binding of only one of the two
antigens on the effector cells does not result in activation of the
effector cell, binding of both antigens on the effector cell
simultaneously leads to activation of the effector cell. This has
the advantages that (1) antigens on effector cells can be used that
do not function individually, but require costimulation of a second
antigen, and (2) the number of different antigens that dictates the
specificity with which a certain cell (such as a cancer cell) is
differentiated from other cells can be increased from two (if the
first and second set of polypeptides have the same targeting
moieties T1 and T2, respectively) to up to four different antigens
(if the first and second set of polypeptides have no targeting
moiety in common).
[0398] Similar effects may be achieved with two sets of
polypeptides with different targeting moieties, but the same
functional domain: These sets of polypeptides are designed to have
a functional domain directed against an effector cell antigen that
normally allows each set of polypeptides by itself to activate the
effector cell. However, both sets of polypeptides are used in a
concentration that is just too low to cause efficient effector cell
activation. If both sets of polypeptides are present simultaneously
(e.g. upon simultaneous administration to a patient) each set of
polypeptides by itself is not capable of activating the effector
cell (due to its low concentration), while the combination of both
sets of polypeptides is (because the effects of the two sets of
polypeptides act synergistically and thus the sum of the effects
caused by the two sets of polypeptides is sufficient to activate
the effector cell).
[0399] As another alternative to recruitment/activation of effector
cells, a "pretargeting" approach can be pursued, as it is well
established for bispecific antibody constructs (Cancer Imaging and
Therapy with Bispecific Antibody Pretargeting. Goldenberg D M,
Chatal J F, Barbet J, Boerman O, Sharkey R M. Update Cancer Ther.
2007 March; 2(1):19-31). To this end, F1 and F2 are substituted by
V.sub.H and V.sub.L fragments of an antibody specific for an
antigen, a carrier molecule (i.e. a molecule/part of a molecule
that is not recognized as foreign by the immune system of the
patient to whom said set of polypeptides is administered or a
molecule that causes no or only a weak immune reaction by a patient
to whom it is administered) or an affinity tag. Subsequently (or
simultaneously) to administering the polypeptides P1 and P2, a
therapeutic or diagnostic compound coupled to said antigen, carrier
molecule or affinity tag is administered. Only cells which carry
both the antigens A1 and A2 at their surface are bound by both
polypeptide P1 and polypeptide P2. Consequently, only at these
cells functional complementation leads to generation of a binding
site capable of recruiting the therapeutic or diagnostic compound
through said antigen, carrier molecule or affinity tag. This
approach allows exclusive addressing of target cells combined with
the possibility of precise administration and dosing of therapeutic
compounds like toxins or radioactive substances or diagnostic
compounds, while cells that do not express the antigens or do
express only one of the antigens are not affected.
[0400] A suitable carrier molecule may for example be a peptide or
a carbohydrate molecule. Preferably, the carrier molecule may be
gelatine, dextrane, or hydroxyethyl starch, which are common plasma
expanders that are metabolically inert, remain in the blood and
are, if they are small enough, renally eliminated. Alternatively,
the carrier molecule may be inulin, a metabolically inert molecule
that is used routinely in the clinic for determination of
glomerular clearance (and, in addition, antibodies exist that
specifically recognize inulin).
[0401] A suitable affinity tag may be, for example, a Flag-tag, a
myc-tag, a glutathione-S-transferase(GST)-tag, a
hemagglutinin(HA)-Tag, a polyhistidine(His)-tag, or a maltose
binding protein(MBP)-tag, a digoxigenin (DIG)-tag.
[0402] The therapeutic compound coupled to the antigen, carrier
molecule or affinity tag may for example be a radioactive compound
or a toxin.
[0403] Suitable radioactive compounds are for example compounds
comprising .sup.90Y, .sup.177Lu, .sup.131I, .sup.32P, .sup.10B, or
.sup.213Bi. Recruitment of the antigen, carrier molecule or
affinity tag linked to the radioactive compound to cells that
express both the first and the second antigen leads to accumulation
of radioactivity onto the tumour site, resulting in specific
destruction of tumour cells/tissue.
[0404] Alternatively, the therapeutic compound coupled to the
antigen, carrier molecule or affinity tag may for example be a
toxic compound that is not able to cross the cell membrane without
prior binding to the cell surface.
[0405] This prerequisite is fulfilled by the A components of
classical AB-toxins derived from a number of pathogenic bacteria
like Clostridium perfringens, C. botulinum, C. difficile, B.
anthracis and others. AB-toxins are two-component protein complexes
that interfere with internal cell functions. The A component is the
"active" component (i.e. it kills a cell upon membrane
penetration), but is not able to cross the cell membrane on its
own. The B component is the "binding" component that by itself is
non-toxic, but is essential for uptake and membrane penetration of
component A.
[0406] For example, Bacillus anthracis protective antigen (PA) is a
classical toxin B component which mediates the uptake of the actual
anthrax exotoxins edema factor and lethal factor (LF). LF without
the PA-component is non-toxic since LF by its own does not
penetrate membranes and thus cannot execute its pathogenic
capabilities (Pezard C, Berche P, Mock M. "Contribution of
individual toxin components to virulence of Bacillus anthracis"
1991 Infect. Immun. 59 (10): 3472). However, when bound to cell
surface molecules, LF is internalised and highly toxic to the
cell.
[0407] Upon dimerization of the polypeptides P1 and P2, the
function of the functional domain F is reconstituted. Through
interaction of the reconstituted functional domain with the
antigen, carrier molecule or affinity tag coupled to the toxin, the
toxin is recruited to the cell membrane of the target cells,
incorporated into the cells and kills the cells. This principle is
easily adapted to the purposes of the invention by the skilled
person, since it is already widely used in so called immunotoxins,
where a targeting moiety, mostly an antibody-like domain or natural
ligand, is coupled to the toxin component (see, e.g., Immunotoxins
for targeted cancer therapy. Kreitman R J, AAPS J. 2006 Aug. 18;
8(3):E532-51). Examples include immunotoxins based on diphtheria
toxin (such as Denileukin diftitox (U.S. trade name Ontak) which
has been approved by FDA for the treatment of some T cell
lymphomas) or based on B. anthracis Lethal Factor (Pastan I, Hassan
R, FitzGerald D J, Kreitman RJ (2007). "Immunotoxin treatment of
cancer". Annu. Rev. Med. 58: 221-37).
[0408] Suitable A components of AB-toxins may for example be B.
anthracis edema factor, B. anthracis lethal factor, C. perfringens
iota toxin, C. botulinum C2 toxin, C. difficile
ADP-ribosyltransferase C. diphtheriae diphteria toxin fragment
A.
[0409] Alternatively, the therapeutic compound may for example be a
cytotoxic compound that is toxic upon entry into a cell and that is
capable of crossing the cell membrane by itself without prior
binding to the cell surface. In this case, the antigen, carrier
molecule or affinity tag that the therapeutic compound is coupled
to is selected such that it prevents the resulting conjugate (i.e.
the therapeutic compound linked to the antigen/carrier
molecule/affinity tag) from crossing cell membranes and entering
cells without prior binding of the conjugate to the cell surface (a
suitable carrier molecule may for example be a hydroxyethyl starch
carrier). Thus, such a conjugate does not enter cells without prior
binding to their cell surface; once such a conjugate binds to the
cell surface, however, it is internalized into the cell and the
toxic compound kills the cell. The conjugate does not bind to
cells, unless it is recruited in the presence of the inventive set
of polypeptides to cells that simultaneously express both antigens
A1 and A2 at their cell surface. Such cells bind and recruit both
polypeptides P1 and P2, and the reconstituted functional domain
specifically binds to and recruits the antigen/carrier
molecule/affinity tag which, in turn, results in internalization of
the therapeutic compound. In consequence, a specific killing of
cells that carry both antigens A1 and A2 at their cell surface is
accomplished. Cytotoxic compounds that may be used in this context
include e.g. auristatin, ricin, saponin, bryodin 1, bouganin,
gelonin, pokeweed antiviral protein (PAP), antifolates, vinca
alkaloides, anthracyclines, calicheamicin, ribonuclease, abrin,
modeccin, or Listeriolysin O.
[0410] The diagnostic compound coupled to the antigen, carrier
molecule or affinity tag may for example be a radioactive compound,
a fluorophore, or a compound capable of mediating
bioluminescence.
[0411] Suitable radioactive compounds are for example compounds
comprising .sup.99mTc, .sup.111In, .sup.82Rb or .sup.201Tl. Such
compounds are detected by well-known medical imaging procedures in
the clinic.
[0412] Alternatively, a fluorescent compound may be used as
diagnostic compound, such as GFP (green fluorescent protein) or a
GFP variant (e.g. BFP (blue fluorescent protein), CFP (cyan
fluorescent protein), or YFP (yellow fluorescent protein)), or a
fluorescent small-molecule compound like FITC (fluorescein
isothiocyanate) or PE (phycoerythrin), alexa fluor dyes (such as
AlexaFluor488 and related dyes sold by Molecular Probes, e.g.) or
cyanine dyes (such as Cy3 (Indocarbocyanine) or Cy5
(Indodicarbocyanine) or related dyes). Alternatively, a compound
capable of mediating bioluminescence may be used as diagnostic
compound, such as a luciferase, for example Gaussia luciferase
(Chopra A. Gaussia princeps luciferase. In: Molecular Imaging and
Contrast Agent Database (MICAD) [database online]. Bethesda (MD):
National Library of Medicine (US), NCBI; 2004-2012. Available from:
http://micad.nih.gov.). The employment of Gaussia luciferase for in
vivo imaging is well established (see, e.g., Santos E B et al.
Sensitive in vivo imaging of T cells using a membrane-bound Gaussia
princeps luciferase. Nat Med. 2009 March; 15(3):338-44. Epub 2009
Feb. 15; or Inoue Y et al. Gaussia luciferase for bioluminescence
tumor monitoring in comparison with firefly luciferase. Mol
Imaging. 2011 Oct. 1; 10(5):377-85. doi: 10.2310/7290.2010.00057.
Epub 2011 Apr. 26; see also below for additional details).
[0413] Moreover, the fragments F1 and F2 (i.e. the V.sub.L and
V.sub.H fragments of anti CD3 Fv in the above-described exemplary
embodiment) can be replaced by V.sub.L and V.sub.H fragments of an
antibody that is specific for a therapeutic or diagnostic compound
(i.e. in this case the functional domain F is capable of directly
binding to the therapeutic or diagnostic compound). Here, the same
therapeutic and diagnostic compounds as described above in the
context of the "pretargeting" approach may be considered.
[0414] Furthermore, the fragments F1 and F2 (i.e. the V.sub.L and
V.sub.H fragments of anti CD3 Fv in the above-described exemplary
embodiment) can be replaced by fragments of a fluorescent or
bioluminescent compound that are biologically inactive on their
own, but regain their function (i.e. their ability to mediate
fluorescence or bioluminescence) upon association of the two
fragments and functional complementation, thus allowing for
specific identification of cells that carry both the antigens A1
and A2.
[0415] A number of fluorescent molecules that may be used in this
context are well known and characterized in the art including, but
are not limited to, GFP (green fluorescent protein), GFP
derivatives (like YFP (yellow fluorescent protein) and CFP (cyan
fluorescent protein), Venus (Nagai T et al., A variant of yellow
fluorescent protein with fast and efficient maturation for
cell-biological applications. Nat Biotechnol. 2002 January;
20(1):87-90), or Cerulean (Enhanced CFP with S72A, Y145A and H148D
substitutions)). For these molecules, split fragments are described
that self-assemble in the situation of close proximity in a process
called bimolecular fluorescence complementation (BiFC).
[0416] For example, GFP, CFP, Venus, Venus with a M153T
substitution, or Cerulean may be split after amino acid 155 (i.e.,
for example, fragment F1 may comprise amino acids 1-155 of GFP,
while fragment F2 may comprise amino acids 156-245 of GFP, or vice
versa). Alternatively, YFP or Venus may be split after amino acid
173. Further details on split GFP and split GFP variants can be
found in Kerppola TK., Visualization of molecular interactions
using bimolecular fluorescence complementation analysis:
characteristics of protein fragment complementation. Chem Soc Rev.
2009; 38:2876-86.
[0417] An example for a molecule that mediates bioluminescence and
that can be used in this context is split luciferase. Particularly
suited is the luciferase of Gaussia princeps, which requires no
cofactors to be active and catalyzes the oxidation of the substrate
coelenterate luciferin (coelenterazine) in a reaction that emits
blue light, or derivatives of Gaussia luciferase (Remy I and
Michnick S, A highly sensitive protein-protein interaction assay
based on Gaussia luciferase. Nature Methods--3, 977-979 (2006)).
For example, fragment F1 may comprise a fragment from the
N-terminus of Gaussia luciferase to Gly-93, while fragment F2 may
comprise a fragment from Glu-94 to the C-terminus of Gaussia
luciferase, or vice versa (see Remy I and Michnick S, Nature
Methods, 2006 for details). Application of the Gaussia split
luciferase system in vivo has been established (Luker et al., In
vivo imaging of ligand receptor binding with Gaussia luciferase
complementation. Nature Medicine 2011, doi:10.1038/nm.2590),
allowing for straightforward adaptation to the purposes of the
present invention by a skilled person.
[0418] Intravital imaging of tumour lesions is of eminent
importance in cases, where cancer cells infiltrate tissues and the
complete elimination of all transformed cells is prerequisite for
cure. A surgeon searching for disseminated cancer cells in the
operation site may use split GFP or split GFP derivatives fused to
the targeting moieties and a laser assisted Multispectral
fluorescence camera system for detection of cells aberrantly
expressing an addressed antigen profile, similar to the
intraoperative use of fluorescence or bioluminescence that is
already exploited in some clinical settings (van Dam G M et al.,
Intraoperative tumor-specific fluorescence imaging in ovarian
cancer by folate receptor-.alpha. targeting: first in-human
results. Nat Med. 2011 Sep. 18; 17(10):1315-9; Luker et al., In
vivo imaging of ligand receptor binding with Gaussia luciferase
complementation. Nature Medicine 2011, doi:10.1038/nm.2590).
[0419] For detection of complemented split luciferase, the
application of a substrate for luciferase, which can be luciferin
or coelenterazine, is mandatory. Coelenterazine is preferred
because coelenterazine emits light independent of ATP and is well
established for in vivo imaging and in vivo applications. A surgeon
will be able to visualize cancer cells after having tagged the
tumor with polypeptide P1 and P2 and injected a non-toxic amount of
coelenterazine intravenously.
[0420] In another exemplary embodiment, the inventive principle is
applied in the context of a patient who suffers from a
haematopoietic tumour and who received a transplantation of healthy
haematopoietic cells from another person (the donor). Here, the set
of polypeptides according to the invention can be used for the
specific elimination (or detection) of remaining malignant
haematopoietic cells of the recipient after transplantation of
healthy haematopoietic cells from the donor.
[0421] To destroy the malignant haematopoietic cells in a patient
suffering from a haematopoietic tumour, the patient may be
subjected to chemotherapy and/or radiation therapy. Subsequently,
the patient receives a transplantation of healthy haematopoietic
cells from a donor.
[0422] To minimize the risk of transplant rejection or graft versus
host disease, transplantation of tissue/cells (e.g. bone marrow)
from a donor who has the same set of MHC (major histocompatibility
complex) molecules is usually preferred. However, often no donor
with the same set of MHC molecules ("HLA-identical donor") can be
identified. Therefore, transplant grafts with one or two mismatches
in the set of MHC variants, unrelated cord blood with up to three
mismatches, or haploidentical transplantations are increasingly
employed. Accordingly, it is common that there is at least one
distinctive difference between the set of MHC molecules expressed
by the cells of the recipient and the cells of the donor.
[0423] In the transplantation according to this exemplary
embodiment of the invention, donor cells are used that are distinct
from the recipient cells with respect to at least one of their HLA
variants. This means that there is at least one "distinguishing
antigen" that is present at the cell surface of the recipient
cells, but not at the cell surface of the donor cells. For example,
the distinguishing antigen may be HLA-A2, if the patient (i.e. the
recipient) is HLA-A2 positive, while the donor is HLA-A2
negative.
[0424] Despite chemotherapy/radiation therapy, individual malignant
haematopoietic cells of the recipient may have escaped eradication.
Since the surviving malignant haematopoietic cells are recipient
cells, they carry the distinguishing antigen that differentiates
recipient cells from donor cells. At the same time, they are cells
of haematopoietic lineage origin and thus have markers of this cell
lineage, such as CD45, at their cell surface. Leukemic blasts and
other haematopoietic cells of the patient are the only cells that
simultaneously display the distinguishing antigen (here HLA-A2) and
markers of haematopoietic cell lineage (here CD45). The set of
polypeptides according to the invention exploits this fact to
specifically eliminate these cells.
[0425] To this end, the first targeting moiety T1 of the first
polypeptide P1 may be a scFv specific for the distinguishing
antigen which is present only on recipient cells (here HLA-A2). As
fragment F1 of the functional domain F, the variable region of the
light chain (V.sub.L) of a CD3.epsilon.-specific antibody may be
chosen. The second targeting moiety T2 of the second polypeptide P2
may be a single chain variable Fragment (scFv) specific for CD45.
As fragment F2 of the functional domain F, the variable region of
the heavy chain (V.sub.H) of said CD3.epsilon.-specific antibody
may be chosen. (Naturally, it is equally possible to use the
variable region of the heavy chain (V.sub.H) of a
CD3.epsilon.-specific antibody as fragment F1 and the variable
region of the light chain (V.sub.L) of said CD3.epsilon.-specific
antibody as fragment F2. As is evident for a person of skill in the
art, this is a general principle, and it is generally possible to
switch the fragments used for fragment F1 and fragment F2.) Neither
is V.sub.L of the CD3.epsilon.-specific antibody capable of
engaging CD3.epsilon. in the absence of V.sub.H, nor is V.sub.H of
the CD3.epsilon.-specific antibody capable of engaging CD3.epsilon.
in the absence of V.sub.L. Accordingly, neither P1 nor P2 is by
itself capable of binding to CD3.epsilon..
[0426] However, if both the distinguishing antigen (e.g. HLA-A2)
and the CD45 antigen are present on one single cell, binding to
their respective antigens brings the two polypeptides P1 and P2
into close proximity. As a consequence, the unpaired V.sub.H and
V.sub.L domains assemble, resulting in heterodimerization of the
polypeptides P1 and P2 and in the formation of a functional
variable antibody fragment Fv from the V.sub.H and V.sub.L domains
that is capable of binding to CD3.epsilon. (see FIG. 2).
[0427] As a result, T cells are recruited and activated through
CD3.epsilon., and the cell carrying both HLA-A2 and CD45 at its
cell surface is specifically eliminated by a cytotoxic T cell
response.
[0428] A person of skill in the art understands that, within the
principle of the present invention, diverse variations to this
exemplary embodiment are possible.
[0429] For example, in polypeptide P2 the scFv fragment recognising
the haematopoietic cell lineage marker CD45 can be replaced by a
scFv fragment recognising a marker of a different cell lineage or
cell type, i.e. the targeting moiety T2 may be a domain that
specifically binds an antigen that is specific for a cell lineage
other than the haematopoietic cell lineage or for a certain cell
type (for a detailed list of various cell lineage markers and cell
type markers that may be used in this context see David J. Dabbs,
Diagnostic immunohistochemistry, Churchill Livingstone, 3rd edition
(2010); or F Lin and J Prichard, Handbook of Practical
Immunohistochemistry: Frequently Asked Questions, Springer, New
York, 1st edition (2011)). To adapt the set of polypeptides to an
alternative cell lineage marker/cell type marker, it is sufficient
to replace the targeting moiety T2 of polypeptide P2 with a
targeting moiety that has binding specificity for the desired
alternative cell lineage marker/cell type marker.
[0430] For example, in the situation of metastatic renal cell
carcinoma (RCC), a person skilled in the art might consult the
above-cited databases for information on cell surface proteins with
restricted expression to kidney cells. Among many other molecules,
he will learn that expression of certain members of the aquaporin
family is confined to kidney cells and erythrocytes. Having
obtained this information, a person skilled in the art will
construct a polypeptide P2 recognising an aquaporin family member
that is confined to kidney cells and erythrocytes fused to the
variable region of the heavy chain (V.sub.H) of a
CD3.epsilon.-specific antibody. In case that the patient suffering
renal cell carcinoma is HLA A2 positive and a kidney transplant
from a healthy donor is HLA A2 negative, the clinician treating the
patient may utilise the two constructs (anti-aquaporin fused to
anti-CD3(V.sub.H) and anti-HLA A2 fused to the light chain
(V.sub.L) of said CD3.epsilon.-specific antibody). In this case,
all cells simultaneously expressing said aquaporin and HLA A2 will
be tagged for lysis by T cells which are renal cell carcinoma cells
and metastatic tissues. Kidney cells donated by the healthy donor
are HLA A2 negative and will not be attacked. Since erythrocytes
loose HLA expression along the process of ontogeny and thus do not
carry HLA molecules on their surfaces, they will be spared despite
expressing large amounts of aquoporins. Again, a conventional,
non-complementing bispecific antibody addressing aquaporin would
mediate killing of all kidney cells from donor and recipient as
well as erythrocytes. A bispecific antibody addressing HLA A2 in a
HLA A2 positive patient most likely would be fatal, since every
recipient cell except erythrocytes express HLA A2 and can be
attacked by the retargeted T cells.
[0431] Another example is hepatocellular carcinoma (HCC).
Hepatocytes are largely involved in a number of metabolic processes
including the trafficking of lipoproteins. To this end, hepatocytes
express receptors for high density lipoproteins (HDL) on their
surfaces (scavenger receptor class B member 1, SCARB1). Treatment
of an HLA A2 positive patient suffering HCC which expresses SCARB1
on the surface of tumor cells and metastases can be accomplished by
a Polypeptide P2 construct comprising a scFv domain addressing
SCARB1 fused to the variable region of the heavy chain (V.sub.H) of
said CD3.epsilon.-specific antibody and a Polypeptide P1 (anti-HLA
A2 scFv fused to the light chain (V.sub.L) of said
CD3.epsilon.-specific antibody) and transplantation of liver cells
from a healthy, HLA A2 negative donor. In this case, all
hepatocytes and hepatocyte-derived malignant cells expressing both,
SCARB1 and HLA A2 will be tagged for lysis by T lymphocytes.
Hepatocytes of the donor lacking HLA A2 will be spared as well as
normal SCARB1 negative donor cells expressing HLA A2. Since SCARB1
expression is also reported for cells participating in steroid
synthesis in the adrenal gland, these cells most likely will also
be destroyed by redirected T cells, resulting in Addison's
disease.
[0432] Various markers that are specific for certain cell types or
cell lineages or a few cell types/lineages are known (for a list of
examples, see above). More information on lineage markers,
differentiation antigens and tissue markers as well as their tissue
distribution are easily accessible from published sources (see,
e.g. David J. Dabbs, Diagnostic immunohistochemistry, Churchill
Livingstone, 3rd edition (2010); or F Lin and J Prichard, Handbook
of Practical Immunohistochemistry: Frequently Asked Questions,
Springer, New York, 1st edition (2011)) and public databases (such
as the Gene Expression Atlas of the European Bioinformatics
Institute (EBI), http://www.ebi.ac.uk/gxa/; or the Gene Expression
Omnibus (GEO) platform, see above). Moreover, such markers can be
identified and/or verified in a straightforward manner by a skilled
person using similar methods as described above for the
identification of tumour-specific combinations of antigens.
[0433] In certain preferred embodiments, an antigen with less than
perfect specificity for a certain cell type or cell lineage is used
(i.e. an antigen is used that is present on more than one, but
preferably only a few, cell types or cell lineages). In some
embodiments, an antigen is used that is expressed by said cell
type/cell lineage at a higher rate or at a higher proportion or
amount than by other cell types/cell lineages, in the sense that
there may be a small but detectable expression of said antigen also
in other cell types/cell lineages.
[0434] The concept can further be adapted to any other HLA
haplotype besides HLA-A2 used in the exemplary embodiment above, as
long as the recipient cells are positive for this HLA antigen and
the donor cells are negative for it. Possible HLA antigens include,
among others, HLA A1, HLA A2, HLA A3, HLA A25, HLA B7, HLA B8, HLA
B35, HLA B44 and HLA Cw3, HLA Cw4, HLA Cw6, HLA Cw7. To adapt the
set of polypeptides to an alternative HLA antigen, it is sufficient
to replace the targeting moiety T1 of polypeptide P1 with a
targeting moiety that has binding specificity for the desired
alternative HLA antigen. By an appropriate choice of the targeting
moiety T1, it is of course also possible to specifically eliminate
donor cells.
[0435] Moreover, instead of a V.sub.L domain and a V.sub.H domain
that upon assembly form a domain capable of binding to CD3.epsilon.
(i.e. fragment F1 and fragment F2 of polypeptides P1 and P2,
respectively), the V.sub.L domain and V.sub.H domain can be
replaced with domains/fragments that upon assembly confer a
different function to the resulting dimer. In this respect, all the
variations described above for the exemplary embodiment relating to
the elimination/detection of tumour cells identified by a specific
combination of two cell surface antigens are equally applicable.
For example, upon assembly the complemented functional domain may
mediate binding/activation of other effector cells than T cells,
may be adapted to a "pretargeting" approach, may bind a therapeutic
or diagnostic compound, or may form a fluorescent molecule/molecule
capable of mediating bioluminescence.
[0436] The diverse options for the choice of the fragments F1 and
F2 and for the choice of the targeting moieties T1 or T2 described
above in the exemplary embodiment relating to application of the
inventive principle for the specific elimination of tumour cells
may of course be considered, as well.
[0437] From the described exemplary embodiments and variations, it
will be clear to a person of skill in the art that the inventive
principle described above can not only be used for the highly
specific identification/elimination of tumour cells or of remaining
malignant recipient cells after a cell transplantation, but also
for the identification/elimination of any other type of cell
carrying a specific combination of two antigens that distinguishes
it from other types of cells.
[0438] In the following, reference is made to the figures:
[0439] FIG. 1 shows the principle of the invention. FIG. 1A:
Antigens and design of polypeptides P1 and P2. FIG. 1B: If a cell
expresses both antigens 1 and 2 at its cell surface, simultaneous
binding of polypeptide P1 and polypeptide P2 to the surface of this
cell brings P1 and P2 in close proximity, causes association of
fragments F1 and F2 and restoration of the biological function of
domain F by complementation. No restoration of biological function
occurs if only antigen A1 (FIG. 1C) or antigen A2 (FIG. 1D) is
present on the cell surface.
[0440] FIG. 2 shows an exemplary embodiment of the invention in an
allogeneic transplantation setting for haematopoietic neoplasias
with mismatched HLA antigens. In this situation, the dual
information of recipient HLA haplotype (HLA.sub.patient) and
haematopoietic lineage origin (CD45) is displayed exclusively on
leukemic blasts and other haematopoietic cells of the patient. The
first polypeptide P1 comprises a single-chain variable fragment
antibody construct directed against the HLA of the patient
(targeting moiety T1) fused to the V.sub.L fragment of anti CD3
(fragment F1). The second polypeptide P2 comprises a single-chain
variable fragment construct specific for the haematopoietic lineage
marker CD45 (targeting moiety T2), fused to the V.sub.H
split-fragment of anti CD3 Fv (fragment F2).
[0441] CD45: antigen specific for haematopoietic cells.
HLA.sub.patient: HLA-antigen specific for patient cells, i.e. an
allelic variant of the human MHC that is present on the surface of
patient cells (=cells of the recipient of cell transplantation),
but absent from the surface of donor cells. .alpha.CD45 scFv: scFv
with binding specificity for CD45. .alpha.HLA.sub.patient scFv:
scFV with binding specificity for HLA.sub.patient. CD3(V.sub.H):
variable region of an immunoglobulin heavy chain of an antibody
with binding specificity for CD3. CD3(V.sub.L): variable region of
an immunoglobulin light chain of an antibody with binding
specificity for CD3.
[0442] Upon binding of the two constructs through their .alpha.CD45
scFv and .alpha.HLA.sub.patient scFv, respectively, to a cell
carrying both the CD45 and the HLA.sub.patient antigen, assembly of
CD3(V.sub.H) with CD3(V.sub.L) leads to functional complementation
of the antibody with binding specificity for CD3, thus allowing for
specific recruitment and activation of T cells through the CD3
molecules at their cell surface.
[0443] FIG. 3 shows the constructs used in the experiments depicted
in FIGS. 4-9. (Construct 85 differs from construct 71 by the fact
that construct 85 has a Flag tag while construct 71 has a myc tag.
Construct 75 differs from construct 82 by the fact that construct
75 has a Flag tag while construct 82 has a myc tag.) V.sub.HCD3:
variable region of the heavy chain of an anti-CD3 antibody;
V.sub.LCD3: variable region of the light chain of an anti-CD3
antibody; V.sub.HA2: variable region of the heavy chain of an
anti-HLA-A2 antibody; V.sub.LA2: variable region of the light chain
of an anti-HLA-A2 antibody; V.sub.L45: variable region of the heavy
chain of an anti-CD45 antibody; V.sub.H45: variable region of the
light chain of an anti-CD45 antibody; L18, L7, L15, L6, L19: linker
of 18, 7, 15, 6, 19 amino acids, respectively.
[0444] FIG. 4 shows conventional tandem bispecific single chain
scFv constructs used to control the assay system. Briefly,
bispecific antibody constructs with specificity for CD3 and HLA A2
were titrated as indicated to a co-culture of U266, a HLA A2
positive, CD45 positive myeloma cell line, and HLA A2 negative T
cells (monocyte depleted peripheral blood mononuclear cells), and
production of interleukin 2 by T cells was determined. Substantial
T cell stimulatory capacity was detected for the two FvCD3-HLA-A2
constructs 85 and 71, which differ by their respective Flag or
Myc-Tags (For domain structure of constructs see FIG. 3.).
Bispecific tandem Fv constructs in HLA-A2-CD3 configuration were
less efficient and single chain constructs addressing either HLA-A2
or CD3 did not stimulate T cells at all. Positive control is
conducted using unspecific PHA-L (phytohemagglutinin)
stimulation.
[0445] FIG. 5 shows exquisite and highly specific T cell
stimulatory capacity if a pair of complementing constructs
according to the invention is used, but not if only one of the two
constructs of a pair is used individually. Briefly,
V.sub.LCD3-scFvHLA-A2 (construct 42),
V.sub.HCD3-scFvCD45(V.sub.L-V.sub.H) (construct 45) and
V.sub.HCD3-scFvCD45(V.sub.H-V.sub.L) (construct 55) were titrated
separately or in the combinations of constructs 42 and 45, or 42
and 55 to co-cultures of U266 and T cells as described. High T cell
stimulatory capacity was demonstrated for the combinations of 42/45
or 42/55 with minute activity, if only one of these constructs was
given separately. These results show that the V.sub.L and V.sub.H
domains of FvCD3 have to cooperate in order to reconstitute or
complement T cell engaging function. Importantly, the scFvCD45
targeting moiety could be switched from (V.sub.L-V.sub.H) to the
(V.sub.H-V.sub.L) configuration, clearly indicating that the
modular character of the constructs allows replacement of a
targeting moiety by another targeting moiety with desired
specificity. The assay system was controlled by the use of single
chain constructs CD45(V.sub.L-V.sub.H) and CD45(V.sub.H-V.sub.L)
which did not stimulate T cells to produce IL2.
[0446] FIG. 6 shows a first of three competitive blocking
experiments. The bispecific tandem construct FvCD3-HLA-A2
(construct 71) was given to co-cultures of U266 and T cells as
described and stimulatory function was determined through induced
IL2 production by T lymphocytes. The T cell stimulating function
was blocked by single chain constructs that occupy the targeted
epitope on the HLA A2 molecule (construct 4, concentration*100).
Intrinsic stimulation of T cells by the HLA A2 or CD3 specific
single chain constructs (construct 4 (concentration*100) or
construct 36 (concentration*9)) was ruled out. PHA-L was used as
positive control.
[0447] FIG. 7 shows that "tridomain constructs" (i.e. constructs
according to the invention) first have to bind on the surface of a
single cell to dimerize and complement T cell engaging functions
the competitive epitope blocking experiments. Briefly, constructs
42 and 45 were given to co-cultures of U266 cells and HLA-A2
negative T lymphocytes and stimulatory capacity was determined by
IL2 production of T cells. In experimental situations where the
epitopes on HLA A2 or CD45 molecules were competitively blocked by
constructs 4 or 46 (both concentrations*100), T cell stimulatory
function was abrogated. These results clearly indicate that the two
respective "tridomain constructs" have to bind simultaneously onto
the surface of a cell in order to restore or to complement T cell
engaging function. Intrinsic stimulatory activity of either
construct (42, 45, 4, 46 and 36) was ruled out using different
concentrations.
[0448] FIG. 8 shows the analogous experiment to FIG. 7 for the
combination of constructs 42 and 55. Again, T cell stimulatory
capacity of the combination of the two "tridomain constructs" was
abrogated by competitive blocking of antigenic epitopes on the HLA
A2 or the CD45 molecule. Importantly, these results again show that
the targeting module can be easily replaced by another module with
appropriate specificity. More importantly, the
V.sub.L-V.sub.H-V.sub.L configuration of construct 42 and the
V.sub.H-V.sub.H-V.sub.L configuration of construct 55 impede homo-
or hetero-dimerization or self-assembling of the constructs without
prior binding to a substrate expressing both, HLA A2 and CD45
antigens.
[0449] FIG. 9 shows lysis of U266 cells by HLA A2 negative T cells
in a sample comprising both V.sub.LCD3-scFvHLA-A2 and
V.sub.HCD3-scFvCD45(V.sub.H-V.sub.L) constructs ("both
constructs"). No significant lysis was observed in control samples
comprising only one of the two constructs.
[0450] FIG. 10 shows the On-target restoration of the polypeptides.
