U.S. patent application number 13/567672 was filed with the patent office on 2013-05-23 for compositions comprising cross-species-specific antibodies and uses thereof.
This patent application is currently assigned to Amgen Research (Munich) GmbH. The applicant listed for this patent is Ronny Cierpka, Roman KISCHEL, Peter Kufer, Doris Rau, Tobias Raum, Bernd Schlereth. Invention is credited to Ronny Cierpka, Roman KISCHEL, Peter Kufer, Doris Rau, Tobias Raum, Bernd Schlereth.
Application Number | 20130129729 13/567672 |
Document ID | / |
Family ID | 37943161 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130129729 |
Kind Code |
A1 |
KISCHEL; Roman ; et
al. |
May 23, 2013 |
COMPOSITIONS COMPRISING CROSS-SPECIES-SPECIFIC ANTIBODIES AND USES
THEREOF
Abstract
The present invention relates to uses of bispecific antibodies
exhibiting cross-species specificity for evaluating the in vivo
safety and/or activity and/or pharmacokinetic profile of the same
in non-human species and humans. The present invention moreover
relates to methods for evaluating the in vivo safety and/or
activity and/or pharmacokinetic profile of said bispecific
antibodies exhibiting cross-species specificity. The present
invention also relates to methods of measuring the biological
activity and/or efficacy of such bispecific antibodies exhibiting
cross-species specificity. In addition, the present invention
relates to pharmaceutical compositions comprising bispecific single
chain antibodies exhibiting cross-species specificity and to
methods for the preparation of pharmaceutical compositions
comprising said bispecific single chain antibodies exhibiting
cross-species specificity for the treatment of diseases.
Inventors: |
KISCHEL; Roman; (Karlsfield,
DE) ; Raum; Tobias; (Munchen, DE) ; Schlereth;
Bernd; (Germering, DE) ; Rau; Doris;
(Unterhaching, DE) ; Cierpka; Ronny; (Munchen,
DE) ; Kufer; Peter; (Moosburg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KISCHEL; Roman
Raum; Tobias
Schlereth; Bernd
Rau; Doris
Cierpka; Ronny
Kufer; Peter |
Karlsfield
Munchen
Germering
Unterhaching
Munchen
Moosburg |
|
DE
DE
DE
DE
DE
DE |
|
|
Assignee: |
Amgen Research (Munich)
GmbH
|
Family ID: |
37943161 |
Appl. No.: |
13/567672 |
Filed: |
August 6, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12083351 |
Jun 3, 2008 |
8236308 |
|
|
PCT/EP2006/009782 |
Oct 10, 2006 |
|
|
|
13567672 |
|
|
|
|
60724781 |
Oct 11, 2005 |
|
|
|
Current U.S.
Class: |
424/135.1 ;
435/320.1; 435/328; 435/69.6; 530/387.3; 536/23.53 |
Current CPC
Class: |
A61K 39/39558 20130101;
C07K 2317/622 20130101; A61P 35/00 20180101; C07K 2317/34 20130101;
C07K 16/2809 20130101; A61P 43/00 20180101; A61P 37/00 20180101;
C07K 2317/56 20130101; C07K 16/30 20130101; A61K 45/06 20130101;
C07K 2317/31 20130101; C07K 16/468 20130101; A61K 2039/505
20130101; C07K 16/40 20130101 |
Class at
Publication: |
424/135.1 ;
530/387.3; 536/23.53; 435/320.1; 435/328; 435/69.6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 45/06 20060101 A61K045/06; C07K 16/46 20060101
C07K016/46 |
Claims
1. A bispecific single chain antibody which comprises (i) a first
binding domain binding to an epitope of human and non-chimpanzee
primate CD3, and (ii) a second binding domain binding to a cell
surface antigen, wherein the epitope comprises the amino acid
sequence "FSEXE" (SEQ ID NO. 204), wherein "X" represents L
(Leucine) or M (Methionine).
2. (canceled)
3. The bispecific single chain antibody of claim 1, wherein said
first binding domain is located C-terminally or N-terminally to the
second binding domain.
4. The bispecific single chain antibody of claim 1, wherein the
second binding domain binds to a human cell surface antigen and to
the non-chimpanzee primate homolog of said cell surface
antigen.
5. The bispecific single chain antibody of claim 1, wherein the
cell surface antigen is a tumor antigen.
6. The bispecific single chain antibody of claim 1, wherein the
first binding domain comprises a VH region having an amino acid
sequence as shown in any of SEQ ID NOs. 2, 110 or 6.
7. The bispecific single chain antibody of claim 1, wherein the
first binding domain comprises a VL region having an amino acid
sequence as shown in any of SEQ ID NOs. 4, 148, 168 or 8.
8. The bispecific single chain antibody of claim 1, wherein the VH
region of the first binding domain comprises or consists of the
amino acid sequence shown in SEQ ID NO. 2 and the VL region of the
first binding domain comprises or consists of the amino acid
sequence shown in SEQ ID NO. 4.
9. The bispecific single chain antibody of claim 1, wherein the VH
region of the first binding domain comprises or consists of the
amino acid sequence shown in SEQ ID NO. 110 and the VL region of
the first binding domain comprises or consists of the amino acid
sequence shown in SEQ ID NO. 148 or SEQ ID NO: 168.
10. The bispecific single chain antibody of claim 1, wherein the VH
region of the first binding domain comprises or consists of the
amino acid sequence shown in SEQ ID NO. 6 and the VL region of the
first binding domain comprises or consists of the amino acid
sequence shown in SEQ ID NO. 8.
11. The bispecific single chain antibody of claim 5, wherein said
tumor antigen is EpCAM, EGFR, EGFRvIII or Carboanhydrase IX (MN/CA
IX).
12. The bispecific single chain antibody of claim 1, wherein the
non-chimpanzee primate is a baboon, marmoset or an old world
monkey.
13. The bispecific single chain antibody of claim 12, wherein the
old world monkey is a monkey of the macaque genus.
14. The bispecific single chain antibody of claim 13, wherein the
monkey of the macaque genus is Assamese macaque (Macaca
assamensis), Barbary macaque (Macaca sylvanus), Bonnet macaque
(Macaca radiata), Booted or Sulawesi-Booted macaque (Macaca
ochreata), Sulawesi-crested macaque (Macaca nigra), Formosan rock
macaque (Macaca cyclopsis), Japanese snow macaque or Japanese
macaque (Macaca fuscata), Cynomolgus monkey or crab-eating macaque
or long-tailed macaque or Java macaque (Macaca fascicularis),
Lion-tailed macaque (Macaca silenus), Pigtailed macaque (Macaca
nemestrina), Rhesus macaque (Macaca mulatta), Tibetan macaque
(Macaca thibetana), Tonkean macaque (Macaca tonkeana), Toque
macaque (Macaca sinica), Stump-tailed macaque or Red-faced macaque
or Bear monkey (Macaca arctoides), or Moor macaque (Macaca
maurus).
15. The bispecific single chain antibody of claim 1, wherein the
non-chimpanzee primate CD3 comprises or consists of an amino acid
sequence shown in SEQ ID NOs. 135, 136, 144 or 145.
16. The bispecific single chain antibody of claim 1, wherein said
bispecific single chain antibody comprises an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
as depicted in any of SEQ ID NOs. 38, 40, 124, 42 or 44; (b) an
amino acid sequence encoded by a nucleic acid sequence as shown in
SEQ ID NOs. 37, 39, 125, 41 or 43; (c) an amino acid sequence
encoded by a nucleic acid sequence hybridising under stringent
conditions to the complementary nucleic acid sequence of (b); (d)
an amino acid sequence encoded by a nucleic acid sequence which is
degenerate as a result of the genetic code to a nucleotide sequence
of (b); and (e) an amino acid sequence at least 85% identical, more
preferred at least 90% identical, most preferred at least 95%
identical to the amino acid sequence of (a) or (b).
17. The bispecific single chain antibody of claim 1, wherein said
bispecific single chain antibody comprises an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
as depicted in any of SEQ ID NOs. 66, 68, 74, 76, 122, 70, 72, 78,
80, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; (b)
an amino acid sequence encoded by a nucleic acid sequence as shown
in SEQ ID NOs. 65, 67, 73, 75, 123, 69, 71, 77, 79, 171, 173, 175,
177, 179, 181, 183, 185, 187, 189, or 191; (c) an amino acid
sequence encoded by a nucleic acid sequence hybridising under
stringent conditions to the complementary nucleic acid sequence of
(b); (d) an amino acid sequence encoded by a nucleic acid sequence
which is degenerate as a result of the genetic code to a nucleotide
sequence of (b); and (e) an amino acid sequence at least 85%
identical, more preferred at least 90% identical, most preferred at
least 95% identical to the amino acid sequence of (a) or (b).
18. The bispecific single chain antibody of claim 1, wherein at
least one of said first or second binding domains is human,
humanized, CDR-grafted and/or deimmunized.
19. An isolated nucleic acid sequence encoding the bispecific
single chain antibody of claim 1.
20. A vector which comprises a nucleic acid sequence of claim 19,
wherein said vector optionally comprises a regulatory sequence
which is operably linked to said nucleic acid sequence.
21.-22. (canceled)
23. A host transformed or transfected with said nucleic acid
sequence of claim 19 or a vector comprising said nucleic acid
sequence.
24. The pharmaceutical composition of claim 26, further comprising
a proteinaceous compound capable of providing an activation signal
for immune effector cells.
25. A process for the production of a bispecific single chain
antibody comprising culturing a host transformed or transfected
with a nucleic acid sequence encoding the bispecific single chain
antibody of claim 1 or a vector comprising said nucleic acid
sequence, under conditions allowing the expression of the
bispecific single chain antibody, and recovering the produced
bispecific single chain antibody from the culture.