Binding of two separate polypeptides (P1 and P2) to their
respective antigens on a target cell, each consisting of a specific
single-chain variable antibody fragment (scFv, V.sub.H-V.sub.L)
fused to the variable light (V.sub.L) or variable heavy chain
domain (V.sub.H) of a CD3-specific antibody (Fragment F1 and F2),
enables V.sub.H/V.sub.L heterodimerization and the formation of a
functional CD3 binding site to engage T cells.
[0451] FIG. 11 shows that CD3 V.sub.H/V.sub.L dimerization engages
T cells and is dual-antigen-restricted. U266 myeloma, primary T
cell pro-lymphocytic leukemia (T-PLL), and THP-1 acute myeloid
leukemia cells, all HLA-A2-positive and CD45-positive, were probed
with HLA-A2-negative donor peripheral blood mononuclear cells
(PBMC) and the polypeptides as indicated. T-cell engagement was
assessed by reactive interleukin-2 (IL-2) production (A) and target
cell lysis (B). The bispecific tandem scFv
(CD3(V.sub.H-V.sub.L)-HLA-A2(V.sub.H-V.sub.L) antibody was used as
a positive control. (C), Binding of the polypeptides on THP-1 cells
is competitively blocked by an excess of scFvCD45 (left) and
scFvHLA-A2 (right) inhibitors (blocking the individual antigen
epitopes on the target cell), as indicated, and reactive IL2
production by donor PBMCs was investigated. (D), The single or
double antigen negative cell lines RAJI and KMS-12-BM were probed
with the polypeptides. PHA-L was used as a nonspecific stimulus
control for PBMCs.
[0452] FIG. 12 shows targeted therapy by conditional
CD3V.sub.H/V.sub.L complementation in vivo. (A), Survival of mice
(n=6 per group) after intraperitoneal injection of 5.times.10.sup.6
THP-1 acute leukemic cells together with 1.25.times.10.sup.5
CMV-specific, HLA-A2-negative donor T cells and the polypeptides
(0.5 .mu.g) as indicated (tumor cells:T-cell ratio=40/1). (B),
Caspase 3 activation was assessed in vitro by flow cytometry in
HLA-A2/CD45 double-positive THP-1 and CD45-positive but
HLA-A2-negative bystander cells after co-culture with donor T cells
and the polypeptides (3 nM) as indicated. The bispecific tandem
scFv (CD3(V.sub.H-V.sub.L)-HLA-A2(V.sub.H-V.sub.L)) antibody was
used as a positive control.
[0453] FIG. 13 shows that EGFR- and EpCAM-directed polypeptides
engage T cells for carcinoma cell destruction. EGFR and EpCAM
double-positive human colon cancer cell line COLO-206F and melanoma
cell line FM-55 (EGFR-positive but EpCAM-negative) were probed with
PBMCs in the presence of polypeptides specific for EGFR
(CD3(V.sub.H)-EGFR(V.sub.H-V.sub.L)) and EpCAM
(CD3(V.sub.L)-EpCAM(V.sub.H-V.sub.L)) as indicated. T cell
engagement was assessed by reactive interferon-.gamma. (IFN.gamma.)
production (A) and activation of caspase 3 in target cells (B).
[0454] FIG. 14 shows that HLA-A2 and CEA directed polypeptides
redirect T cells for tumor cell destruction. Human colon cancer
cell line COLO-206F, melanoma cell line FM-55 and ovarian cancer
cell line OVCAR were probed with PBMCs in the presence of
polypeptides specific for HLA-A2
(CD3(V.sub.L)-HLA-A2(V.sub.H-V.sub.L)) and CEA
(CD3(V.sub.H)-CEA(V.sub.H-V.sub.L)) as indicated. T cell engagement
was assessed by reactive IFN.gamma. production. Samples were run
and analyzed as duplicates.
[0455] FIG. 15 shows that HLA-A2 and EGFR directed polypeptides
redirect T cells for tumor cell destruction. Human cell lines
COLO-206F, FM-55 and OVCAR were probed with PBMCs in the presence
of polypeptides specific for HLA-A2
(CD3(V.sub.L)-HLA-A2(V.sub.H-V.sub.L)) and EGFR
(CD3(V.sub.H)-EGFR(V.sub.H-V.sub.L)) as indicated. T cell
engagement was assessed by reactive IFN.gamma. production. Samples
were run and analyzed as duplicates.
[0456] FIG. 16 shows that HLA-A2 and Her2 directed polypeptides
redirect T cells for tumor cell destruction. Human cell lines
COLO-206F, FM-55 and OVCAR were probed with PBMCs in the presence
of polypeptides specific for HLA-A2
(CD3(V.sub.L)-HLA-A2(V.sub.H-V.sub.L)) and Her2
(CD3(V.sub.H)-Her2(V.sub.H-V.sub.L)) as indicated. T cell
engagement was assessed by reactive IFN.gamma. production. Samples
were run and analyzed as duplicates.
[0457] FIG. 17 shows that CD45 and HLA-A2 directed polypeptides
redirect T cells for tumor cell destruction. In this experiment the
split antiCD3 fragments (CD3(V.sub.H) and CD3(V.sub.L)) for the
anti-CD45 and anti-HLA-A2 targeting moieties were exchanged,
compared to the CD45 and HLA-A2 polypeptides used in FIG. 5,7-9,
11,12, 14-16. Human myeloma cell line U266 was probed with PBMCs in
the presence of polypeptides specific for CD45
(CD3(V.sub.L)-CD45(V.sub.H-V.sub.L)) and HLA-A2
(CD3(V.sub.H)-HLA-A2(V.sub.H-V.sub.L)) as indicated. T cell
engagement was assessed by reactive IFN.gamma. production. Samples
were run and analyzed as duplicates.
[0458] FIG. 18 shows that EGFR and EpCAM directed polypeptides
redirect T cells for tumor cell destruction. Human colon cancer
cell lines COLO-206F and CX-1 and ovarian cancer cell line OVCAR
were probed with PBMCs in the presence of polypeptides specific for
EpCAM (CD3(V.sub.L)-EpCAM(V.sub.H-V.sub.L)) and EGFR
(CD3(V.sub.H)-EGFR(V.sub.H-V.sub.L)) as indicated. T cell
engagement was assessed by reactive IFN.gamma. production. Samples
were run and analyzed as duplicates.
[0459] FIG. 19 shows that Her2 and EpCAM directed polypeptides
redirect T cells for tumor cell destruction. Human ovarian cancer
cell line OVCAR were probed with PBMCs in the presence of
polypeptides specific for EpCAM
(CD3(V.sub.L)-EpCAM(V.sub.H-V.sub.L)) and Her2
(CD3(V.sub.H)-Her2(V.sub.H-V.sub.L)) as indicated. T cell
engagement was assessed by reactive IFN.gamma. production. Samples
were run and analyzed as duplicates.
[0460] FIG. 20 shows that CD45 and CD138 directed polypeptides
redirect T cells for tumor cell destruction. Human myeloma cell
line AMO-1 was probed with PBMCs in the presence of polypeptides
specific for CD45 (CD3(V.sub.L)-CD45(V.sub.H-V.sub.L) upper panel,
CD3(V.sub.H)-CD45(V.sub.H-V.sub.L) lower panel) and CD138
(CD3(V.sub.H)-CD138(V.sub.H-V.sub.L) upper panel,
CD3(V.sub.L)-CD138(V.sub.H-V.sub.L) lower panel) as indicated. T
cell engagement was assessed by reactive IFN.gamma. production.
Samples were run and analyzed as duplicates.
[0461] FIG. 21 shows that targeting a single antigen (CD138) with
CD138 directed polypeptides redirect T cells for tumor cell
destruction. Human myeloma cell line AMO-1 was probed with PBMCs in
the presence of polypeptides specific for CD138
(CD3(V.sub.L)-CD138(V.sub.H-V.sub.L) and
(CD3(V.sub.H)-CD138(V.sub.H-V.sub.L)) as indicated. T cell
engagement was assessed by reactive IFN.gamma. production. Samples
were run and analyzed as duplicates.
[0462] FIG. 22 shows that targeting a single antigen (CD45) with
CD45 directed polypeptides redirect T cells for tumor cell
destruction. Human myeloma cell lines AMO-1 and U266 were probed
with PBMCs in the presence of polypeptides specific for CD45
(CD3(V.sub.L)-CD45(V.sub.H-V.sub.L) and
(CD3(V.sub.H)-CD45(V.sub.H-V.sub.L)) as indicated. T cell
engagement was assessed by reactive IFN.gamma. production. Samples
were run and analyzed as duplicates.
[0463] FIG. 23 shows the On-target restoration of two polypeptides
directed against a single antigen on the cell surface, targeting
two different epitopes (upper part) or the same epitope (lower
part) on the antigen. Binding of two separate polypeptides (P1 and
P2) to their respective epitope, on the same antigen, on a target
cell. For targeting two different epitopes, the targeting moiety of
each polypeptide consists of a specific single-chain variable
antibody fragment (scFv). For targeting the same epitope, the
targeting moiety of each polypeptide consists of the same
single-chain variable antibody fragment (scFv). The targeting
moieties are fused via peptide linkers to the variable light
(V.sub.L) or variable heavy chain domain (V.sub.H) of a
CD3-specific antibody (Fragment F1 and F2), enables V.sub.H/V.sub.L
heterodimerization and the formation of a functional CD3 binding
site (functional domain) to engage T cells.
[0464] FIG. 24 shows the possibility to use different effector ways
to kill a target cell with a kit of polypeptide parts. To this end,
the anti-CD3 module (F1 and F2) is replaced by an anti-HIS
(hexa-histidine) module which, after simultaneous binding of
polypeptide 1 and 2, complements a hexa-histidine binding site and
thus binds histidine labeled payloads (eg. a HIS-tagged toxin). The
targeting moiety T1 (V.sub.H-V.sub.L) of polypeptide P1
specifically binds to HLA-A2, the targeting moiety T2
(V.sub.H-V.sub.L) of polypeptide P2 specifically binds to CD45. The
fragment F1 of polypeptide P1 comprises of a V.sub.H domain of an
antibody against a hexahistidine-tag and fragment F2 of polypeptide
P2 comprises a V.sub.L domain of the same antibody. Human myeloid
leukemia cell line THP-1 was probed with a histidine (His) tagged
Clostridium perfringens Iota toxin component Ia at 0.01 .mu.g/ml in
combination with indicated polypeptides. After 48 hours in culture
the cell viability was measured using the alamarBlue.RTM. assay.
The results show a reduction of viability against the background of
the assay for cells probed with the combination, but not with
individual polypeptides. Control THP-1 cells were grown
simultaneously in culture without toxin. Samples were run and
analyzed as duplicates.
[0465] FIG. 25 shows that HLA-A2 and CD45 directed polypeptides,
comprising of a split antibody against a His-tag, kill tumor cells
using a histidine (His) tagged Shiga toxin subunit A at a
concentration of 0.01 .mu.g/ml. The same experimental setup was
used as in figure F24.
[0466] FIG. 26 shows that HLA-A2 and CD45 directed polypeptides,
comprising of a split antibody against a His-tag, kill tumor cells
using a histidine (His) tagged Shiga toxin subunit A at a
concentration of 0.1 .mu.g/ml. The same experimental setup was used
as in figure F24/25.
[0467] FIG. 27 shows that EGFR and EpCAM directed polypeptides,
comprising of a functional domain F with F1 and F2 are V.sub.H and
H.sub.L of a antibody specific for digoxigenin (aDig), mark tumor
cells using a digoxigenin labeled horse radish peroxidase (HRP)
molecule. The targeting moiety T1 (V.sub.H-V.sub.L) of polypeptide
P1 specifically binds to EGFR, the targeting moiety T2
(V.sub.H-V.sub.L) of polypeptide P2 specifically binds to EpCAM.
The fragment F1 of polypeptide P1 comprises of a V.sub.H domain of
an antibody against digoxigenin and fragment F2 of polypeptide P2
comprises a V.sub.L domain of the same antibody. Human colon cancer
cell line Colo-206F was first probed with indicated polypeptides
followed by probing with digoxigenin labeled HRP. The samples were
analyzed using the (Invitrogen.TM., ELISA Kit) and the absorbance
was read with a BioRAD-micro plate reader. For analysis the
chromogen blank sample (no Digoxigenin-HRP) was set to 0. Samples
were run and analyzed as duplicates.
[0468] FIG. 28 shows that CD45 and HLA-CW6 directed polypeptides
redirect T cells for patient cell destruction. Primary patient
cells with known HLA-haplotypes were used. A51=cells of a patient
with MDS (myelodysplastic syndrom), homozygous for the HLA-Cw6
haplotype. A49=cells of a patient after allogeneic bone marrow
transplantation, heterozygous for the HLA-Cw6 haplotype. Patient
cells were incubated with healthy PBMCs for 30 hours, in the
presence of polypeptides specific for CD45
(CD3(V.sub.L)-CD45(V.sub.H-V.sub.L) and HLA-Cw6
(CD3(V.sub.H)-HLA-CW6(V.sub.H-V.sub.L)) as indicated. T cell
engagement was assessed by reactive IFN.gamma. production. Samples
were run and analyzed as duplicates.
[0469] FIG. 29 shows that EGFR and EpCAM directed polypeptides
redirect T cells for primary cancer patient cell destruction. A44
tumor cells were collected from the malignant ascites of a 48 years
old male patient with metastatic pancreatic cancer. Patient tumor
cells were incubated with patients own PBMCs (collected by
phlebotomy) for 30 hours, in the presence of polypeptides specific
for EpCAM (CD3(V.sub.L)-EpCAM(V.sub.H-V.sub.L) and EGFR
(CD3(V.sub.H)-EGFR(V.sub.H-V.sub.L)) as indicated. T cell
engagement was assessed by reactive IFN.gamma. production. Samples
were run and analyzed as duplicates.
[0470] FIG. 30 shows that CD45 and HLA-A2 directed polypeptides
redirect CMV restricted CD8+ T cells for tumor cell destruction.
Human tumor cells THP-1 and U266 were incubated with CMV restricted
T-cells from a HLA-A2 negative healthy donor for 30 hours, in the
presence of polypeptides specific for HLA-A2
(CD3(V.sub.L)-HLA-A2(V.sub.H-V.sub.L) and CD45
(CD3(V.sub.H)-CD45(V.sub.H-V.sub.L)) as indicated. The bispecific
tandem scFv
(CD3(V.sub.H-V.sub.L).times.HLA-A2(V.sub.H-V.sub.L))-antibody was
used as a positive control. T cell engagement was assessed by
reactive IFN.gamma. production. Samples were run and analyzed as
duplicates.
[0471] FIG. 31 shows the principle idea to eliminate autoimmune or
hypersensitivity disorder causing B-cell clones with a kit of
polypeptide parts, consisting of an allergen specific polypeptide
and a cell type specific polypeptide. The first polypeptide P1 has
at its targeting moiety an allergen (eg. Betv-1A, Der-f2,
Conglutin-7, Feld-di). The second polypeptide P2 has at its
targeting moiety a specific single-chain variable antibody fragment
(scFv, V.sub.H-V.sub.L) targeting a cell surface protein (eg. CD19,
CD138, CD38). Both targeting moieties are fused to either the
variable light (V.sub.L) or variable heavy chain domain (V.sub.H)
of a CD3-specific antibody (Fragment F1 and F2).
[0472] In the following, reference is made to certain (human) genes
or proteins also referred to in the specification, the appended
examples and figures as well as (partially) in the claims. Herein
below, corresponding (exemplary) gene accession numbers are
provided. Further accession numbers are also provided in the
specification elsewhere herein as well as the appended examples.
[0473] CD45: Gene ID: 5788, updated on 13 Jan. 2013, 3.
Protein=P08575-1=Isoform 1, Last modified Jul. 19, 2003. Version 2
[0474] CD34: Protein: P28906-1/2 Last modified Jul. 15, 1998.
Version 2. [0475] CD33: Gene ID: 945, updated on 30 Dec. 2012:
Protein: P20138 [UniParc]. Last modified Oct. 17, 2006. Version 2.
Checksum: 1C73E588240FBAD8 [0476] CD138: Gene ID: 6382, updated on
6 Jan. 2013, 4. Protein=P18827 [UniParc]. Last modified May 5,
2009. Version 3. [0477] CD15: Gene ID: 2526, updated on 5 Jan. 2013
[0478] CD1a: Gene ID: 909, updated on 30 Dec. 2012, P06126
[UniParc]. Last modified Feb. 9, 2010. Version 4. Checksum:
C575C3C538F0AA29 [0479] CD2: Gene ID: 914, updated on 5 Jan. 2013;
P06729 [UniParc]. Last modified Oct. 23, 2007. Version 2. Checksum:
A03D853C3B618917 [0480] CD3e: Gene ID: 916, updated on 5 Jan. 2013,
P07766 [UniParc]. Last modified Feb. 1, 1996. Version 2. Checksum:
A1603D010E9957D7 [0481] CD4: Gene ID: 920, updated on 13 Jan. 2013;
P01730 [UniParc]. Last modified Nov. 1, 1988. Version 1. Checksum:
20ED893F9E56D236 [0482] CD5: Gene ID: 921, updated on 30 Dec. 2012;
P06127 [UniParc]. Last modified Nov. 30, 2010. Version 2. Checksum:
9131AEC9683EE1D3 [0483] CD8a: Gene ID: 925, updated on 30 Dec.
2012; Isoform 1/2 (membrane) P01732-1/2 (mCD8alpha) [UniParc]. Last
modified Jul. 21, 1986. Version 1. Checksum: FCCA29BAA73726BB
[0484] CD20: Gene ID: 931, updated on 6 Jan. 2013; P11836
[UniParc]. Last modified Oct. 1, 1989. Version 1. Checksum:
AC5420F8B626BDD1 [0485] CD23: Gene ID: 2208, updated on 4 Jan.
2013; P06734 [UniParc]. Last modified Jan. 1, 1988. Version 1.
Checksum: F86708C0E6515B87 [0486] CD31: Gene ID: 5175, updated on
13 Jan. 2013; Isoform Long [UniParc]. Last modified Apr. 1, 1990.
Version 1. Checksum: C57BBFA200A407A6, P16284-1/2/3/4/5/6=Isoforms
1-6 [0487] CD43: Gene ID: 6693, updated on 30 Dec. 2012; P16150
[UniParc]. Last modified Apr. 1, 1990. Version 1. Checksum:
C9C9AB8435D5E1FE [0488] CD56: Gene ID: 4684, updated on 30 Dec.
2012; Isoform 1 [UniParc]. Last modified Jul. 22, 2008. Version 3.
Checksum: FD3B9DE80D802554, P13591-2/1/3/4/4/6, Isoforms 1-6 [0489]
CD57: Gene ID: 27087, updated on 5 Jan. 2013 [0490] CD68: Gene ID:
968, updated on 6 Jan. 2013; Isoform Long (CD68.1) [UniParc]. Last
modified May 15, 2007. Version 2. Checksum: 69E68D69EDE8EFB0,
P34810-1/2, Isoform 1/2 [0491] CD79a: Gene ID: 973, updated on 5
Jan. 2013; Isoform 1 (Long) [UniParc]. Last modified Jun. 1, 1994.
Version 2., Checksum: 6E5B837409969292, P11912-1/2, Isoform 1/2
[0492] CD146: Gene ID: 4162, updated on 30 Dec. 2012; Isoform 1
[UniParc]. Last modified Jan. 10, 2006. Version 2. Checksum:
E46CB8AC7BA0738E, P43121-1/2, Isoform 1/2. [0493] surfactant
proteins (A and B): [0494] Gene ID: 6440, updated on 30 Dec. 2012
and Gene ID: 6439, updated on 30 Dec. 2012, P07988 [UniParc]. Last
modified May 1, 1992. Version 3. [0495] Checksum: 9FD7F66678A35153,
and Isoform 1 [UniParc]. Last modified Apr. 1, 1990. Version 2.
Checksum: C26A21E33C60AA78, P11686-1/2, Isoform 1/2 [0496]
synaptophysin: [0497] Gene ID: 6855, updated on 30 Dec. 2012,
P08247 [UniParc]. Last modified Aug. 1, 1991. Version 3. Checksum:
592289C43B12EFA7 [0498] nicotinic acetylcholine receptors: [0499]
Gene ID: 1138, updated on 30 Dec. 2012, Gene ID: 1136, updated on 6
Jan. 2013, Gene ID: 1139, updated on 13 Jan. 2013, Gene ID: 1137,
updated on 30 Dec. 2012, Gene ID: 1141, updated on 5 Jan. 2013
[0500] muscle-specific kinase MUSK: [0501] Gene ID: 4593, updated
on 8 Jan. 2013, Isoform 1 [UniParc]. Last modified Jan. 1, 1998.
Version 1. Checksum: 3DDC20E179FA010C, 015146-1/2, Isoform 1/2
[0502] voltage-gated calcium channel (P/Q-type): [0503] Gene ID:
773, updated on 5 Jan. 2013; Isoform 1 (1A-1) (BI-1-GGCAG)
[UniParc]. Last modified Jul. 15, 1999. Version 2. Checksum:
2F2F378ACE02FD56, 000555-1/2/3/4/5/6/7, Isoforms 1-7, Gene 1D:
25398, updated on 11 Jan. 2013, J3 KP41 [UniParc]. Last modified
Oct. 3, 2012. Version 1. Checksum: AEDF4D2A5E49263F [0504]
voltage-gated potassium channel (VGKC): [0505] Gene ID: 3737,
updated on 30 Dec. 2012, Gene ID: 3736, updated on 8 Jan. 2013,
Gene ID: 3742, updated on 8 Jan. 2013 [0506] N-methyl-D-aspartate
receptor (NMDA): [0507] Gene ID: 2904, updated on 5 Jan. 2013,
Q13224 [UniParc]. Last modified Jun. 20, 2001. Version 3. Checksum:
40AEB12BE6E50CEF; Gene ID: 2902, updated on 30 Dec. 2012, Isoform 3
(Long) (NR1-3) [UniParc]. Last modified Jun. 1, 1994. Version 1.
Checksum: CDF5402769E530AB, Q05586-1/2/3/4/5, Isoforms 1-5 [0508]
TSHR: Gene ID: 7253, updated on 4 Jan. 2013, Isoform Long
[UniParc]. Last modified Mar. 29, 2005. Version 2. Checksum:
D2EE9CEBFD64A65F, P16473-1/2/3, Isoforms 1-3 [0509] Amphiphysin:
[0510] Gene ID: 273, updated on 8 Jan. 2013, Isoform 1 (128 kDa)
[UniParc]. Last modified Feb. 1, 1996. Version 1., Checksum:
78B4F75AB75BA357, P49418-1/2, Isoform 1-2 [0511] ganglioside GQ1B:
Gene ID: 29906, updated on 30 Dec. 2012 [0512] GD3: Gene ID:
117189, updated on 22 Jun. 2012 [0513] Ca-125: Gene ID: 94025,
updated on 30 Dec. 2012, Q8WXI7 [UniParc]. Last modified Mar. 1,
2003. Version 2. Checksum: B3E7BDF19997A440 [0514] Her-2/neu: Gene
ID: 2064, updated on 13 Jan. 2013, 4.
Protein=P04626-1/2/3/4=Isoform 1-4, Last modified Aug. 13, 1987.
Version 1. [0515] gross cystic disease fluid protein 15; Gene ID:
5304, updated on 30 Dec. 2012 [0516] CD117: Gene ID: 3815, updated
on 6 Jan. 2013 [0517] CD30: Gene ID: 943, updated on 6 Jan. 2013;
Isoform Long [UniParc]. Last modified Dec. 1, 1992. Version 1.
Checksum: 7A407CC78A6E0BC8, P28908-1/2, Isoform 1/2 [0518] Platelet
derived growth factor receptor PDGFR alpha: [0519] Gene ID: 5159,
updated on 13 Jan. 2013, Gene ID: 5156, updated on 13 Jan. 2013,
Isoform 1 [UniParc]. Last modified Apr. 1, 1990. Version 1. [0520]
Checksum: 5E3FB9940ACD1BE8, P16234-1/2/3, Isoforms 1-3; P09619
[UniParc]. Last modified Jul. 1, 1989. Version 1. Checksum:
038C15E531D6E89D [0521] Melanoma associated marker/Mart 1: [0522]
Gene ID: 2315, updated on 30 Dec. 2012; Q16655 [UniParc]. Last
modified Nov. 1, 1996. Version 1. Checksum: B755BFF39CFCB16E [0523]
CD133: Gene ID: 8842, updated on 13 Jan. 2013; Isoform 1 (AC133-1)
(S2) [UniParc]. [0524] Last modified Jun. 1, 1998. Version 1.
Checksum: D21CBC05ADB2DEDF, 043490-1/2/3/4/5/6/7, Isoforms 1-7
[0525] In the following, reference is made to the examples which
are given to illustrate, not to limit the present invention.
EXAMPLES
Example 1
Cloning of Recombinant Antibody Constructs
[0526] DNA sequences derived from hybridoma cells and coding for
the variable domains of anti-CD3, anti-CD45 and anti-HLA A2
antibodies, respectively, were used to generate the antibody
constructs depicted in FIG. 3 by standard methods of molecular
biology (see, e.g. Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, New York (2001)). The
constructs were designed to carry different affinity tags to
facilitate identification and purification upon expression of
recombinant proteins (Myc-, Flag, His-Tag). For details on domain
arrangement, affinity tags and linkers of the constructs, see FIG.
3.
[0527] pelB Leader codes for an amino acid sequence that directs a
protein expressed in bacteria to the bacterial periplasm. The
leader sequence is cleaved by bacterial enzymes and the protein can
be isolated.
Example 2
Expression and Purification of Recombinant Antibodies
Periplasmic Protein Expression:
[0528] Recombinant antibody constructs were expressed in the
periplasm of E. coli strain TG1 using an appropriate prokaryotic
expression vector. Two litres of 2.times.TY medium including 0.1%
glucose and 100 .mu.g/ml ampicillin were inoculated with 20 ml of
an overnight culture of transformed TG1 and grown to exponential
phase (OD600 0.8-0.9) at 37.degree. C. Since the antibody fragments
are under control of the lactose promotor, protein expression was
induced by addition of 1 mM IPTG followed by incubation at RT (room
temperature) with shaking for additional 3 h. Cells were harvested
by centrifugation for 10 min at 2,750.times.g and 4.degree. C. and
were resuspended in 100 ml or an appropriate buffer. Cell lysis was
performed by adding 50 .mu.g/ml freshly dissolved lysozyme [Roche
Diagnostics] and incubating for 25 min on ice. Following, 10 mM
MgSO.sub.4 were added to stabilise spheroblasts, and cells were
centrifuged for 10 min at 6,200.times.g and 4.degree. C. Finally,
the supernatant obtained, containing the periplasmic protein, was
dialysed against PBS overnight at 4.degree. C. and was centrifuged
again for 15 min as stated above. Afterwards, recombinant proteins
were purified by Ni-NTA-IMAC (Nickel Nitrilo-triacetic acid
Immobilised Metal Affinity Chromatography).
Immobilised-Metal Affinity Chromatography (IMAC):
[0529] For purification of recombinant proteins with a His.sub.6
tag, an IMAC was performed by means of immobilised
nickel-nitrilotriacetic acid (NTA) agarose beads [Qiagen]. First, a
column of 1 ml Ni-NTA agarose needed to be equilibrated with
approximately 10 ml of sterile PBS or a sodium phosphate buffered
solution with 20 mM imidazole. Then, crude protein, either
precipitated from cytoplasmic expression or dialysed from
periplasmic expression, was gradually applied to the column. After
washing with about 20 ml of an appropriate IMAC wash buffer (sodium
phosphate buffered solution containing 20-35 mM imidazole) until no
more protein was detectable in the flow, bound protein was eluted
from the column in 500 .mu.l fractions with a sodium
phosphate-buffered solution including 250 mM imidazole.
[0530] All collected wash and elution fractions were tested for
presence of protein by a qualitative Bradford assay by adding 10
.mu.l of each sample to 90 .mu.l of 1.times. Bradford solution.
Verification of the purification process was performed by an
SDS-PAGE analysis. For this purpose, eluted fractions were run in
parallel with crude protein, flow, and wash fraction under reducing
conditions. Finally, positive fractions determined by the
colorimetric reaction were pooled into peak and minor fractions and
dialysed against PBS overnight at 4.degree. C. For usage in
stimulation assays, purified proteins needed to be sterile
filtrated, and their concentration has been determined. In
addition, after protein quantification, 2 .mu.g of further used
fractions were also analysed by SDS-PAGE and Western blotting under
reducing and non-reducing conditions.
[0531] In an alternative of Example 2, DNA coding for
(V.sub.H)CD3-EGFR(V.sub.H-V.sub.L),
(V.sub.H)CD3-CEA(V.sub.H-V.sub.L),
(V.sub.H)CD3-Her2(V.sub.H-V.sub.L),
(V.sub.H)CD3-HLA-A2(V.sub.H-V.sub.L), (V.sub.H)CD3-HLA-CW6
(V.sub.H-V.sub.L) (V.sub.H)CD3-CD138(V.sub.H-V.sub.L),
(V.sub.H)antiDig-EGFR(V.sub.H-V.sub.L),
(V.sub.H)antiHis-HLA-A2(V.sub.H-V.sub.L),
(V.sub.L)CD3-CEA(V.sub.H-V.sub.L),
(V.sub.L)CD3-EpCAM(V.sub.H-V.sub.L),
(V.sub.L)antiDig-EpCAM(V.sub.H-V.sub.L),
(V.sub.L)antiHis-CD45(V.sub.H-V.sub.L),
(V.sub.L)CD3-CD45(V.sub.H-V.sub.L) were synthesised and proteins
were produced and isolated by GenScript (Piscataway, N.J., USA).
The DNA was codon optimized for E. coli expression (vector E3),
expression optimized, grown in 2 litres standard LB-medium, protein
was obtained from inclusion bodies or periplasm (pelB leader) in
one step by Ni-HiTrap column. Bacterial endotoxins were removed by
dialysis against 5 litres 1.times. phosphate buffered saline (PBS).
The concentration was measured by Bradford protein assay with
bovine serum albumin (BSA) as standard. The purity was estimated by
densitometric analysis of a Coomassie Blue-stained SDS-PAGE gel.
Aliquots were stored at -80.degree. C. or +4.degree. C. Storage
buffer was used 1.times.PBS, 5% Glycerol, 0.5% sodium lauroyl
sarcosine, pH 7.4.
Example 3
Cell Culture Techniques
Cell Cultivation:
[0532] Mammalian cells were cultivated in T75 tissue culture flasks
in 20 ml of the appropriate culture medium at 37.degree. C. with 5%
CO.sub.2. Cells were split every 2-3 days. Adherent cells first
needed to be detached with 1.times. trypsin-EDTA. Cells were
counted using a vital stain, eosin or trypan blue. For storage,
cells of 60-80% confluence were harvested by centrifugation for 5
min at 450.times.g, resuspended in FCS with 10% DMSO, aliquoted in
cryovials, and gradually frozen to a temperature of -80.degree. C.
Cells were thawed quickly at 37.degree. C. in a water bath and
cautiously added to 5 ml medium. In order to remove DMSO, cells
were centrifuged again, resuspended in fresh medium and transferred
into a tissue culture flask.
Preparation of Peripheral Blood Mononuclear Cells (PBMC):
[0533] PBMC, comprising lymphocytes and monocytes, were previously
isolated from the buffy coat of a healthy human donor by density
centrifugation using the Ficoll based lymphocyte separation
solution LSM 1077 (PAA Laboratories, Pasching, Austria). Since,
during usage, these PBMC nevertheless appeared as an inhomogeneous
cell population, the separation from remaining erythrocytes,
granulocytes, and thrombocytes was repeated as follows. Thawed
PBMC, resuspended in 30 ml RPMI 1640 medium containing 10% FCS and
Pen-Strep, were cautiously layered onto 10 ml of LSM 1077 and
centrifuged for 5 min at 800.times.g without braking. After
discarding the upper phase, PBMC concentrated in the interphase
were transferred into a fresh tube, resuspended in 30 ml of medium,
and centrifuged for 5 min at 450.times.g. Monocytes were removed by
cultivating PBMC in a O10 cm tissue culture plate overnight,
allowing adherence of monocytes to the plate. Finally, PBMC,
remaining in solution, were harvested.
[0534] In an alternative of Example 3, Primary human cancer cells
from a patient with metastatic pancreatic cancer were extracted
from the ascites bags of the patient (FIG. 29). 4 litres with fresh
collected malignant ascites were stored in 2 litres glass bottles
at 4.degree. C. over night. The next day the cell pellet from the
glass bottom was washed in 1.times.PBS and resuspended in culture
medium (DMED supplemented with 200 .mu.M 1-glutamine, 10% heat
inactivated FBS, penicillin (200 U/mL), streptomycin (200 .mu.g/mL)
and sodium pyruvate (1 mM) (Gibco.RTM.)). Adherend cells were
cultured in incubator 36.degree. C., 5% CO.sub.2, 90% humidity. The
same day the ascites was collected from the patient, 20 ml
peripheral blood for PBMC extraction was collected. Primary
leukemic cells were obtained from a 71 year old male patient with
T-cell-prolymphocytic leukemia (T-PLL) (FIG. 11A) relapsing 32 days
after matched allogeneic stem cell transplantation. The leukemic
T-PLL cells were extracted as PBMCs from the peripheral blood of
the patients. At the time the sample was drawn the patient had
>90% leukemic blast in his blood count in routine clinic
diagnostic. From all patients an informed consent, approved by the
University hospital of Wurzburg ethical committee, was signed.
[0535] In an alternative of Example 3, generation of
cytomegalievirus (CMV)-specific human T-cells: Briefly, dendritic
cells (DC) were generated from plastic adherent monocytes from PBMC
of HLA-A0201 negative, B0702+ donor. After 72 h of culture in
GM-CSF/IL4-containing DC medium (Cellgenix), DC were matured in
medium containing IL4(100 ng/ml), GM-CSF (800 IU/ml), LPS (10
ng/ml) and IFN.gamma. (100 U/ml) plus 2.5 ug/ml CMV pp65 derived
peptide TPRVTGGG. After 16 h, DC were irradiated (30Gy) and
co-incubated with CD45RO.sup.-, CD57.sup.- naive CD8.sup.+ T-cells
at a 1:4 ratio in medium containing 5% AB serum and IL21 (10
ng/ml). Fresh medium, IL7 and IL15 was added on days 3, 5 and 7 of
culture, before evaluation on day 10-12. Cells were cultured in
Cellgenix DC medium. Human AB serum was used from PAA. One single
batch was used throughout all experiments. IL4, IL7, IL15, IL21
were either purchased from Peprotech or Cellgenix (with identical
results). GM-CSF was purchased from Gentaur. LPS (E. coli O:15) was
purchased from Sigma. The HLA-B0702-restricted CMV-specific peptide
TPRVTGGG was purchased from jpt. For in vivo experiments,
CMV-specific T-cells were further purified using APC-labelled
MHC-multimers (Immudex). MHC multimer staining was performed at
room temperature, followed by isolation of MHC-multimer+ T-cells
with anti-APC-beads (Miltenyi).
Example 4
Functional Assays
Flow Cytometry:
[0536] Binding of antibody fusion proteins to antigen-presenting
tumour cells and/or T lymphocytes was tested by flow cytometry. For
this purpose, 2.5-5.times.10.sup.5 cells were incubated with 10
.mu.g/ml of scFv or 0.004-4 .mu.g/ml of titrated fusion proteins in
100 .mu.l of a suitable buffer solution (such as PBS+bovine serum
albumin, or other acceptable buffer solution) per well on a 96-well
V-shaped plate at 4.degree. C. for 2 h. After washing three times
with 150 .mu.l of a suitable buffer solution, cells were incubated
with FITC-conjugated anti-His.sub.6 tag or anti-Flag Tag or
anti-myc Tag antibody at RT for 30 min and washed again two times.
For gating and testing for background staining, additionally two
samples of each cell type were prepared, one of unstained cells and
one stained with FITC-conjugated anti-His.sub.6 tag antibody
without any protein. Finally, cells were resuspended in 500 .mu.l
of a suitable buffer solution, transferred into FACS tubes, and
analysed by flow cytometry.
PBMC Stimulation Assay:
[0537] Stimulatory properties of recombinant proteins were tested
in a cell-based stimulation assay. Thereby, T-cell activation
mediated by bispecific antibodies and "tridomain constructs" was
determined by measuring PBMC stimulation in terms of the IL-2
release induced.
Measurement of Stimulatory Activity of Constructs:
[0538] CD45 pos/HLA A2 myeloma cell line U266 were seeded in a
flat-bottomed 96-well cell culture plate at a density of 105 cells
per well in 100 .mu.l of culture medium. Titrated stimulatory
proteins were added as indicated in 100 .mu.l medium per well and
were preincubated for 1 h at 37.degree. C. and 5% CO2 to ensure
sufficient binding. Unstimulated PBMC, thawed and isolated the day
before, were then added at indicated density and incubated for 24 h
at 37.degree. C. and 5% CO.sub.2. Finally, plates were centrifuged
for 5 min at 450.times.g to harvest cell-free supernatants for IL-2
quantification in ELISA.
IL-2 Sandwich ELISA:
[0539] As an indicator for the stimulatory activity, T-cell
activation induced by bispecific antibodies was measured in terms
of the IL-2 release. Upon PBMC stimulation, concentration of
secreted IL-2 in the supernatant was determined by an IL-2 sandwich
ELISA.
[0540] First, a 96-well ELISA plate was coated with 400 ng/100
.mu.l per well of mouse anti-human IL-2 antibody overnight at
4.degree. C., followed by saturation of nonspecific binding sites
with a suitable blocking buffer for 2 h at RT. In the meantime,
serial 1:2 dilutions of an IL-2 standard were prepared in duplicate
in reagent diluent starting with a maximum IL-2 concentration of
1,000 pg/ml. Then, supernatants containing IL-2 were 1:3 diluted in
RPMI 1640 medium containing 10% FCS and Pen-Strep
(Penicillin-Streptomycine). Both diluted supernatants and standards
were transferred into the ELISA plate and incubated for 2 h at RT.
Following, IL-2 was detected by incubation with 17.5 ng/100 .mu.l
per well of biotinylated goat anti-human IL-2 antibody for 2 h at
RT. Finally, 100 .mu.l of HRP-conjugated streptavidin, 1:200
diluted in reagent diluent, was added per well and incubated for 20
min at RT. Each plate was developed using a TMB substrate solution.
In order to achieve a background signal, at least 2 wells on each
plate were incubated with reagent diluent or medium only and the
detecting antibody plus TMB. Between each incubation step, the
plate was washed three times with PBS containing 0.05% Tween-20 and
once with PBS only.
[0541] A seven point standard curve was created by plotting the
absorbance signals of each standard sample against the IL-2
concentration. Thus, the amount of IL-2 of each supernatant could
be determined by interpolation of the standard curve fitted with
the nonlinear regression equation for one phase exponential
association using GraphPad Prism.RTM..
IFN-.gamma. ELISA (Alternative of Example 4):
[0542] In 100 .mu.l cell culture supernatant the IFN-.gamma.
concentration was measured using the human IFN-.gamma. ELISA Kit
(Invitrogen.TM.) after manufacturer's protocol. Briefly 50 .mu.L of
Incubation Buffer was added to each well of a precoated 96-well
plat. 50 .mu.L of the Standard Diluent Buffer to zero wells. 50
.mu.L of standards and samples to each well. 50 .mu.L of
biotinylated Hu IFN-.gamma. Biotin Conjugate solution into each
well. Taped gently on the side of the plate to mix. Covered plate
with plate cover and incubate for 1 hour and 30 minutes at room
temperature. Thoroughly aspirated solution from wells and discarded
the liquid. Washed wells 4 times. Added 100 .mu.L Streptavidin-HRP
Working Solution to each well. Covered plate with the plate cover
and incubated for 45 minutes at room temperature. Thoroughly
aspirated solution from wells and discarded the liquid. Added 100
.mu.L of Stabilized Chromogen to each well. The liquid in wells
turned blue. We incubated for 15-30 minutes at room temperature and
in the dark. Added 100 .mu.L of Stop Solution to each well. Taped
side of plate gently to mix. The solution in the wells changed from
blue to yellow. The absorbance of each well was read with a BioRad
plate reader at 450 nm.
Cytotoxicity Assay:
[0543] The HLA-A2/CD45 positive cell line U266 or myeloma cell line
U266 was labelled with 10 .mu.M CFSE (Invitrogen Vybrant CFDA SE
Cell Tracer Kit) in 350 .mu.l PBS for 10 min at room temperature
(RT) in the dark. The labelling reaction was stopped by the
addition of 5 ml fetal calf serum (FCS), followed by a 1-minute
incubation at RT. After 2 washes, the CFSE-labelled target cells
were resuspended in assay medium and co-incubated with Peripheral
Blood Mononuclear Cells (PBMC) from a HLA-A2 negative healthy donor
at a ration of 1:10 (5*10.sup.5 U266 and 5*10.sup.6 PBMCs in 2 ml)
and 27 nM of antibody constructs as indicated. A sample treated
with Triton was used as positive control (100% lysis) and a sample
without antibody construct as negative control (0% lysis). After 24
h, apoptotic cells were visualized by 7AAD stain (Biozol, 10 min at
RT) and % specific Lysis of CFSE labelled U266 cells was calculated
employing flow cytometry techniques.
Caspase-3 Assay (Alternative of Example 4):
[0544] Staining was performed after co-incubating of the target
cells with T-cells (tumor cells:T-cells ratio 2:1) with or without
the specific polypeptides for 4 h. Surface staining for HLA-A2 and
CD45 was performed first, followed by fixation and permeabilization
(Fix+Perm, BD Biosciences). Activated Caspase-3 antibody was then
added for 30 min. (BD Biosciences). Cells were washed with
1.times.PBS+5% human serum (HS, PAA Laboratories) and analyzed on a
BD-FACS Canto-II. % specific apoptosis was calculated as (%
experimental value-% spontaneous release)/(100%-% spontaneous
release)*100.
Alamar Blue Assay (Alternative of Example 4):
[0545] The alamarBlue.RTM. assay (Abd Serotec) was used to measure
proliferation and viability of cells after exposure to toxins.
Briefly, cells were grown in 100 .mu.l cell culture medium per well
(96 well plate). For analysis 10 .mu.l alamarBlue was added per
well and incubated in the incubator for 30-120 minutes. The
absorbance was read with a BioRad plate reader at 570 nM and 600
nM. For blank media only was used. The percent difference in
reduction of cell proliferation between the different polypeptide
groups was calculated as indicated by the manufacturer, using cells
growing in culture without toxin as control.
Digoxigenin Assay (Alternative of Example 4):
[0546] First peroxidise from horseradish (HRP, Sigma-Aldrich Chemie
gmbH) was labelled with digoxigenin NHS-ester (Sigma-Aldrich Chemie
gmbH) in a 1/3 molar ratio. Dig-HRP was cleaned up with micro
Bio-Spin.TM. chromatography columns (BioRad and stored at 4.degree.
C. in the dark. Colo-206F cells were first incubated with indicated
polypeptides at various concentrations for 90 minutes. Cells were
washed with PBS and resuspended in cell culture medium with Dig-HRP
and incubated for 30 minutes. Afterward cells were washed twice
with PBS and resuspended in 50 .mu.l PBS. 50 .mu.L of Stabilized
Chromogen (Invitrogen.TM.) was added for 15-30 minutes at room
temperature in the dark. 50 .mu.L of Stop Solution was added and
the absorbance was read with a BioRad plate reader at 450 nm.
Mice (Alternative of Example 4):
[0547] The HLA.A2 transgenic, immunodeficient mice (NodScid IL-2rg
-/- HLA.A2/B2m tg; Stock number 14570, The Jackson Laboratory, Bar
Harbor, Me., USA) for the in vivo experiment (FIG. 12A) were
maintained in our certified animal facility (ZEMM, Center for
experimental molecular medicine, University hospital Wurzburg) in
accordance with European guidelines. Female Mice, 6-10 weeks old,
were divided into five groups, six mice per group (n=30).
5.times.10.sup.6 THP-1 cells, 1.25.times.10.sup.5CMV specific CD8+
T-cells (tumour cell:T-cell ratio 40/1) and the 0.5 .mu.g of the
polypeptides were injected intraperitoneally (i.p.) as indicated.
After injection, mice were monitored by daily inspection. A second
injection of 1.16.times.10.sup.5 CMV-specific CD8+ T-cells/mouse
was given at day 13 and injections of the polypeptides were
repeated every three days a week. The animals were sacrificed when
the increase in body weight was greater 80% or if they appeared
moribund according to institutional guidelines.
[0548] Domain structure, affinity tags and linkers of the
constructs or polypeptides used in Examples 5-9 or FIGS. 4-11 are
shown in FIG. 3. These constructs and all constructs or
polypeptides used in FIGS. 4-30 were prepared as described in
Examples 1 and 2. Cell culture and functional assays in Examples
5-9 and culture, functional assays and in vivo work as to FIGS.
4-30 were carried out as described in Examples 3 and 4.
Example 5
[0549] The CD45 and HLA A2 positive myeloma target cell line U266
was co-incubated with HLA A2 negative T cells (monocyte depleted
PBMCs (peripheral blood mononuclear cells) from a healthy donor and
varying amounts of HLA A2 and CD3 bispecific antibody constructs as
indicated (Numbers 85, 82, 75 and 71). PHA-L (phytohemagglutinin, a
lectin that causes unspecific stimulation of T cells; 1 .mu.g/ml
final concentration) was used as positive control and single chain
scFv constructs with specificity for HLA A2 (Number 4) or CD3
(Number 36) were investigated. IL2 (Interleukin-2) production by T
cells was measured by ELISA techniques. No IL2 production was found
in experimental situations without any constructs. Data obtained is
depicted in FIG. 4.
Example 6
[0550] The CD45 and HLA A2 positive myeloma target cell line U266
was co-incubated with HLA A2 negative T cells (monocyte depleted
PBMCs) from a healthy donor and varying amounts of "tridomain
constructs" added either separately (Numbers 42, 45, 55; numbers
referring to constructs as depicted in FIG. 3) or in combinations
(42+45 or 42+55). PHA-L and single chain scFv constructs with
specificity for CD45 (Numbers 46 and 17) were given as controls.
IL2 production by T cells was measured by ELISA techniques. No IL
production was found in experimental situations without any
constructs. Data obtained is depicted in FIG. 5.
Example 7
[0551] The CD45 and HLA A2 positive myeloma target cell line U266
was co-incubated with HLA A2 negative T cells (monocyte depleted
PBMCs) from a healthy donor and the HLA A2 and CD3 bispecific
antibody construct alone (number 71, 27 nM) or in combination with
single chain scFv constructs that block the antigenic epitopes on
HLA A2 (Number 4, hundredfold excess compared to the concentration
of construct 71, i.e. 2700 nM) or CD3 (Number 36, ninefold excess
compared to the concentration of construct 71, i.e. 243 nM). IL2
production by T cells was measured by ELISA techniques and PHA-L is
given as control. Data obtained is depicted in FIG. 6.
Example 8
[0552] The CD45 and HLA A2 positive myeloma target cell line U266
was co-incubated with HLA A2 negative T cells (monocyte depleted
PBMCs) from a healthy donor and the combination of constructs 42
and 45. T cell stimulatory function was blocked by single chain
constructs specific for HLA A2 (number 4) or CD45 (number 46).
Complementation of T cell stimulatory function was tested by
assaying constructs 42 and 45 separately or the single chain scFv
construct directed against CD3 (number 36). IL2 production by T
cells was measured by ELISA techniques and PHA-L is given as
control. Concentration of constructs was 27 nM, unless indicated
otherwise. ("9.times." indicates a concentration of 243 nM,
"100.times." a concentration of 2700 nM.) Data obtained is depicted
in FIG. 7.
Example 9
[0553] The CD45 and HLA A2 positive myeloma target cell line U266
was co-incubated with HLA A2 negative T cells (monocyte depleted
PBMCs) from a healthy donor and the combination of constructs 42
and 55. T cell stimulatory function was blocked by single chain
constructs specific for HLA A2 (number 4) or CD45 (number 46).
Complementation of T cell stimulatory function was tested by
assaying constructs 42 and 55 separately or the single chain scFv
construct directed against CD3 (number 36). IL2 production by T
cells was measured by ELISA techniques and PHA-L is given as
control. Concentration of constructs was 27 nM, unless indicated
otherwise. ("9.times." indicates a concentration of 243 nM,
"100.times." a concentration of 2700 nM.) Data obtained is depicted
in FIG. 8.
[0554] The results of the preceding Examples clearly demonstrate
that two constructs (42+45) or (42+55) first have to bind their
ligands on the surface of a single cell in order to subsequently
complement T cell engaging function.
Example 10
[0555] Lysis of the CD45 and HLA A2 positive myeloma target cell
line U266 by HLA A2 negative T cells (monocyte depleted PBMCs) in
the presence of V.sub.LCD3-scFvHLA A2 (27 nMol) or V.sub.H-scFvCD45
(27 nMol) or the combination of both of these constructs (27 nMol
each) was determined using flow cytometry based techniques. Percent
lysis was calculated by apoptotic U266 cells divided through total
U266 cells and background apoptosis was subtracted. Data obtained
is depicted in FIG. 9.
Example 11
[0556] As parts of the final bipartite construct, two polypeptides
were designed, each composed of an antigen-binding single-chain
variable fragment (scFv) and either the variable light (V.sub.L) or
variable heavy chain (V.sub.H) domain of a T cell-activating
anti-CD3 antibody (FIG. 10). When these two polypeptides bind their
respective antigens on the surface of a single cell, the V.sub.L
and V.sub.H domains interact with each other to reconstitute the
original anti-CD3 binding site. The thus on-target formed
trispecific heterodimer engages and stimulates T cells for tumor
cell destruction.
[0557] This scenario is fully validated in vitro when T lymphocytes
are confronted with target cells that have been incubated with the
two different polypeptides. As proof of principle, major
histocompatibility antigen HLA-A2 and the hematopoetic lineage
marker CD45 were targeted as first and second antigens, which both
are expressed on U266 myeloma cells, primary cells from a patient
with pro-lymphocytic leukemia of the T cell lineage (T-PLL), and
THP-1 acute myeloid leukemic blasts (FIG. 11). Due to the described
V.sub.L/V.sub.H interaction, the now trispecific heterodimer
potently stimulates T cells to secrete interleukin-2 (IL-2) (FIG.
11a) and to lyse the labeled tumor cells at nanomolar concentration
(FIG. 11b), the cytotoxic efficacy being quite similar to that of a
bispecific T cell-activating antibody, which was employed as a
positive control (FIG. 11A, left panel), Mack, 1995, Proc Natl Acad
Sci 92, 7021-7025. When the polypeptides were added separately from
each other, they did not induce T lymphocytes to lyse target cells.
These results are in line with structural data indicating that
both, V.sub.H and V.sub.L domains are required to confer sufficient
affinity to the target antigen (FIG. 11A, B), Colman, 1987, Nature
326, 358-363; Amit, 1986, Science 233, 747-753. Moreover, the
results reveal that possible homodimerization of either V.sub.H or
V.sub.L arms results in a negligible measurable biological
effect.
[0558] To demonstrate that the two molecules must first bind their
antigens on the surface of the target cell for V.sub.H/V.sub.L
heterodimerization to occur, single-chain variable fragments
specific for HLA-A2 and CD45 were used to block the respective
epitopes on the target. As shown in FIG. 11c, when present in great
excess, these inhibitors prevented the two polypeptides from
triggering T cells in a dose-dependent manner. Furthermore, T cells
were not stimulated when the target cells were omitted (data not
shown) or when target cells were probed that express CD45 only
(RAJI cells, FIG. 11D) or neither target molecule (KMS-12-BM, FIG.
11D).
Example 12
[0559] For in vivo proof of concept, a model of allogeneic mismatch
stem cell transplantation was resorted in which a patient's
residual leukemic and hematopoietic cells, all HLA-A2 and
CD45-positive, must be eliminated to give the allogeneic donor stem
cells (HLA-A2-negative, CD45-positive) a chance to engraft and to
reconstitute hematopoesis (see FIG. 2). To put the specificity of
the bipartite construct to the test, immunodeficient mice
expressing the human HLA-A2 transgene on virtually all nucleated
cells were used, the question being whether HLA-A2-positive but
CD45-negative murine tissues would suffer collateral damage. THP-1
cells were injected intraperitoneally with or without CD8 T
lymphocytes from an HLA-A2-negative donor, which had been selected
for specificity to cytomegalovirus (CMV) to avoid human anti-murine
immune reactivity. Intraperitoneal tumors developed rapidly in mice
that did not receive the polypeptides, and in mice treated either
with single molecule types or with the combination of both
polypeptides but without T cells. In all instances, fatal
disseminated disease developed within 3 to 4 weeks (FIG. 12A). In
stark contrast, all tumor-bearing mice treated with T cells and
repeated injections of both polypeptides survived the end of the
experiment on day 31, albeit with palpable tumors at the injection
site. These results clearly show that the bipartite construct truly
redirects T cells irrespective of their specificity at tumor cells
that simultaneously express both target molecules (HLA-A2 and CD45)
in vivo. As an aside, a T cell recruiting bispecific antibodies
against HLA-A2 would wreak havoc by redirecting T cells against all
HLA-A2 positive murine tissues. Likewise, a CD45-binding bispecific
antibody would have mediated lysis of all hematopoietic cells,
including THP-1 leukemic blasts and T cells from the donor. In our
set-up, however, injection of HLA-A2-specific polypeptide into the
HLA-A2 transgenic animals caused no apparent toxicity.
[0560] To further examine possible toxicity to bystanders, we
employed a highly sensitive apoptosis assay on THP-1 cells and
HLA-A2-negative but CD45-positive monocytes, the latter
representing the healthy bystander compartment. As depicted in FIG.
12B, we observed caspase-3 activation in THP-1 cells but not in
monocytes treated in the same well with the combination of the
polypeptides or the bispecific positive control and donor T cells.
THP-1 cells cultured with T cells and individual polypeptides were
unaffected. These observations again clearly show initiation of
apoptosis exclusively in the double antigen positive target
population, while the HLA-A2-negative bystander cells are spared.
These experiments model quite accurately the dire clinical
situation of leukemia patients with a HLA-mismatched stem cell
transplant. The combinatorial approach of using a distinctive HLA
molecule and CD45 aims at enhancing the desired graft versus
leukemia effects by retargeting the donor's T cells against
leukemic blasts of both, myeloid and lymphoid origin.
Example 13
[0561] To venture into solid tumors, we targeted the combinatorial
approach to epithelial cell adhesion molecule (EpCAM) and epidermal
growth factor receptor (EGFR) antigens. Both antigens are
over-expressed in various carcinomas and have been extensively
studied in clinical phase II and III trials. The expression of EGFR
is closely associated with cell proliferation, while EpCAM is
present at the basolateral surface of virtually all simple
epithelia and was recently found to act like a signaling protein in
the Wnt pathway, Maetzel, 2009, Nat Cell Biol 11, 162-171. As FIG.
13a illustrates, the two polypeptides trigger the release of
interferon-.gamma. (IFN.gamma.) from co-incubated donor lymphocytes
and mediate apoptosis of the double-positive cancer cell line
COLO-206F at nanomolar concentrations (FIG. 13a, b), but only when
given in combination and not with either part alone. As a
descendant of neuroepithelial tissue, the melanoma cell line FM-55
lacks EpCAM, and therefore was completely resistant to the
polypeptides (FIG. 13a, b). Though the expression of EGFR and EpCAM
overlaps broadly on proliferating carcinoma cells,
non-proliferating epithelial cells, e.g., of liver and pancreas
solely expressing EGFR or EpCAM antigens, respectively, should be
less susceptible to or protected from the two-pronged attack.
Notably, hepatic and pancreatic toxicities have been dose-limiting
for high-affinity monoclonal EpCAM antibodies in clinical trials
(for review see, Munz, 2010, Cancer Cell Int 10:44).
Example 14
[0562] The further validation of the bipartite functional
complementation strategy was performed by extensive in vitro
experiments, using a combination of different polypeptides,
targeting various cell surface antigens on different human cell
lines.
[0563] The HLA A2 positive human tumor cell lines FM-55 (myeloma),
Colo-206F (colon cancer) and OVCAR (ovarian cancer) were
co-incubated with HLA-A2 negative PBMCs from a healthy donor,
polypeptide against HLA-A2 (CD3(V.sub.L)-HLA-A2(V.sub.H-V.sub.L))
and with a second polypeptide targeting either CEA
(CD3(V.sub.H)-CEA(V.sub.H-V.sub.L)), EGFR
(CD3(VH)-EGFR(V.sub.H-V.sub.L)) or Her2
(CD3(V.sub.H)-Her2(V.sub.H-V.sub.L)). IL2 or IFN-.gamma. production
by lymphocytes was measured by ELISA techniques. These data
demonstrate that (i) a specific combination of antigens, an antigen
signature, can be expressed on carcinomas of various origin (skin,
neuroepithelial, gut and ovary tissue), (ii) the antigen signature
is approachable with our bipartite functional complementation
strategy using a set of polypeptides specific for the antigen
signature. Data obtained are depicted in FIGS. 14, 15 and 16.
Example 15
[0564] To demonstrate the exchangeability of the functional domain,
the fragments F1 and F2 of a set of polypeptides were exchanged
with each other, retaining their specific complementation ability
for on target restoration of their original antibody domain to
engage T cells. Therefore the set of polypeptides against the CD45
and HLA-A2 target antigen was used. The polypeptide against CD45
had CD3(V.sub.L) as fragment F1 and the polypeptide against HLA-A2
had CD3(V.sub.H) as fragment F2. The CD45 and HLA-A2 positive
myeloma cell line U266 was co-incubated with HLA-A2 negative T
cells from a healthy donor and polypeptides against CD45
(CD3(V.sub.L)-CD45(V.sub.H-V.sub.L)) and HLA-A2
(CD3(V.sub.H)-HLA-A2(V.sub.H-V.sub.L)) in varying amounts. T cell
engagement was assessed by reactive IFN.gamma. production, measured
by ELISA techniques. No IFN.gamma. production was found in
experimental situations without any polypeptides. Data obtained is
depicted in FIG. 17.
Example 16
[0565] The bipartite functional complementation strategy was
further tested by targeting a set of antigens, already used as
targets for antibody therapy of cancer (EGFR, EpCAM and Her2) (Her2
is a target for Trastuzumab in breast cancer, EGFR is a target for
Cetuximab in colorectal cancer and EpCAM is a target for
Catumazumab for the treatment of neoplastic ascites). The EGFR,
EpCAM and Her2 positive cells (Colo-206F, CX-1 and OVCAR) were
co-incubated with PBMCs from a healthy donor and the combination of
polypeptides against EGFR (CD3(V.sub.H)-EGFR(V.sub.H+V.sub.L)),
EpCAM (CD3(V.sub.L)-EpCAM(V.sub.H+V.sub.L)) and Her2
(CD3(V.sub.H)-Her2(V.sub.H+V.sub.L)). Complementation of lymphocyte
stimulatory function was assessed by reactive IFN.gamma.
production, measured by ELISA techniques. No IFN.gamma. production
was found in experimental situations without any polypeptides. Data
obtained is depicted in FIGS. 18 and 19.
Example 17
[0566] To test an antigen combination with close clinical
correlation, the combination CD45 and CD138 was used to target
human multiple myeloma (MM) cells. The majority of human MM cells
are positive for CD45 and CD138. A T cell recruiting bispecific
antibodies against CD45 would kill all hematopoetic cells of a
patient and against CD138 would cause severe side effects because
of its expression on various normal tissues (epithelial cells,
endothelia, trophoblastic cells and glandular cells of the GI
tract, The Human Protein Atlas, Version: 10.0, Atlas updated: 2012
Sep. 12). In contrast the combination of CD45 and CD138 is found
exclusively on plasma cells and MM cells and is therefore a good
antigen signature for the targeted therapy approach. The CD45 and
CD138 positive human multiple myeloma cell line AMO-1 was
co-incubated with PBMCs from a healthy donor and the combination of
polypeptides against CD45 (CD3(V.sub.L)-CD45(V.sub.H+V.sub.L)) and
CD138 (CD3(V.sub.H)-CD138(V.sub.H+V.sub.L)). Complementation of
lymphocyte stimulatory function was assessed by reactive IFN.gamma.
production, measured by ELISA techniques. No IFN.gamma. production
was found in experimental situations with single polypeptides or
without any polypeptides. Data obtained is depicted in FIG. 20.
Example 18
[0567] A further application of the bipartite functional
complementation strategy is to target single antigens on the cell
surface and to kill single antigen positive tumor cells. One major
drawback for T cell recruiting bispecific antibodies with
functional antiCD3 binding sides are severe side effects caused by
unspecific T-cell activation and cytokine release (Linke, R. et al.
Catumaxomab: clinical development and future directions. MAbs 2,
129-136 (2010)). The advantage of this bipartite functional
complementation strategy is the fact, antibodies that the T-cell
activating antiCD3 functional domain is exclusively restored on the
target cell. Without the target cell, no T-cell activating domain
is present. The CD45 and CD138 positive human multiple myeloma
cells AMO-1 and U266 were co-incubated with PBMCs from a healthy
donor and the combination of polypeptides against a single target
antigen, either CD138
(CD3(V.sub.H)-CD138(V.sub.H+V.sub.L)+CD3(V.sub.L)-CD138(V.sub.H+V.sub.L))
or CD45
(CD3(V.sub.H)-CD45(V.sub.H+V.sub.L)+CD3(V.sub.L)-CD45(V.sub.H+V.s-
ub.L)). Complementation of lymphocyte stimulatory function was
assessed by reactive IFN.gamma. production, measured by ELISA
techniques. No IFN.gamma. production was found in experimental
situations with single polypeptides or without any polypeptides.
Data obtained are depicted in FIGS. 21 and 22. In FIG. 23 the
single antigen approach is illustrated, by using a set of
polypeptides targeting two different epitopes (upper part) or the
same epitope (lower part) on the target antigen A1.
Example 19
[0568] This is an example to demonstrate that the functional
complementation strategy can be further elaborated for targeted
payload delivery and that different effector ways are possible to
kill a target cell. By complementing the F1 and F2 fragments of a
set of bound polypeptides on target, the newly formed antibody
binding site can bind any molecule it is specific for. In order to
direct a HIS-tagged payload precisely to a target cell, the V.sub.H
and V.sub.L fragments of an anti-HIS(hexa-histidine)-antibody were
used. After simultaneous binding of polypeptide 1
(antiHis(V.sub.L)-CD45(V.sub.H-V.sub.L) and polypeptide 2
(antiHis(V.sub.H)-HLA-A2(V.sub.H-V.sub.L) to their specific target
antigens CD45 and HLA-A2, a hexa-histidine binding site is
complemented on target that binds histidine labeled payloads with
high high affinity. The payload be a HIS-tagged toxin as given in
this example here. The CD45 and HLA-A2 positive cells THP-1 were
co-incubated with a histidine(His)-tagged Clostridium perfringens
Iota toxin component Ia (FIG. 24) or a histidine(His)-tagged Shiga
toxin subunit A (FIGS. 25, 26) in combination with polypeptides
against CD45 (antiHis(V.sub.L)-CD45(V.sub.H-V.sub.L)) and HLA-A2
(antiHis(V.sub.H)-HLA-A2(V.sub.H-V.sub.L)). Complementation of
his-tagged toxin binding and subsequent target cell killing was
assessed by measuring cell viability using an alamarBlue.RTM.
assay. At the highest concentration of polypeptides used (80 nM), a
clear difference in target cell killing, measured as reduction in
cell viability, was found in experimental situations with a
combination of both polypeptides compared to single
polypeptides.
Example 20
[0569] To further demonstrate the versatility, flexibility and the
exchangeability of the bipartite functional complementation
strategy, the V.sub.H and V.sub.L fragments of an anti-Digoxigenin
antibody were used to identify and mark double antigen positive
cells with Digoxigenin-labeled HRP (horse raddish peroxidase). EGFR
and EpCAM positive Colo-206F cells were co-incubated with
polypeptides against EGFR (antiDig(V.sub.H)-EGFR(V.sub.H+V.sub.L))
and EpCAM (antiDig(V.sub.L)-EpCAM(V.sub.H+V.sub.L)). On target
complementation of the functional domain anti-Digoxigenin,
indicated by Digoxigenin-HRP labelling of Colo-206F cells, was
assessed by measuring the peroxidase activity, using a standard
ELISA Kit (Invitrogen.TM.). A clear difference in Dig-HRP labeled
target cells was found in experimental situation with a combination
of both polypeptides compared to single polypeptides. Data obtained
are depicted in FIG. 27.
Example 21
[0570] Using the human leucocytic antigens (HLA) as one arm for
dual antigen restricted bipartite functional complementation, this
haplotype strategy was further validated by exchanging the
functional domains of the polypeptides with V.sub.H and V.sub.L
fragments of an anti-HLA-Cw6 antibody. HLA-Cw6 positive primary
patient PBMCs were co-incubated with HLA-Cw6 negative PBMCs from a
healthy donor, polypeptide against CD45
(CD3(V.sub.L)-CD45(V.sub.H-V.sub.L)) and HLA-Cw6
(CD3(V.sub.H)-HLA-Cw6(V.sub.H-V.sub.L)). IFN.gamma. production by
lymphocytes was measured by ELISA techniques. These data
demonstrate that hematopoietic cells of patients with other
haplotypes than HLA-A2 can be targeted simply by exchanging one
targeting domain (anti HLA-A2, FIG. 5, 7-9, 11-12) by another (anti
HLA-Cw6). Data obtained are depicted in FIG. 28.
Example 22
[0571] The dual antigen-induced bipartite functional
complementation strategy was further validated in an in vitro
patient assay, using freshly isolated primary patient cancer cells
and antigen targets already used for cancer therapy in clinic or
clinical trials (EGFR, EpCAM, CEA and Her2). Malignant cells of a
48 years old male patient with metastatic pancreatic cancer were
co-incubated with the patients own peripheral blood lymphocytes and
the combination of polypeptides against EGFR
(CD3(V.sub.H)-EGFR(V.sub.H+V.sub.L)), EpCAM
(CD3(V.sub.L)-EpCAM(V.sub.H+V.sub.L)), Her2
(CD3(V.sub.H)-Her2(V.sub.H+V.sub.L)), CEA
(CD3(V.sub.H)-CEA(V.sub.H-V.sub.L)) and HLA-A2
(CD3(V.sub.L)-HLA-A2(V.sub.H-V.sub.L)). Complementation of
lymphocyte stimulatory function was assessed by reactive IFN.gamma.
production, measured by ELISA techniques. No IFN.gamma. production
was found in experimental situations without any polypeptides.
These data demonstrate the potential of this strategy to use
patients own immune cells to target and kill his malignant
transformed cells. Data obtained are depicted in FIG. 29.
Example 23
[0572] A highly enriched CD3/CD8 positive CMV restricted T-cell
population was used to show that any T cell, irrespective of its
specificity, can serve as effector cell an kill double antigen
positive tumor cells by this complementation strategy. The CD45 and
HLA-A2 positive U266 and THP-1 cells were co-incubated with
cytomegalievirus (CMV) specific T-cells from a HLA-A2 negative
healthy donor and polypeptides against CD45
(CD3(V.sub.H)-CD45(V.sub.H-V.sub.L)) and HLA-A2
(CD3(V.sub.L)-HLA-A2(V.sub.H-V.sub.L)) in varying amounts. The
bispecific tandem scFv
(CD3(V.sub.H-V.sub.L).times.HLA-A2(V.sub.H-V.sub.L))-antibody was
used as a positive control. T cell engagement was assessed by
reactive IFN.gamma. production, measured by ELISA techniques. No
IFN.gamma. production was found in experimental situations with
single polypeptides or without any polypeptides. Data obtained are
depicted in FIG. 30. Cells from the same frozen aliquot batch, CMV
specific T-cells and THP-1 cells, were used for the in vivo murine
model (FIG. 12A).