26. A pharmaceutical composition comprising the bispecific single
chain antibody of claim 1, which is further comprises suitable
formulations of carriers, stabilizers and/or excipients.
27.-30. (canceled)
31. A method for the prevention, treatment or amelioration of a
disease in a subject in the need thereof, said method comprising
the administration of an effective amount of a pharmaceutical
composition of claim 26.
32. The method of claim 31, wherein said disease is a proliferative
disease, a tumorous disease, or an immunological disorder.
33. The method of claim 32, wherein said tumorous disease is a
malignant disease, preferably cancer.
34. The method of claim 31, wherein said pharmaceutical composition
comprising the bispecific single chain antibody is administered in
combination with an additional drug.
35. The method of claim 34, wherein said drug is a
non-proteinaceous compound or a proteinaceous compound.
36. The method of claim 35, comprising the administration of a
proteinaceous compound capable of providing an activation signal
for immune effector cells.
37. The method of claim 35, wherein said proteinaceous compound or
non-proteinaceous compound is administered simultaneously or
non-simultaneously with the pharmaceutical composition comprising
the bispecific single chain antibody.
38. The method of claim 31, wherein said subject is a human.
39. A kit comprising a bispecific single chain antibody of claim
1.
40.-43. (canceled)
Description
[0001] The present invention relates to uses of bispecific
antibodies exhibiting cross-species specificity for evaluating the
in vivo safety and/or activity and/or pharmacokinetic profile of
the same in non-human species and humans. The present invention
moreover relates to methods for evaluating the in vivo safety
and/or activity and/or pharmacokinetic profile of said bispecific
antibodies exhibiting cross-species specificity. The present
invention also relates to methods of measuring the biological
activity and/or efficacy of such bispecific antibodies exhibiting
cross-species specificity. In addition, the present invention
relates to pharmaceutical compositions comprising bispecific single
chain antibodies exhibiting cross-species specificity and to
methods for the preparation of pharmaceutical compositions
comprising said bispecific single chain antibodies exhibiting
cross-species specificity for the treatment of diseases.
[0002] In order to be marketed, any new candidate medication must
pass through rigorous testing. Roughly, this testing can be
subdivided into preclinical and clinical phases: Whereas the
latter--further subdivided into the generally known clinical phases
I, II and III--is performed in human patients, the former is
performed in animals. Generally, the aim of pre-clinical testing is
to prove that the drug candidate works and is efficacious and safe.
Specifically, the purpose of these animal studies is to prove that
the drug is not carcinogenic, mutagenic or teratogenic, as well as
to understand the pharmacokinetic of the drug. Only when the safety
in animals and possible effectiveness of the drug candidate has
been established in preclinical testing will this drug candidate be
approved for clinical testing in humans.
[0003] The behavior of a small molecule drug candidate, e.g. a new
anthracycline-based antineoplastic agent, in animals will in many
cases be indicative of the expected behavior of this drug candidate
upon administration to humans. As a result, the data obtained from
such preclinical testing will therefore generally have a high
predictive power for the human case. However, such compatibility is
not to be expected with all types of drug candidates; certain
molecular formats would be expected to behave one way in animals
and another way in humans. In such cases, the predictive power of
preclinical tests--and hence the likelihood of approval of the drug
candidate for clinical testing--is greatly reduced.
[0004] One format of drug candidate which often acts differently in
animals than in humans is an antibody. Generally, antibodies
function by way of highly specific recognition of--usually
proteinaceous--target molecules. Most antibody drug candidates are
monoclonal antibodies; they recognize only a single site, or
epitope, on their target molecule. However, while this
discriminatory ability inherent to monoclonal antibodies and
fragments thereof makes these compounds very interesting candidates
for drug development, it also complicates their preclinical
testing. This is because of species-dependent variations in the
sequence of the target molecule bound by such antibodies. A
monoclonal antibody or fragment thereof which specifically
recognizes and binds to, say, molecule Y via epitope X in humans,
will often fail to specifically recognize and bind to the
corresponding molecule Y' in a non-human species since the
corresponding epitope X' may be different from its human
counterpart X. Thus, monoclonal antibodies (e.g. against human
antigens) by design tend to have limited reactivity to
phylogenetically distant species such as rodents, except in the
very rare cases in which the antigen is highly conserved. Even
among the group of monoclonal antibodies with reactivity to human
and primate antigens, there are numerous examples of antibodies
which react only with the human and chimpanzee antigen homologs.
This has also been observed for anti-CD3 monoclonal antibodies. One
of the most widely used and best characterized monoclonal
antibodies specific for the CD3 complex is OKT-3 which reacts with
chimpanzee CD3 but not with the CD3 homolog of other primates, such
as macaques, or with dog CD3 (Sandusky et al., J. Med. Primatol. 15
(1986), 441-451). The anti-CD3 monoclonal antibody UCHT-1 is also
reactive with CD3 from chimpanzee but not with CD3 from macaques
(own data; see the following Examples). On the other hand, there
are also examples of monoclonal antibodies which recognize macaque
antigens, but not their human counterparts. One example of this
group is monoclonal antibody FN-18 directed to CD3 from macaques
(Uda et al., J. Med. Primatol. 30 (2001), 141-147). Interestingly,
it has been found that peripheral lymphocytes from about 12% of
cynomolgus monkeys lacked reactivity with anti-rhesus monkey CD3
monoclonal antibody (FN-18) due to a polymorphism of the CD3
antigen in macaques. Uda et al. described a substitution of two
amino acids in the CD3 sequence of cynomolgus monkeys which are not
reactive with FN-18 antibodies, as compared to CD3 derived from
animals which are reactive with FN-18 antibodies (Uda et al., J
Med. Primatol. 32 (2003), 105-10; Uda et al., J Med. Primatol. 33
(2004), 34-7).
[0005] Similar difficulties with the high specificity of monoclonal
antibodies in preclinical animal testing are observed with
bispecific antibodies, for example a recombinant bispecific single
chain antibody of the general type disclosed in, for example, U.S.
Pat. No. 5,260,203. This added difficulty is due to the fact that a
bispecific antibody, for example a bispecific single chain
antibody, comprises two distinct binding domains, either one of
which--or both--may fail to recognize the non-human counterpart of
its human target molecule. Effectively, the risk that e.g. a
bispecific single chain antibody, will fail to recognize its
intended respective target molecules in an animal is twice as high
as with a monospecific antibody or fragment thereof.
[0006] There exist several known strategies for countering such
problems.
[0007] One known approach is to perform preclinical testing of the
(bispecific) antibody drug candidate or fragment thereof in a
chimpanzee model. The chimpanzee is the closest genetic relative to
human, identical to the latter in over 99% of its genome, so the
chimpanzee variant of a molecule specifically bound by a
(bispecific) antibody drug candidate or fragment thereof is very
likely to be identical to the human variant of this molecule. The
danger of non-recognition of this molecule by the (bispecific)
antibody drug candidate or fragment thereof in chimpanzee is
therefore minimized. However, testing in chimpanzees is very
expensive and fraught with ethical problems. Furthermore,
chimpanzees are endangered animals so that the number of animals
which can be used in experimentation is very limited. For most
developers of (bispecific) antibody therapeutics, such preclinical
testing in chimpanzees is therefore precluded.
[0008] The above approach is described e.g. in Schlereth et al.
(Cancer Immunol. Immunother. 20 (2005), 1-12). In this study, the
biological activity of a clinical drug candidate, bispecific single
chain antibody CD19.times.CD3, has been tested in chimpanzee.
CD19.times.CD3 antibody, previously described in WO 99/54440 for
therapeutic administration in humans, is a bispecific single chain
antibody specifically binding to human B cell antigen CD19 and
human T cell antigen CD3. The authors of this paper found that this
bispecific single chain antibody bound to both human and chimpanzee
variants of the CD3 and CD19 molecule. However, no reactivity of
said bispecific single chain antibody to B and T cells from other
species, i.e. mouse, beagle dog, and non-chimpanzee primates
(cynomolgus, rhesus and baboon), could be found, again confirming
the extreme species sensitivity of monoclonal antibodies.
[0009] Another approach adapts the molecule used in preclinical
testing to the animal used for this testing. According to this
approach, the requisite safety information is obtained in
preclinical studies by constructing so-called "surrogate"
antibodies for administration to test animals. Generally, such a
surrogate antibody is an antibody which has been modified so as to
specifically recognize and bind to the test animal counterpart of
the target molecule bound by the non-surrogate antibody, i.e. the
actual drug candidate in humans. Thus, in approaches using such
"surrogate" antibodies, two different molecules have to be
separately developed and investigated: the clinical drug candidate
and a candidate for preclinical testing in an animal species
corresponding to the target specificity of the clinical candidate.
The major drawback of such surrogate approaches is that the
surrogate antibody for preclinical testing has been modified
vis-a-vis the actual drug candidate antibody. Therefore, the data
obtained in preclinical testing using a surrogate antibody are
often not directly applicable to the human case. As explained
above, this reduced applicability ultimately reduces the predictive
power of any preclinical study using these approaches.
[0010] While the above approach adapts the drug candidate to match
the animal used for testing, other known approaches do exactly the
converse; according to these other known approaches, the animal
used for testing is adapted to the drug candidate intended for
administration to humans.
[0011] One example of the adaptation of the test animal to the drug
candidate intended for administration to humans, is the creation of
a transgenic animal expressing the human molecule specifically
bound by the (bispecific) antibody or fragment thereof instead of
the non-human molecule which is endogenous to its own species. In
this way, the (bispecific) antibody or fragment thereof
administered in preclinical trials will encounter and bind to the
human antigen in the transgenic test animal. For example, in a
study designed by Bugelski et al. (Bugelski et al., Hum Exp
Toxicol. 19 (2000), 230-243), preclinical safety assessment of
monoclonal antibody Keliximab has been carried out in a human CD4
transgenic mouse in order to support chronic treatment of
rheumatoid arthritis in human patients. Keliximab is a monoclonal
antibody with specificity for human and chimpanzee CD4. The authors
conclude that the use of transgenic mice expressing human proteins
provides a useful alternative to studies in chimpanzees with
biopharmaceutical agents having limited cross-species specificity
(Bugelski et al., Hum Exp Toxicol. 19 (2000), 230-243). However,
creation of transgenic animals for test purposes is very labor- and
therefore cost-intensive.