Example 24
[0573] This illustration depicts the potential to target
allergen/autoimmune specific B-cell clones with the bipartite
functional complementation strategy. By using a synthetic allergen
as targeting moiety, the allergen linked polypeptide will bind
specifically to its clonotypic B-cell receptor expressed on the
surface of the allergen specific B-cell clone. The second arm of
the bipartite strategy will use a B-cell specific polypeptide
(CD19, CD20, CD38, CD138), restricting the followed complementation
of the effector domain with subsequent target cell killing to the
allergen specific B-cell clone. The ultimate goal of this strategy
is to eliminate the B cell clone that causes and allergic or
autoimmune disease (upper part of FIG. 31) whilst sparing B cells
with other specificities or cells other than B cells (eg. mast
cells or basophilic cells) which bind the antibody responsible for
the disease via Fc-receptors (lower part of FIG. 31).
[0574] The features of the present invention disclosed in the
specification, the claims, and/or in the accompanying drawings may,
both separately and in any combination thereof, be material for
realizing the invention in various forms thereof.
Sequence CWU 1
1
2011119PRTArtificial SequenceVH anti-CD3 1Asp Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Thr Thr Asp Lys Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr
Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp
Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
2111PRTArtificial SequenceVL anti-CD3 2Asp Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp
Thr Ser Lys Val Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Asn Ser Leu Glu Ala Glu
65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser
Ala Ala Ala 100 105 110 3119PRTArtificial SequenceVH anti-CD3 3Asp
Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10
15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr
Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys
Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp
His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr
Val Ser Ser 115 4106PRTArtificial SequenceVL anti-CD3 4Asp Ile Gln
Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu
Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25
30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser
Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser
Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys 100 105 5122PRTArtificial SequencehuMAb anti-CD variant 9
VH 5Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr
Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser
Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Ser Val
Asp Lys Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly
Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 6113PRTArtificial
SequencehuMAb anti-CD variant 1 VL 6Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr
Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu
Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Ile Lys Arg 100 105 110 Thr 7119PRTArtificial SequenceAnti-CD3
VH (L2K) 7Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro
Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr
Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly
Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu
Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110
Thr Thr Leu Thr Val Ser Ser 115 8106PRTArtificial SequenceAnti-CD3
VL (L2K) 8Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser
Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser
Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser
Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Val Ala Ser Gly
Val Pro Tyr Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr
Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr
Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly
Ala Gly Thr Lys Leu Glu Leu Lys 100 105 9119PRTArtificial
SequenceAnti-CD3 VH (145.2C11) 9Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Ser Cys Glu
Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val
Arg Gln Ala Pro Gly Arg Gly Leu Glu Ser Val 35 40 45 Ala Tyr Ile
Thr Ser Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala Val 50 55 60 Lys
Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Leu Leu Phe 65 70
75 80 Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly Gln Gly
Thr Met Val 100 105 110 Thr Val Ser Ser Ala Lys Thr 115
10110PRTArtificial SequenceAnti-CD3 VL (145.2C11) 10Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly 1 5 10 15 Asp Arg
Val Thr Ile Asn Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Asn Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Arg Asp Ser Ser Phe Thr Ile Ser Ser
Leu Glu Ser 65 70 75 80 Glu Asp Ile Gly Ser Tyr Tyr Cys Gln Gln Tyr
Tyr Asn Tyr Pro Trp 85 90 95 Thr Phe Gly Pro Gly Thr Lys Leu Glu
Ile Lys Arg Ala Asp 100 105 110 11114PRTArtificial SequenceAnti-HIS
VH 11Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Asp Val Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Ser Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly Gly
Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr
Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser
Leu Thr Ser Glu Asp Ser Ser Val Tyr Tyr Cys 85 90 95 Glu Ser Gln
Ser Gly Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110 Ser
Ala 12116PRTArtificial SequenceAnti-HIS VL 12Asp Tyr Lys Asp Ile
Leu Met Thr Gln Thr Pro Ser Ser Leu Pro Val 1 5 10 15 Ser Leu Gly
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile 20 25 30 Val
His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro 35 40
45 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
50 55 60 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr 65 70 75 80 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly
Val Tyr Tyr Cys 85 90 95 Phe Gln Gly Ser His Val Pro Phe Thr Phe
Gly Ser Gly Thr Lys Leu 100 105 110 Glu Ile Lys Arg 115
13128PRTArtificial SequenceAnti-DIG VH 13Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu
Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ala Met
Ser Trp Ile Arg Gln Thr Pro Glu Asn Arg Leu Glu Trp Val 35 40 45
Ala Ser Ile Asn Ile Gly Ala Thr Tyr Ala Tyr Tyr Pro Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Gly Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95 Ala Arg Pro Gly Ser Pro Tyr Glu Tyr Asp Lys
Ala Tyr Tyr Ser Met 100 105 110 Ala Tyr Trp Gly Pro Gly Thr Ser Val
Thr Val Ser Ser Ala Lys Thr 115 120 125 14118PRTArtificial
SequenceAnti-DIG VL 14Asp Val Gln Met Thr Gln Ser Thr Ser Ser Leu
Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala
Ser Gln Asp Ile Lys Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Ser Ser Thr
Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser
Gly Thr Asp Phe Ser Leu Thr Ile Thr Asn Leu Glu Arg 65 70 75 80 Glu
Asp Ile Ala Thr Tyr Phe Cys Gln Gln Ser Ile Thr Leu Pro Pro 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110 Pro Thr Val Ser Ile Phe 115 1510PRTArtificial
SequenceAnti-CD3 VH CDR1 (WT) 15Gly Tyr Thr Phe Thr Arg Tyr Thr Met
His 1 5 10 1617PRTArtificial SequenceAnti-CD3 VH CDR2 (VH5) 16Tyr
Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 1710PRTArtificial SequenceAnti-CD3 VH CDR3 (WT) 17Tyr Tyr
Asp Asp His Tyr Cys Leu Asp Tyr 1 5 10 1810PRTArtificial
SequenceAnti-CD3 VK CDR1 (WT) 18Arg Ala Ser Gln Ser Val Ser Tyr Met
Asn 1 5 10 197PRTArtificial SequenceAnti-CD3 VK CDR2 (WT) 19Asp Thr
Ser Lys Val Ala Ser 1 5 209PRTArtificial SequenceAnti-CD3 VK CDR3
(WT) 20Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 2110PRTArtificial
SequenceAnti-CD3 VH CDR1 (WT) 21Gly Tyr Thr Phe Thr Arg Tyr Thr Met
His 1 5 10 2217PRTArtificial SequenceAnti-CD3 VH CDR2 (WT) 22Tyr
Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10
15 Asp 2310PRTArtificial SequenceAnti-CD3 VH CDR3 (WT) 23Tyr Tyr
Asp Asp His Tyr Cys Leu Asp Tyr 1 5 10 2410PRTArtificial
SequenceAnti-CD3 VK CDR1 (WT) 24Arg Ala Ser Ser Ser Val Ser Tyr Met
Asn 1 5 10 257PRTArtificial SequenceAnti-CD3 VK CDR2 (WT) 25Asp Thr
Ser Lys Val Ala Ser 1 5 269PRTArtificial SequenceAnti-CD3 VK CDR3
(WT) 26Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 2710PRTArtificial
SequenceAnti-CD3 VH CDR1 (UCHT-1) 27Gly Tyr Ser Phe Thr Gly Tyr Thr
Met Asn 1 5 10 2817PRTArtificial SequenceAnti-CD3 VH CDR2 (UCHT-1)
28Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys 1
5 10 15 Asp 2911PRTArtificial SequenceAnti-CD3 VH CDR3 (UCHT-1)
29Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val 1 5 10
3011PRTArtificial SequenceAnti-CD3 VL CDR1 (UCHT-1) 30Arg Ala Ser
Gln Asp Ile Arg Asn Tyr Leu Asn 1 5 10 317PRTArtificial
SequenceAnti-CD3 VL CDR2 (UCHT-1) 31Tyr Thr Ser Arg Leu Glu Ser 1 5
329PRTArtificial SequenceAnti-CD3 VL CDR3 (UCHT-1) 32Gln Gln Gly
Asn Thr Leu Pro Trp Thr 1 5 338PRTArtificial SequenceAnti-CD3 VH
CDR 1 (L2K) 33Gly Tyr Thr Phe Thr Arg Tyr Thr 1 5 348PRTArtificial
SequenceAnti-CD3 VH CDR 2 (L2K) 34Ile Asn Pro Ser Arg Gly Tyr Thr 1
5 3512PRTArtificial SequenceAnti-CD3 VH CDR 3 (L2K) 35Ala Arg Tyr
Tyr Asp Asp His Tyr Cys Leu Asp Tyr 1 5 10 365PRTArtificial
SequenceAnti-CD3 VL CDR 1 (L2K) 36Ser Ser Val Ser Tyr 1 5
379PRTArtificial SequenceAnti-CD3 VL CDR 3 (L2K) 37Gln Gln Trp Ser
Ser Asn Pro Leu Thr 1 5 388PRTArtificial SequenceAnti-CD3 VH CDR 1
(145-2C11) 38Gly Phe Thr Phe Ser Gly Tyr Gly 1 5 398PRTArtificial
SequenceAnti-CD3 VH CDR 2 (145-2C11) 39Ile Thr Ser Ser Ser Ile Asn
Ile 1 5 409PRTArtificial SequenceAnti-CD3 VH CDR 3 (145-2C11) 40Ala
Arg Phe Asp Trp Asp Lys Asn Tyr 1 5 416PRTArtificial
SequenceAnti-CD3 VL CDR 1 (145-2C11) 41Gln Asp Ile Ser Asn Tyr 1 5
429PRTArtificial SequenceAnti-CD3 VL CDR 3 (145-2C11) 42Gln Gln Tyr
Tyr Asn Tyr Pro Trp Thr 1 5 438PRTArtificial SequenceAnti-HIS VH
CDR1 43Gly Tyr Thr Phe Thr Asp Tyr Tyr 1 5 448PRTArtificial
SequenceAnti-HIS VH CDR2 44Ile Asn Pro Asn Asn Gly Gly Thr 1 5
457PRTArtificial SequenceAnti-HIS VH CDR3 45Glu Ser Gln Ser Gly Ala
Tyr 1 5 4611PRTArtificial SequenceAnti-HIS VL CDR1 46Gln Ser Ile
Val His Ser Asn Gly Asn Thr Tyr 1 5 10 479PRTArtificial
SequenceAnti-HIS VL CDR3 47Phe Gln Gly Ser His Val Pro Phe Thr 1 5
488PRTArtificial SequenceAnti-DIG VH CDR1 48Gly Phe Thr Phe Ser Asp
Tyr Ala 1 5 498PRTArtificial SequenceAnti-DIG VH CDR2 49Ile Asn Ile
Gly Ala Thr Tyr Ala 1 5 506PRTArtificial SequenceAnti-DIG VL CDR1
50Gln Asp Ile Lys Asn Tyr 1 5 51123PRTArtificial
SequenceAnti-HLA-A2 VH 51Gln Val Gln Leu Val Gln Ser Gly Gly Gly
Val Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Val Thr Leu Ser Asp
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Ala Phe Ile Arg Asn Asp Gly Ser Asp Lys Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Lys Thr Val Ser 65 70 75 80 Leu Gln Met Ser Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Gly Glu
Ser Gly Pro Leu Asp Tyr Trp Tyr Phe Asp Leu 100 105 110 Trp Gly Arg
Gly Thr Leu Val Thr Val Ser Ser 115 120 52108PRTArtificial
SequenceAnti-HLA-A2 VL 52Asp Val Val Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln
Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser
Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Phe Pro Leu 85
90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 100 105
53118PRTArtificial SequenceAnti-HLA-Cw6 VH 53Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Ser Phe Ser Trp Phe Asp Val
Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala 115
54108PRTArtificial SequenceAnti-HLA-Cw6 VL 54Asp Ile Glu Leu Thr
Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg
Ile Ser Cys Ser Gly Asp Ala Leu Gly Asp Lys Tyr Ala 20 25 30 Ser
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40
45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln
Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asn
Phe Asp Ser Pro 85 90 95 Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly 100 105 55120PRTArtificial SequenceAnti-EpCAM VH 55Glu Val
Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 1 5 10 15
Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn 20
25 30 Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu
Trp 35 40 45 Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr
Asn Glu Lys 50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys
Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Thr Phe
Glu Asp Ser Ala Val Tyr Phe 85 90 95 Cys Ala Arg Leu Arg Asn Trp
Asp Glu Pro Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr
Val Ser Ser 115 120 56113PRTArtificial SequenceAnti-EpCAM VL 56Glu
Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10
15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro
Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp
Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 100 105 110 Lys
57120PRTArtificial SequenceAnti-HER2 VH 57Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met
Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115
120 58107PRTArtificial SequenceAnti-HER2 VL 58Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr
Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 59119PRTArtificial SequenceAnti-EGFR-1 VH 59Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly 20 25
30 Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu
35 40 45 Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn
Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser
Lys Thr Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Arg Asp Arg Val Thr Gly
Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser
Ser 115 60107PRTArtificial SequenceAnti-EGFR-1 VL 60Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe
Asp His Leu Pro Leu 85 90 95 Ala Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105 61127PRTArtificial SequenceAnti-CEA VH 61Ser Arg
Val Ala Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala 1 5 10 15
Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr 20
25 30 Phe Thr Thr Tyr Thr Ile His Trp Val Arg Gln Arg Pro Gly His
Asp 35 40 45 Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Ser Gly Tyr
Ser Asp Tyr 50 55 60 Asn Gln Asn Phe Lys Gly Lys Thr Thr Leu Thr
Ala Asp Lys Ser Ser 65 70 75 80 Asn Thr Ala Tyr Met Gln Leu Asn Ser
Leu Thr Ser Glu Asp Ser Ala 85 90 95 Val Tyr Tyr Cys Ala Arg Arg
Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr 100 105 110 Trp Phe Ala Tyr Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125
62110PRTArtificial SequenceAnti-CEA VL 62Asp Ile Glu Leu Thr Gln
Ser Pro Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Asn
Val Thr Tyr Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Ala
Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile 35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln
Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr
Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg Ala Asp 100 105 110 63120PRTArtificial SequenceAnti-CD45 VH
63Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg
Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Thr Ser Ser Thr Ile Asn
Phe Thr Pro Ser Leu 50 55 60 Lys Asp Lys Val Phe Ile Ser Arg Asp
Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Lys Val Arg
Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Gly Asn Tyr
Tyr Arg Tyr Gly Asp Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr
Ser Val Thr Val Ser 115 120 64111PRTArtificial SequenceAnti-CD45 VL
64Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr
Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly
Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu
Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala
Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Phe Thr
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110
65121PRTArtificial SequenceVH anti-CD138 65Gln Val Gln Leu Gln Gln
Ser Gly Ser Glu Leu Met Pro Gly Ala Ser 1 5 10 15 Val Lys Ile Ser
Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn Tyr Trp 20 25 30 Ile Glu
Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly 35 40 45
Glu Ile Leu Pro Gly Thr Gly Arg Thr Ile Tyr Asn Glu Lys Phe Lys 50
55 60 Gly Lys Ala Thr Phe Thr Ala Asp Ile Ser Ser Asn Thr Val Gln
Met 65 70 75 80 Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys Ala 85 90 95 Arg Glu Gln Tyr Tyr Gly Asn Phe Tyr Tyr Ala
Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser
115 120 66110PRTArtificial SequenceVL anti-CD138 66Asp Ile Gln Met
Thr Gln Ser Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg
Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Asn Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Glu Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn
Leu Glu Pro 65 70 75 80 Glu Asp Ile Gly Thr Tyr Tyr Cys Gln Gln Tyr
Ser Lys Leu Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg Thr Val 100 105 110 67246PRTArtificial
SequenceAnti-HLA-A2 scFv 67Gln Val Gln Leu Val Gln Ser Gly Gly Gly
Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Val Ser Cys Ala Ala
Ser Gly Val Thr Leu Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Ala Phe Ile Arg
Asn Asp Gly Ser Asp Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Ser 65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Asn Gly Glu Ser Gly Pro Leu Asp Tyr Trp Tyr Phe Asp
Leu 100 105 110 Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
Val Val Met Thr Gln 130 135 140 Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly Asp Arg Val Thr Ile Thr 145 150 155 160 Cys Gln Ala Ser Gln Asp
Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln 165 170 175 Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu 180 185 190 Glu Thr
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 210
215 220 Tyr Cys Gln Gln Tyr Ser Ser Phe Pro Leu Thr Phe Gly Gly Gly
Thr 225 230 235 240 Lys Val Asp Ile Lys Arg 245 68245PRTArtificial
SequenceAnti-HLA-Cw6 scFv 68Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser
Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Tyr Ser Phe Ser Trp Phe Asp Val Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser Ala Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly
Gly Gly Ser Asp Ile Glu Leu Thr Gln Pro 130 135 140 Pro Ser Val Ser
Val Ala Pro Gly Gln Thr Ala Arg Ile Ser Cys Ser 145 150 155 160 Gly
Asp Ala Leu Gly Asp Lys Tyr Ala Ser Trp Tyr Gln Gln Lys Pro 165 170
175 Gly Gln Ala Pro Val Leu Val Ile Tyr Asp Asp Ser Asp Arg Pro Ser
180 185 190 Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr
Ala Thr 195 200 205 Leu Thr Ile Ser Gly Thr Gln Ala Glu Asp Glu Ala
Asp Tyr Tyr Cys 210 215 220 Gln Ser Tyr Asp Asn Phe Asp Ser Pro Val
Phe Gly Gly Gly Thr Lys 225 230 235 240 Leu Thr Val Leu Gly 245
69248PRTArtificial SequenceAnti-EpCAM scFv 69Glu Val Gln Leu Leu
Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 1 5 10 15 Thr Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn 20 25 30 Tyr
Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp 35 40
45 Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys
50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser
Ala Val Tyr Phe 85 90 95 Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro
Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly
Ser Glu Leu Val Met Thr Gln Ser Pro Ser 130 135 140 Ser Leu Thr Val
Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser 145 150 155 160 Ser
Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp 165 170
175 Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala
180 185 190 Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser
Gly Ser 195 200 205 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln
Ala Glu Asp Leu 210 215 220 Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser
Tyr Pro Leu Thr Phe Gly 225 230 235 240 Ala Gly Thr Lys Leu Glu Ile
Lys 245 70242PRTArtificial SequenceAnti-HER2 scFv 70Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr
Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly
Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Ser Leu Ser
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 145 150 155 160
Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly 165
170 175 Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser
Gly 180 185 190 Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp
Phe Thr Leu 195 200 205 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln 210 215 220 Gln His Tyr Thr Thr Pro Pro Thr Phe
Gly Gln Gly Thr Lys Val Glu 225 230 235 240 Ile Lys
71241PRTArtificial SequenceAnti-EGFR(1) scFv 71Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly 20 25 30 Asp
Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu 35 40
45 Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser
50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser Lys Thr
Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Ile Tyr Tyr 85 90 95 Cys Val Arg Asp Arg Val Thr Gly Ala Phe
Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser
Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser 145 150 155 160 Gln
Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170
175 Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val
180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Phe Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr
Phe Cys Gln His 210 215 220 Phe Asp His Leu Pro Leu Ala Phe Gly Gly
Gly Thr Lys Val Glu Ile 225 230 235 240 Lys 72252PRTArtificial
SequenceAnti-CEA scFv 72Ser Arg Val Ala Gln Val Gln Leu Gln Gln Ser
Gly Ala Glu Leu Ala 1 5 10 15 Arg Pro Gly Ala Ser Val Lys Met Ser
Cys Lys Ala Ser Gly Tyr Thr 20 25 30 Phe Thr Thr Tyr Thr Ile His
Trp Val Arg Gln Arg Pro Gly His Asp 35 40 45 Leu Glu Trp Ile Gly
Tyr Ile Asn Pro Ser Ser Gly Tyr Ser Asp Tyr 50 55 60 Asn Gln Asn
Phe Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser 65 70 75 80 Asn
Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala 85 90
95 Val Tyr Tyr Cys Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr
100 105 110 Trp Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Asp Ile 130 135 140 Glu Leu Thr Gln Ser Pro Lys Phe Met Ser
Thr Ser Val Gly Asp Arg 145 150 155 160 Val Asn Val Thr Tyr Lys Ala
Ser Gln Asn Val Gly Thr Asn Val Ala 165 170 175 Trp Phe Gln Gln Lys
Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Ser 180 185 190 Ala Ser Tyr
Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 195 200 205 Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp 210 215
220 Leu Ala Glu Tyr Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe
225 230 235 240 Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp 245
250 73250PRTArtificial SequenceAnti-CD45 scFv 73Asp Ile Val Leu Thr
Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala
Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly
Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40
45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn
Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys
Gln His Ser Arg 85 90 95 Glu Leu Pro Phe Thr Phe Gly Ser Gly Thr
Lys Leu Glu Ile Lys Lys 100 105 110 Ile Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Ser Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro 130 135 140 Gly Gly Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser 145 150 155 160 Arg
Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 165 170
175 Trp Ile Gly Glu Ile Asn Pro Thr Ser Ser Thr Ile Asn Phe Thr Pro
180 185 190 Ser Leu Lys Asp Lys Val Phe Ile Ser Arg Asp Asn Ala Lys
Asn Thr 195 200 205 Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp
Thr Ala Leu Tyr 210 215 220 Tyr Cys Ala Arg Gly Asn Tyr Tyr Arg Tyr
Gly Asp Ala Met Asp Tyr 225 230 235 240 Trp Gly Gln Gly Thr Ser Val
Thr Val Ser 245 250 74246PRTArtificial SequenceAnti-CD138 scFv
74Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Met Pro Gly Ala Ser 1
5 10 15 Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn Tyr
Trp 20 25 30 Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu
Trp Ile Gly 35 40 45 Glu Ile Leu Pro Gly Thr Gly Arg Thr Ile Tyr
Asn Glu Lys Phe Lys 50 55 60 Gly Lys Ala Thr Phe Thr Ala Asp Ile
Ser Ser Asn Thr Val Gln Met 65 70 75 80 Gln Leu Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Gln Tyr Tyr
Gly Asn Phe Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr
Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Thr 130 135
140 Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Ser
145 150 155 160 Ala Ser Gln Gly Ile Asn Asn Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro 165 170 175 Asp Gly Thr Val Glu Leu Leu Ile Tyr Tyr Thr
Ser Thr Leu Gln Ser 180 185 190 Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Tyr Ser 195 200 205 Leu Thr Ile Ser Asn Leu Glu
Pro Glu Asp Ile Gly Thr Tyr Tyr Cys 210 215 220 Gln Gln Tyr Ser Lys
Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu 225 230 235 240 Glu Ile
Lys Arg Thr Val 245 758PRTArtificial SequenceAnti-HLA-A2 VH CDR1
75Gly Val Thr Leu Ser Asp Tyr Gly 1 5 768PRTArtificial
SequenceAnti-HLA-A2 VH CDR2 76Ile Arg Asn Asp Gly Ser Asp Lys 1 5
7716PRTArtificial SequenceAnti-HLA-A2 VH CDR3 77Ala Lys Asn Gly Glu
Ser Gly Pro Leu Asp Tyr Trp Tyr Phe Asp Leu 1 5 10 15
786PRTArtificial SequenceAnti-HLA-A2 VL CDR1 78Gln Asp Ile Ser Asn
Tyr 1 5 799PRTArtificial SequenceAnti-HLA-A2 VL CDR3 79Gln Gln Tyr
Ser Ser Phe Pro Leu Thr 1 5 808PRTArtificial SequenceAnti-HLA-Cw6
VH CDR1 80Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 818PRTArtificial
SequenceAnti-HLA-Cw6 VH CDR2 81Ile Ser Gly Ser Gly Gly Ser Thr 1 5
8210PRTArtificial SequenceAnti-HLA-Cw6 VH CDR3 82Ala Arg Tyr Ser
Phe Ser Trp Phe Asp Val 1 5 10 836PRTArtificial
SequenceAnti-HLA-Cw6 VL CDR1 83Ala Leu Gly Asp Lys Tyr 1 5
8410PRTArtificial SequenceAnti-HLA-Cw6 VL CDR3 84Gln Ser Tyr Asp
Asn Phe Asp Ser Pro Val 1 5 10 858PRTArtificial SequenceAnti-EpCAM
CDR1 VH 85Gly Tyr Ala Phe Thr Asn Tyr Trp 1 5 868PRTArtificial
SequenceAnti-EpCAM CDR2 VH 86Ile Phe Pro Gly Ser Gly Asn Ile 1 5
8712PRTArtificial SequenceAnti-EpCAM CDR3 VH 87Ala Arg Leu Arg Asn
Trp Asp Glu Pro Met Asp Tyr 1 5 10 8812PRTArtificial
SequenceAnti-EpCAM CDR1 VL 88Gln Ser Leu Leu Asn Ser Gly Asn Gln
Lys Asn Tyr 1 5 10 899PRTArtificial SequenceAnti-EpCAM CDR3 VL
89Gln Asn Asp Tyr Ser Tyr Pro Leu Thr 1 5 908PRTArtificial
SequenceAnti-HER2 VH CDR1 90Gly Phe Asn Ile Lys Asp Thr Tyr 1 5
918PRTArtificial SequenceAnti-HER2 VH CDR2 91Ile Tyr Pro Thr Asn
Gly Tyr Thr 1 5 9213PRTArtificial SequenceAnti-HER2 VH CDR3 92Ser
Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 1 5 10
936PRTArtificial SequenceAnti-HER2 VL CDR1 93Gln Asp Val Asn Thr
Ala 1 5 949PRTArtificial SequenceAnti-HER2 VL CDR3 94Gln Gln His
Tyr Thr Thr Pro Pro Thr 1 5 9510PRTArtificial SequenceAnti-EGFR-1
VH CDR1 95Gly Gly Ser Val Ser Ser Gly Asp Tyr Tyr 1 5 10
967PRTArtificial SequenceAnti-EGFR-1 VH CDR2 96Ile Tyr Tyr Ser Gly
Asn Thr 1 5 9711PRTArtificial SequenceAnti-EGFR-1 VH CDR3 97Val Arg
Asp Arg Val Thr Gly Ala Phe Asp Ile 1 5 10 986PRTArtificial
SequenceAnti-EGFR-1 VL CDR1 98Gln Asp Ile Ser Asn Tyr 1 5
999PRTArtificial SequenceAnti-EGFR-1 VL CDR3 99Gln His Phe Asp His
Leu Pro Leu Ala 1 5 1008PRTArtificial SequenceAnti-CEA VH CDR1
100Gly Tyr Thr Phe Thr Thr Tyr Thr 1 5 1018PRTArtificial
SequenceAnti-CEA VH CDR2 101Ile Asn Pro Ser Ser Gly Tyr Ser 1 5