[0012] In the same vein, an alternative approach often employed is
to inject a non-transgenic test animal with human cells expressing
the molecule to be specifically bound by the (bispecific) antibody
or fragment thereof being tested. However, while avoiding the costs
and time associated with constructing transgenic animal species,
this approach presents other problems. For one, in approaches using
e.g. immunocompetent mice, foreign cells introduced into the animal
are often recognized by the immune system of the test animal and
are systematically eliminated. Although immunodeficient mice allow
the injection and growth of non-syngeneic cells, for instance in
xenograft tumor models, the applicability of the data obtained for
the drug candidate in such studies is limited due to the
phylogenetic distance between rodents and humans. In addition,
multiple blood extractions are problematic in lower animals, say a
mouse. However, such multiple blood extractions are essential for
the determination of pharmacokinetic parameters and the continuous
testing of blood parameters for evaluating the biological effects
of a drug candidate in preclinical animal testing.
[0013] In summary, there are two main approaches of obtaining
preclinical data on safety and toxicity of a drug candidate for
administration in humans. One way is the application of the
clinical drug candidate to transgenic animal models, mostly mouse
models. However, preclinical data are of limited explanatory power
due to the fact that rodents are less related to humans compared to
primates. Another way is the testing of surrogate molecules in a
relevant animal species. These surrogate molecules are specific for
the animals used and are therefore different from the clinical drug
candidate developed for administration in humans. The problem is
that the clinical drug candidate cannot directly be applied in an
animal other than chimpanzees which is closely related to humans
and has highly predictive power when used in preclinical testing.
Existing methods for obtaining meaningful preclinical data
regarding a (bispecific) antibody or fragment thereof undergoing
testing as a drug candidate either match this antibody to the test
animal, in which case the data obtained are often of only limited
applicability for the drug candidate or, conversely, match the test
animal to the antibody, in which case ethical and/or cost
difficulty arise/s and, in the worst case, the applicability of the
data obtained for the drug candidate may still be limited.
[0014] It is therefore an aim of the invention to provide a
solution to the problems outlined above.
[0015] The solution to these problems is the provision of
bispecific single chain antibodies exhibiting cross-species
specificity which bind to human and non-chimpanzee primate target
molecules and therefore can be used both for preclinical evaluation
of safety, activity and/or pharmacokinetic profile of said
bispecific antibody in primates and--in the identical form--as
drugs in humans.
[0016] Accordingly, one aspect of the invention relates to the use
of a bispecific single chain antibody comprising a first binding
domain binding to a non-chimpanzee primate CD3, and a second
binding domain binding to a cell surface antigen antigen, wherein
said first binding domain binds to human and non-chimpanzee primate
CD3, for evaluating the (in vivo) safety and/or activity and/or
pharmacokinetic profile of said bispecific single chain antibody in
humans, comprising (i) administering said bispecific single chain
antibody to a non-chimpanzee primate, (ii) measuring said (in vivo)
safety and/or activity and/or pharmacokinetic profile of said
bispecific single chain antibody in said non-chimpanzee primate,
and (iii) evaluating the (in vivo) safety and/or activity and/or
pharmacokinetic profile of said bispecific single chain antibody in
humans.
[0017] In another aspect, the invention relates to a method for
evaluating the biological activity/safety/toxicity of a bispecific
single chain antibody as defined above, comprising
(i) administering said bispecific single chain antibody to a
non-chimpanzee primate, (ii) measuring the in vivo safety and/or
activity and/or pharmacokinetic profile of said bispecific single
chain antibody in said non-chimpanzee primate, (iii) evaluating the
in vivo safety and/or activity and/or pharmacokinetic profile of
said bispecific single chain antibody in the non-chimpanzee
primate, and (iv) determining an effective and non-toxic dose of
said bispecific single chain antibody and administering said dose
to humans.
[0018] In particular, it is an aim of the invention to provide
means and methods which improve the predictive value of data
obtained in preclinical animal testing for the administration of
the drug candidate to humans.
[0019] As used herein, a "bispecific single chain antibody" denotes
a single polypeptide chain comprising two binding domains. Each
binding domain comprises one variable region from an antibody heavy
chain ("VH region"), wherein the VH region of the first binding
domain specifically binds to said first molecule, i.e. the CD3
molecule, and the VH region of the second binding domain
specifically binds to a cell surface antigen, as defined in more
detail below. The two binding domains are optionally linked to one
another by a short polypeptide spacer generally comprising on the
order of 5 amino acids. Each binding domain may additionally
comprise one variable region from an antibody light chain ("VL
region"), the VH region and VL region within each of the first and
second binding domains being linked to one another via a
polypeptide linker, for example of the type disclosed and claimed
in EP 623679 B1, but in any case long enough to allow the VH region
and VL region of the first binding domain and the VH region and VL
region of the second binding domain to pair with one another such
that, together, they are able to specifically bind to the
respective first and second molecules.
[0020] As used herein, the term "binds" or related expressions such
as "binding" or "reactivity with/to" etc. refer to the ability of
the first and/or second binding domains of the bispecific single
chain antibody as defined herein to discriminate between the
respective first and/or second molecule to such an extent that,
from a pool of a plurality of different molecules as potential
binding partners, only said respective first and/or second molecule
is/are bound, or is/are significantly bound. Such binding
measurements can be routinely performed e.g. on a Biacore
apparatus.
[0021] More specifically, the first binding domain of the
bispecific single chain antibody as defined herein binds to human
CD3 and to non-chimpanzee primate CD3. The term "non-chimpanzee
primate" is explained in more detail below. As evident to the
person skilled in the art, it is not excluded from the scope of the
invention that the first binding domain of the bispecific single
chain antibodies exhibiting cross-species specificity as defined
herein may also bind, e.g., to chimpanzee CD3. On the other hand,
it is apparent that binding domains which only bind to human CD3,
but not to non-chimpanzee primate CD3, are excluded from the scope
of the invention. This applies mutatis mutandis to binding domains
which only bind to non-chimpanzee primate CD3, but not to human
CD3, such as e.g. those of monoclonal antibody FN-18.
[0022] The second binding domain of the bispecific single chain
antibodies as defined herein binds to a cell surface antigen,
preferably a tumor antigen, as set forth below. Preferably, both
binding molecules of the bispecific single chain antibodies as
defined herein are binding to their respective human and
non-chimpanzee primate target molecules. The second binding domain,
thus, binds to a human cell surface antigen and to the
corresponding homolog of the cell surface antigen in a
non-chimpanzee primate. The identification and determination of
homologs of human cell surface antigens in non-chimpanzee primates
is well known to the person skilled in the art and can be carried
out e.g. by sequence alignments.
[0023] The term "cross-species specificity" or "interspecies
specificity" as used herein means binding of at least one of the
two binding domains, preferably of both binding domains, of the
bispecific single chain antibody described herein to the same
target molecule in humans and non-chimpanzee primates. Thus,
"cross-species specificity" or "interspecies specificity" is to be
understood as an interspecies reactivity to the same molecule X,
but not to a molecule other than X. Cross-species specificity of a
monoclonal antibody recognizing e.g. human CD3, to a non-chimpanzee
primate CD3, e.g. macaque CD3, can be determined, for instance, by
FACS analysis. The FACS analysis is carried out in a way that the
respective monoclonal antibody is tested for binding to human and
non-chimpanzee primate cells, e.g. macaque cells, expressing said
human and non-chimpanzee primate CD3 antigens, respectively. An
appropriate assay is shown in the following examples. For the
generation of the first binding domain of the bispecific single
chain antibodies as defined herein, e.g. monoclonal antibodies
binding to both the human and non-chimpanzee CD3 (e.g. macaque CD3)
can be used. Similarly, for the generation of the second binding
domain of the bispecific single chain antibodies as defined herein,
monoclonal antibodies binding to both of the respective human and
non-chimpanzee primate cell surface antigens can be utilized.
Appropriate binding domains for the bispecific single chain
antibodies as defined herein can be derived from cross-species
specific monoclonal antibodies by recombinant methods described in
the art. A monoclonal antibody binding to a human cell surface
antigen and to the homolog of said cell surface antigen in a
non-chimpanzee primate can be tested by FACS assays as set forth
above. It is evident to those skilled in the art that cross-species
specific monoclonal antibodies can also be generated by hybridoma
techniques described in the literature (Milstein and Kohler, Nature
256 (1975), 495-7). For example, mice may be alternately immunized
with human and non-chimpanzee primate CD3. From these mice,
cross-species specific antibody-producing hybridoma cells are
isolated via hybridoma technology and analysed by FACS as set forth
above. The generation and analysis of bispecific single chain
antibodies exhibiting cross-species specificity as described herein
is shown in the following examples. The advantages of the
bispecific single chain antibodies exhibiting cross-species
specificity include the points enumerated below.
[0024] As used herein, "human" and "man" refers to the species Homo
sapiens. A "human" molecule is therefore the variant of that
molecule as it is naturally expressed in Homo sapiens. As far as
the medical uses of the constructs described herein are concerned,
human patients are to be treated with the same.