10216PRTArtificial SequenceAnti-CEA VH CDR3 102Ala Arg Arg Ala Asp
Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr 1 5 10 15
1036PRTArtificial SequenceAnti-CEA VL CDR1 103Gln Asn Val Gly Thr
Asn 1 5 1049PRTArtificial SequenceAnti-CEA VL CDR3 104Gln Gln Tyr
His Thr Tyr Pro Leu Thr 1 5 1055PRTArtificial SequenceAnti-CD45 VH
CDR1 105Gly Phe Asp Phe Ser 1 5 1069PRTArtificial SequenceAnti-CD45
VH CDR2 106Glu Ile Asn Pro Thr Ser Ser Thr Ile 1 5
10710PRTArtificial SequenceAnti-CD45 VL CDR1 107Lys Ser Val Ser Thr
Ser Gly Tyr Ser Tyr 1 5 10 1089PRTArtificial SequenceAnti-CD45 VL
CDR3 108Gln His Ser Arg Glu Leu Pro Phe Thr 1 5 1098PRTArtificial
SequenceAnti-CD138 VH CDR1 109Gly Tyr Thr Phe Ser Asn Tyr Trp 1 5
1108PRTArtificial SequenceAnti-CD138 VH CDR2 110Ile Leu Pro Gly Thr
Gly Arg Thr 1 5 11115PRTArtificial SequenceAnti-CD138 VH CDR3
111Ala Arg Glu Gln Tyr Tyr Gly Asn Phe Tyr Tyr Ala Met Asp Tyr 1 5
10 15 1126PRTArtificial SequenceAnti-CD138 VL CDR1 112Gln Gly Ile
Asn Asn Tyr 1 5 1139PRTArtificial SequenceAnti-CD138 VL CDR3 113Gln
Gln Tyr Ser Lys Leu Pro Arg Thr 1 5 114388PRTArtificial
SequencepelB-CD3VL-scFvEPCAM(VH-VL)-6His 114Met Lys Tyr Leu Leu Pro
Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala
Met Ala Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser
Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45
Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser 50
55 60 Pro Lys Arg Trp Ile Tyr
Asp Thr Ser Lys Val Ala Ser Gly Val Pro 65 70 75 80 Tyr Arg Phe Ser
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser
Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110
Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115
120 125 Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly
Ala 130 135 140 Glu Leu Val Arg Pro Gly Thr Ser Val Lys Ile Ser Cys
Lys Ala Ser 145 150 155 160 Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly
Trp Val Lys Gln Arg Pro 165 170 175 Gly His Gly Leu Glu Trp Ile Gly
Asp Ile Phe Pro Gly Ser Gly Asn 180 185 190 Ile His Tyr Asn Glu Lys
Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp 195 200 205 Lys Ser Ser Ser
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu 210 215 220 Asp Ser
Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro 225 230 235
240 Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly
245 250 255 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Leu Val 260 265 270 Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala
Gly Glu Lys Val 275 280 285 Thr Met Ser Cys Lys Ser Ser Gln Ser Leu
Leu Asn Ser Gly Asn Gln 290 295 300 Lys Asn Tyr Leu Thr Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro Lys 305 310 315 320 Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg 325 330 335 Phe Thr Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 340 345 350 Val
Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser 355 360
365 Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys His His
370 375 380 His His His His 385 115404PRTArtificial
SequencepelB-CD3VH-scFvHer2-6HIS 115Met Lys Tyr Leu Leu Pro Thr Ala
Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu 20 25 30 Leu Ala Arg Pro
Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly 35 40 45 Tyr Thr
Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly 50 55 60
Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr 65
70 75 80 Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr
Asp Lys 85 90 95 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp 100 105 110 Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr
Asp Asp His Tyr Cys Leu 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Thr
Leu Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 145 150 155 160 Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 165 170 175 Ser
Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp 180 185
190 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr
195 200 205 Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly
Arg Phe 210 215 220 Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
Leu Gln Met Asn 225 230 235 240 Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ser Arg Trp Gly 245 250 255 Gly Asp Gly Phe Tyr Ala Met
Asp Tyr Trp Gly Gln Gly Thr Leu Val 260 265 270 Thr Val Ser Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 275 280 285 Gly Gly Ser
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 290 295 300 Ser
Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 305 310
315 320 Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys 325 330 335 Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
Pro Ser Arg 340 345 350 Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser 355 360 365 Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln His Tyr Thr 370 375 380 Thr Pro Pro Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys His His 385 390 395 400 His His His His
116403PRTArtificial SequencepelB-CD3VH-scFvEGFR(1)-6HIS 116Met Lys
Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15
Ala Gln Pro Ala Met Ala Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu 20
25 30 Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser
Gly 35 40 45 Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln
Arg Pro Gly 50 55 60 Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro
Ser Arg Gly Tyr Thr 65 70 75 80 Asn Tyr Asn Gln Lys Phe Lys Asp Lys
Ala Thr Leu Thr Thr Asp Lys 85 90 95 Ser Ser Ser Thr Ala Tyr Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp 100 105 110 Ser Ala Val Tyr Tyr
Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu 115 120 125 Asp Tyr Trp
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu 145 150
155 160 Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser
Leu 165 170 175 Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly Asp
Tyr Tyr Trp 180 185 190 Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu
Glu Trp Ile Gly His 195 200 205 Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr
Asn Pro Ser Leu Lys Ser Arg 210 215 220 Leu Thr Ile Ser Ile Asp Thr
Ser Lys Thr Gln Phe Ser Leu Lys Leu 225 230 235 240 Ser Ser Val Thr
Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Val Arg Asp 245 250 255 Arg Val
Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr 260 265 270
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275
280 285 Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser 290 295 300 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln
Asp Ile Ser 305 310 315 320 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu 325 330 335 Leu Ile Tyr Asp Ala Ser Asn Leu
Glu Thr Gly Val Pro Ser Arg Phe 340 345 350 Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu 355 360 365 Gln Pro Glu Asp
Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu 370 375 380 Pro Leu
Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys His His His 385 390 395
400 His His His 117414PRTArtificial SequencepelB-CD3VH-scFvCEA-6HIS
117Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15 Ala Gln Pro Ala Met Ala Asp Ile Lys Leu Gln Gln Ser Gly
Ala Glu 20 25 30 Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys
Lys Thr Ser Gly 35 40 45 Tyr Thr Phe Thr Arg Tyr Thr Met His Trp
Val Lys Gln Arg Pro Gly 50 55 60 Gln Gly Leu Glu Trp Ile Gly Tyr
Ile Asn Pro Ser Arg Gly Tyr Thr 65 70 75 80 Asn Tyr Asn Gln Lys Phe
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys 85 90 95 Ser Ser Ser Thr
Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp 100 105 110 Ser Ala
Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu 115 120 125
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly 130
135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Arg Val
Ala 145 150 155 160 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala
Arg Pro Gly Ala 165 170 175 Ser Val Lys Met Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Thr Tyr 180 185 190 Thr Ile His Trp Val Arg Gln Arg
Pro Gly His Asp Leu Glu Trp Ile 195 200 205 Gly Tyr Ile Asn Pro Ser
Ser Gly Tyr Ser Asp Tyr Asn Gln Asn Phe 210 215 220 Lys Gly Lys Thr
Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 225 230 235 240 Met
Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 245 250
255 Ala Arg Arg Ala Asp Tyr Gly Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr
260 265 270 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
Gly Ser 275 280 285 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile
Glu Leu Thr Gln 290 295 300 Ser Pro Lys Phe Met Ser Thr Ser Val Gly
Asp Arg Val Asn Val Thr 305 310 315 320 Tyr Lys Ala Ser Gln Asn Val
Gly Thr Asn Val Ala Trp Phe Gln Gln 325 330 335 Lys Pro Gly Gln Ser
Pro Lys Val Leu Ile Tyr Ser Ala Ser Tyr Arg 340 345 350 Tyr Ser Gly
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 355 360 365 Phe
Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr 370 375
380 Phe Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr
385 390 395 400 Lys Leu Glu Ile Lys Arg Ala Asp His His His His His
His 405 410 118392PRTArtificial SequencepelB-CD3VL-scFvCEA-6HIS
118Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15 Ala Gln Pro Ala Met Ala Asp Ile Gln Leu Thr Gln Ser Pro
Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr
Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln
Gln Lys Ser Gly Thr Ser 50 55 60 Pro Lys Arg Trp Ile Tyr Asp Thr
Ser Lys Val Ala Ser Gly Val Pro 65 70 75 80 Tyr Arg Phe Ser Gly Ser
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Met Glu
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser
Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125
Ser Gly Gly Gly Gly Ser Ser Arg Val Ala Gln Val Gln Leu Gln Gln 130
135 140 Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser
Cys 145 150 155 160 Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Thr Ile
His Trp Val Arg 165 170 175 Gln Arg Pro Gly His Asp Leu Glu Trp Ile
Gly Tyr Ile Asn Pro Ser 180 185 190 Ser Gly Tyr Ser Asp Tyr Asn Gln
Asn Phe Lys Gly Lys Thr Thr Leu 195 200 205 Thr Ala Asp Lys Ser Ser
Asn Thr Ala Tyr Met Gln Leu Asn Ser Leu 210 215 220 Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys Ala Arg Arg Ala Asp Tyr 225 230 235 240 Gly
Asn Tyr Glu Tyr Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Thr 245 250
255 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
260 265 270 Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Lys Phe
Met Ser 275 280 285 Thr Ser Val Gly Asp Arg Val Asn Val Thr Tyr Lys
Ala Ser Gln Asn 290 295 300 Val Gly Thr Asn Val Ala Trp Phe Gln Gln
Lys Pro Gly Gln Ser Pro 305 310 315 320 Lys Val Leu Ile Tyr Ser Ala
Ser Tyr Arg Tyr Ser Gly Val Pro Asp 325 330 335 Arg Phe Thr Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 340 345 350 Asn Val Gln
Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr His 355 360 365 Thr
Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 370 375
380 Ala Asp His His His His His His 385 390 119414PRTArtificial
SequencepelB-(aCD3)VH-scFvHLA-Cw6-myc-6His 119Met Lys Tyr Leu Leu
Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro
Ala Met Ala Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu 20 25 30 Leu
Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly 35 40
45 Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly
50 55 60 Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly
Tyr Thr 65 70 75 80 Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu
Thr Thr Asp Lys 85 90 95 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser
Ser Leu Thr Ser Glu Asp 100 105 110 Ser Ala Val Tyr Tyr Cys Ala Arg
Tyr Tyr Asp Asp His Tyr Cys Leu 115 120 125 Asp Tyr Trp Gly Gln Gly
Thr Thr Leu Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly
Gly Gly Ser Gly Gly Glu Val Gln Leu Val Glu Ser 145 150 155 160 Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 165 170
175 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln
180 185 190 Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly
Ser Gly 195 200 205 Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser 210 215 220 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg 225 230 235 240 Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala Arg Tyr Ser Phe Ser Trp 245 250 255 Phe Asp Val Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gly 260 265 270 Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly
Ser Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro 290 295
300 Gly Gln Thr Ala Arg Ile Ser Cys Ser Gly Asp Ala Leu Gly Asp Lys
305 310 315 320 Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Val Leu Val 325 330
335 Ile Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser
340 345 350 Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly
Thr Gln 355 360 365 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Asn Phe Asp 370 375 380 Ser Pro Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Glu Gln 385 390 395 400 Lys Leu Ile Ser Glu Glu Asp
Leu His His His His His His 405 410 120391PRTArtificial
SequencepelB-CD3VL-scFvCD138-6His 120Met Lys Tyr Leu Leu Pro Thr
Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met
Ala Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala
Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser
Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser 50 55
60 Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro
65 70 75 80 Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
Thr Ile 85 90 95 Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gln Val Gln Leu Gln 130 135 140 Gln Ser Gly Ser Glu Leu
Met Pro Gly Ala Ser Val Lys Ile Ser Cys 145 150 155 160 Lys Ala Thr
Gly Tyr Thr Phe Ser Asn Tyr Trp Ile Glu Trp Val Lys 165 170 175 Gln
Arg Pro Gly His Gly Leu Glu Trp Ile Gly Glu Ile Leu Pro Gly 180 185
190 Thr Gly Arg Thr Ile Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Phe
195 200 205 Thr Ala Asp Ile Ser Ser Asn Thr Val Gln Met Gln Leu Ser
Ser Leu 210 215 220 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
Glu Gln Tyr Tyr 225 230 235 240 Gly Asn Phe Tyr Tyr Ala Met Asp Tyr
Trp Gly Gln Gly Thr Ser Val 245 250 255 Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly 260 265 270 Gly Gly Ser Asp Ile
Gln Met Thr Gln Ser Thr Ser Ser Leu Ser Ala 275 280 285 Ser Leu Gly
Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile 290 295 300 Asn
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Glu 305 310
315 320 Leu Leu Ile Tyr Tyr Thr Ser Thr Leu Gln Ser Gly Val Pro Ser
Arg 325 330 335 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
Ile Ser Asn 340 345 350 Leu Glu Pro Glu Asp Ile Gly Thr Tyr Tyr Cys
Gln Gln Tyr Ser Lys 355 360 365 Leu Pro Arg Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg Thr 370 375 380 Val His His His His His His
385 390 121408PRTArtificial SequencepelB-CD3VH-scFvCD138-6His
121Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15 Ala Gln Pro Ala Met Ala Asp Ile Lys Leu Gln Gln Ser Gly
Ala Glu 20 25 30 Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys
Lys Thr Ser Gly 35 40 45 Tyr Thr Phe Thr Arg Tyr Thr Met His Trp
Val Lys Gln Arg Pro Gly 50 55 60 Gln Gly Leu Glu Trp Ile Gly Tyr
Ile Asn Pro Ser Arg Gly Tyr Thr 65 70 75 80 Asn Tyr Asn Gln Lys Phe
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys 85 90 95 Ser Ser Ser Thr
Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp 100 105 110 Ser Ala
Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu 115 120 125
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly 130
135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln
Leu 145 150 155 160 Gln Gln Ser Gly Ser Glu Leu Met Pro Gly Ala Ser
Val Lys Ile Ser 165 170 175 Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn
Tyr Trp Ile Glu Trp Val 180 185 190 Lys Gln Arg Pro Gly His Gly Leu
Glu Trp Ile Gly Glu Ile Leu Pro 195 200 205 Gly Thr Gly Arg Thr Ile
Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr 210 215 220 Phe Thr Ala Asp
Ile Ser Ser Asn Thr Val Gln Met Gln Leu Ser Ser 225 230 235 240 Leu
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Gln Tyr 245 250
255 Tyr Gly Asn Phe Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
260 265 270 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly 275 280 285 Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Thr
Ser Ser Leu Ser 290 295 300 Ala Ser Leu Gly Asp Arg Val Thr Ile Ser
Cys Ser Ala Ser Gln Gly 305 310 315 320 Ile Asn Asn Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Asp Gly Thr Val 325 330 335 Glu Leu Leu Ile Tyr
Tyr Thr Ser Thr Leu Gln Ser Gly Val Pro Ser 340 345 350 Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser 355 360 365 Asn
Leu Glu Pro Glu Asp Ile Gly Thr Tyr Tyr Cys Gln Gln Tyr Ser 370 375
380 Lys Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
385 390 395 400 Thr Val His His His His His His 405
122404PRTArtificial SequencepelB-(aHis)VH-scFvHLA-A2(VH-VL)-myc
122Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15 Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Gln Ser Gly
Pro Glu 20 25 30 Asp Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys
Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Asp Tyr Tyr Met Asn Trp
Val Lys Gln Ser Pro Gly 50 55 60 Lys Gly Leu Glu Trp Ile Gly Asp
Ile Asn Pro Asn Asn Gly Gly Thr 65 70 75 80 Ser Tyr Asn Gln Lys Phe
Lys Gly Arg Ala Thr Leu Thr Val Asp Lys 85 90 95 Ser Ser Ser Thr
Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp 100 105 110 Ser Ser
Val Tyr Tyr Cys Glu Ser Gln Ser Gly Ala Tyr Trp Gly Gln 115 120 125
Gly Thr Thr Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly 130
135 140 Gly Ser Gly Gly Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val
Val 145 150 155 160 Gln Pro Gly Gly Ser Leu Arg Val Ser Cys Ala Ala
Ser Gly Val Thr 165 170 175 Leu Ser Asp Tyr Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly 180 185 190 Leu Glu Trp Met Ala Phe Ile Arg
Asn Asp Gly Ser Asp Lys Tyr Tyr 195 200 205 Ala Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 210 215 220 Lys Thr Val Ser
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 225 230 235 240 Val
Tyr Tyr Cys Ala Lys Asn Gly Glu Ser Gly Pro Leu Asp Tyr Trp 245 250
255 Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly
260 265 270 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asp Val 275 280 285 Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg 290 295 300 Val Thr Ile Thr Cys Gln Ala Ser Gln Asp
Ile Ser Asn Tyr Leu Asn 305 310 315 320 Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr Asp 325 330 335 Ala Ser Asn Leu Glu
Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 340 345 350 Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp 355 360 365 Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Phe Pro Leu Thr Phe 370 375
380 Gly Gly Gly Thr Lys Val Asp Ile Lys Arg Glu Gln Lys Leu Ile Ser
385 390 395 400 Glu Glu Asp Leu 123410PRTArtificial
SequencepelB-(aHis)VL-scFvCD45(VL-VH)-myc 123Met Lys Tyr Leu Leu
Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro
Ala Met Ala Asp Tyr Lys Asp Ile Leu Met Thr Gln Thr 20 25 30 Pro
Ser Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys 35 40
45 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu
50 55 60 Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
Tyr Lys 65 70 75 80 Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser Gly 85 90 95 Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp 100 105 110 Leu Gly Val Tyr Tyr Cys Phe Gln
Gly Ser His Val Pro Phe Thr Phe 115 120 125 Gly Ser Gly Thr Lys Leu
Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser
Gly Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser 145 150 155 160 Leu
Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser 165 170
175 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln
180 185 190 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser
Asn Leu 195 200 205 Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp 210 215 220 Phe Thr Leu Asn Ile His Pro Val Glu Glu
Glu Asp Ala Ala Thr Tyr 225 230 235 240 Tyr Cys Gln His Ser Arg Glu
Leu Pro Phe Thr Phe Gly Ser Gly Thr 245 250 255 Lys Leu Glu Ile Lys
Lys Ile Ser Gly Gly Gly Gly Ser Gly Gly Gly 260 265 270 Gly Ser Gly
Gly Gly Gly Ser Ser Gln Val Gln Leu Val Glu Ser Gly 275 280 285 Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala 290 295
300 Ser Gly Phe Asp Phe Ser Arg Tyr Trp Met Ser Trp Val Arg Gln Ala
305 310 315 320 Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro
Thr Ser Ser 325 330 335 Thr Ile Asn Phe Thr Pro Ser Leu Lys Asp Lys
Val Phe Ile Ser Arg 340 345 350 Asp Asn Ala Lys Asn Thr Leu Tyr Leu
Gln Met Ser Lys Val Arg Ser 355 360 365 Glu Asp Thr Ala Leu Tyr Tyr
Cys Ala Arg Gly Asn Tyr Tyr Arg Tyr 370 375 380 Gly Asp Ala Met Asp
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 385 390 395 400 Glu Gln
Lys Leu Ile Ser Glu Glu Asp Leu 405 410 124415PRTArtificial
SequencepelB-(aCD3)VH-scFvHLA-A2(VH-VL)-myc-6His 124Met Lys Tyr Leu
Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln
Pro Ala Met Ala Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu 20 25 30
Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly 35
40 45 Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro
Gly 50 55 60 Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg
Gly Tyr Thr 65 70 75 80 Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr
Leu Thr Thr Asp Lys 85 90 95 Ser Ser Ser Thr Ala Tyr Met Gln Leu
Ser Ser Leu Thr Ser Glu Asp 100 105 110 Ser Ala Val Tyr Tyr Cys Ala
Arg Tyr Tyr Asp Asp His Tyr Cys Leu 115 120 125 Asp Tyr Trp Gly Gln
Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gln Val Gln Leu Val Gln Ser 145 150 155 160
Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Val Ser Cys Ala 165
170 175 Ala Ser Gly Val Thr Leu Ser Asp Tyr Gly Met His Trp Val Arg
Gln 180 185 190 Ala Pro Gly Lys Gly Leu Glu Trp Met Ala Phe Ile Arg
Asn Asp Gly 195 200 205 Ser Asp Lys Tyr Tyr Ala Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser 210 215 220 Arg Asp Asn Ser Lys Lys Thr Val Ser
Leu Gln Met Ser Ser Leu Arg 225 230 235 240 Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Lys Asn Gly Glu Ser Gly 245 250 255 Pro Leu Asp Tyr
Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val 260 265 270 Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 275 280 285
Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 290
295 300 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp
Ile 305 310 315 320 Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys 325 330 335 Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu
Thr Gly Val Pro Ser Arg 340 345 350 Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser Ser 355 360 365 Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser 370 375 380 Phe Pro Leu Thr
Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg Glu 385 390 395 400 Gln
Lys Leu Ile Ser Glu Glu Asp Leu His His His His His His 405 410 415
125406PRTArtificial SequencepelB-(aCD3)VL-scFvCD45(VL-VH)-myc-6His
125Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15 Ala Gln Pro Ala Met Ala Asp Ile Gln Leu Thr Gln Ser Pro
Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr
Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln
Gln Lys Ser Gly Thr Ser 50 55 60 Pro Lys Arg Trp Ile Tyr Asp Thr
Ser Lys Val Ala Ser Gly Val Pro 65 70 75 80 Tyr Arg Phe Ser Gly Ser
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Met Glu
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser
Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Val Leu
130
135 140 Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala
Thr 145 150 155 160 Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser
Gly Tyr Ser Tyr 165 170 175 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro Lys Leu Leu Ile 180 185 190 Tyr Leu Ala Ser Asn Leu Glu Ser
Gly Val Pro Ala Arg Phe Ser Gly 195 200 205 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Asn Ile His Pro Val Glu Glu 210 215 220 Glu Asp Ala Ala
Thr Tyr Tyr Cys Gln His Ser Arg Glu Leu Pro Phe 225 230 235 240 Thr
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Lys Ile Ser Gly Gly 245 250
255 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gln Val
260 265 270 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
Ser Leu 275 280 285 Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser
Arg Tyr Trp Met 290 295 300 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Ile Gly Glu 305 310 315 320 Ile Asn Pro Thr Ser Ser Thr
Ile Asn Phe Thr Pro Ser Leu Lys Asp 325 330 335 Lys Val Phe Ile Ser
Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 340 345 350 Met Ser Lys
Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Arg 355 360 365 Gly
Asn Tyr Tyr Arg Tyr Gly Asp Ala Met Asp Tyr Trp Gly Gln Gly 370 375
380 Thr Ser Val Thr Val Ser Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
385 390 395 400 His His His His His His 405 126415PRTArtificial
SequencepelB-VHaDIG-scFvEGFR-FLAG-6HIS 126Met Lys Tyr Leu Leu Pro
Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala
Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly 20 25 30 Leu Val
Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Val Ser Gly 35 40 45
Phe Thr Phe Ser Asp Tyr Ala Met Ser Trp Ile Arg Gln Thr Pro Glu 50
55 60 Asn Arg Leu Glu Trp Val Ala Ser Ile Asn Ile Gly Ala Thr Tyr
Ala 65 70 75 80 Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn 85 90 95 Ala Lys Asn Thr Leu Phe Leu Gln Met Ser Ser
Leu Gly Ser Glu Asp 100 105 110 Thr Ala Met Tyr Tyr Cys Ala Arg Pro
Gly Ser Pro Tyr Glu Tyr Asp 115 120 125 Lys Ala Tyr Tyr Ser Met Ala
Tyr Trp Gly Pro Gly Thr Ser Val Thr 130 135 140 Val Ser Ser Ala Lys
Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 165 170 175
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly 180
185 190 Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu
Glu 195 200 205 Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr
Asn Pro Ser 210 215 220 Leu Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr
Ser Lys Thr Gln Phe 225 230 235 240 Ser Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Ile Tyr Tyr 245 250 255 Cys Val Arg Asp Arg Val
Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly 260 265 270 Thr Met Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 275 280 285 Ser Gly
Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 290 295 300
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser 305
310 315 320 Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys 325 330 335 Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu
Glu Thr Gly Val 340 345 350 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Phe Thr 355 360 365 Ile Ser Ser Leu Gln Pro Glu Asp
Ile Ala Thr Tyr Phe Cys Gln His 370 375 380 Phe Asp His Leu Pro Leu
Ala Phe Gly Gly Gly Thr Lys Val Glu Ile 385 390 395 400 Lys Asp Tyr
Lys Asp Asp Asp Asp Lys His His His His His His 405 410 415
127412PRTArtificial SequencepelB-VLaDIG-scFvEpCAM-myc-6HIS 127Met
Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10
15 Ala Gln Pro Ala Met Ala Asp Val Gln Met Thr Gln Ser Thr Ser Ser
20 25 30 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