[0025] As used herein, a "non-chimpanzee primate" or "non-chimp
primate" or grammatical variants thereof refers to any primate
other than chimpanzee, i.e. other than an animal of belonging to
the genus Pan, and including the species Pan paniscus and Pan
troglodytes, also known as Anthropopithecus troglodytes or Simia
satyrus. A "primate", "primate species", "primates" or grammatical
variants thereof denote/s an order of eutherian mammals divided
into the two suborders of prosimians and anthropoids and comprising
man, apes, monkeys and lemurs. Specifically, "primates" as used
herein comprises the suborder Strepsirrhini (non-tarsier
prosimians), including the infraorder Lemuriformes (itself
including the superfamilies Chemogaleoidea and Lemuroidea), the
infraorder Chiromyiformes (itself including the family
Daubentoniidae) and the infraorder Lorisiformes (itself including
the families Lorisidae and Galagidae). "Primates" as used herein
also comprises the suborder Haplorrhini, including the infraorder
Tarsiiformes (itself including the family Tarsiidae), the
infraorder Simiiformes (itself including the Platyrrhini, or New
World monkeys, and the Catarrhini, including the Cercopithecidea,
or Old-World Monkeys).
[0026] The non-chimpanzee primate species may be understood within
the meaning of the invention to be a lemur, a tarsier, a gibbon, a
marmoset (belonging to New World Monkeys of the family Cebidae) or
an Old-World Monkey (belonging to the superfamily
Cercopithecoidea).
[0027] As used herein, an "Old-World Monkey" comprises any monkey
falling in the superfamily Cercopithecoidea, itself subdivided into
the families: the Cercopithecinae, which are mainly African but
include the diverse genus of macaques which are Asian and North
African; and the Colobinae, which include most of the Asian genera
but also the African colobus monkeys.
[0028] Specifically, within the subfamily Cercopithecinae, an
advantageous non-chimpanzee primate may be from the Tribe
Cercopithecini, within the genus Allenopithecus (Allen's Swamp
Monkey, Allenopithecus nigroviridis); within the genus Miopithecus
(Angolan Talapoin, Miopithecus talapoin; Gabon Talapoin,
Miopithecus ogouensis); within the genus Erythrocebus (Patas
Monkey, Erythrocebus patas); within the genus Chlorocebus (Green
Monkey, Chlorocebus sabaceus; Grivet, Chlorocebus aethiops; Bale
Mountains Vervet, Chlorocebus djamdjamensis; Tantalus Monkey,
Chlorocebus tantalus; Vervet Monkey, Chlorocebus pygerythrus;
Malbrouck, Chlorocebus cynosuros); or within the genus
Cercopithecus (Dryas Monkey or Salongo Monkey, Cercopithecus dryas;
Diana Monkey, Cercopithecus diana; Roloway Monkey, Cercopithecus
roloway; Greater Spot-nosed Monkey, Cercopithecus nictitans; Blue
Monkey, Cercopithecus mitis; Silver Monkey, Cercopithecus doggetti;
Golden Monkey, Cercopithecus kandti; Sykes's Monkey, Cercopithecus
albogularis; Mona Monkey, Cercopithecus mona; Campbell's Mona
Monkey, Cercopithecus campbelli; Lowe's Mona Monkey, Cercopithecus
lowei; Crested Mona Monkey, Cercopithecus pogonias; Wolfs Mona
Monkey, Cercopithecus wolfi; Dent's Mona Monkey, Cercopithecus
denti; Lesser Spot-nosed Monkey, Cercopithecus petaurista;
White-throated Guenon, Cercopithecus erythrogaster; Sclater's
Guenon, Cercopithecus sclateri; Red-eared Guenon, Cercopithecus
erythrotis; Moustached Guenon, Cercopithecus cephus; Red-tailed
Monkey, Cercopithecus ascanius; L'Hoest's Monkey, Cercopithecus
lhoesti; Preuss's Monkey, Cercopithecus preussi; Sun-tailed Monkey,
Cercopithecus solatus; Hamlyn's Monkey or Owl-faced Monkey,
Cercopithecus hamlyni; De Brazza's Monkey, Cercopithecus
neglectus).
[0029] Alternatively, an advantageous non-chimpanzee primate, also
within the subfamily Cercopithecinae but within the Tribe
Papionini, may be from within the genus Macaca (Barbary Macaque,
Macaca sylvanus; Lion-tailed Macaque, Macaca silenus; Southern
Pig-tailed Macaque or Beruk, Macaca nemestrina; Northern Pig-tailed
Macaque, Macaca leonina; Pagai Island Macaque or Bokkoi, Macaca
pagensis; Siberut Macaque, Macaca siberu; Moor Macaque, Macaca
maura; Booted Macaque, Macaca ochreata; Tonkean Macaque, Macaca
tonkeana; Heck's Macaque, Macaca hecki; Gorontalo Macaque, Macaca
nigriscens; Celebes Crested Macaque or Black "Ape", Macaca nigra;
Cynomolgus monkey or Crab-eating Macaque or Long-tailed Macaque or
Kera, Macaca fascicularis; Stump-tailed Macaque or Bear Macaque,
Macaca arctoides; Rhesus Macaque, Macaca mulatta; Formosan Rock
Macaque, Macaca cyclopis; Japanese Macaque, Macaca fuscata; Toque
Macaque, Macaca sinica; Bonnet Macaque, Macaca radiata; Barbary
Macaque, Macaca sylvanmus; Assam Macaque, Macaca assamensis;
Tibetan Macaque or Milne-Edwards' Macaque, Macaca thibetana;
Arunachal Macaque or Munzala, Macaca munzala); within the genus
Lophocebus (Gray-cheeked Mangabey, Lophocebus albigena; Lophocebus
albigena albigena; Lophocebus albigena osmani; Lophocebus albigena
johnstoni; Black Crested Mangabey, Lophocebus aterrimus;
Opdenbosch's Mangabey, Lophocebus opdenboschi; Highland Mangabey,
Lophocebus kipunji); within the genus Papio (Hamadryas Baboon,
Papio hamadryas; Guinea Baboon, Papio papio; Olive Baboon, Papio
anubis; Yellow Baboon, Papio cynocephalus; Chacma Baboon, Papio
ursinus); within the genus Theropithecus (Gelada, Theropithecus
gelada); within the genus Cercocebus (Sooty Mangabey, Cercocebus
atys; Cercocebus atys atys; Cercocebus atys lunulatus; Collared
Mangabey, Cercocebus torquatus; Agile Mangabey, Cercocebus agilis;
Golden-bellied Mangabey, Cercocebus chrysogaster; Tana River
Mangabey, Cercocebus galeritus; Sanje Mangabey, Cercocebus sanjei);
or within the genus Mandrillus (Mandrill, Mandrillus sphinx; Drill,
Mandrillus leucophaeus).
[0030] Most preferred is Macaca fascicularis (also known as
Cynomolgus monkey and, therefore, in the Examples named
"Cynomolgus") and Macaca mulatta (rhesus monkey, named
"rhesus").
[0031] Within the subfamily Colobinae, an advantageous
non-chimpanzee primate may be from the African group, within the
genus Colobus (Black Colobus, Colobus satanas; Angola Colobus,
Colobus angolensis; King Colobus, Colobus polykomos; Ursine
Colobus, Colobus vellerosus; Mantled Guereza, Colobus guereza);
within the genus Piliocolobus (Western Red Colobus, Piliocolobus
badius; Piliocolobus badius badius; Piliocolobus badius temminckii;
Piliocolobus badius waldronae; Pennant's Colobus, Piliocolobus
pennantii; Piliocolobus pennantii pennantii; Piliocolobus pennantii
epieni; Piliocolobus pennantii bouvieri; Preuss's Red Colobus,
Piliocolobus preussi; Thollon's Red Colobus, Piliocolobus tholloni;
Central African Red Colobus, Piliocolobus foai; Piliocolobus foai
foai; Piliocolobus foai ellioti; Piliocolobus foai oustaleti;
Piliocolobus foai semlikiensis; Piliocolobus foai parmentierorum;
Ugandan Red Colobus, Piliocolobus tephrosceles; Uzyngwa Red
Colobus, Piliocolobus gordonorum; Zanzibar Red Colobus,
Piliocolobus kirkii; Tana River Red Colobus, Piliocolobus
rufomitratus); or within the genus Procolobus (Olive Colobus,
Procolobus verus).
[0032] Within the subfamily Colobinae, an advantageous
non-chimpanzee primate may alternatively be from the Langur (leaf
monkey) group, within the genus Semnopithecus (Nepal Gray Langur,
Semnopithecus schistaceus; Kashmir Gray Langur, Semnopithecus ajax;
Tarai Gray Langur, Semnopithecus hector; Northern Plains Gray
Langur, Semnopithecus entellus; Black-footed Gray Langur,
Semnopithecus hypoleucos; Southern Plains Gray Langur,
Semnopithecus dussumieri; Tufted Gray Langur, Semnopithecus priam);
within the T. vetulus group or the genus Trachypithecus
(Purple-faced Langur, Trachypithecus vetulus; Nilgiri Langur,
Trachypithecus johnii); within the T. cristatus group of the genus
Trachypithecus (Javan Lutung, Trachypithecus auratus; Silvery Leaf
Monkey or Silvery Lutung, Trachypithecus cristatus; Indochinese
Lutung, Trachypithecus germaini; Tenasserim Lutung, Trachypithecus
barbei); within the T. obscurus group of the genus Trachypithecus
(Dusky Leaf Monkey or Spectacled Leaf Monkey, Trachypithecus
obscurus; Phayre's Leaf Monkey, Trachypithecus phayrei); within the
T. pileatus group of the genus Trachypithecus (Capped Langur,
Trachypithecus pileatus; Shortridge's Langur, Trachypithecus
shortridgei; Gee's Golden Langur, Trachypithecus geei); within the
T. francoisi group of the genus Trachypithecus (Francois' Langur,
Trachypithecus francoisi; Hatinh Langur, Trachypithecus
hatinhensis; White-headed Langur, Trachypithecus poliocephalus;
Laotian Langur, Trachypithecus laotum; Delacour's Langur,
Trachypithecus delacouri; Indochinese Black Langur, Trachypithecus
ebenus); or within the genus Presbytis (Sumatran Surili, Presbytis
melalophos; Banded Surili, Presbytis femoralis; Sarawak Surili,
Presbytis chrysomelas; White-thighed Surili, Presbytis siamensis;
White-fronted Surili, Presbytis frontata; Javan Surili, Presbytis
comata; Thomas's Langur, Presbytis thomasi; Hose's Langur,
Presbytis hosei; Maroon Leaf Monkey, Presbytis rubicunda; Mentawai
Langur or Joja, Presbytis potenziani; Natuna Island Surili,
Presbytis natunae).