Ala Ser 35 40 45 Gln Asp Ile Lys Asn Tyr Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Gly 50 55 60 Thr Val Lys Leu Leu Ile Tyr Tyr Ser Ser
Thr Leu Leu Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Arg Gly
Ser Gly Thr Asp Phe Ser Leu Thr 85 90 95 Ile Thr Asn Leu Glu Arg
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 Ser Ile Thr Leu
Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Gly Gly Ser Gly 130 135 140
Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu 145
150 155 160 Val Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr 165 170 175 Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln
Arg Pro Gly His 180 185 190 Gly Leu Glu Trp Ile Gly Asp Ile Phe Pro
Gly Ser Gly Asn Ile His 195 200 205 Tyr Asn Glu Lys Phe Lys Gly Lys
Ala Thr Leu Thr Ala Asp Lys Ser 210 215 220 Ser Ser Thr Ala Tyr Met
Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser 225 230 235 240 Ala Val Tyr
Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp 245 250 255 Tyr
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly 260 265
270 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr
275 280 285 Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val
Thr Met 290 295 300 Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly
Asn Gln Lys Asn 305 310 315 320 Tyr Leu Thr Trp Tyr Gln Gln Lys Pro
Gly Gln Pro Pro Lys Leu Leu 325 330 335 Ile Tyr Trp Ala Ser Thr Arg
Glu Ser Gly Val Pro Asp Arg Phe Thr 340 345 350 Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln 355 360 365 Ala Glu Asp
Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro 370 375 380 Leu
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Glu Gln Lys Leu 385 390
395 400 Ile Ser Glu Glu Asp Leu His His His His His His 405 410
128400PRTArtificial SequencepelB-murineCD3VH-scFvEpCAM-6His 128Met
Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10
15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly
20 25 30 Leu Val Gln Pro Gly Lys Ser Leu Lys Leu Ser Cys Glu Ala
Ser Gly 35 40 45 Phe Thr Phe Ser Gly Tyr Gly Met His Trp Val Arg
Gln Ala Pro Gly 50 55 60 Arg Gly Leu Glu Ser Val Ala Tyr Ile Thr
Ser Ser Ser Ile Asn Ile 65 70 75 80 Lys Tyr Ala Asp Ala Val Lys Gly
Arg Phe Thr Val Ser Arg Asp Asn 85 90 95 Ala Lys Asn Leu Leu Phe
Leu Gln Met Asn Ile Leu Lys Ser Glu Asp 100 105 110 Thr Ala Met Tyr
Tyr Cys Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp 115 120 125 Gly Gln
Gly Thr Met Val Thr Val Ser Ser Ala Lys Thr Ser Ser Gly 130 135 140
Gly Gly Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg 145
150 155 160 Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
Ala Phe 165 170 175 Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro
Gly His Gly Leu 180 185 190 Glu Trp Ile Gly Asp Ile Phe Pro Gly Ser
Gly Asn Ile His Tyr Asn 195 200 205 Glu Lys Phe Lys Gly Lys Ala Thr
Leu Thr Ala Asp Lys Ser Ser Ser 210 215 220 Thr Ala Tyr Met Gln Leu
Ser Ser Leu Thr Phe Glu Asp Ser Ala Val 225 230 235 240 Tyr Phe Cys
Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp 245 250 255 Gly
Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 260 265
270 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln Ser
275 280 285 Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met
Ser Cys 290 295 300 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln
Lys Asn Tyr Leu 305 310 315 320 Thr Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro Lys Leu Leu Ile Tyr 325 330 335 Trp Ala Ser Thr Arg Glu Ser
Gly Val Pro Asp Arg Phe Thr Gly Ser 340 345 350 Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu 355 360 365 Asp Leu Ala
Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr 370 375 380 Phe
Gly Ala Gly Thr Lys Leu Glu Ile Lys His His His His His His 385 390
395 400 129384PRTArtificial SequencepelB-murineCD3VL-scFvEGFR1-6His
129Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15 Ala Gln Pro Ala Met Ala Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser 20 25 30 Leu Pro Ala Ser Leu Gly Asp Arg Val Thr Ile Asn
Cys Gln Ala Ser 35 40 45 Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu Ile Tyr Tyr
Thr Asn Lys Leu Ala Asp Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Arg Asp Ser Ser Phe Thr 85 90 95 Ile Ser Ser Leu
Glu Ser Glu Asp Ile Gly Ser Tyr Tyr Cys Gln Gln 100 105 110 Tyr Tyr
Asn Tyr Pro Trp Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile 115 120 125
Lys Arg Ala Asp Ser Ser Gly Gly Gly Gln Val Gln Leu Gln Glu Ser 130
135 140 Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
Thr 145 150 155 160 Val Ser Gly Gly Ser Val Ser Ser Gly Asp Tyr Tyr
Trp Thr Trp Ile 165 170 175 Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp
Ile Gly His Ile Tyr Tyr 180 185 190 Ser Gly Asn Thr Asn Tyr Asn Pro
Ser Leu Lys Ser Arg Leu Thr Ile 195 200 205 Ser Ile Asp Thr Ser Lys
Thr Gln Phe Ser Leu Lys Leu Ser Ser Val 210 215 220 Thr Ala Ala Asp
Thr Ala Ile Tyr Tyr Cys Val Arg Asp Arg Val Thr 225 230 235 240 Gly
Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 245 250
255 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
260 265 270 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly Asp 275 280 285 Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile
Ser Asn Tyr Leu 290 295 300 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr 305 310 315 320 Asp Ala Ser Asn Leu Glu Thr
Gly Val Pro Ser Arg Phe Ser Gly Ser 325 330 335 Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu 340 345 350 Asp Ile Ala
Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu Ala 355 360 365 Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys His His His His His His 370 375
380 130558PRTArtificial SequencepelB-ta(DIG*EpCAM)-Myc-6HIS 130Met
Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10
15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly
20 25 30 Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Val
Ser Gly 35 40 45 Phe Thr Phe Ser Asp Tyr Ala Met Ser Trp Ile Arg
Gln Thr Pro Glu 50 55 60 Asn Arg Leu Glu Trp Val Ala Ser Ile Asn
Ile Gly Ala Thr Tyr Ala 65 70 75 80 Tyr Tyr Pro Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ala Lys Asn Thr Leu Phe
Leu Gln Met Ser Ser Leu Gly Ser Glu Asp 100 105 110 Thr Ala Met Tyr
Tyr Cys Ala Arg Pro Gly Ser Pro Tyr Glu Tyr Asp 115 120 125 Lys Ala
Tyr Tyr Ser Met Ala Tyr Trp Gly Pro Gly Thr Ser Val Thr 130 135 140
Val Ser Ser Ala Lys Thr Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 145
150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Asp Val Gln Met Thr Gln
Ser Thr 165 170 175 Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr
Ile Ser Cys Arg 180 185 190 Ala Ser Gln Asp Ile Lys Asn Tyr Leu Asn
Trp Tyr Gln Gln Lys Pro 195 200 205 Gly Gly Thr Val Lys Leu Leu Ile
Tyr Tyr Ser Ser Thr Leu Leu Ser 210 215 220 Gly Val Pro Ser Arg Phe
Ser Gly Arg Gly Ser Gly Thr Asp Phe Ser 225 230 235 240 Leu Thr Ile
Thr Asn Leu Glu Arg Glu Asp Ile Ala Thr Tyr Phe Cys 245 250 255 Gln
Gln Ser Ile Thr Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu 260 265
270 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Gly Gly
275 280 285 Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser
Gly Ala 290 295 300 Glu Leu Val Arg Pro Gly Thr Ser Val Lys Ile Ser
Cys Lys Ala Ser 305 310 315 320 Gly Tyr Ala Phe Thr Asn Tyr Trp Leu
Gly Trp Val Lys Gln Arg Pro 325 330 335 Gly His Gly Leu Glu Trp Ile
Gly Asp Ile Phe Pro Gly Ser Gly Asn 340 345 350 Ile His Tyr Asn Glu
Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp 355 360 365 Lys Ser Ser
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu 370 375
380 Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro
385 390 395 400 Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser Gly Gly 405 410 415 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Leu Val 420 425 430 Met Thr Gln Ser Pro Ser Ser Leu Thr
Val Thr Ala Gly Glu Lys Val 435 440 445 Thr Met Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asn Ser Gly Asn Gln 450 455 460 Lys Asn Tyr Leu Thr
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys 465 470 475 480 Leu Leu
Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg 485 490 495
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 500
505 510 Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr
Ser 515 520 525 Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
Lys Glu Gln 530 535 540 Lys Leu Ile Ser Glu Glu Asp Leu His His His
His His His 545 550 555 1319PRTArtificial SequenceAnti-DIG VL CDR3
131Gln Gln Ser Ile Thr Leu Pro Pro Thr 1 5 1328PRTArtificial
SequenceAnti-CD45 VH CDR1 132Gly Phe Asp Phe Ser Arg Tyr Trp 1 5
1338PRTArtificial SequenceAnti-CD45 VH CDR2 133Ile Asn Pro Thr Ser
Ser Thr Ile 1 5 13414PRTArtificial SequenceAnti-CD45 VH CDR3 134Ala
Arg Gly Asn Tyr Tyr Arg Tyr Gly Asp Ala Met Asp Tyr 1 5 10
1351167DNAArtificial SequenceNucleotide sequence encoding
pelB-CD3VL- scFvEPCAM(VH-VL)-6His 135atgaaatacc tgctgccgac
cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60atggccgaca ttcagctgac
ccagtctcca gcaatcatgt ctgcatctcc aggggagaag 120gtcaccatga
cctgcagagc cagttcaagt gtaagttaca tgaactggta ccagcagaag
180tcaggcacct cccccaaaag atggatttat gacacatcca aagtggcttc
tggagtccct 240tatcgcttca gtggcagtgg gtctgggacc tcatactctc
tcacaatcag cagcatggag 300gctgaagatg ctgccactta ttactgccaa
cagtggagta gtaacccgct cacgttcggt 360gctgggacca agctggagct
gaaatccgga ggtggtggat ccgaggtgca gctgctcgag 420cagtctggag
ctgagctggt aaggcctggg acttcagtga agatatcctg caaggcttct
480ggatacgcct tcactaacta ctggctaggt tgggtaaagc agaggcctgg
acatggactt 540gagtggattg gagatatttt ccctggaagt ggtaatatcc
actacaatga gaagttcaag 600ggcaaagcca cactgactgc agacaaatct
tcgagcacag cctatatgca gctcagtagc 660ctgacatttg aggactctgc
tgtctatttc tgtgcaagac tgaggaactg ggacgagcct 720atggactact
ggggccaagg gaccacggtc accgtctcct caggtggtgg tggttctggc
780ggcggcggct ccggtggtgg tggttctgag ctcgtgatga cacagtctcc
atcctccctg 840actgtgacag caggagagaa ggtcactatg agctgcaagt
ccagtcagag tctgttaaac 900agtggaaatc aaaagaacta cttgacctgg
taccagcaga aaccagggca gcctcctaaa 960ctgttgatct actgggcatc
cactagggaa tctggggtcc ctgatcgctt cacaggcagt 1020ggatctggaa
cagatttcac tctcaccatc agcagtgtgc aggctgaaga cctggcagtt
1080tattactgtc agaatgatta tagttatccg ctcacgttcg gtgctgggac
caagcttgag 1140atcaaacatc atcaccatca tcattag
11671361215DNAArtificial SequenceNucleotide sequence encoding
pelB-CD3VH-scFvHer2/neu-6HIS 136atgaaatacc tgctgccgac cgctgctgct
ggtctgctgc tcctcgctgc ccagccggcg 60atggccgata tcaaactgca gcagtcaggg
gctgaactgg caagacctgg ggcctcagtg 120aagatgtcct gcaagacttc
tggctacacc tttactaggt acacgatgca ctgggtaaaa 180cagaggcctg
gacagggtct ggaatggatt ggatacatta atcctagccg tggttatact
240aattacaatc agaagttcaa ggacaaggcc acattgacta cagacaaatc
ctccagcaca 300gcctacatgc aactgagcag cctgacatct gaggactctg
cagtctatta ctgtgcaaga 360tattatgatg atcattactg ccttgactac
tggggccaag gcaccactct cacagtctcc 420tcaggtggtg gtggttctgg
cggcggcggc tccggtggtg gtggttctga ggttcagctg 480gtggagtctg
gcggtggcct ggtgcagcca gggggctcac tccgtttgtc ctgtgcagct
540tctggcttca acattaaaga cacctatata cactgggtgc gtcaggcccc
gggtaagggc 600ctggaatggg ttgcaaggat ttatcctacg aatggttata
ctagatatgc cgatagcgtc 660aagggccgtt tcactataag cgcagacaca
tccaaaaaca cagcctacct gcagatgaac 720agcctgcgtg ctgaggacac
tgccgtctat tattgttcta ggtggggagg ggacggcttc 780tatgctatgg
actattgggg tcaaggaacc ctggtcactg tctcctccgg tggtggtggt
840tctggcggcg gcggctccgg tggtggtggt tctgatatcc agatgaccca
gtccccgagc 900tccctgtccg cctctgtggg cgatagggtc accatcacct
gccgtgccag tcaggatgtg 960aatactgctg tagcctggta tcaacagaaa
ccaggaaaag ctccgaaact actgatttac 1020tcggcatcct tcctctactc
tggagtccct tctcgcttct ctggatccag atctgggacg 1080gatttcactc
tgaccatcag cagtctgcag ccggaagact tcgcaactta ttactgtcag
1140caacattata ctactcctcc cacgttcgga cagggtacca aggtggagat
caaacatcat 1200caccatcatc attag 12151371212DNAArtificial
SequenceNucleotide sequence encoding pelB-CD3VH- scFvEGFR1-6HIS
137atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc
ccagccggcg 60atggccgata tcaaactgca gcagtcaggg gctgaactgg caagacctgg
ggcctcagtg 120aagatgtcct gcaagacttc tggctacacc tttactaggt
acacgatgca ctgggtaaaa 180cagaggcctg gacagggtct ggaatggatt
ggatacatta atcctagccg tggttatact 240aattacaatc agaagttcaa
ggacaaggcc acattgacta cagacaaatc ctccagcaca 300gcctacatgc
aactgagcag cctgacatct gaggactctg cagtctatta ctgtgcaaga
360tattatgatg atcattactg ccttgactac tggggccaag gcaccactct
cacagtctcc 420tcaggtggtg gtggttctgg cggcggcggc tccggtggtg
gtggttctca ggtgcagctg 480caggagtcgg gcccaggact ggtgaagcct
tcggagaccc tgtccctcac ctgcactgtc 540tctggtggct ccgtcagcag
tggtgattac tactggacct ggatccggca gtccccaggg 600aagggactgg
agtggattgg acacatctat tacagtggga acaccaatta taacccctcc
660ctcaagagcc gactcaccat atcaattgac acgtccaaga ctcagttctc
cctgaagctg 720agttctgtga ccgctgcgga cacggccatt tattactgtg
tgcgagatcg agtgactggt 780gcttttgata tctggggcca agggacaatg
gtcaccgtct cttccggtgg tggtggttct 840ggcggcggcg gctccggtgg
tggtggttct gacatccaga tgacccagtc tccatcctcc 900ctgtctgcat
ctgtcggaga cagagtcacc atcacttgcc aggcgagtca ggacatcagc
960aactatttaa attggtatca gcagaaacca gggaaagccc ctaaactcct
gatctacgat 1020gcatccaatt tggaaacagg ggtcccatca aggttcagtg
gaagtggatc tgggacagat 1080tttactttca ccatcagcag cctgcagcct
gaagatattg caacatattt ctgtcaacac 1140tttgatcatc tcccgctcgc
tttcggcgga gggaccaagg tggagatcaa acatcatcac 1200catcatcatt ag
12121381245DNAArtificial SequenceNucleotide sequence encoding
pelB-CD3VH- scFvCEA-6HIS 138atgaaatacc tgctgccgac cgctgctgct
ggtctgctgc tcctcgctgc ccagccggcg 60atggccgata tcaaactgca gcagtcaggg
gctgaactgg caagacctgg ggcctcagtg 120aagatgtcct gcaagacttc
tggctacacc tttactaggt acacgatgca ctgggtaaaa 180cagaggcctg
gacagggtct ggaatggatt ggatacatta atcctagccg tggttatact
240aattacaatc agaagttcaa ggacaaggcc acattgacta cagacaaatc
ctccagcaca 300gcctacatgc aactgagcag cctgacatct gaggactctg
cagtctatta ctgtgcaaga 360tattatgatg atcattactg ccttgactac
tggggccaag gcaccactct cacagtctcc 420tcaggtggtg gtggttctgg
cggcggcggc tccggtggtg gtggttcttc tagagtggcc 480caggtgcaac
tgcagcagtc aggggctgag ctggctagac ctggggcttc agtgaagatg
540tcctgcaagg cttctggcta cacctttact acctacacaa tacactgggt
aagacagagg 600cctggacacg atctggaatg gattggatac attaatccta
gcagtggata ttctgactac 660aatcaaaact tcaagggcaa gaccacattg
actgcagaca agtcctccaa cacagcctac 720atgcaactga acagcctgac
atctgaggac tctgcggtct attactgtgc aagaagagcg 780gactatggta
actacgaata tacctggttt gcttactggg gccaagggac cacggtcacc
840gtctcctcag gtggaggcgg ttcaggcgga ggtggctctg gcggtggcgg
atcggacatc 900gagctcactc agtctccaaa attcatgtcc acatcagtag
gagacagggt caacgtcacc 960tacaaggcca gtcagaatgt gggtactaat
gtagcctggt ttcaacaaaa accagggcaa 1020tctcctaaag ttctgattta
ctcggcatct taccgataca gtggagtccc tgatcgcttc 1080acaggcagtg
gatctggaac agatttcact ctcaccatca gcaatgtgca gtctgaagac
1140ttggcagagt atttctgtca gcaatatcac acctatcctc tcacgttcgg
agggggcacc 1200aagctggaaa tcaaacgggc ggatcatcat caccatcatc attag
12451391179DNAArtificial SequenceNucleotide sequence encoding
pelB-CD3VL- scFvCEA-6HIS 139atgaaatacc tgctgccgac cgctgctgct
ggtctgctgc tcctcgctgc ccagccggcg 60atggccgaca ttcagctgac ccagtctcca
gcaatcatgt ctgcatctcc aggggagaag 120gtcaccatga cctgcagagc
cagttcaagt gtaagttaca tgaactggta ccagcagaag 180tcaggcacct
cccccaaaag atggatttat gacacatcca aagtggcttc tggagtccct
240tatcgcttca gtggcagtgg gtctgggacc tcatactctc tcacaatcag
cagcatggag 300gctgaagatg ctgccactta ttactgccaa cagtggagta
gtaacccgct cacgttcggt 360gctgggacca agctggagct gaaatccgga
ggtggtggat cctctagagt ggcccaggtg 420caactgcagc agtcaggggc
tgagctggct agacctgggg cttcagtgaa gatgtcctgc 480aaggcttctg
gctacacctt tactacctac acaatacact gggtaagaca gaggcctgga
540cacgatctgg aatggattgg atacattaat cctagcagtg gatattctga
ctacaatcaa 600aacttcaagg gcaagaccac attgactgca gacaagtcct
ccaacacagc ctacatgcaa 660ctgaacagcc tgacatctga ggactctgcg
gtctattact gtgcaagaag agcggactat 720ggtaactacg aatatacctg
gtttgcttac tggggccaag ggaccacggt caccgtctcc 780tcaggtggag
gcggttcagg cggaggtggc tctggcggtg gcggatcgga catcgagctc
840actcagtctc caaaattcat gtccacatca gtaggagaca gggtcaacgt
cacctacaag 900gccagtcaga atgtgggtac taatgtagcc tggtttcaac
aaaaaccagg gcaatctcct 960aaagttctga tttactcggc atcttaccga
tacagtggag tccctgatcg cttcacaggc 1020agtggatctg gaacagattt
cactctcacc atcagcaatg tgcagtctga agacttggca 1080gagtatttct
gtcagcaata tcacacctat cctctcacgt tcggaggggg caccaagctg
1140gaaatcaaac gggcggatca tcatcaccat catcattag
11791401245DNAArtificial SequenceNucleotide sequence encoding
pelB-(aCD3)VH- scFvHLA-Cw6-myc-6His 140atgaaatacc tgctgccgac
cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60atggccgata tcaaactgca
gcagtcaggg gctgaactgg caagacctgg ggcctcagtg 120aagatgtcct
gcaagacttc tggctacacc tttactaggt acacgatgca ctgggtaaaa
180cagaggcctg gacagggtct ggaatggatt ggatacatta atcctagccg
tggttatact 240aattacaatc agaagttcaa ggacaaggcc acattgacta
cagacaaatc ctccagcaca 300gcctacatgc aactgagcag cctgacatct
gaggactctg cagtctatta ctgtgcaaga 360tattatgatg atcattactg
ccttgactac tggggccaag gcaccactct cacagtctcc 420tcaggcggcg
gcggcagcgg cggcggcggc agcggcggcg aagtgcagct ggtggaaagc
480ggcggcggcc tggtgcagcc gggcggcagc ctgcgcctga gctgcgcggc
gagcggcttt 540acctttagca gctatgcgat gagctgggtg cgccaggcgc
cgggcaaagg cctggaatgg 600gtgagcgcga ttagcggcag cggcggcagc
acctattatg cggatagcgt gaaaggccgc 660tttaccatta gccgcgataa
cagcaaaaac accctgtatc tgcagatgaa cagcctgcgc 720gcggaagata
ccgcggtgta ttattgcgcg cgctatagct ttagctggtt tgatgtgtgg
780ggccagggca ccctggtgac cgtgagcagc gcgggcggcg gcagcggcgg
cggcggcagc 840ggcggcggcg gcagcggcgg cggcggcagc gatattgaac
tgacccagcc gccgagcgtg 900agcgtggcgc cgggccagac cgcgcgcatt
agctgcagcg gcgatgcgct gggcgataaa 960tatgcgagct ggtatcagca
gaaaccgggc caggcgccgg tgctggtgat ttatgatgat 1020agcgatcgcc
cgagcggcat tccggaacgc tttagcggca gcaacagcgg caacaccgcg
1080accctgacca ttagcggcac ccaggcggaa gatgaagcgg attattattg
ccagagctat 1140gataactttg atagcccggt gtttggcggc ggcaccaaac
tgaccgtgct gggcgaacaa 1200aaactcatct cagaagagga tctgcatcat
caccatcatc attag 12451411176DNAArtificial SequenceNucleotide
sequence encoding pelB-CD3VL- scFvCD138-6His 141atgaaatacc
tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60atggccgaca
ttcagctgac ccagtctcca gcaatcatgt ctgcatctcc aggggagaag
120gtcaccatga cctgcagagc cagttcaagt gtaagttaca tgaactggta
ccagcagaag 180tcaggcacct cccccaaaag atggatttat gacacatcca
aagtggcttc tggagtccct 240tatcgcttca gtggcagtgg gtctgggacc
tcatactctc tcacaatcag cagcatggag 300gctgaagatg ctgccactta
ttactgccaa cagtggagta gtaacccgct cacgttcggt 360gctgggacca
agctggagct gaaatccgga ggtggtggat ccggaggtgg tggatcccag
420gtgcagctgc agcagagcgg cagcgaactg atgccgggcg cgagcgtgaa
aattagctgc 480aaagcgaccg gctatacctt tagcaactat tggattgaat
gggtgaaaca gcgcccgggc 540catggcctgg aatggattgg cgaaattctg
ccgggcaccg gccgcaccat ttataacgaa 600aaatttaaag gcaaagcgac
ctttaccgcg gatattagca gcaacaccgt gcagatgcag 660ctgagcagcc
tgaccagcga agatagcgcg gtgtattatt gcgcgcgcga acagtattat
720ggcaactttt attatgcgat ggattattgg ggccagggca ccagcgtgac
cgtgagcagc 780ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg
gcagcgatat tcagatgacc 840cagagcacca gcagcctgag cgcgagcctg
ggcgatcgcg tgaccattag ctgcagcgcg 900agccagggca ttaacaacta
tctgaactgg tatcagcaga aaccggatgg caccgtggaa 960ctgctgattt
attataccag caccctgcag agcggcgtgc cgagccgctt tagcggcagc
1020ggcagcggca ccgattatag cctgaccatt agcaacctgg aaccggaaga
tattggcacc 1080tattattgcc agcagtatag caaactgccg cgcacctttg
gcggcggcac caaactggaa 1140attaaacgca ccgtgcatca tcaccatcat cattag
11761421227DNAArtificial SequenceNucleotide sequence encoding
pelB-CD3VH- scFvCD138-6His 142atgaaatacc tgctgccgac cgctgctgct
ggtctgctgc tcctcgctgc ccagccggcg 60atggccgata tcaaactgca gcagtcaggg
gctgaactgg caagacctgg ggcctcagtg 120aagatgtcct gcaagacttc
tggctacacc tttactaggt acacgatgca ctgggtaaaa 180cagaggcctg
gacagggtct ggaatggatt ggatacatta atcctagccg tggttatact
240aattacaatc agaagttcaa ggacaaggcc acattgacta cagacaaatc
ctccagcaca 300gcctacatgc aactgagcag cctgacatct gaggactctg
cagtctatta ctgtgcaaga 360tattatgatg atcattactg ccttgactac
tggggccaag gcaccactct cacagtctcc 420tcaggtggtg gtggttctgg
cggcggcggc tccggtggtg gtggttctca ggtgcagctg 480cagcagagcg
gcagcgaact gatgccgggc gcgagcgtga aaattagctg caaagcgacc
540ggctatacct ttagcaacta ttggattgaa tgggtgaaac agcgcccggg
ccatggcctg 600gaatggattg gcgaaattct gccgggcacc ggccgcacca
tttataacga aaaatttaaa 660ggcaaagcga cctttaccgc ggatattagc
agcaacaccg tgcagatgca gctgagcagc 720ctgaccagcg aagatagcgc
ggtgtattat tgcgcgcgcg aacagtatta tggcaacttt 780tattatgcga
tggattattg gggccagggc accagcgtga ccgtgagcag cggcggcggc
840ggcagcggcg gcggcggcag cggcggcggc ggcagcgata ttcagatgac
ccagagcacc 900agcagcctga gcgcgagcct gggcgatcgc gtgaccatta
gctgcagcgc gagccagggc 960attaacaact atctgaactg gtatcagcag
aaaccggatg gcaccgtgga actgctgatt 1020tattatacca gcaccctgca
gagcggcgtg ccgagccgct ttagcggcag cggcagcggc 1080accgattata
gcctgaccat tagcaacctg gaaccggaag atattggcac ctattattgc
1140cagcagtata gcaaactgcc gcgcaccttt ggcggcggca ccaaactgga
aattaaacgc 1200accgtgcatc atcaccatca tcattag
12271431212DNAArtificial SequenceNucleotide sequence encoding
pelB-(aHis)VH- scFvHLA-A2(VH-VL)-myc 143atgaaatacc tgctgccgac
cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60atggcccagg tgcagctgca
gcagagcggc ccggaagatg tgaaaccggg cgcgagcgtg 120aaaattagct
gcaaagcgag cggctatacc tttaccgatt attatatgaa ctgggtgaaa
180cagagcccgg gcaaaggcct ggaatggatt ggcgatatta acccgaacaa
cggcggcacc 240agctataacc agaaatttaa aggccgcgcg accctgaccg
tggataaaag cagcagcacc 300gcgtatatgg aactgcgcag cctgaccagc
gaagatagca gcgtgtatta ttgcgaaagc 360cagagcggcg cgtattgggg
ccagggcacc accgtgaccg tgagcgcggg cggcggcggc 420agcggcggcg
gcggcagcgg cggccaggtg cagctggtgc agtctggggg aggcgtggtc
480cagcctgggg ggtccctgag agtctcctgt gcagcgtctg gggtcaccct
cagtgattat 540ggcatgcatt gggtccgcca ggctccaggc aaggggctgg
agtggatggc ttttatacgg 600aatgatggaa gtgataaata ttatgcagac
tccgtgaagg gccgattcac catctccaga 660gacaactcca agaaaacagt
gtctctgcaa atgagcagtc tcagagctga agacacggct 720gtgtattact
gtgcgaaaaa tggcgaatct gggcctttgg actactggta cttcgatctc
780tggggccgtg gcaccctggt caccgtgtcg agtggtggag gcggttcagg
cggaggtggc 840tctggcggtg gcggatcgga tgttgtgatg actcagtctc
catcctccct gtctgcatct 900gtaggagaca gagtcaccat cacttgccag
gcgagtcagg acattagcaa ctatttaaat 960tggtatcagc agaaaccagg
gaaagcccct aagctcctga tctacgatgc atccaatttg 1020gaaacagggg
tcccatcaag gttcagtgga agtggatctg ggacagattt tactttcacc
1080atcagcagcc tgcagcctga ggattttgca acttattact gccaacaata
tagtagtttt 1140ccgctcactt tcggcggagg gaccaaagtg gatatcaaac
gtgaacaaaa actcatctca 1200gaagaggatc tg 12121441230DNAArtificial
SequenceNucleotide sequence encoding pelB-(aHis)VL-
scFvCD45(VL-VH)-myc 144atgaaatacc tgctgccgac cgctgctgct ggtctgctgc
tcctcgctgc ccagccggcg 60atggccgatt ataaagatat tctgatgacc cagaccccga
gcagcctgcc ggtgagcctg 120ggcgatcagg cgagcattag ctgccgcagc
agccagagca ttgtgcatag caacggcaac 180acctatctgg aatggtatct
gcagaaaccg ggccagagcc cgaaactgct gatttataaa 240gtgagcaacc
gctttagcgg cgtgccggat cgctttagcg gcagcggcag cggcaccgat
300tttaccctga aaattagccg cgtggaagcg gaagatctgg gcgtgtatta
ttgctttcag 360ggcagccatg tgccgtttac ctttggcagc ggcaccaaac
tggaaattaa acgcggcggc 420ggcggcagcg gcggcggcgg cagcggcggc
gatattgttc tgacccagag cccggcgagc 480ctggcggtta gcctgggtca
gcgtgccacc attagctgcc gtgcgagcaa aagcgtgagc 540accagcggct
atagctatct gcattggtat cagcagaaac cgggccagcc tccaaaactg
600ctgatttatc tggccagcaa cctggaaagc ggtgtgccgg cccgttttag
cggcagcggc 660agcggtaccg attttaccct gaacattcat ccggtggaag
aagaagatgc ggcgacctat 720tattgccagc atagccgtga actgccgttt
acctttggca gcggcaccaa actggaaatt 780aaaaagatct ctggtggcgg
cggctcgggt ggtggtgggt cgggcggcgg cggctcgagc 840caggtgcagc
tggtggaaag cggtggcgga ctggtgcagc cgggcggcag cctgaaactg
900agctgtgccg ccagcggttt tgattttagc cgttattgga tgagctgggt
gcgtcaggcg 960ccgggcaaag gcctggaatg gattggcgaa attaacccga
ccagcagcac cattaacttt 1020accccgagcc tgaaagataa agtgtttatt
agccgtgata acgcgaaaaa caccctgtat 1080ctgcagatga gcaaagtgcg
tagcgaagat accgcgctgt attattgcgc gcgtggcaac 1140tattatcgtt
atggcgatgc gatggattat tggggccagg gcaccagcgt gaccgtgagc
1200gaacaaaaac tcatctcaga agaggatctg 12301451248DNAArtificial
SequenceNucleotide sequence encoding
pelB-(aCD3)VH-scFvHLA-A2(VH-VL)-myc-6His 145atgaaatacc
tgctgccgac
cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60atggccgata tcaaactgca
gcagtcaggg gctgaactgg caagacctgg ggcctcagtg 120aagatgtcct
gcaagacttc tggctacacc tttactaggt acacgatgca ctgggtaaaa
180cagaggcctg gacagggtct ggaatggatt ggatacatta atcctagccg
tggttatact 240aattacaatc agaagttcaa ggacaaggcc acattgacta
cagacaaatc ctccagcaca 300gcctacatgc aactgagcag cctgacatct
gaggactctg cagtctatta ctgtgcaaga 360tattatgatg atcattactg
ccttgactac tggggccaag gcaccactct cacagtctcc 420tcaggcggcg
gcggcagcgg cggcggcggc agcggcggcc aggtgcagct ggtgcagtct
480gggggaggcg tggtccagcc tggggggtcc ctgagagtct cctgtgcagc
gtctggggtc 540accctcagtg attatggcat gcattgggtc cgccaggctc
caggcaaggg gctggagtgg 600atggctttta tacggaatga tggaagtgat
aaatattatg cagactccgt gaagggccga 660ttcaccatct ccagagacaa
ctccaagaaa acagtgtctc tgcaaatgag cagtctcaga 720gctgaagaca
cggctgtgta ttactgtgcg aaaaatggcg aatctgggcc tttggactac
780tggtacttcg atctctgggg ccgtggcacc ctggtcaccg tgtcgagtgg
tggaggcggt 840tcaggcggag gtggctctgg cggtggcgga tcggatgttg
tgatgactca gtctccatcc 900tccctgtctg catctgtagg agacagagtc
accatcactt gccaggcgag tcaggacatt 960agcaactatt taaattggta
tcagcagaaa ccagggaaag cccctaagct cctgatctac 1020gatgcatcca
atttggaaac aggggtccca tcaaggttca gtggaagtgg atctgggaca
1080gattttactt tcaccatcag cagcctgcag cctgaggatt ttgcaactta
ttactgccaa 1140caatatagta gttttccgct cactttcggc ggagggacca
aagtggatat caaacgtgaa 1200caaaaactca tctcagaaga ggatctgcat
catcaccatc atcattag 12481461221DNAArtificial SequenceNucleotide
sequence encoding pelB-(aCD3)VL-scFvCD45(VL-VH)-myc-6His
146atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc
ccagccggcg 60atggccgaca ttcagctgac ccagtctcca gcaatcatgt ctgcatctcc
aggggagaag 120gtcaccatga cctgcagagc cagttcaagt gtaagttaca
tgaactggta ccagcagaag 180tcaggcacct cccccaaaag atggatttat
gacacatcca aagtggcttc tggagtccct 240tatcgcttca gtggcagtgg
gtctgggacc tcatactctc tcacaatcag cagcatggag 300gctgaagatg
ctgccactta ttactgccaa cagtggagta gtaacccgct cacgttcggt
360gctgggacca agctggagct gaaaggcggc ggcggcagcg gcggcggcgg
cagcggcggc 420gatattgttc tgacccagag cccggcgagc ctggcggtta
gcctgggtca gcgtgccacc 480attagctgcc gtgcgagcaa aagcgtgagc
accagcggct atagctatct gcattggtat 540cagcagaaac cgggccagcc
tccaaaactg ctgatttatc tggccagcaa cctggaaagc 600ggtgtgccgg
cccgttttag cggcagcggc agcggtaccg attttaccct gaacattcat
660ccggtggaag aagaagatgc ggcgacctat tattgccagc atagccgtga
actgccgttt 720acctttggca gcggcaccaa actggaaatt aaaaagatct
ctggtggcgg cggctcgggt 780ggtggtgggt cgggcggcgg cggctcgagc
caggtgcagc tggtggaaag cggtggcgga 840ctggtgcagc cgggcggcag
cctgaaactg agctgtgccg ccagcggttt tgattttagc 900cgttattgga
tgagctgggt gcgtcaggcg ccgggcaaag gcctggaatg gattggcgaa
960attaacccga ccagcagcac cattaacttt accccgagcc tgaaagataa
agtgtttatt 1020agccgtgata acgcgaaaaa caccctgtat ctgcagatga
gcaaagtgcg tagcgaagat 1080accgcgctgt attattgcgc gcgtggcaac
tattatcgtt atggcgatgc gatggattat 1140tggggccagg gcaccagcgt
gaccgtgagc gaacaaaaac tcatctcaga agaggatctg 1200catcatcacc
atcatcatta g 12211471248DNAArtificial SequenceNucleotide sequence
encoding pelB-VHaDIG- scFvEGFR-FLAG-6HIS 147atgaaatacc tgctgccgac
cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60atggccgaag tgcagctggt
ggaaagcggc ggcggcctgg tgaaaccggg cggcagcctg 120aaactgagct
gcgcggtgag cggctttacc tttagcgatt atgcgatgag ctggattcgc
180cagaccccgg aaaaccgcct ggaatgggtg gcgagcatta acattggcgc
gacctatgcg 240tattatccgg atagcgtgaa aggccgcttt accattagcc
gcgataacgc gaaaaacacc 300ctgtttctgc agatgagcag cctgggcagc
gaagataccg cgatgtatta ttgcgcgcgc 360ccgggcagcc cgtatgaata
tgataaagcg tattatagca tggcgtattg gggcccgggc 420accagcgtga
ccgtgagcag cgcgaaaacc ggtggtggtg gttctggcgg cggcggctcc
480caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac
cctgtccctc 540acctgcactg tctctggtgg ctccgtcagc agtggtgatt
actactggac ctggatccgg 600cagtccccag ggaagggact ggagtggatt
ggacacatct attacagtgg gaacaccaat 660tataacccct ccctcaagag
ccgactcacc atatcaattg acacgtccaa gactcagttc 720tccctgaagc
tgagttctgt gaccgctgcg gacacggcca tttattactg tgtgcgagat
780cgagtgactg gtgcttttga tatctggggc caagggacaa tggtcaccgt
ctcttccggt 840ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt
ctgacatcca gatgacccag 900tctccatcct ccctgtctgc atctgtcgga
gacagagtca ccatcacttg ccaggcgagt 960caggacatca gcaactattt
aaattggtat cagcagaaac cagggaaagc ccctaaactc 1020ctgatctacg
atgcatccaa tttggaaaca ggggtcccat caaggttcag tggaagtgga
1080tctgggacag attttacttt caccatcagc agcctgcagc ctgaagatat
tgcaacatat 1140ttctgtcaac actttgatca tctcccgctc gctttcggcg
gagggaccaa ggtggagatc 1200aaagactaca aggatgacga tgacaaacat
catcaccatc atcattag 12481481239DNAArtificial SequenceNucleotide
sequence encoding pelB-VLaDIG- scFvEpCAM-myc-6HIS 148atgaaatacc
tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60atggccgatg
tgcagatgac ccagagcacc agcagcctga gcgcgagcct gggcgatcgc
120gtgaccatta gctgccgcgc gagccaggat attaaaaact atctgaactg
gtatcagcag 180aaaccgggcg gcaccgtgaa actgctgatt tattatagca
gcaccctgct gagcggcgtg 240ccgagccgct ttagcggccg cggcagcggc
accgatttta gcctgaccat taccaacctg 300gaacgcgaag atattgcgac
ctatttttgc cagcagagca ttaccctgcc gccgaccttt 360ggcggcggca
ccaaactgga aattaaacgc gcggatgcgg cgccgaccgt gagcattttt
420ggtggttccg gaggtggtgg atccgaggtg cagctgctcg agcagtctgg
agctgagctg 480gtaaggcctg ggacttcagt gaagatatcc tgcaaggctt
ctggatacgc cttcactaac 540tactggctag gttgggtaaa gcagaggcct
ggacatggac ttgagtggat tggagatatt 600ttccctggaa gtggtaatat
ccactacaat gagaagttca agggcaaagc cacactgact 660gcagacaaat
cttcgagcac agcctatatg cagctcagta gcctgacatt tgaggactct
720gctgtctatt tctgtgcaag actgaggaac tgggacgagc ctatggacta
ctggggccaa 780gggaccacgg tcaccgtctc ctcaggtggt ggtggttctg
gcggcggcgg ctccggtggt 840ggtggttctg agctcgtgat gacacagtct
ccatcctccc tgactgtgac agcaggagag 900aaggtcacta tgagctgcaa
gtccagtcag agtctgttaa acagtggaaa tcaaaagaac 960tacttgacct
ggtaccagca gaaaccaggg cagcctccta aactgttgat ctactgggca
1020tccactaggg aatctggggt ccctgatcgc ttcacaggca gtggatctgg
aacagatttc 1080actctcacca tcagcagtgt gcaggctgaa gacctggcag
tttattactg tcagaatgat 1140tatagttatc cgctcacgtt cggtgctggg
accaagcttg agatcaaaga acagaaactg 1200atctctgaag aagacctgca
tcatcaccat catcattag 12391491203DNAArtificial SequenceNucleotide
sequence encoding pelB-murineCD3VH- scFvEpCaAM-6His 149atgaaatatc
tgctgccgac cgcggcggcg ggcctgctgc tgctggcggc gcagccggcg 60atggcggaag
tgcagctggt ggaaagcggc ggcggcctgg tgcagccggg caaaagcctg
120aaactgagct gcgaagcgag cggctttacc tttagcggct atggcatgca
ttgggtgcgc 180caggcgccgg gccgcggcct ggaaagcgtg gcgtatatta
ccagcagcag cattaacatt 240aaatatgcgg atgcggtgaa aggccgcttt
accgtgagcc gcgataacgc gaaaaacctg 300ctgtttctgc agatgaacat
tctgaaaagc gaagataccg cgatgtatta ttgcgcgcgc 360tttgattggg
ataaaaacta ttggggccag ggcaccatgg tgaccgtgag cagcgcgaaa
420accagcagcg gcggcggcga ggtgcagctg ctcgagcagt ctggagctga
gctggtaagg 480cctgggactt cagtgaagat atcctgcaag gcttctggat
acgccttcac taactactgg 540ctaggttggg taaagcagag gcctggacat
ggacttgagt ggattggaga tattttccct 600ggaagtggta atatccacta
caatgagaag ttcaagggca aagccacact gactgcagac 660aaatcttcga
gcacagccta tatgcagctc agtagcctga catttgagga ctctgctgtc
720tatttctgtg caagactgag gaactgggac gagcctatgg actactgggg
ccaagggacc 780acggtcaccg tctcctcagg tggtggtggt tctggcggcg
gcggctccgg tggtggtggt 840tctgagctcg tgatgacaca gtctccatcc
tccctgactg tgacagcagg agagaaggtc 900actatgagct gcaagtccag
tcagagtctg ttaaacagtg gaaatcaaaa gaactacttg 960acctggtacc
agcagaaacc agggcagcct cctaaactgt tgatctactg ggcatccact
1020agggaatctg gggtccctga tcgcttcaca ggcagtggat ctggaacaga
tttcactctc 1080accatcagca gtgtgcaggc tgaagacctg gcagtttatt
actgtcagaa tgattatagt 1140tatccgctca cgttcggtgc tgggaccaag
cttgagatca aacatcatca ccatcatcat 1200tag 12031501155DNAArtificial
SequenceNucleotide sequence encoding pelB-murineCD3VL-
scFvEGFR-6His 150atgaaatatc tgctgccgac cgcggcggcg ggcctgctgc
tgctggcggc gcagccggcg 60atggcggata ttcagatgac ccagagcccg agcagcctgc
cggcgagcct gggcgatcgc 120gtgaccatta actgccaggc gagccaggat
attagcaact atctgaactg gtatcagcag 180aaaccgggca aagcgccgaa
actgctgatt tattatacca acaaactggc ggatggcgtg 240ccgagccgct
ttagcggcag cggcagcggc cgcgatagca gctttaccat tagcagcctg
300gaaagcgaag atattggcag ctattattgc cagcagtatt ataactatcc
gtggaccttt 360ggcccgggca ccaaactgga aattaaacgc gcggatagca
gcggcggcgg ccaggtgcag 420ctgcaggagt cgggcccagg actggtgaag
ccttcggaga ccctgtccct cacctgcact 480gtctctggtg gctccgtcag
cagtggtgat tactactgga cctggatccg gcagtcccca 540gggaagggac
tggagtggat tggacacatc tattacagtg ggaacaccaa ttataacccc
600tccctcaaga gccgactcac catatcaatt gacacgtcca agactcagtt
ctccctgaag 660ctgagttctg tgaccgctgc ggacacggcc atttattact
gtgtgcgaga tcgagtgact 720ggtgcttttg atatctgggg ccaagggaca
atggtcaccg tctcttccgg tggtggtggt 780tctggcggcg gcggctccgg
tggtggtggt tctgacatcc agatgaccca gtctccatcc 840tccctgtctg
catctgtcgg agacagagtc accatcactt gccaggcgag tcaggacatc
900agcaactatt taaattggta tcagcagaaa ccagggaaag cccctaaact
cctgatctac 960gatgcatcca atttggaaac aggggtccca tcaaggttca
gtggaagtgg atctgggaca 1020gattttactt tcaccatcag cagcctgcag
cctgaagata ttgcaacata tttctgtcaa 1080cactttgatc atctcccgct
cgctttcggc ggagggacca aggtggagat caaacatcat 1140caccatcatc attag
11551511677DNAArtificial SequenceNucleotide sequence encoding
pelB-ta (DIG*EpCAM)-Myc-6HIS 151atgaaatacc tgctgccgac cgctgctgct
ggtctgctgc tcctcgctgc ccagccggcg 60atggccgaag tgcagctggt ggaaagcggc
ggcggcctgg tgaaaccggg cggcagcctg 120aaactgagct gcgcggtgag
cggctttacc tttagcgatt atgcgatgag ctggattcgc 180cagaccccgg
aaaaccgcct ggaatgggtg gcgagcatta acattggcgc gacctatgcg
240tattatccgg atagcgtgaa aggccgcttt accattagcc gcgataacgc
gaaaaacacc 300ctgtttctgc agatgagcag cctgggcagc gaagataccg
cgatgtatta ttgcgcgcgc 360ccgggcagcc cgtatgaata tgataaagcg
tattatagca tggcgtattg gggcccgggc 420accagcgtga ccgtgagcag
cgcgaaaacc tcctcaggtg gtggtggttc tggcggcggc 480ggctccggtg
gtggtggttc tggtgatgtg cagatgaccc agagcaccag cagcctgagc
540gcgagcctgg gcgatcgcgt gaccattagc tgccgcgcga gccaggatat
taaaaactat 600ctgaactggt atcagcagaa accgggcggc accgtgaaac
tgctgattta ttatagcagc 660accctgctga gcggcgtgcc gagccgcttt
agcggccgcg gcagcggcac cgattttagc 720ctgaccatta ccaacctgga
acgcgaagat attgcgacct atttttgcca gcagagcatt 780accctgccgc
cgacctttgg cggcggcacc aaactggaaa ttaaacgcgc ggatgcggcg
840ccgaccgtga gcatttttgg tggttccgga ggtggtggat ccgaggtgca
gctgctcgag 900cagtctggag ctgagctggt aaggcctggg acttcagtga
agatatcctg caaggcttct 960ggatacgcct tcactaacta ctggctaggt
tgggtaaagc agaggcctgg acatggactt 1020gagtggattg gagatatttt
ccctggaagt ggtaatatcc actacaatga gaagttcaag 1080ggcaaagcca
cactgactgc agacaaatct tcgagcacag cctatatgca gctcagtagc
1140ctgacatttg aggactctgc tgtctatttc tgtgcaagac tgaggaactg
ggacgagcct 1200atggactact ggggccaagg gaccacggtc accgtctcct
caggtggtgg tggttctggc 1260ggcggcggct ccggtggtgg tggttctgag
ctcgtgatga cacagtctcc atcctccctg 1320actgtgacag caggagagaa
ggtcactatg agctgcaagt ccagtcagag tctgttaaac 1380agtggaaatc
aaaagaacta cttgacctgg taccagcaga aaccagggca gcctcctaaa
1440ctgttgatct actgggcatc cactagggaa tctggggtcc ctgatcgctt
cacaggcagt 1500ggatctggaa cagatttcac tctcaccatc agcagtgtgc
aggctgaaga cctggcagtt 1560tattactgtc agaatgatta tagttatccg
ctcacgttcg gtgctgggac caagcttgag 1620atcaaagaac agaaactgat
ctctgaagaa gacctgcatc atcaccatca tcattag 1677152124PRTArtificial
SequenceAnti-CD19 VH 152Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro
Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys
Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90
95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp
100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
153112PRTArtificial SequenceAnti-CD19 VL 153Asp Ile Gln Leu Thr Gln
Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr
Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp
Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45
Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50
55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile
His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln
Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Ser 100 105 110 154251PRTArtificial SequencescFv
anti CD19 (VH-linker-VL) 154Gln Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp
Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly
Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85
90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met
Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly
Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asp Ile Gln Leu Thr 130 135 140 Gln Ser Pro Ala Ser Leu Ala Val Ser
Leu Gly Gln Arg Ala Thr Ile 145 150 155 160 Ser Cys Lys Ala Ser Gln
Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu 165 170 175 Asn Trp Tyr Gln
Gln Ile Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 180 185 190 Asp Ala
Ser Asn Leu Val Ser Gly Ile Pro Pro Arg Phe Ser Gly Ser 195 200 205
Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Lys Val 210
215 220 Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr Glu Asp Pro Trp
Thr 225 230 235 240 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser 245
250 1558PRTArtificial SequenceAnti-CD19 VH CDR1 155Gly Tyr Ala Phe
Ser Ser Tyr Trp 1 5 1568PRTArtificial SequenceAnti-CD19 VH CDR2
156Ile Trp Pro Gly Asp Gly Asp Thr 1 5 15717PRTArtificial
SequenceAnti-CD19 VH CDR3 157Ala Arg Arg Glu Thr Thr Thr Val Gly
Arg Tyr Tyr Tyr Ala Met Asp 1 5 10 15 Tyr 15810PRTArtificial
SequenceAnti-CD19 VL CDR1 158Gln Ser Val Asp Tyr Asp Gly Asp Ser
Tyr 1 5 10 1599PRTArtificial SequenceAnti-CD19 VL CDR3 159Gln Gln
Ser Thr Glu Asp Pro Trp Thr 1 5 160451PRTArtificial
SequenceClostridium perfringens Iota toxin component Ia
(a.a.23-454) 6x histidine tag two protease cleavage sites 160Met
Ala Ser Thr Thr His His His His His His Asp Thr Asp Ile Pro 1 5 10
15 Thr Thr Gly Gly Gly Ser Arg Pro Asp Asp Asp Asp Lys Glu Asn Leu
20 25 30 Tyr Phe Gln Gly His Met Ala Phe Ile Glu Arg Pro Glu Asp
Phe Leu 35 40 45 Lys Asp Lys Glu Asn Ala Ile Gln Trp Glu Lys Lys
Glu Ala Glu Arg 50 55 60 Val Glu Lys Asn Leu Asp Thr Leu Glu Lys
Glu Ala Leu Glu Leu Tyr 65 70 75 80 Lys Lys Asp Ser Glu Gln Ile Ser
Asn Tyr Ser Gln Thr Arg Gln Tyr 85 90 95 Phe Tyr Asp Tyr Gln Ile
Glu Ser Asn Pro Arg Glu Lys Glu Tyr Lys 100 105 110 Asn Leu Arg Asn
Ala Ile Ser Lys Asn Lys Ile Asp Lys Pro Ile Asn 115 120 125 Val Tyr
Tyr Phe Glu Ser Pro Glu Lys Phe Ala Phe Asn Lys Glu Ile 130 135 140
Arg Thr Glu Asn Gln Asn Glu Ile Ser Leu Glu Lys Phe Asn Glu Leu 145
150 155 160 Lys Glu Thr Ile Gln Asp Lys Leu Phe Lys Gln Asp Gly Phe
Lys Asp 165 170 175 Val Ser Leu Tyr Glu Pro Gly Asn Gly Asp Glu Lys
Pro Thr Pro Leu 180 185 190 Leu Ile His Leu Lys Leu Pro Lys Asn Thr
Gly Met Leu Pro Tyr Ile 195 200 205 Asn Ser Asn Asp
Val Lys Thr Leu Ile Glu Gln Asp Tyr Ser Ile Lys 210 215 220 Ile Asp
Lys Ile Val Arg Ile Val Ile Glu Gly Lys Gln Tyr Ile Lys 225 230 235
240 Ala Glu Ala Ser Ile Val Asn Ser Leu Asp Phe Lys Asp Asp Val Ser
245 250 255 Lys Gly Asp Leu Trp Gly Lys Glu Asn Tyr Ser Asp Trp Ser
Asn Lys 260 265 270 Leu Thr Pro Asn Glu Leu Ala Asp Val Asn Asp Tyr
Met Arg Gly Gly 275 280 285 Tyr Thr Ala Ile Asn Asn Tyr Leu Ile Ser
Asn Gly Pro Leu Asn Asn 290 295 300 Pro Asn Pro Glu Leu Asp Ser Lys
Val Asn Asn Ile Glu Asn Ala Leu 305 310 315 320 Lys Leu Thr Pro Ile
Pro Ser Asn Leu Ile Val Tyr Arg Arg Ser Gly 325 330 335 Pro Gln Glu
Phe Gly Leu Thr Leu Thr Ser Pro Glu Tyr Asp Phe Asn 340 345 350 Lys
Ile Glu Asn Ile Asp Ala Phe Lys Glu Lys Trp Glu Gly Lys Val 355 360
365 Ile Thr Tyr Pro Asn Phe Ile Ser Thr Ser Ile Gly Ser Val Asn Met
370 375 380 Ser Ala Phe Ala Lys Arg Lys Ile Ile Leu Arg Ile Asn Ile
Pro Lys 385 390 395 400 Asp Ser Pro Gly Ala Tyr Leu Ser Ala Ile Pro
Gly Tyr Ala Gly Glu 405 410 415 Tyr Glu Val Leu Leu Asn His Gly Ser
Lys Phe Lys Ile Asn Lys Val 420 425 430 Asp Ser Tyr Lys Asp Gly Thr
Val Thr Lys Leu Ile Leu Asp Ala Thr 435 440 445 Leu Ile Asn 450
161300PRTArtificial SequenceBurkholderia sp. CCGE1002 Shiga toxin
subunit A (a.a.24-285) 6x histidine tag two protease cleavage sites
161Met Ala Ser Thr Thr His His His His His His Asp Thr Asp Ile Pro
1 5 10 15 Thr Thr Gly Gly Gly Ser Arg Pro Asp Asp Asp Asp Lys Glu
Asn Leu 20 25 30 Tyr Phe Gln Gly His Met Glu Phe Ser Val Asp Phe
Thr Ser Pro Gln 35 40 45 Lys Tyr Val Gln Ser Leu Gly Ala Ile Arg
Ala Ala Met Gly Asp Ala 50 55 60 Met Ser Leu Thr Asn Ile Pro Gly
Asn Lys Ile Leu Tyr Gln Leu Arg 65 70 75 80 Pro Asp Ala Ser Asn Ile
Val Glu Gly Val Thr Ile Glu Ile Ile Gly 85 90 95 Val Gly Arg Asn
Asn Ser Pro Ser Asn Arg Asp Val Arg Phe Val Ile 100 105 110 Asn Pro
Ser Asp Leu Tyr Leu Thr Gly Phe Ile Val Gly Arg Ile Phe 115 120 125
Tyr Arg Phe Ser Asp Phe Ser Asp Thr Ala Ser Gly Arg Val Gln Val 130
135 140 Asn Ala Pro Arg His Leu Val Asp Phe Thr Ile Asp Met Thr Val
Asp 145 150 155 160 Ser Ser Tyr Leu Ser Leu Ala Arg Ser Ala Gly Val
Ser Ala Asp Arg 165 170 175 Thr Asp Leu Ser Ile Asp Arg Tyr Ser Leu
Met Lys Gly Tyr Arg Asp 180 185 190 Leu Ile Asn His Val Ser Ser Thr
Arg Thr Ile Asn Gly Ala Glu Ala 195 200 205 Arg Ala Leu Leu Ser Tyr
Ala Thr Val Leu Ser Glu Ala Val Arg Phe 210 215 220 Arg Ser Ile Gln
Gly Asn Phe Ala Ser Thr Ala Leu Gly Asp Asp Ala 225 230 235 240 Phe
Thr Pro Tyr Arg Leu Ser Leu Glu Asp Ser Asn Arg Thr Thr Arg 245 250
255 Trp Asp Arg Leu Ser Asp Glu Ile Arg Lys Ala His Tyr Gly Ala Ile
260 265 270 Lys Ile Ala Thr His Gly Ala Ala Pro Ile Leu Leu Ala Asn
Val Arg 275 280 285 Asp Val Phe Gly Met Thr Thr Cys Thr Ser Lys Lys
290 295 300 162521PRTArtificial SequenceAnthrax lethal factor
endopeptidase no PA-binding region (34-295) 6x histidine tag 162Gln
Arg Met Leu Ala Arg Tyr Glu Lys Trp Glu Lys Ile Lys Gln His 1 5 10
15 Tyr Gln His Trp Ser Asp Ser Leu Ser Glu Glu Gly Arg Gly Leu Leu
20 25 30 Lys Lys Leu Gln Ile Pro Ile Glu Pro Lys Lys Asp Asp Ile
Ile His 35 40 45 Ser Leu Ser Gln Glu Glu Lys Glu Leu Leu Lys Arg
Ile Gln Ile Asp 50 55 60 Ser Ser Asp Phe Leu Ser Thr Glu Glu Lys
Glu Phe Leu Lys Lys Leu 65 70 75 80 Gln Ile Asp Ile Arg Asp Ser Leu
Ser Glu Glu Glu Lys Glu Leu Leu 85 90 95 Asn Arg Ile Gln Val Asp
Ser Ser Asn Pro Leu Ser Glu Lys Glu Lys 100 105 110 Glu Phe Leu Lys
Lys Leu Lys Leu Asp Ile Gln Pro Tyr Asp Ile Asn 115 120 125 Gln Arg
Leu Gln Asp Thr Gly Gly Leu Ile Asp Ser Pro Ser Ile Asn 130 135 140
Leu Asp Val Arg Lys Gln Tyr Lys Arg Asp Ile Gln Asn Ile Asp Ala 145
150 155 160 Leu Leu His Gln Ser Ile Gly Ser Thr Leu Tyr Asn Lys Ile
Tyr Leu 165 170 175 Tyr Glu Asn Met Asn Ile Asn Asn Leu Thr Ala Thr
Leu Gly Ala Asp 180 185 190 Leu Val Asp Ser Thr Asp Asn Thr Lys Ile
Asn Arg Gly Ile Phe Asn 195 200 205 Glu Phe Lys Lys Asn Phe Lys Tyr
Ser Ile Ser Ser Asn Tyr Met Ile 210 215 220 Val Asp Ile Asn Glu Arg
Pro Ala Leu Asp Asn Glu Arg Leu Lys Trp 225 230 235 240 Arg Ile Gln
Leu Ser Pro Asp Thr Arg Ala Gly Tyr Leu Glu Asn Gly 245 250 255 Lys
Leu Ile Leu Gln Arg Asn Ile Gly Leu Glu Ile Lys Asp Val Gln 260 265
270 Ile Ile Lys Gln Ser Glu Lys Glu Tyr Ile Arg Ile Asp Ala Lys Val
275 280 285 Val Pro Lys Ser Lys Ile Asp Thr Lys Ile Gln Glu Ala Gln
Leu Asn 290 295 300 Ile Asn Gln Glu Trp Asn Lys Ala Leu Gly Leu Pro
Lys Tyr Thr Lys 305 310 315 320 Leu Ile Thr Phe Asn Val His Asn Arg
Tyr Ala Ser Asn Ile Val Glu 325 330 335 Ser Ala Tyr Leu Ile Leu Asn
Glu Trp Lys Asn Asn Ile Gln Ser Asp 340 345 350 Leu Ile Lys Lys Val
Thr Asn Tyr Leu Val Asp Gly Asn Gly Arg Phe 355 360 365 Val Phe Thr
Asp Ile Thr Leu Pro Asn Ile Ala Glu Gln Tyr Thr His 370 375 380 Gln
Asp Glu Ile Tyr Glu Gln Val His Ser Lys Gly Leu Tyr Val Pro 385 390
395 400 Glu Ser Arg Ser Ile Leu Leu His Gly Pro Ser Lys Gly Val Glu
Leu 405 410 415 Arg Asn Asp Ser Glu Gly Phe Ile His Glu Phe Gly His
Ala Val Asp 420 425 430 Asp Tyr Ala Gly Tyr Leu Leu Asp Lys Asn Gln
Ser Asp Leu Val Thr 435 440 445 Asn Ser Lys Lys Phe Ile Asp Ile Phe
Lys Glu Glu Gly Ser Asn Leu 450 455 460 Thr Ser Tyr Gly Arg Thr Asn
Glu Ala Glu Phe Phe Ala Glu Ala Phe 465 470 475 480 Arg Leu Met His
Ser Thr Asp His Ala Glu Arg Leu Lys Val Gln Lys 485 490 495 Asn Ala
Pro Lys Thr Phe Gln Phe Ile Asn Asp Gln Ile Lys Phe Ile 500 505 510
Ile Asn Ser His His His His His His 515 520 163389PRTArtificial
SequenceCorynebacterium diphtheria toxin 6x histidine tag 163Met
Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu 1 5 10
15 Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile
20 25 30 Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn
Tyr Asp 35 40 45 Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys
Tyr Asp Ala Ala 50 55 60 Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu
Ser Gly Lys Ala Gly Gly 65 70 75 80 Val Val Lys Val Thr Tyr Pro Gly
Leu Thr Lys Val Leu Ala Leu Lys 85 90 95 Val Asp Asn Ala Glu Thr
Phe Lys Lys Glu Leu Gly Leu Ser Leu Thr 100 105 110 Glu Pro Leu Met
Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe 115 120 125 Gly Asp
Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly 130 135 140
Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu 145
150 155 160 Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg
Gly Gln 165 170 175 Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala
Gly Asn Arg Val 180 185 190 Arg Arg Ser Val Gly Ser Ser Leu Ser Cys
Ile Asn Leu Asp Trp Asp 195 200 205 Val Ile Arg Asp Lys Thr Lys Thr
Lys Ile Glu Ser Leu Lys Glu His 210 215 220 Gly Pro Ile Lys Asn Lys
Met Ser Glu Ser Pro Asn Lys Thr Val Ser 225 230 235 240 Glu Glu Lys
Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu 245 250 255 Glu
His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro 260 265
270 Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln
275 280 285 Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr
Ala Ala 290 295 300 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly
Ile Ala Asp Gly 305 310 315 320 Ala Val His His Asn Thr Glu Glu Ile
Val Ala Gln Ser Ile Ala Leu 325 330 335 Ser Ser Leu Met Val Ala Gln
Ala Ile Pro Leu Val Gly Glu Leu Val 340 345 350 Asp Ile Gly Phe Ala
Ala Tyr Asn Phe Val Glu Asp Ser Ile Ile Arg 355 360 365 Thr Gly Phe
Gln Gly Glu Ser Gly His Lys Thr Gln Pro His Met His 370 375 380 His
His His His His 385 164506PRTArtificial SequenceClostridium
perfringens str. 13 pfoA perfringolysin O 6x histidine tag 164Met
Ile Arg Phe Lys Lys Thr Lys Leu Ile Ala Ser Ile Ala Met Ala 1 5 10
15 Leu Cys Leu Phe Ser Gln Pro Val Ile Ser Phe Ser Lys Asp Ile Thr
20 25 30 Asp Lys Asn Gln Ser Ile Asp Ser Gly Ile Ser Ser Leu Ser
Tyr Asn 35 40 45 Arg Asn Glu Val Leu Ala Ser Asn Gly Asp Lys Ile
Glu Ser Phe Val 50 55 60 Pro Lys Glu Gly Lys Lys Thr Gly Asn Lys
Phe Ile Val Val Glu Arg 65 70 75 80 Gln Lys Arg Ser Leu Thr Thr Ser
Pro Val Asp Ile Ser Ile Ile Asp 85 90 95 Ser Val Asn Asp Arg Thr
Tyr Pro Gly Ala Leu Gln Leu Ala Asp Lys 100 105 110 Ala Phe Val Glu
Asn Arg Pro Thr Ile Leu Met Val Lys Arg Lys Pro 115 120 125 Ile Asn
Ile Asn Ile Asp Leu Pro Gly Leu Lys Gly Glu Asn Ser Ile 130 135 140
Lys Val Asp Asp Pro Thr Tyr Gly Lys Val Ser Gly Ala Ile Asp Glu 145
150 155 160 Leu Val Ser Lys Trp Asn Glu Lys Tyr Ser Ser Thr His Thr
Leu Pro 165 170 175 Ala Arg Thr Gln Tyr Ser Glu Ser Met Val Tyr Ser
Lys Ser Gln Ile 180 185 190 Ser Ser Ala Leu Asn Val Asn Ala Lys Val
Leu Glu Asn Ser Leu Gly 195 200 205 Val Asp Phe Asn Ala Val Ala Asn
Asn Glu Lys Lys Val Met Ile Leu 210 215 220 Ala Tyr Lys Gln Ile Phe
Tyr Thr Val Ser Ala Asp Leu Pro Lys Asn 225 230 235 240 Pro Ser Asp
Leu Phe Asp Asp Ser Val Thr Phe Asn Asp Leu Lys Gln 245 250 255 Lys
Gly Val Ser Asn Glu Ala Pro Pro Leu Met Val Ser Asn Val Ala 260 265
270 Tyr Gly Arg Thr Ile Tyr Val Lys Leu Glu Thr Thr Ser Ser Ser Lys
275 280 285 Asp Val Gln Ala Ala Phe Lys Ala Leu Ile Lys Asn Thr Asp
Ile Lys 290 295 300 Asn Ser Gln Gln Tyr Lys Asp Ile Tyr Glu Asn Ser
Ser Phe Thr Ala 305 310 315 320 Val Val Leu Gly Gly Asp Ala Gln Glu
His Asn Lys Val Val Thr Lys 325 330 335 Asp Phe Asp Glu Ile Arg Lys
Val Ile Lys Asp Asn Ala Thr Phe Ser 340 345 350 Thr Lys Asn Pro Ala
Tyr Pro Ile Ser Tyr Thr Ser Val Phe Leu Lys 355 360 365 Asp Asn Ser
Val Ala Ala Val His Asn Lys Thr Asp Tyr Ile Glu Thr 370 375 380 Thr
Ser Thr Glu Tyr Ser Lys Gly Lys Ile Asn Leu Asp His Ser Gly 385 390
395 400 Ala Tyr Val Ala Gln Phe Glu Val Ala Trp Asp Glu Val Ser Tyr
Asp 405 410 415 Lys Glu Gly Asn Glu Val Leu Thr His Lys Thr Trp Asp
Gly Asn Tyr 420 425 430 Gln Asp Lys Thr Ala His Tyr Ser Thr Val Ile
Pro Leu Glu Ala Asn 435 440 445 Ala Arg Asn Ile Arg Ile Lys Ala Arg
Glu Cys Thr Gly Leu Ala Trp 450 455 460 Glu Trp Trp Arg Asp Val Ile
Ser Glu Tyr Asp Val Pro Leu Thr Asn 465 470 475 480 Asn Ile Asn Val
Ser Ile Trp Gly Thr Thr Leu Tyr Pro Gly Ser Ser 485 490 495 Ile Thr
Tyr Asn His His His His His His 500 505 165273PRTArtificial
SequenceRicin A chain 6x histidine tag 165Ile Phe Pro Lys Gln Tyr
Pro Ile Ile Asn Phe Thr Thr Ala Gly Ala 1 5 10 15 Thr Val Gln Ser
Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg Leu 20 25 30 Thr Thr
Gly Ala Asp Val Arg His Glu Ile Pro Val Leu Pro Asn Arg 35 40 45
Val Gly Leu Pro Ile Asn Gln Arg Phe Ile Leu Val Glu Leu Ser Asn 50
55 60 His Ala Glu Leu Ser Val Thr Leu Ala Leu Asp Val Thr Asn Ala
Tyr 65 70 75 80 Val Val Gly Tyr Arg Ala Gly Asn Ser Ala Tyr Phe Phe
His Pro Asp 85 90 95 Ala Gln Glu Asp Ala Glu Ala Ile Thr His Leu
Phe Thr Asp Val Gln 100 105 110 Asn Arg Tyr Thr Phe Ala Phe Gly Gly
Asn Tyr Asp Arg Leu Glu Gln 115 120 125 Leu Ala Gly Asn Leu Arg Glu
Asn Ile Glu Leu Gly Asn Gly Pro Leu 130 135 140 Glu Glu Ala Ile Ser
Ala Leu Tyr Tyr Tyr Ser Thr Gly Gly Thr Gln 145 150 155 160 Leu Pro
Thr Leu Ala Arg Ser Phe Ile Ile Cys Ile Gln Met Ile Ser 165 170 175
Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly Glu Met Arg Thr Arg Ile 180
185 190 Arg Tyr Asn Arg Arg Ser Ala Pro Asp Pro Ser Val Ile Thr Leu
Glu 195 200 205 Asn Ser Trp Gly Arg Leu Ser Thr Ala Ile Gln Glu Ser
Asn Gln Gly 210 215 220 Ala Phe Ala Ser Pro Ile Gln Leu Gln Arg Arg
Asn Gly Ser Lys Phe 225 230 235 240 Ser Val Tyr Asp Val Ser Ile Leu
Ile Pro Ile Ile Ala Leu Met Val 245 250 255 Tyr Arg Cys Ala Pro Pro
Pro Ser Ser Gln Phe His His His His His 260 265 270 His
166285PRTArtificial SequenceRicin A chain linker
peptide 6x histidine tag 166Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn
Phe Thr Thr Ala Gly Ala 1 5 10 15 Thr Val Gln Ser Tyr Thr Asn Phe
Ile Arg Ala Val Arg Gly Arg Leu 20 25 30 Thr Thr Gly Ala Asp Val
Arg His Glu Ile Pro Val Leu Pro Asn Arg 35 40 45 Val Gly Leu Pro
Ile Asn Gln Arg Phe Ile Leu Val Glu Leu Ser Asn 50 55 60 His Ala
Glu Leu Ser Val Thr Leu Ala Leu Asp Val Thr Asn Ala Tyr 65 70 75 80
Val Val Gly Tyr Arg Ala Gly Asn Ser Ala Tyr Phe Phe His Pro Asp 85
90 95 Ala Gln Glu Asp Ala Glu Ala Ile Thr His Leu Phe Thr Asp Val
Gln 100 105 110 Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn Tyr Asp Arg
Leu Glu Gln 115 120 125 Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu Leu
Gly Asn Gly Pro Leu 130 135 140 Glu Glu Ala Ile Ser Ala Leu Tyr Tyr
Tyr Ser Thr Gly Gly Thr Gln 145 150 155 160 Leu Pro Thr Leu Ala Arg
Ser Phe Ile Ile Cys Ile Gln Met Ile Ser 165 170 175 Glu Ala Ala Arg
Phe Gln Tyr Ile Glu Gly Glu Met Arg Thr Arg Ile 180 185 190 Arg Tyr
Asn Arg Arg Ser Ala Pro Asp Pro Ser Val Ile Thr Leu Glu 195 200 205
Asn Ser Trp Gly Arg Leu Ser Thr Ala Ile Gln Glu Ser Asn Gln Gly 210
215 220 Ala Phe Ala Ser Pro Ile Gln Leu Gln Arg Arg Asn Gly Ser Lys
Phe 225 230 235 240 Ser Val Tyr Asp Val Ser Ile Leu Ile Pro Ile Ile
Ala Leu Met Val 245 250 255 Tyr Arg Cys Ala Pro Pro Pro Ser Ser Gln
Phe Ser Leu Leu Ile Arg 260 265 270 Pro Val Val Pro Asn Phe Asn His
His His His His His 275 280 285 167256PRTArtificial SequencePlant
RIP bouganin with reduced immunogenic potential 6x histidine tag
167Tyr Asn Thr Val Ser Phe Asn Leu Gly Glu Ala Tyr Glu Tyr Pro Thr
1 5 10 15 Phe Ile Gln Asp Leu Arg Asn Glu Leu Ala Lys Gly Thr Pro
Val Cys 20 25 30 Gln Leu Pro Val Thr Leu Gln Thr Ile Ala Asp Asp
Lys Arg Phe Val 35 40 45 Leu Val Asp Ile Thr Thr Thr Ser Lys Lys
Thr Val Lys Val Ala Ile 50 55 60 Asp Val Thr Asp Val Tyr Val Val
Gly Tyr Gln Asp Lys Trp Asp Gly 65 70 75 80 Lys Asp Arg Ala Val Phe
Leu Asp Lys Val Pro Thr Val Ala Thr Ser 85 90 95 Lys Leu Phe Pro
Gly Val Thr Asn Arg Val Thr Leu Thr Phe Asp Gly 100 105 110 Ser Tyr
Gln Lys Leu Val Asn Ala Ala Lys Ala Asp Arg Lys Ala Leu 115 120 125
Glu Leu Gly Val Asn Lys Leu Glu Phe Ser Ile Glu Ala Ile His Gly 130
135 140 Lys Thr Ile Asn Gly Gln Glu Ala Ala Lys Phe Phe Leu Ile Val
Ile 145 150 155 160 Gln Met Val Ser Glu Ala Ala Arg Phe Lys Tyr Ile
Glu Thr Glu Val 165 170 175 Val Asp Arg Gly Leu Tyr Gly Ser Phe Lys
Pro Asn Phe Lys Val Leu 180 185 190 Asn Leu Glu Asn Asn Trp Gly Asp
Ile Ser Asp Ala Ile His Lys Ser 195 200 205 Ser Pro Gln Cys Thr Thr
Ile Asn Pro Ala Leu Gln Leu Ile Ser Pro 210 215 220 Ser Asn Asp Pro
Trp Val Val Asn Lys Val Ser Gln Ile Ser Pro Asp 225 230 235 240 Met
Gly Ile Leu Lys Phe Lys Ser Ser Lys His His His His His His 245 250
255 168149PRTArtificial SequenceHuman RNASE3 ribonuclease (RNase A
family, 3) protein without N-terminal signal peptide but with a
N-terminal nuclear localisation sequence 6x histidine tag 168Pro
Lys Lys Lys Arg Lys Val Glu Ala Ser Arg Pro Pro Gln Phe Thr 1 5 10
15 Arg Ala Gln Trp Phe Ala Ile Gln His Ile Ser Leu Asn Pro Pro Arg
20 25 30 Cys Thr Ile Ala Met Arg Ala Ile Asn Asn Tyr Arg Trp Arg
Cys Lys 35 40 45 Asn Gln Asn Thr Phe Leu Arg Thr Thr Phe Ala Asn
Val Val Asn Val 50 55 60 Cys Gly Asn Gln Ser Ile Arg Cys Pro His
Asn Arg Thr Leu Asn Asn 65 70 75 80 Cys His Arg Ser Arg Phe Arg Val
Pro Leu Leu His Cys Asp Leu Ile 85 90 95 Asn Pro Gly Ala Gln Asn
Ile Ser Asn Cys Thr Tyr Ala Asp Arg Pro 100 105 110 Gly Arg Arg Phe
Tyr Val Val Ala Cys Asp Asn Arg Asp Pro Arg Asp 115 120 125 Ser Pro
Arg Tyr Pro Val Val Pro Val His Leu Asp Thr Thr Ile His 130 135 140
His His His His His 145 16918PRTArtificial SequenceAntigen for
human myeloma cell line U266 antibody IgE-ND 169Leu Ser Pro His Leu
Leu Trp Asp Leu Phe Arg Val Gly Leu Pro Gly 1 5 10 15 Ala Ala
170146PRTDermatophagoides farinae 170Met Ile Ser Lys Ile Leu Cys
Leu Ser Leu Leu Val Ala Ala Val Val 1 5 10 15 Ala Asp Gln Val Asp
Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys 20 25 30 Val Met Val
Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg 35 40 45 Gly
Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr 50 55
60 Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile
65 70 75 80 Asp Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys
Cys Pro 85 90 95 Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr
Trp Asn Val Pro 100 105 110 Lys Ile Ala Pro Lys Ser Glu Asn Val Val