[0033] Within the subfamily Colobinae, an advantageous
non-chimpanzee primate may alternatively be from the Odd-Nosed
group, within the genus Pygathrix (Red-shanked Douc, Pygathrix
nemaeus; Black-shanked Douc, Pygathrix nigripes; Gray-shanked Douc,
Pygathrix cinerea); within the genus Rhinopithecus (Golden
Snub-nosed Monkey, Rhinopithecus roxellana; Black Snub-nosed
Monkey, Rhinopithecus bieti; Gray Snub-nosed Monkey, Rhinopithecus
brelichi; Tonkin Snub-nosed Langur, Rhinopithecus avunculus);
within the genus Nasalis (Proboscis Monkey, Nasalis larvatus); or
within the genus Simias (Pig-tailed Langur, Simias concolor).
[0034] As used herein, the term "marmoset" denotes any New World
Monkeys of the genus Callithrix, for example belonging to the
Atlantic marmosets of subgenus Callithrix (sic!) (Common Marmoset,
Callithrix (Callithrix) jacchus; Black-tufted Marmoset, Callithrix
(Callithrix) penicillata; Wied's Marmoset, Callithrix (Callithrix)
kuhlii; White-headed Marmoset, Callithrix (Callithrix) geoffroyi;
Buffy-headed Marmoset, Callithrix (Callithrix) flaviceps;
Buffy-tufted Marmoset, Callithrix (Callithrix) aurita); belonging
to the Amazonian marmosets of subgenus Mico (Rio Acari Marmoset,
Callithrix (Mico) acariensis; Manicore Marmoset, Callithrix (Mico)
manicorensis; Silvery Marmoset, Callithrix (Mico) argentata; White
Marmoset, Callithrix (Mico) leucippe; Emilia's Marmoset, Callithrix
(Mico) emiliae; Black-headed Marmoset, Callithrix (Mico) nigriceps;
Marca's Marmoset, Callithrix (Mico) marcai; Black-tailed Marmoset,
Callithrix (Mico) melanura; Santarem Marmoset, Callithrix (Mico)
humeralifera; Maues Marmoset, Callithrix (Mico) mauesi;
Gold-and-white Marmoset, Callithrix (Mico) chrysoleuca;
Hershkovitz's Marmoset, Callithrix (Mico) intermedia; Salere
Marmoset, Callithrix (Mico) saterei); Roosmalens' Dwarf Marmoset
belonging to the subgenus Callibella (Callithrix (Callibella)
humilis); or the Pygmy Marmoset belonging to the subgenus Cebuella
(Callithrix (Cebuella) pygmaea).
[0035] As used herein, CD3 denotes a molecule expressed as part of
the T cell receptor and has the meaning as typically ascribed to it
in the prior art. In human, it encompasses in individual or
independently combined form all known CD3 subunits, for example CD3
epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta.
The non-chimpanzee primate CD3 antigens as referred to herein are,
for example, Macaca fascicularis CD3 and Macaca mulatta CD3. In
Macaca fascicularis, it encompasses CD3 epsilon FN-18 negative and
CD3 epsilon FN-18 positive, CD3 gamma and CD3 delta. In Macaca
mulatta, it encompasses CD3 epsilon, CD3 gamma and CD3 delta.
Preferably, said CD3 as used herein is CD3 epsilon. The human CD3
epsilon is indicated in GenBank Accession No. NM.sub.--000733 and
comprises SEQ ID NO. 134. The human CD3 gamma is indicated in
GenBank Accession No. NM.sub.--000073 and comprises SEQ ID NO. 142.
The human CD3 delta is indicated in GenBank Accession No.
NM.sub.--000732 and comprises SEQ ID NO. 143.
[0036] The CD3 epsilon "FN-18 negative" of Macaca fascicularis
(i.e. CD3 epsilon not recognized by monoclonal antibody FN-18 due
to a polymorphism as set forth above) is indicated in GenBank
Accession No. AB073994 and comprises SEQ ID NO. 136. The CD3
epsilon "FN-18 positive" of Macaca fascicularis (i.e. CD3 epsilon
recognized by monoclonal antibody FN-18) is indicated in GenBank
Accession No. AB073993 and comprises SEQ ID NO. 135. The CD3 gamma
of Macaca fascicularis is indicated in GenBank Accession No.
AB073992 and comprises SEQ ID NO. 144. The CD3 delta of Macaca
fascicularis is indicated in GenBank Accession No. AB073991 and
comprises SEQ ID NO. 145.
[0037] The nucleic acid sequences and amino acid sequences of the
respective CD3 epsilon, gamma and delta homologs of Macaca mulatta
can be identified and isolated by recombinant techniques described
in the art (Sambrook et al. Molecular Cloning: A Laboratory Manual;
Cold Spring Harbor Laboratory Press, 3.sup.rd edition 2001). This
applies mutatis mutandis to the CD3 epsilon, gamma and delta
homologs of other non-chimpanzee primates as defined herein.
[0038] As pointed out above and as disclosed herein, it is
envisaged that the first binding domain of the bispecific single
chain antibody comprised in the inventive pharmaceutical
composition leads to an epitope of human and non-chimpanzee primate
CD3 which comprises the amino acid sequence "phenylalanine
(F)-serine (S)-glutamic acid (E)". The person skilled in the art is
readily in the position to deduce an epitope detected by a given
antibody/binding molecule and/or (as in the present invention) a
given "binding domain" of a single chain construct by methods known
in the art, said methods are also illustrated in the appended
examples and may comprise Western blot analysis, epitope mapping or
pepspot analysis and the like.
[0039] The epitope to be detected by said first binding domain is
preferably in the range of 15 amino acids +/-3 amino acids.
Envisaged are (but not limiting) 18, 17, 16, 15, 14, 13, 12, 11,
10, 9, 8, 7, 6, 5, 4 or 3 amino acids in said epitope comprising
the "F-S-E" stretch/"F-S-E" core epitope.
[0040] As shown in the following Examples, the minimum core epitope
of human and non-chimpanzee primate CD3 bound by the first binding
domain of the bispecific single chain antibody as defined herein is
an epitope comprising the amino acid residues "FSE". More
specifically, the minimum epitope comprises the amino acid residues
"FSEXE" (SEQ ID NOs. 202 and 204), wherein the substitution of
methionine to leucine is a conserved amino acid substitution
between two neutral, non-polar amino acid residues. The minimum
epitope may be part of a discontinuous epitope. As used herein, the
term "discontinuous epitope" is to be understood as at least two
non-adjacent amino acid sequence stretches within a given
polypeptide chain, here e.g. CD3 (preferably CD3 epsilon), which
are simultaneously bound by an antibody. These amino acid stretches
might be of different length and may also be involved in the
interaction of antibody and antigen. Accordingly, in addition to
the minimum (core) epitope as defined above, the bispecific single
chain antibody may simultaneously bind to one, two or even more
non-adjacent epitopes. This (these) non-adjacent epitope(s) in
combination with the minimal (core) epitope could represent the
contact site between antigen and antibody. According to this
definition, such simultaneous binding may be of the polypeptide in
linear form. Here, one may imagine the polypeptide forming an
extended loop, in one region of which the two sequences for example
are more or less in parallel and in proximity of one another.
Non-adjacent epitopes in the linear sequence could form a three
dimensional structure leading to a close proximity of these
epitopes. In this state they are simultaneously bound by the
bispecific single chain antibody as defined herein. According to
this definition, simultaneous binding of the at least two sequence
stretches of the polypeptide indicated above (including the minimum
(core) epitope) may also take the form of antibody binding to a
conformational epitope. Here, the mature polypeptide has already
formed its tertiary conformation as it normally exists in vivo. In
this tertiary conformation, the polypeptide chain is folded in such
a manner as to bring the at least two sequence stretches indicated
above into spatial proximity, for example, on the outer surface of
a particular region of mature, folded polypeptide, where they are
then recognized by virtue of their three-dimensional conformation
in the context of the surrounding polypeptide sequences.
[0041] The term "cell surface antigen" as used herein denotes a
molecule which is displayed on the surface of a cell. In most
cases, this molecule will be located in or on the plasma membrane
of the cell such that at least part of this molecule remains
accessible from outside the cell in tertiary form. A non-limiting
example of a cell surface molecule which is located in the plasma
membrane is a transmembrane protein comprising, in its tertiary
conformation, regions of hydrophilicity and hydrophobicity. Here,
at least one hydrophobic region allows the cell surface molecule to
be embedded, or inserted in the hydrophobic plasma membrane of the
cell while the hydrophilic regions extend on either side of the
plasma membrane into the cytoplasm and extracellular space,
respectively. Non-limiting examples of a cell surface molecules
which are located on the plasma membrane are proteins which have
been modified at a cysteine residue to bear a palmitoyl group,
proteins modified at a C-terminal cysteine residue to bear a
farnesyl group or proteins which have been modified at the
C-terminus to bear a glycosyl phosphatidyl inositol ("GPI") anchor.
These groups allow covalent attachment of proteins to the outer
surface of the plasma membrane, where they remain accessible for
recognition by extracellular molecules such as antibodies.