Val Thr Val Lys Leu Ile 115 120 125 Gly Asp Asn Gly Val Leu Ala Cys
Ala Ile Ala Thr His Gly Lys Ile 130 135 140 Arg Asp 145
171320PRTDermatophagoides pteronyssinus 171Met Lys Ile Val Leu Ala
Ile Ala Ser Leu Leu Ala Leu Ser Ala Val 1 5 10 15 Tyr Ala Arg Pro
Ser Ser Ile Lys Thr Phe Glu Glu Tyr Lys Lys Ala 20 25 30 Phe Asn
Lys Ser Tyr Ala Thr Phe Glu Asp Glu Glu Ala Ala Arg Lys 35 40 45
Asn Phe Leu Glu Ser Val Lys Tyr Val Gln Ser Asn Gly Gly Ala Ile 50
55 60 Asn His Leu Ser Asp Leu Ser Leu Asp Glu Phe Lys Asn Arg Phe
Leu 65 70 75 80 Met Ser Ala Glu Ala Phe Glu His Leu Lys Thr Gln Phe
Asp Leu Asn 85 90 95 Ala Glu Thr Asn Ala Cys Ser Ile Asn Gly Asn
Ala Pro Ala Glu Ile 100 105 110 Asp Leu Arg Gln Met Arg Thr Val Thr
Pro Ile Arg Met Gln Gly Gly 115 120 125 Cys Gly Ser Cys Trp Ala Phe
Ser Gly Val Ala Ala Thr Glu Ser Ala 130 135 140 Tyr Leu Ala Tyr Arg
Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu 145 150 155 160 Val Asp
Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg 165 170 175
Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr Tyr 180
185 190 Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln
Arg 195 200 205 Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn
Val Asn Lys 210 215 220 Ile Arg Glu Ala Leu Ala Gln Thr His Ser Ala
Ile Ala Val Ile Ile 225 230 235 240 Gly Ile Lys Asp Leu Asp Ala Phe
Arg His Tyr Asp Gly Arg Thr Ile 245 250 255 Ile Gln Arg Asp Asn Gly
Tyr Gln Pro Asn Tyr His Ala Val Asn Ile 260 265 270 Val Gly Tyr Ser
Asn Ala Gln Gly Val Asp Tyr Trp Ile Val Arg Asn 275 280 285 Ser Trp
Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala Ala 290 295 300
Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val Val Ile Leu 305
310 315 320 172141PRTTyrophagus putrescentiae 172Met Lys Phe Leu
Ile Leu Phe Ala Leu Val Ala Val Ala Ala Ala Gly 1 5 10 15 Gln Val
Lys Phe Thr Asp Cys Gly Lys Lys Glu Ile Ala Ser Val Ala 20 25 30
Val Asp Gly Cys Glu Gly Asp Leu Cys Val Ile His Lys Ser Lys Pro 35
40 45 Val His Val Ile Ala Glu Phe Thr Ala Asn Gln Asp Thr Cys Lys
Ile 50 55 60 Glu Val Lys Val Thr Gly Gln Leu Asn Gly Leu Glu Val
Pro Ile Pro 65 70 75 80 Gly Ile Glu Thr Asp Gly Cys Lys Val Leu Lys
Cys Pro Leu Lys Lys 85 90 95 Gly Thr Lys Tyr Thr Met Asn Tyr Ser
Val Asn Val Pro Ser Val Val 100 105 110 Pro Asn Ile Lys Thr Val Val
Lys Leu Leu Ala Thr Gly Glu His Gly 115 120 125 Val Leu Ala Cys Gly
Ala Val Asn Thr Asp Val Lys Pro 130 135 140 173109PRTFelis catus
173Met Arg Gly Ala Leu Leu Val Leu Ala Leu Leu Val Thr Gln Ala Leu
1 5 10 15 Gly Val Lys Met Ala Glu Thr Cys Pro Ile Phe Tyr Asp Val
Phe Phe 20 25 30 Ala Val Ala Asn Gly Asn Glu Leu Leu Leu Asp Leu
Ser Leu Thr Lys 35 40 45 Val Asn Ala Thr Glu Pro Glu Arg Thr Ala
Met Lys Lys Ile Gln Asp 50 55 60 Cys Tyr Val Glu Asn Gly Leu Ile
Ser Arg Val Leu Asp Gly Leu Val 65 70 75 80 Met Thr Thr Ile Ser Ser
Ser Lys Asp Cys Met Gly Glu Ala Val Gln 85 90 95 Asn Thr Val Glu
Asp Leu Lys Leu Asn Thr Leu Gly Arg 100 105 17492PRTFelis catus
174Met Lys Gly Ala Cys Val Leu Val Leu Leu Trp Ala Ala Leu Leu Leu
1 5 10 15 Ile Ser Gly Gly Asn Cys Glu Ile Cys Pro Ala Val Lys Arg
Asp Val 20 25 30 Asp Leu Phe Leu Thr Gly Thr Pro Asp Glu Tyr Val
Glu Gln Val Ala 35 40 45 Gln Tyr Lys Ala Leu Pro Val Val Leu Glu
Asn Ala Arg Ile Leu Lys 50 55 60 Asn Cys Val Asp Ala Lys Met Thr
Glu Glu Asp Lys Glu Asn Ala Leu 65 70 75 80 Ser Val Leu Asp Lys Ile
Tyr Thr Ser Pro Leu Cys 85 90 175146PRTFelis catus 175Glu Ile Cys
Pro Ala Val Lys Arg Asp Val Asp Leu Phe Leu Thr Gly 1 5 10 15 Thr
Pro Asp Glu Tyr Val Glu Gln Val Ala Gln Tyr Lys Ala Leu Pro 20 25
30 Val Val Leu Glu Asn Ala Arg Ile Leu Lys Asn Cys Val Asp Ala Lys
35 40 45 Met Thr Glu Glu Asp Lys Glu Asn Ala Leu Ser Leu Leu Asp
Lys Ile 50 55 60 Tyr Thr Ser Pro Leu Cys Val Lys Met Ala Glu Thr
Cys Pro Ile Phe 65 70 75 80 Tyr Asp Val Phe Phe Ala Val Ala Asn Gly
Asn Glu Leu Leu Leu Asp 85 90 95 Leu Ser Leu Thr Lys Val Asn Ala
Thr Glu Pro Glu Arg Thr Ala Met 100 105 110 Lys Lys Ile Gln Asp Cys
Tyr Val Glu Asn Gly Leu Ile Ser Arg Val 115 120 125 Leu Asp Gly Leu
Val Met Thr Thr Ile Ser Ser Ser Lys Asp Cys Met 130 135 140 Gly Glu
145 176172PRTArachis hypogaea 176Met Ala Lys Leu Thr Ile Leu Val
Ala Leu Ala Leu Phe Leu Leu Ala 1 5 10 15 Ala His Ala Ser Ala Arg
Gln Gln Trp Glu Leu Gln Gly Asp Arg Arg 20 25 30 Cys Gln Ser Gln
Leu Glu Arg Ala Asn Leu Arg Pro Cys Glu Gln His 35 40 45 Leu Met
Gln Lys Ile Gln Arg Asp Glu Asp Ser Tyr Gly Arg Asp Pro 50 55 60
Tyr Ser Pro Ser Gln Asp Pro Tyr Ser Pro Ser Gln Asp Pro Asp Arg 65
70 75 80 Arg Asp Pro Tyr Ser Pro Ser Pro Tyr Asp Arg Arg Gly Ala
Gly Ser 85 90 95 Ser Gln His Gln Glu Arg Cys Cys Asn Glu Leu Asn
Glu Phe Glu Asn 100 105 110 Asn Gln Arg Cys Met Cys Glu Ala Leu Gln
Gln Ile Met Glu Asn Gln 115 120 125 Ser Asp Arg Leu Gln Gly Arg Gln
Gln Glu Gln Gln Phe Lys Arg Glu 130 135 140 Leu Arg Asn Leu Pro Gln
Gln Cys Gly Leu Arg Ala Pro Gln Arg Cys 145 150 155 160 Asp Leu Glu
Val Glu Ser Gly Gly Arg Asp Arg Tyr 165 170 177157PRTAlternaria
alternata (Alternaria rot fungus) 177Met Gln Phe Thr Thr Ile Ala
Ser Leu Phe Ala Ala Ala Gly Leu Ala 1 5 10 15 Ala Ala Ala Pro Leu
Glu Ser Arg Gln Asp Thr Ala Ser Cys Pro Val 20 25 30 Thr Thr Glu
Gly Asp Tyr Val Trp Lys Ile Ser Glu Phe Tyr Gly Arg 35 40 45 Lys
Pro Glu Gly Thr Tyr Tyr Asn Ser Leu Gly Phe Asn Ile Lys Ala 50 55
60 Thr Asn Gly Gly Thr Leu Asp Phe Thr Cys Ser Ala Gln Ala Asp Lys
65 70 75 80 Leu Glu Asp His Lys Trp Tyr Ser Cys Gly Glu Asn Ser Phe
Met Asp 85 90 95 Phe Ser Phe Asp Ser Asp Arg Ser Gly Leu Leu Leu
Lys Gln Lys Val 100 105 110 Ser Asp Asp Ile Thr Tyr Val Ala Thr Ala
Thr Leu Pro Asn Tyr Cys 115 120 125 Arg Ala Gly Gly Asn Gly Pro Lys
Asp Phe Val Cys Gln Gly Val Ala 130 135 140 Asp Ala Tyr Ile Thr Leu
Val Thr Leu Pro Lys Ser Ser 145 150 155 178174PRTCanis familiaris
178Met Lys Thr Leu Leu Leu Thr Ile Gly Phe Ser Leu Ile Ala Ile Leu
1 5 10 15 Gln Ala Gln Asp Thr Pro Ala Leu Gly Lys Asp Thr Val Ala
Val Ser 20 25 30 Gly Lys Trp Tyr Leu Lys Ala Met Thr Ala Asp Gln
Glu Val Pro Glu 35 40 45 Lys Pro Asp Ser Val Thr Pro Met Ile Leu
Lys Ala Gln Lys Gly Gly 50 55 60 Asn Leu Glu Ala Lys Ile Thr Met
Leu Thr Asn Gly Gln Cys Gln Asn 65 70 75 80 Ile Thr Val Val Leu His
Lys Thr Ser Glu Pro Gly Lys Tyr Thr Ala 85 90 95 Tyr Glu Gly Gln
Arg Val Val Phe Ile Gln Pro Ser Pro Val Arg Asp 100 105 110 His Tyr
Ile Leu Tyr Cys Glu Gly Glu Leu His Gly Arg Gln Ile Arg 115 120 125
Met Ala Lys Leu Leu Gly Arg Asp Pro Glu Gln Ser Gln Glu Ala Leu 130
135 140 Glu Asp Phe Arg Glu Phe Ser Arg Ala Lys Gly Leu Asn Gln Glu
Ile 145 150 155 160 Leu Glu Leu Ala Gln Ser Glu Thr Cys Ser Pro Gly
Gly Gln 165 170 179286PRTTriticum aestivum 179Met Lys Thr Phe Leu
Ile Leu Val Leu Leu Ala Ile Val Ala Thr Thr 1 5 10 15 Ala Thr Thr
Ala Val Arg Phe Pro Val Pro Gln Leu Gln Pro Gln Asn 20 25 30 Pro
Ser Gln Gln Gln Pro Gln Glu Gln
Val Pro Leu Val Gln Gln Gln 35 40 45 Gln Phe Leu Gly Gln Gln Gln
Pro Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60 Gln Pro Gln Pro Phe
Pro Ser Gln Leu Pro Tyr Leu Gln Leu Gln Pro 65 70 75 80 Phe Pro Gln
Pro Gln Leu Pro Tyr Ser Gln Pro Gln Pro Phe Arg Pro 85 90 95 Gln
Gln Pro Tyr Pro Gln Pro Gln Pro Gln Tyr Ser Gln Pro Gln Gln 100 105
110 Pro Ile Ser Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
115 120 125 Gln Gln Gln Gln Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln
Leu Ile 130 135 140 Pro Cys Met Asp Val Val Leu Gln Gln His Asn Ile
Ala His Gly Arg 145 150 155 160 Ser Gln Val Leu Gln Gln Ser Thr Tyr
Gln Leu Leu Gln Glu Leu Cys 165 170 175 Cys Gln His Leu Trp Gln Ile
Pro Glu Gln Ser Gln Cys Gln Ala Ile 180 185 190 His Asn Val Val His
Ala Ile Ile Leu His Gln Gln Gln Lys Gln Gln 195 200 205 Gln Gln Pro
Ser Ser Gln Val Ser Phe Gln Gln Pro Leu Gln Gln Tyr 210 215 220 Pro
Leu Gly Gln Gly Ser Phe Arg Pro Ser Gln Gln Asn Pro Gln Ala 225 230
235 240 Gln Gly Ser Val Gln Pro Gln Gln Leu Pro Gln Phe Glu Glu Ile
Arg 245 250 255 Asn Leu Ala Leu Gln Thr Leu Pro Ala Met Cys Asn Val
Tyr Ile Pro 260 265 270 Pro Tyr Cys Thr Ile Ala Pro Phe Gly Ile Phe
Gly Thr Asn 275 280 285 180491PRTBlattella germanica 180Ala Ile Glu
Phe Leu Asn Asn Ile His Asp Leu Leu Gly Ile Pro His 1 5 10 15 Ile
Pro Val Thr Ala Arg Lys His His Arg Arg Gly Val Gly Ile Thr 20 25
30 Gly Leu Ile Asp Asp Ile Ile Ala Ile Leu Pro Val Asp Asp Leu Tyr
35 40 45 Ala Leu Phe Gln Glu Lys Leu Glu Thr Ser Pro Glu Phe Lys
Ala Leu 50 55 60 Tyr Asp Ala Ile Arg Ser Pro Glu Phe Gln Ser Ile
Val Gly Thr Leu 65 70 75 80 Glu Ala Met Pro Glu Tyr Gln Asn Leu Ile
Gln Lys Leu Lys Asp Lys 85 90 95 Gly Val Asp Val Asp His Ile Ile
Glu Leu Ile His Gln Ile Phe Asn 100 105 110 Ile Val Arg Asp Thr Arg
Gly Leu Pro Glu Asp Leu Gln Asp Phe Leu 115 120 125 Ala Leu Ile Pro
Thr Asp Gln Val Leu Ala Ile Ala Ala Asp Tyr Leu 130 135 140 Ala Asn
Asp Ala Glu Val Lys Ala Ala Val Glu Tyr Leu Lys Ser Asp 145 150 155
160 Glu Phe Glu Thr Ile Val Val Thr Val Asp Ser Leu Pro Glu Phe Lys
165 170 175 Asn Phe Leu Asn Phe Leu Gln Thr Asn Gly Leu Asn Ala Ile
Glu Phe 180 185 190 Leu Asn Asn Ile His Asp Leu Leu Gly Ile Pro His
Ile Pro Val Thr 195 200 205 Ala Arg Lys His Leu Arg Arg Gly Val Gly
Ile Thr Gly Leu Ile Asp 210 215 220 Asp Ile Ile Ala Ile Leu Pro Val
Asp Asp Leu Tyr Ala Leu Phe Gln 225 230 235 240 Glu Lys Leu Glu Thr
Ser Pro Glu Phe Lys Ala Leu Tyr Asp Ala Ile 245 250 255 Arg Ser Pro
Glu Phe Gln Ser Ile Val Glu Thr Leu Lys Ala Met Pro 260 265 270 Glu
Tyr Gln Ser Leu Ile Gln Lys Leu Lys Asp Lys Gly Val Asp Val 275 280
285 Asp His Ile Ile Glu Leu Ile His Gln Ile Phe Asn Ile Val Arg Asp
290 295 300 Thr Arg Gly Leu Pro Glu Asp Leu Gln Asp Phe Leu Ala Leu
Ile Pro 305 310 315 320 Ile Asp Gln Ile Leu Ala Ile Ala Ala Asp Tyr
Leu Ala Asn Asp Ala 325 330 335 Glu Val Gln Ala Ala Val Glu Tyr Leu
Lys Ser Asp Glu Phe Glu Thr 340 345 350 Ile Val Val Thr Val Asp Ser
Leu Pro Glu Phe Lys Asn Phe Leu Asn 355 360 365 Phe Leu Gln Thr Asn
Gly Leu Asn Ala Ile Glu Phe Ile Asn Asn Ile 370 375 380 His Asp Leu
Leu Gly Ile Pro His Ile Pro Ala Thr Gly Arg Lys His 385 390 395 400
Val Arg Arg Gly Val Gly Ile Asn Gly Leu Ile Asp Asp Val Ile Ala 405
410 415 Ile Leu Pro Val Asp Glu Leu Tyr Ala Leu Phe Gln Glu Lys Leu
Glu 420 425 430 Ser Ser Pro Glu Phe Lys Ala Leu Tyr Asp Ala Ile Arg
Ser Pro Glu 435 440 445 Phe Gln Ser Ile Val Gln Thr Leu Lys Ala Met
Pro Glu Tyr Gln Asp 450 455 460 Leu Ile Gln Arg Leu Lys Asp Lys Gly
Val Asp Val Asp His Phe Ile 465 470 475 480 Glu Leu Ile Lys Lys Leu
Phe Gly Leu Ser His 485 490 181160PRTBetula pendula (Betula
verrucosa) 181Met Gly Val Phe Asn Tyr Glu Thr Glu Thr Thr Ser Val
Ile Pro Ala 1 5 10 15 Ala Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly
Asp Asn Leu Phe Pro 20 25 30 Lys Val Ala Pro Gln Ala Ile Ser Ser
Val Glu Asn Ile Glu Gly Asn 35 40 45 Gly Gly Pro Gly Thr Ile Lys
Lys Ile Ser Phe Pro Glu Gly Phe Pro 50 55 60 Phe Lys Tyr Val Lys
Asp Arg Val Asp Glu Val Asp His Thr Asn Phe 65 70 75 80 Lys Tyr Asn
Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Thr Leu 85 90 95 Glu
Lys Ile Ser Asn Glu Ile Lys Ile Val Ala Thr Pro Asp Gly Gly 100 105
110 Ser Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Asp His Glu
115 120 125 Val Lys Ala Glu Gln Val Lys Ala Ser Lys Glu Met Gly Glu
Thr Leu 130 135 140 Leu Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser
Asp Ala Tyr Asn 145 150 155 160 182160PRTBetula pendula (Betula
verrucosa) 182Met Gly Val Phe Asn Tyr Glu Thr Glu Ala Thr Ser Val
Ile Pro Ala 1 5 10 15 Ala Arg Met Phe Lys Ala Phe Ile Leu Asp Gly
Asp Lys Leu Val Pro 20 25 30 Lys Val Ala Pro Gln Ala Ile Ser Ser
Val Glu Asn Ile Glu Gly Asn 35 40 45 Gly Gly Pro Gly Thr Ile Lys
Lys Ile Asn Phe Pro Glu Gly Phe Pro 50 55 60 Phe Lys Tyr Val Lys
Asp Arg Val Asp Glu Val Asp His Thr Asn Phe 65 70 75 80 Lys Tyr Asn
Tyr Ser Val Ile Glu Gly Gly Pro Val Gly Asp Thr Leu 85 90 95 Glu
Lys Ile Ser Asn Glu Ile Lys Ile Val Ala Thr Pro Asp Gly Gly 100 105
110 Cys Val Leu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Asn His Glu
115 120 125 Val Lys Ala Glu Gln Val Lys Ala Ser Lys Glu Met Gly Glu
Thr Leu 130 135 140 Leu Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser
Asp Ala Tyr Asn 145 150 155 160 183133PRTBetula pendula (Betula
verrucosa) 183Met Ser Trp Gln Thr Tyr Val Asp Glu His Leu Met Cys
Asp Ile Asp 1 5 10 15 Gly Gln Ala Ser Asn Ser Leu Ala Ser Ala Ile
Val Gly His Asp Gly 20 25 30 Ser Val Trp Ala Gln Ser Ser Ser Phe
Pro Gln Phe Lys Pro Gln Glu 35 40 45 Ile Thr Gly Ile Met Lys Asp
Phe Glu Glu Pro Gly His Leu Ala Pro 50 55 60 Thr Gly Leu His Leu
Gly Gly Ile Lys Tyr Met Val Ile Gln Gly Glu 65 70 75 80 Ala Gly Ala
Val Ile Arg Gly Lys Lys Gly Ser Gly Gly Ile Thr Ile 85 90 95 Lys
Lys Thr Gly Gln Ala Leu Val Phe Gly Ile Tyr Glu Glu Pro Val 100 105
110 Thr Pro Gly Gln Cys Asn Met Val Val Glu Arg Leu Gly Asp Tyr Leu
115 120 125 Ile Asp Gln Gly Leu 130 184263PRTPhleum pratense 184Met
Ala Ser Ser Ser Ser Val Leu Leu Val Val Val Leu Phe Ala Val 1 5 10
15 Phe Leu Gly Ser Ala Tyr Gly Ile Pro Lys Val Pro Pro Gly Pro Asn
20 25 30 Ile Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser
Thr Trp 35 40 45 Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys Asp Asn
Gly Gly Ala Cys 50 55 60 Gly Tyr Lys Asp Val Asp Lys Pro Pro Phe
Ser Gly Met Thr Gly Cys 65 70 75 80 Gly Asn Thr Pro Ile Phe Lys Ser
Gly Arg Gly Cys Gly Ser Cys Phe 85 90 95 Glu Ile Lys Cys Thr Lys
Pro Glu Ala Cys Ser Gly Glu Pro Val Val 100 105 110 Val His Ile Thr
Asp Asp Asn Glu Glu Pro Ile Ala Pro Tyr His Phe 115 120 125 Asp Leu
Ser Gly His Ala Phe Gly Ala Met Ala Lys Lys Gly Asp Glu 130 135 140
Gln Lys Leu Arg Ser Ala Gly Glu Leu Glu Leu Gln Phe Arg Arg Val 145
150 155 160 Lys Cys Lys Tyr Pro Glu Gly Thr Lys Val Thr Phe His Val
Glu Lys 165 170 175 Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys
Tyr Val Asn Gly 180 185 190 Asp Gly Asp Val Val Ala Val Asp Ile Lys
Glu Lys Gly Lys Asp Lys 195 200 205 Trp Ile Glu Leu Lys Glu Ser Trp
Gly Ala Ile Trp Arg Ile Asp Thr 210 215 220 Pro Asp Lys Leu Thr Gly
Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly 225 230 235 240 Gly Thr Lys
Thr Glu Ala Glu Asp Val Ile Pro Glu Gly Trp Lys Ala 245 250 255 Asp
Thr Ser Tyr Glu Ser Lys 260 185122PRTPhleum pratense 185Met Ser Met
Ala Ser Ser Ser Ser Ser Ser Leu Leu Ala Met Ala Val 1 5 10 15 Leu
Ala Ala Leu Phe Ala Gly Ala Trp Cys Val Pro Lys Val Thr Phe 20 25
30 Thr Val Glu Lys Gly Ser Asn Glu Lys His Leu Ala Val Leu Val Lys
35 40 45 Tyr Glu Gly Asp Thr Met Ala Glu Val Glu Leu Arg Glu His
Gly Ser 50 55 60 Asp Glu Trp Val Ala Met Thr Lys Gly Glu Gly Gly
Val Trp Thr Phe 65 70 75 80 Asp Ser Glu Glu Pro Leu Gln Gly Pro Phe
Asn Phe Arg Phe Leu Thr 85 90 95 Glu Lys Gly Met Lys Asn Val Phe
Asp Asp Val Val Pro Glu Lys Tyr 100 105 110 Thr Ile Gly Ala Thr Tyr
Ala Pro Glu Glu 115 120 186159PRTMalus domestica 186Met Gly Val Tyr
Thr Phe Glu Asn Glu Phe Thr Ser Glu Ile Pro Pro 1 5 10 15 Ser Arg
Leu Phe Lys Ala Phe Val Leu Asp Ala Asp Asn Leu Ile Pro 20 25 30
Lys Ile Ala Pro Gln Ala Ile Lys Gln Ala Glu Ile Leu Glu Gly Asn 35
40 45 Gly Gly Pro Gly Thr Ile Lys Lys Ile Thr Phe Gly Glu Gly Ser
Gln 50 55 60 Tyr Gly Tyr Val Lys His Arg Ile Asp Ser Ile Asp Glu
Ala Ser Tyr 65 70 75 80 Ser Tyr Ser Tyr Thr Leu Ile Glu Gly Asp Ala
Leu Thr Asp Thr Ile 85 90 95 Glu Lys Ile Ser Tyr Glu Thr Lys Leu
Val Ala Cys Gly Ser Gly Ser 100 105 110 Thr Ile Lys Ser Ile Ser His
Tyr His Thr Lys Gly Asn Ile Glu Ile 115 120 125 Lys Glu Glu His Val
Lys Val Gly Lys Glu Lys Ala His Gly Leu Phe 130 135 140 Lys Leu Ile
Glu Ser Tyr Leu Lys Asp His Pro Asp Ala Tyr Asn 145 150 155
18796PRTDactylis glomerata 187Val Lys Val Thr Phe Lys Val Glu Lys
Gly Ser Asp Pro Lys Lys Leu 1 5 10 15 Val Leu Asp Ile Lys Tyr Thr
Arg Pro Gly Asp Thr Leu Ala Glu Val 20 25 30 Glu Leu Arg Gln His
Gly Ser Glu Glu Trp Glu Pro Leu Thr Lys Lys 35 40 45 Gly Asn Leu
Trp Glu Val Lys Ser Ser Lys Pro Leu Thr Gly Pro Phe 50 55 60 Asn
Phe Arg Phe Met Ser Lys Gly Gly Met Arg Asn Val Phe Asp Glu 65 70
75 80 Val Ile Pro Thr Ala Phe Lys Ile Gly Thr Thr Tyr Thr Pro Glu
Glu 85 90 95 188269PRTPhalaris aquatica 188Met Met Lys Met Val Cys
Ser Ser Ser Ser Ser Ser Leu Leu Val Val 1 5 10 15 Ala Ala Leu Leu
Ala Val Phe Val Gly Ser Ala Gln Gly Ile Ala Lys 20 25 30 Val Pro
Pro Gly Pro Asn Ile Thr Ala Glu Tyr Gly Asp Lys Trp Leu 35 40 45
Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys 50
55 60 Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys Ala Pro
Phe 65 70 75 80 Asn Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys
Asp Gly Arg 85 90 95 Gly Cys Gly Ser Cys Phe Glu Leu Lys Cys Ser
Lys Pro Glu Ser Cys 100 105 110 Ser Gly Glu Pro Ile Thr Val His Ile
Thr Asp Asp Asn Glu Glu Pro 115 120 125 Ile Ala Pro Tyr His Phe Asp
Leu Ser Gly His Ala Phe Gly Ser Met 130 135 140 Ala Lys Lys Gly Glu
Glu Glu Asn Val Arg Gly Ala Gly Glu Leu Glu 145 150 155 160 Leu Gln
Phe Arg Arg Val Lys Cys Lys Tyr Pro Asp Gly Thr Lys Pro 165 170 175
Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu 180
185 190 Val Lys Tyr Val Asp Gly Asp Gly Asp Val Val Ala Val Asp Ile
Lys 195 200 205 Glu Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser
Trp Gly Ala 210 215 220 Ile Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr
Gly Pro Phe Thr Val 225 230 235 240 Arg Tyr Thr Thr Glu Gly Gly Thr
Lys Ala Glu Phe Glu Asp Val Ile 245 250 255 Pro Glu Gly Trp Lys Ala
Asp Thr His Asp Ala Ser Lys 260 265 189246PRTCynodon dactylon
189Ala Ile Gly Asp Lys Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Ser
1 5 10 15 Lys Trp Leu Glu Ala Arg Ala Thr Phe Tyr Gly Ser Asn Pro
Arg Gly 20 25 30 Ala Ala Pro Asp Asp His Gly Gly Ala Cys Gly Tyr
Lys Asp Val Asp 35 40 45 Lys Pro Pro Phe Asp Gly Met Thr Ala Cys
Gly Asn Glu Pro Ile Phe 50 55 60 Lys Asp Gly Leu Gly Cys Arg Ala
Cys Tyr Glu Ile Lys Cys Lys Glu 65 70 75 80 Pro Val Glu Cys Ser Gly
Glu Pro Val Leu Val Lys Ile Thr Asp Lys 85 90 95 Asn Tyr Glu His
Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Lys Ala 100 105 110 Phe Gly
Ala Met Ala Lys Lys Gly Gln Glu Asp Lys Leu Arg Lys Ala 115 120 125
Gly Glu Leu Thr Leu Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro Ser 130
135 140 Gly Thr Lys Ile Thr Phe His Ile Glu Lys Gly Ser Asn Asp His
Tyr 145 150
155 160 Leu Ala Leu Leu Val Lys Tyr Ala Ala Gly Asp Gly Asn Ile Val
Ala 165 170 175 Val Asp Ile Lys Pro Arg Asp Ser Asp Glu Phe Ile Pro
Met Lys Ser 180 185 190 Ser Trp Gly Ala Ile Trp Arg Ile Asp Pro Lys
Lys Pro Leu Lys Gly 195 200 205 Pro Phe Ser Ile Arg Leu Thr Ser Glu
Gly Gly Ala His Leu Val Gln 210 215 220 Asp Asp Val Ile Pro Ala Asn
Trp Lys Pro Asp Thr Val Tyr Thr Ser 225 230 235 240 Lys Leu Gln Phe
Gly Ala 245 190214PRTBos primigenius 190Met Lys Leu Leu Ile Leu Thr
Cys Leu Val Ala Val Ala Leu Ala Arg 1 5 10 15 Pro Lys His Pro Ile
Lys His Gln Gly Leu Pro Gln Glu Val Leu Asn 20 25 30 Glu Asn Leu
Leu Arg Phe Phe Val Ala Pro Phe Pro Glu Val Phe Gly 35 40 45 Lys
Glu Lys Val Asn Glu Leu Ser Lys Asp Ile Gly Ser Glu Ser Thr 50 55
60 Glu Asp Gln Ala Met Glu Asp Ile Lys Gln Met Glu Ala Glu Ser Ile
65 70 75 80 Ser Ser Ser Glu Glu Ile Val Pro Asn Ser Val Glu Gln Lys
His Ile 85 90 95 Gln Lys Glu Asp Val Pro Ser Glu Arg Tyr Leu Gly
Tyr Leu Glu Gln 100 105 110 Leu Leu Arg Leu Lys Lys Tyr Lys Val Pro
Gln Leu Glu Ile Val Pro 115 120 125 Asn Ser Ala Glu Glu Arg Leu His
Ser Met Lys Glu Gly Ile His Ala 130 135 140 Gln Gln Lys Glu Pro Met
Ile Gly Val Asn Gln Glu Leu Ala Tyr Phe 145 150 155 160 Tyr Pro Glu
Leu Phe Arg Gln Phe Tyr Gln Leu Asp Ala Tyr Pro Ser 165 170 175 Gly
Ala Trp Tyr Tyr Val Pro Leu Gly Thr Gln Tyr Thr Asp Ala Pro 180 185
190 Ser Phe Ser Asp Ile Pro Asn Pro Ile Gly Ser Glu Asn Ser Glu Lys
195 200 205 Thr Thr Met Pro Leu Trp 210 191142PRTBos primigenius
191Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala
1 5 10 15 Thr Gln Ala Glu Gln Leu Thr Lys Cys Glu Val Phe Arg Glu
Leu Lys 20 25 30 Asp Leu Lys Gly Tyr Gly Gly Val Ser Leu Pro Glu
Trp Val Cys Thr 35 40 45 Thr Phe His Thr Ser Gly Tyr Asp Thr Gln
Ala Ile Val Gln Asn Asn 50 55 60 Asp Ser Thr Glu Tyr Gly Leu Phe
Gln Ile Asn Asn Lys Ile Trp Cys 65 70 75 80 Lys Asp Asp Gln Asn Pro
His Ser Ser Asn Ile Cys Asn Ile Ser Cys 85 90 95 Asp Lys Phe Leu
Asp Asp Asp Leu Thr Asp Asp Ile Met Cys Val Lys 100 105 110 Lys Ile
Leu Asp Lys Val Gly Ile Asn Tyr Trp Leu Ala His Lys Ala 115 120 125
Leu Cys Ser Glu Lys Leu Asp Gln Trp Leu Cys Glu Lys Leu 130 135 140
192386PRTGallus gallus 192Met Gly Ser Ile Gly Ala Ala Ser Met Glu
Phe Cys Phe Asp Val Phe 1 5 10 15 Lys Glu Leu Lys Val His His Ala
Asn Glu Asn Ile Phe Tyr Cys Pro 20 25 30 Ile Ala Ile Met Ser Ala
Leu Ala Met Val Tyr Leu Gly Ala Lys Asp 35 40 45 Ser Thr Arg Thr
Gln Ile Asn Lys Val Val Arg Phe Asp Lys Leu Pro 50 55 60 Gly Phe
Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val Asn Val 65 70 75 80
His Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro Asn Asp 85
90 95 Val Tyr Ser Phe Ser Leu Ala Ser Arg Leu Tyr Ala Glu Glu Arg
Tyr 100 105 110 Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val Lys Glu Leu
Tyr Arg Gly 115 120 125 Gly Leu Glu Pro Ile Asn Phe Gln Thr Ala Ala
Asp Gln Ala Arg Glu 130 135 140 Leu Ile Asn Ser Trp Val Glu Ser Gln
Thr Asn Gly Ile Ile Arg Asn 145 150 155 160 Val Leu Gln Pro Ser Ser
Val Asp Ser Gln Thr Ala Met Val Leu Val 165 170 175 Asn Ala Ile Val
Phe Lys Gly Leu Trp Glu Lys Ala Phe Lys Asp Glu 180 185 190 Asp Thr
Gln Ala Met Pro Phe Arg Val Thr Glu Gln Glu Ser Lys Pro 195 200 205
Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser Met Ala 210
215 220 Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly Thr
Met 225 230 235 240 Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly
Leu Glu Gln Leu 245 250 255 Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr
Glu Trp Thr Ser Ser Asn 260 265 270 Val Met Glu Glu Arg Lys Ile Lys
Val Tyr Leu Pro Arg Met Lys Met 275 280 285 Glu Glu Lys Tyr Asn Leu
Thr Ser Val Leu Met Ala Met Gly Ile Thr 290 295 300 Asp Val Phe Ser
Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Glu 305 310 315 320 Ser
Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn 325 330
335 Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val Asp Ala
340 345 350 Ala Ser Val Ser Glu Glu Phe Arg Ala Asp His Pro Phe Leu
Phe Cys 355 360 365 Ile Lys His Ile Ala Thr Asn Ala Val Leu Phe Phe
Gly Arg Cys Val 370 375 380 Ser Pro 385 193147PRTGallus gallus
193Met Arg Ser Leu Leu Ile Leu Val Leu Cys Phe Leu Pro Leu Ala Ala
1 5 10 15 Leu Gly Lys Val Phe Gly Arg Cys Glu Leu Ala Ala Ala Met
Lys Arg 20 25 30 His Gly Leu Asp Asn Tyr Arg Gly Tyr Ser Leu Gly
Asn Trp Val Cys 35 40 45 Ala Ala Lys Phe Glu Ser Asn Phe Asn Thr
Gln Ala Thr Asn Arg Asn 50 55 60 Thr Asp Gly Ser Thr Asp Tyr Gly
Ile Leu Gln Ile Asn Ser Arg Trp 65 70 75 80 Trp Cys Asn Asp Gly Arg
Thr Pro Gly Ser Arg Asn Leu Cys Asn Ile 85 90 95 Pro Cys Ser Ala
Leu Leu Ser Ser Asp Ile Thr Ala Ser Val Asn Cys 100 105 110 Ala Lys
Lys Ile Val Ser Asp Gly Asn Gly Met Asn Ala Trp Val Ala 115 120 125
Trp Arg Asn Arg Cys Lys Gly Thr Asp Val Gln Ala Trp Ile Arg Gly 130
135 140 Cys Arg Leu 145 194187PRTEquus caballus 194Met Lys Leu Leu
Leu Leu Cys Leu Gly Leu Ile Leu Val Cys Ala Gln 1 5 10 15 Gln Glu
Glu Asn Ser Asp Val Ala Ile Arg Asn Phe Asp Ile Ser Lys 20 25 30
Ile Ser Gly Glu Trp Tyr Ser Ile Phe Leu Ala Ser Asp Val Lys Glu 35
40 45 Lys Ile Glu Glu Asn Gly Ser Met Arg Val Phe Val Asp Val Ile
Arg 50 55 60 Ala Leu Asp Asn Ser Ser Leu Tyr Ala Glu Tyr Gln Thr
Lys Val Asn 65 70 75 80 Gly Glu Cys Thr Glu Phe Pro Met Val Phe Asp
Lys Thr Glu Glu Asp 85 90 95 Gly Val Tyr Ser Leu Asn Tyr Asp Gly
Tyr Asn Val Phe Arg Ile Ser 100 105 110 Glu Phe Glu Asn Asp Glu His
Ile Ile Leu Tyr Leu Val Asn Phe Asp 115 120 125 Lys Asp Arg Pro Phe
Gln Leu Phe Glu Phe Tyr Ala Arg Glu Pro Asp 130 135 140 Val Ser Pro
Glu Ile Lys Glu Glu Phe Val Lys Ile Val Gln Lys Arg 145 150 155 160
Gly Ile Val Lys Glu Asn Ile Ile Asp Leu Thr Lys Ile Asp Arg Cys 165
170 175 Phe Gln Leu Arg Gly Asn Gly Val Ala Gln Ala 180 185
195228PRTEquus caballus 195Met Leu Lys Val Ser Cys Leu Phe Val Leu
Leu Cys Gly Leu Leu Val 1 5 10 15 Pro Ser Ser Ala Gln Gln Ile Pro
Pro Glu Val Ser Ser Gln Ile Thr 20 25 30 Asp Ala Leu Thr Gln Gly
Leu Leu Asp Gly Asn Phe Leu Ser Leu Leu 35 40 45 Asn Ala Ile Asn
Leu Glu Gly Leu Leu Asn Thr Ile Leu Asp Gln Val 50 55 60 Thr Gly
Leu Leu Asn Ile Leu Val Gly Pro Leu Leu Gly Pro Ser Asp 65 70 75 80
Ala Glu Ile Lys Leu Gln Asp Thr Arg Leu Leu Gln Leu Ser Leu Glu 85
90 95 Phe Ser Pro Asp Ser Lys Gly Ile Asp Ile Trp Ile Pro Leu Glu
Leu 100 105 110 Ser Val Tyr Leu Lys Leu Leu Ile Leu Glu Pro Leu Thr
Leu Tyr Val 115 120 125 Arg Thr Asp Ile Arg Val Gln Leu Arg Leu Glu
Ser Asp Glu Asp Gly 130 135 140 Lys Tyr Arg Leu Ala Phe Gly His Cys
Ser Leu Leu Pro Arg Ala Ile 145 150 155 160 Glu Leu Gln Ser Gly Asn
Pro Leu Ser Leu Pro Val Asn Ala Val Leu 165 170 175 Gly Thr Ile Glu
Asn Ala Leu Gly Asn Phe Ile Thr Glu Asp Leu Gly 180 185 190 Ala Gly
Leu Cys Pro Thr Leu Asn Ser Leu Val Ser Asn Leu Asp Leu 195 200 205
Gln Leu Val Asn Asn Leu Ile Asn Leu Ile Leu Asp Arg Ala Asn Val 210
215 220 Asp Leu Ser Val 225 196558DNAArtificial SequenceNucleotide
sequence encoding pelB-CD3(VL) - FLAG-BirA-U266Ant-6His
196atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc
ccagccggcg 60atggccgaca ttcagctgac ccagtctcca gcaatcatgt ctgcatctcc
aggggagaag 120gtcaccatga cctgcagagc cagttcaagt gtaagttaca
tgaactggta ccagcagaag 180tcaggcacct cccccaaaag atggatttat
gacacatcca aagtggcttc tggagtccct 240tatcgcttca gtggcagtgg
gtctgggacc tcatactctc tcacaatcag cagcatggag 300gctgaagatg
ctgccactta ttactgccaa cagtggagta gtaacccgct cacgttcggt
360gctgggacca agctggagct gaaatccgga ggtggtggat ccgactacaa
ggatgacgat 420gacaaaggcg gcggcctgaa cgatattttt gaagcgcaga
aaattgaatg gcatctgagc 480ccgcatctgc tgtgggatct gtttcgcgtg
ggcctgccgg gcgcggcggg cggcggccat 540catcaccatc atcattag
558197185PRTArtificial SequencepelB-CD3(VL) -
FLAG-BirA-U266Ant-6His 197Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala
Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Asp Ile
Gln Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly
Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser
Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser 50 55 60 Pro Lys
Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro 65 70 75 80
Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85
90 95 Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
Trp 100 105 110 Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
Glu Leu Lys 115 120 125 Ser Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp
Asp Asp Lys Gly Gly 130 135 140 Gly Leu Asn Asp Ile Phe Glu Ala Gln
Lys Ile Glu Trp His Leu Ser 145 150 155 160 Pro His Leu Leu Trp Asp
Leu Phe Arg Val Gly Leu Pro Gly Ala Ala 165 170 175 Gly Gly Gly His
His His His His His 180 185 19818PRTArtificial SequenceAlternative
linker 198Gly Glu Gly Thr Ser Thr Gly Ser Gly Gly Ser Gly Gly Ser
Gly Gly 1 5 10 15 Ala Asp 1994PRTArtificial SequenceLinker sequence
199Gly Gly Gly Ser 1 2005PRTArtificial SequenceLinker sequence
200Gly Gly Gly Gly Ser 1 5 2018PRTArtificial SequenceCMV pp65
derived peptide 201Thr Pro Arg Val Thr Gly Gly Gly 1 5
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References