[0042] The "tumor antigen" as used herein may be understood as
those antigens that are presented on tumor cells. These antigens
can be presented on the cell surface with an extracellular part
which is often combined with a transmembrane and cytoplasmic part
of the molecule. These antigens can sometimes be presented only by
tumor cells and never by the normal ones. Tumor antigens can be
exclusively expressed on tumor cells or might represent a tumor
specific mutation compared to normal cells. In this case, they are
called tumor-specific antigens. More common are antigens that are
presented by tumor cells and normal cells, and they are called
tumor-associated antigens. These tumor-associated antigens can be
overexpressed compared to normal cells or are accessible for
antibody binding in tumor cells due to the less compact structure
of the tumor tissue compared to normal tissue. Non-limiting
examples of tumor antigens as used herein are EpCAM (Naundorf, Int.
J. Cancer 100/1 (2002), 101-110), EGFR (Liu, Br. J. Cancer 82/12
(2000), 1991-1999; Bonner, Semin. Radiat. Oncol. 12 (2002), 11-20;
Kiyota, Oncology 63/1 (2002), 92-98; Kuan, Brain Tumor Pathol. 17/2
(2000), 71-78), EGFRvIII (Kuan, Brain Tumor Pathol. 17/2 (2000),
71-78), or Carboanhydrase IX (MN/CA IX) (Uemura, Br. J. Cancer 81/4
(1999), 741-746; Longcaster, Cancer Res. 61/17 (2001), 6394-6399;
Chia, J. Clin. Oncol. 19/16 (2001), 3660-3668; Beasley, Cancer Res.
61/13 (2001), 5262-5267).
[0043] The corresponding sequences of the human and non-chimpanzee
primate nucleic acid and amino acid sequences can be found e.g. in
NCBI databases.
[0044] A cross-species-specific monoclonal antibody binding to a
human cell surface antigen (preferably a tumor antigen) and to the
homolog of said cell surface antigen (preferably a tumor antigen)
in a non-chimpanzee primate can be generated as set out above.
"Homologs" as used herein refer to genes (encoding e.g. CD3, CD3
epsilon, cell surface antigens or tumor antigens) which encode gene
products with similar or identical biological function in different
species and which genes can be attributed to a common precursor
gene. Cross-species specificity of said monoclonal antibody to the
human and non-chimpanzee primate tumor antigen can be tested by
FACS assays as set forth above. Alternatively,
immunohistochemistry, radioimmunoassay, or ELISA assays may be used
as known to the person skilled in the art. The second binding
domain of the bispecific single chain antibody exhibiting
cross-species specificity as described herein can for example be
derived from such cross-species specific monoclonal antibodies by
recombinant techniques described in the following examples.
[0045] The term "evaluating the in vivo safety and/or activity
and/or pharmacokinetic profile" of the bispecific single chain
antibody as used herein may be understood as set forth below.
Before a new candidate medication can be marketed it must pass
through rigorous testing, which may be roughly subdivided into
preclinical testing in animals and clinical phases in human
patients. The aim of preclinical testing in animals is to prove
that the drug candidate is safe and efficacious (see e.g. the
Preclinical safety evaluation of biotechnology-derived
pharmaceuticals; ICH Harmonised Tripartite Guideline; ICH Steering
Committee meeting on Jul. 16, 1997).
[0046] The term "drug", "drug candidate" or "pharmaceutical
composition" as used herein refers to bispecific single chain
antibodies defined herein.
[0047] The biological activity of the bispecific single chain
antibody as defined herein can be determined for instance by
cytotoxicity assays, as described in the following examples, in WO
99/54440 or by Schlereth et al. (Cancer Immunol. Immunother. 20
(2005), 1-12). "Efficacy" or "in vivo efficacy" as used herein
refers to the response to therapy by the bispecific single chain
antibody as defined herein, using e.g. standardized NCI response
criteria. The success or in vivo efficacy of the therapy using a
bispecific single chain antibody as defined herein refers to the
effectiveness of the bispecific single chain antibody as defined
herein for its intended purpose, i.e. the ability of the bispecific
antibody to cause its desired effect, i.e. depletion of pathologic
cells, e.g. tumor cells. The in vivo efficacy may be monitored by
established standard methods for the respective disease entities
including, but not limited to white blood cell counts,
differentials, Fluorescence Activated Cell Sorting, bone marrow
aspiration. In addition, various disease specific clinical
chemistry parameters and other established standard methods may be
used. Furthermore, computer-aided tomography, X-ray, nuclear
magnetic resonance tomography (e.g. for National Cancer
Institute-criteria based response assessment [Cheson B D, Horning S
J, Coiffier B, Shipp M A, Fisher R I, Connors J M, Lister T A, Vose
J, Grillo-Lopez A, Hagenbeek A, Cabanillas F, Klippensten D,
Hiddemann W, Castellino R, Harris N L, Armitage J O, Carter W,
Hoppe R, Canellos G P. Report of an international workshop to
standardize response criteria for non-Hodgkin's lymphomas. NCI
Sponsored International Working Group. J Clin Oncol. 1999 April;
17(4): 1244]), positron-emission tomography scanning, white blood
cell counts, differentials, Fluorescence Activated Cell Sorting,
bone marrow aspiration, lymph node biopsies/histologies, and
various lymphoma specific clinical chemistry parameters (e.g.
lactate dehydrogenase) and other established standard methods may
be used.
[0048] Another major challenge in the development of drugs is the
predictable modulation of pharmacokinetic properties. To this end,
a pharmacokinetic profile of the drug candidate, i.e. a profile of
the pharmacokinetic parameters that effect the ability of a
particular drug to treat a given condition, is established.
Pharmacokinetic parameters of the drug influencing the ability of a
drug for treating a certain disease entity include, but are not
limited to: half-life, volume of distribution, hepatic first-pass
metabolism and the degree of blood serum binding. The efficacy of a
given drug agent can be influenced by each of the parameters
mentioned above.
[0049] "Half-life" means the time where 50% of an administered drug
are eliminated through biological processes, e.g. metabolism,
excretion, etc.
[0050] By "hepatic first-pass metabolism" is meant the propensity
of a drug to be metabolized upon first contact with the liver, i.e.
during its first pass through the liver.
[0051] "Volume of distribution" means the degree of retention of a
drug throughout the various compartments of the body, like e.g.
intracellular and extracellular spaces, tissues and organs, etc.
and the distribution of the drug within these compartments.
[0052] "Degree of blood serum binding" means the propensity of a
drug to interact with and bind to blood serum proteins, such as
albumin, leading to a reduction or loss of biological activity of
the drug.
[0053] Pharmacokinetic parameters also include bioavailability, lag
time (Tlag), Tmax, absorption rates, more onset and/or Cmax for a
given amount of drug administered.
[0054] "Bioavailability" means the amount of a drug in the blood
compartment.
[0055] "Lag time" means the time delay between the administration
of the drug and its detection and measurability in blood or
plasma.
[0056] "Tmax" is the time after which maximal blood concentration
of the drug is reached, and
[0057] "Cmax" is the blood concentration maximally obtained with a
given drug. The time to reach a blood or tissue concentration of
the drug which is required for its biological effect is influenced
by all parameters. Pharmacokinetik parameters of the bispecific
single chain antibodies exhibiting cross-species specificity which
may be determined in preclinical animal testing in non-chimpanzee
primates as outlined above are also set forth e.g. in the
publication by Schlereth et al. (Cancer Immunol. Immunother. 20
(2005), 1-12).
[0058] The term "toxicity" as used herein refers to the toxic
effects of a drug manifested in adverse events or severe adverse
events. These side events might refer to a lack of tolerability of
the drug in general and/or a lack of local tolerance after
administration. Toxicity could also include teratogenic or
carcinogenic effects caused by the drug.
[0059] The term "safety", "in vivo safety" or "tolerability" as
used herein defines the administration of a drug without inducing
severe adverse events directly after administration (local
tolerance) and during a longer period of application of the drug.
"Safety", "in vivo safety" or "tolerability" can be evaluated e.g.
at regular intervals during the treatment and follow-up period.
Measurements include clinical evaluation, e.g. organ
manifestations, and screening of laboratory abnormalities. Clinical
evaluation may be carried out and deviating to normal findings
recorded/coded according to NCI-CTC and/or MedDRA standards. Organ
manifestations may include criteria such as allergy/immunology,
blood/bone marrow, cardiac arrhythmia, coagulation and the like, as
set forth e.g. in the Common Terminology Criteria for adverse
events v3.0 (CTCAE). Laboratory parameters which may be tested
include for instance haematology, clinical chemistry, coagulation
profile and urine analysis and examination of other body fluids
such as serum, plasma, lymphoid or spinal fluid, liquor and the
like. Safety can thus be assessed e.g. by physical examination,
imaging techniques (i.e. ultrasound, x-ray, CT scans, Magnetic
Resonance Imaging (MRI), other measures with technical devices
(i.e. electrocardiogram), vital signs, by measuring laboratory
parameters and recording adverse events. For example, adverse
events in non-chimpanzee primates in the uses and methods according
to the invention may be examined by histopathological and/or
histochemical methods.
[0060] The term "effective and non-toxic dose" as used herein
refers to a tolerable dose of the bispecific single chain antibody
as defined herein which is high enough to cause depletion of
pathologic cells, tumor elimination, tumor shrinkage or
stabilisation of disease without or essentially without major toxic
effects. Such effective and non-toxic doses may be determined e.g.
by dose escalation studies described in the art and should be below
the dose inducing severe adverse side events (dose limiting
toxicity, DLT).
[0061] The above terms are also referred to e.g. in the Preclinical
safety evaluation of biotechnology-derived pharmaceuticals S6; ICH
Harmonised Tripartite Guideline; ICH Steering Committee meeting on
Jul. 16, 1997.
[0062] It has been surprisingly found that it is possible to
generate bispecific antibody-based therapeutics for humans wherein
the identical molecule can also be used in preclinical animal
testing. This is due to the unexpected identification of bispecific
single chain antibodies which, in addition to binding to human
antigens (and due to genetic similarity likely to chimpanzee
counterparts), also bind to the homologs of said antigens of
non-chimpanzee primates, such as macaques. Thus, the need to
construct a surrogate bispecific single chain antibody for testing
in a phylogenetic distant (from humans) species disappears. As a
result, the very same bispecific single chain antibody can be used
in animal preclinical testing as is intended to be administered to
humans in clinical testing as well as following market approval.
The ability to use the same molecule for preclinical animal testing
as in later administration to humans virtually eliminates, or at
least greatly reduces, the danger that the data obtained in
preclinical animal testing are not applicable to the human case. In
short, obtaining preclinical safety data in animals using the same
molecule as will actually be administered to humans does much to
ensure the applicability of the data to a human-relevant scenario.
In contrast, in conventional approaches using surrogate molecules,
said surrogate antibodies have to be molecularly adapted to the
animal test system used for preclinical safety assessment. Thus,
the surrogate antibody to be used in human therapy in fact differs
in sequence and also likely in structure from the one used in
preclinical testing in pharmacokinetic parameters and/or biological
activity, with the consequence that data obtained in preclinical
animal testing have limited applicability/transferability to the
human case. The use of surrogate molecules requires the
construction, production, purification and characterization of a
completely new antibody construct. This leads to additional
development costs and time necessary to obtain that molecule. In
sum, surrogates have to be developed separately in addition to the
actual drug to be used in human therapy, so that two lines of
development for two bispecific single chain antibody molecules have
to be carried out. Therefore, a major advantage of the bispecific
antibody-based constructs exhibiting cross-species specificity
described herein is that the identical molecule can be used for
therapeutics in humans and in preclinical animal testing.
[0063] On the other hand, it is also no longer necessary to adapt
the test animal to the bispecific antibody-drug candidate intended
for administration to humans, such as e.g. the creation of
transgenic animals producing the human molecules bound by the
bispecific antibody. The bispecific single chain antibodies
exhibiting cross-species specificity according to the uses and
methods of invention can be directly used for preclinical testing
in non-chimpanzee primates, without any genetic manipulation of the
animals. As well known to those skilled in the art, approaches in
which the test animal is adapted to the drug candidate always bear
the risk that the results obtained in the preclinical safety
testing are less representative and predictive for humans due to
the modification of the animal. For example, in transgenic animals,
the proteins encoded by the transgenes are often highly
over-expressed. Thus, data obtained for the biological activity of
an antibody against this protein antigen may be limited in their
predictive value for humans in which the protein is expressed at
much lower, more physiological levels.
[0064] A further advantage of the uses of the bispecific single
chain antibody exhibiting cross-species specificity of the
invention lies in the avoidance of chimpanzee as a species for
animal testing. Chimpanzees are the closest relatives to humans and
were recently grouped into the family of hominids based on the
genome sequencing data (Wildman et al., PNAS 100 (2003), 7181).
Therefore, data obtained with chimpanzee is generally considered to
be highly predictive for humans. However, due to their status as
endangered species, the number of chimpanzees which can be used for
medical experiments is highly restricted. As stated above,
maintenance of chimpanzees for animal testing is therefore both
costly and ethically problematic. The uses of the bispecific single
chain antibody of the invention avoids both financial burden and
ethical objection during preclinical testing without prejudicing
the quality, i.e. applicability, of the animal testing data
obtained. In light of this, the uses of bispecific single chain
antibodies exhibiting cross-species specificity and methods
according to the invention for preclinical animal testing in
non-chimpanzee primates provides for a reasonable alternative for
studies in chimpanzees.
[0065] A further advantage of the bispecific single chain antibody
of the invention is the ability of extracting multiple blood
samples when using it as part of animal preclinical testing, for
example in the course of pharmacokinetic animal studies. Multiple
blood extractions can be much more readily obtained with a
non-chimpanzee primate than with lower animals, say a mouse. The
extraction of multiple blood samples allows continuous testing of
blood parameters for the determination of the biological effects
induced by the bispecific single chain antibody of the invention.
Furthermore, the extraction of multiple blood samples enables the
researcher to evaluate the pharmacokinetic profile of the
bispecific single chain antibody as defined herein. In addition,
potential side effects which may be induced by said bispecific
single chain antibody reflected in blood parameters can be measured
in different blood samples extracted during the course of the
administration of said antibody. This allows the determination of
the potential toxicity profile of the bispecific single chain
antibody as defined herein.
[0066] The advantages of the pharmaceutical compositions comprising
bispecific single chain antibodies exhibiting cross-species
specificity, uses of said bispecific antibodies and methods
according to the invention may be briefly summarized as
follows:
[0067] First, the bispecific single chain antibody exhibiting
cross-species specificity used in preclinical testing is the same
as the one used in human therapy. Thus, it is no longer necessary
to develop two independent molecules which may differ in their
pharmacokinetic properties and biological activity. This is highly
advantageous in that e.g. the pharmacokinetic results are more
directly transferable and applicable to the human setting than e.g.
in conventional surrogate approaches.
[0068] Second, the uses of the bispecific antibody exhibiting
cross-species specificity and methods according to the invention
for the preparation of therapeutics in human is less cost- and
labor-intensive than surrogate approaches.
[0069] Third, chimpanzee as a species for animal testing is
avoided.
[0070] Fourth, multiple blood samples can be extracted for
extensive pharmacokinetic studies.
[0071] A further aspect of the invention relates to a method of
determining the biological activity and/or efficacy of a bispecific
single chain antibody as defined above, wherein said bispecific
single chain antibody is administered to a non-chimpanzee primate
and the in vivo activity is measured.
[0072] Preferably, said in vivo activity is T cell activation,
tumor target cell depletion, cytotoxicity, toxicity, occurrence of
adverse side effects, and/or cytokine release. Methods for the
determination of said in vivo activity are set forth e.g. in WO
99/54440.
[0073] The present invention in another aspect also provides for a
pharmaceutical composition for the treatment of a human patient,
comprising a bispecific single chain antibody which comprises
[0074] (i) a first binding domain binding to a non-chimpanzee
primate CD3, and
[0075] (ii) a second binding domain binding to a cell surface
antigen, wherein said first binding domain binds to human and
non-chimpanzee primate CD3.
[0076] In accordance with this invention, the term "pharmaceutical
composition" relates to a composition for administration to a
patient, preferably a human patient. Preferably, the pharmaceutical
composition comprises suitable formulations of carriers,
stabilizers and/or excipients. In a preferred embodiment, the
pharmaceutical composition comprises a composition for parenteral,
transdermal, intraluminal, intraarterial, intrathecal and/or
intranasal administration or by direct injection into tissue. It is
in particular envisaged that said composition is administered to a
patient via infusion or injection. Administration of the suitable
compositions may be effected by different ways, e.g., by
intravenous, intraperitoneal, subcutaneous, intramuscular, topical
or intradermal administration. The composition of the present
invention may further comprise a pharmaceutically acceptable
carrier. Examples of suitable pharmaceutical carriers are well
known in the art and include phosphate buffered saline solutions,
water, emulsions, such as oil/water emulsions, various types of
wetting agents, sterile solutions, liposomes, etc. Compositions
comprising such carriers can be formulated by well known
conventional methods. These compositions can be administered to the
subject at a suitable dose which can be determined e.g. by dose
escalating studies by administration of increasing doses of the
bispecific single chain antibody exhibiting cross-species
specificity described herein to non-chimpanzee primates, for
instance macaques. As set forth above, the bispecific single chain
antibody exhibiting cross-species specificity described herein can
be advantageously used in identical form in preclinical testing in
non-chimpanzee primates and as drug in humans. These compositions
can also be administered in combination with other proteinaceous
and non-proteinaceous drugs. These drugs may be administered
simultaneously with the composition comprising the bispecific
single chain antibody as defined herein or separately before or
after administration of said bispecific antibody in timely defined
intervals and doses. The dosage regimen will be determined by the
attending physician and clinical factors. As is well known in the
medical arts, dosages for any one patient depend upon many factors,
including the patient's size, body surface area, age, the
particular compound to be administered, sex, time and route of
administration, general health, and other drugs being administered
concurrently. Preparations for parenteral administration include
sterile aqueous or non-aqueous solutions, suspensions, and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives may also
be present such as, for example, antimicrobials, anti-oxidants,
chelating agents, inert gases and the like. In addition, the
composition of the present invention might comprise proteinaceous
carriers, like, e.g., serum albumin or immunoglobulin, preferably
of human origin. It is envisaged that the composition of the
invention might comprise, in addition to the bispecific single
chain antibody as defined herein, further biologically active
agents, depending on the intended use of the composition.
[0077] Such agents might be drugs acting on the gastro-intestinal
system, drugs acting as cytostatica, drugs preventing
hyperurikemia, drugs inhibiting immunoreactions (e.g.
corticosteroids), drugs modulating the inflammatory response, drugs
acting on the circulatory system and/or agents such as cytokines
known in the art.
[0078] According to a preferred embodiment of the pharmaceutical
composition of the invention, the first binding domain of the
bispecific single chain antibody as defined herein binds to an
epitope of human and non-chimpanzee primate CD3 comprising the
amino acid sequence "FSE". The minimum core epitope comprising the
amino acid residues "FSE", the minimum epitope comprising the amino
acid sequence "FSEXE" (SEQ ID NOs. 202 and 204; wherein "X"
corresponds to a leucine (L) or to a methionine (M)) or
non-adjacent epitopes as defined herein are preferably 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acid residues
in length. Preferably, said epitopes are 13 amino acid residues in
length. Even more preferrred, the epitope with the "FSEXE" (SEQ ID
NOs. 202 and 204; wherein "X" corresponds to a leucine (L) or to a
methionine (M))--motif comprises the amino acid sequence
"EFSELEQSGYYVC" (SEQ ID NO. 195) of human CD3 epsilon. In
cynomolgus CD3 epsilon, the corresponding epitope reads
"EFSEMEQSGYYVC" (SEQ ID NO. 201). The substitution of methionine to
leucine is a conserved amino acid substitution between two neutral,
non-polar amino acid residues. The corresponding sequence of the
preferred epitope "EFSEXEQSGYYVC" wherein X represents L (Leucine)
or M (Methionine) is depicted in SEQ ID NO. 207. As shown in the
following Examples, the bispecific single chain antibody as defined
herein not only binds to this epitope, but also to amino-acid
stretches non-adjacent to said minimal epitope. For example, the
bispecific single chain antibody as defined herein in addition to
the minimum core epitope may simultaneously bind to (an) epitope(s)
of human CD3 epsilon contained in said CD3 epsilon chain.
Accordingly, said epitope may additionally comprise the amino acid
sequence "QYPGSEILWQHND" (SEQ ID NO. 203). Also additional or
(further) epitopes of cynomolgus CD3 epsilon contained in said
chain may be detected by the binding molecule or molecule
comprising the binding domains as defined therein. These additional
or further sequences may comprise the amino acid sequence
"QDGNEEMGSITQT" (SEQ ID NO. 199) and "YYVSYPRGSNPED" (SEQ ID NO.
200). Thus, the minimal epitope is most likely part of a
discontinuous epitope or a conformational epitope. As evident to a
person skilled in the art, the scope of the present invention
includes bispecific single chain antibodies not only binding to
this minimum (core) epitope, but also to one, two or even more
non-adjacent amino acid sequence stretches within CD3 (preferably
CD3 epsilon). Based on the results shown in the following Examples
it is concluded that cross-species specific anti-CD3 antibodies
contact CD3 epsilon in the area of amino acid residues 57-61 of
both cynomolgus and human CD3 epsilon comprising the amino acid
stretches FSEME (SEQ ID NO. 206) and FSELE (SEQ ID NO. 205) of
cynomolgus and human CD3 epsilon, respectively, with the motif FSE
forming the epitope core. This result--although plausible because
of the accessibility of the E-F-loop (amino acids 56-62) of human
CD3 epsilon (Kjer-Nielsen et al., PNAS 101 (2004), p. 7675-80)
comprising the amino acids FSELE (SEQ ID NO. 205) or FSEME (SEQ ID
NO. 206)--is surprising since there is no overlap of this newly
defined epitope with the known epitope on the CD3 epsilon-chain of
anti-CD3 antibodies OKT-3 and UCHT-1 (Kjer-Nielsen et al., loc.cit;
Arnett et al., PNAS 101 (2004), p. 16268-73) which have so far been
regarded as representative of all anti-CD3 antibodies thought to
form a single family with the same or a very similar epitope. In
summary, the epitopes "FSE" and "FSEXE" (SEQ ID NO. 204) are
distinct from the epitopes recognized by UCHT-1 or OKT-3
(Kjer-Nielsen et al., PNAS 101 (2004), p. 7675-80; Arnett et al.,
PNAS 101 (2004), p. 16268-73) and are unique for cross-species
specific anti-CD3 antibodies binding to human and macaque CD3.
Preferably, the minimum epitope comprises the amino acid sequence
"FSEXE" (SEQ ID NO. 204), wherein X represents L (Leucine) or M
(Methionine) and stands for a substitution of non-polar, neutral
amino acid residues.
[0079] It is envisaged that in the pharmaceutical composition of
the invention, said first binding domain of the bispecific single
chain antibody of the pharmaceutical composition of the invention
is located C-terminally to the second binding domain. However, also
part of this invention is a bispecific construct, wherein the
"first binding domain to a non-chimpanzee primate CD3" is located
N-terminally to the herein defined "second binding domain to a cell
surface antigen".
[0080] As shown in the following examples, the advantages as
described hereinabove are realizable not only when the first
binding domain (binding to CD3) is located C-terminally to the
second binding domain, i.e. closer to the C-terminus of the
bispecific single chain antibody than the second binding domain,
but also when the first binding domain (binding to CD3) is located
N-terminally to the second binding domain, i.e. closer to the
N-terminus of the bispecific single chain antibody than the second
binding domain. The arrangement of the binding domains in the
bispecific single chain antibody defined herein may therefore be
one in which the first binding domain is located C-terminally to
the second binding domain. The arrangement of the V chains may be
VH (cell surface antigen)-VL (cell surface
antigen)-VL(CD3)-VH(CD3), VH (cell surface antigen)-VL (cell
surface antigen)-VH(CD3)-VL(CD3), VL (cell surface antigen)-VH
(cell surface antigen)-VL(CD3)-VH(CD3) or VL (cell surface
antigen)-VH (cell surface antigen)-VH(CD3)-VL(CD3). For an
arrangement, in which the first binding domain is located
N-terminally to the second binding domain, the following orders are
possible: VH (CD3)-VL(CD3)-VL (cell surface antigen)-VH (cell
surface antigen), VH(CD3)-VL(CD3)-VH (cell surface antigen)-VL
(cell surface antigen), VL(CD3)-VH(CD3)-VL (cell surface
antigen)-VH (cell surface antigen) or VL(CD3)-VH(CD3)-VH (cell
surface antigen)-VL (cell surface antigen). As used herein,
"N-terminally to" or "C-terminally to" and grammatical variants
thereof denote relative location within the primary amino acid
sequence rather than placement at the absolute N- or C-terminus of
the bispecific single chain antibody. Hence, as a non-limiting
example, a first binding domain which is "located C-terminally to
the second binding domain" simply denotes that the first binding
domain is located to the carboxyl side of the second binding domain
within the bispecific single chain antibody, and does not exclude
the possibility that an additional sequence, for example a His-tag,
or another proteinaceous or non-proteinaceous compound such as a
radioisotope, is located at the ultimate C-terminus of the
bispecific single chain antibody.
[0081] In another preferred embodiment of the pharmaceutical
composition, the second binding domain binds to a cell surface
antigen and to the non-chimpanzee primate homolog of said cell
surface antigen.
[0082] According to this embodiment of the invention, both the
first and second binding domains of the bispecific single chain
antibody described herein specifically bind to both human and
non-chimpanzee primate variants of said first and second molecules,
respectively. In light of the above statements, this is
particularly advantageous since sufficient (cross-species)
specificity exists on both sides of the bispecific single chain
antibody, thus ensuring interspecies compatibility with respect to
both first and second molecules and hence optimal extrapolability
of the data obtained in preclinical animal studies to the case of
administration in humans.
[0083] Preferably, said cell surface antigen is a tumor antigen.
Even more preferred, said tumor antigen is EpCAM (Naundorf, Int. J.
Cancer 100/1 (2002), 101-110), EGFR (Liu, Br. J. Cancer 82/12
(2000), 1991-1999; Bonner, Semin. Radiat. Oncol. 12 (2002), 11-20;
Kiyota, Oncology 63/1 (2002), 92-98; Kuan, Brain Tumor Pathol. 17/2
(2000), 71-78), EGFRvIII (Kuan, Brain Tumor Pathol. 17/2 (2000),
71-78), or Carboanhydrase IX (MN/CA IX) (Uemura, Br. J. Cancer 81/4
(1999), 741-746; Longcaster, Cancer Res. 61/17 (2001), 6394-6399;
Chia, J. Clin. Oncol. 19/16 (2001), 3660-3668; Beasley, Cancer Res.
61/13 (2001), 5262-5267).
[0084] Particularly preferred as cell surface antigen and/or tumor
antigen is EpCAM. As shown in the following Examples, the present
application for the first time provides for the nucleic acid and
amino acid sequences of the extracellular domain of cynomolgus
EpCAM shown in SEQ ID NOs. 47 and 48, respectively. Said sequences
are essential tools for the generation and characterization of the
bispecific single chain antibodies as defined herein exhibiting
cross-species specificity to human and cynomolgus EpCAM.
[0085] In a further preferred embodiment of the pharmaceutical
composition of the invention, the first binding domain comprises a
VH region having an amino acid sequence as shown in any of SEQ ID
NOs. 2, 110 or 6. It is envisaged and preferred that the VH region
of the first binding domain comprises at least a third CDR (CDR-H3)
comprising an amino acid sequence as set out in SEQ ID NO. 112 or
CDR-H3* comprising an amino acid sequence as set out in SEQ ID NO.
113. The first binding domain may additionally comprise a second
CDR (CDR-H2) comprising an amino acid sequence as set out in SEQ ID
NO. 114. Further, the first binding domain may in addition comprise
a first CDR (CDR-H1) comprising an amino acid sequence as set out
in SEQ ID NO. 115. The VH region of the first binding domain thus
may comprise one, two or all three of the mentioned CDRs. The
mentioned CDRs are included for example in the VH regions shown in
SEQ ID NOs. 2 and 110.
[0086] Alternatively, it is envisaged that the VH region of the
first binding domain comprises a third CDR (CDR-H3) comprising an
amino acid sequence as set out in SEQ ID NO. 119. Preferably, the
first binding domain additionally comprises a second CDR (CDR-H2)
comprising an amino acid sequence as set out in SEQ ID NO. 120.
Especially preferred, the first binding domain additionally
comprises a first CDR (CDR-H1) comprising an amino acid sequence as
set out in SEQ ID NO. 121. The VH region of the first binding
domain thus may comprise one, two or all three of the mentioned
CDRs. The above-indicated CDRs are included for example in the VH
region shown in SEQ ID NO. 6.
[0087] In another preferred embodiment of the pharmaceutical
composition, the first binding domain comprises a VL region having
an amino acid sequence as shown in any of SEQ ID NOs. 4, 148, 168
or 8. It is envisaged and preferred that the VL region of the first
binding domain comprises at least a third CDR (CDR-L3) comprising
an amino acid sequence as set out in SEQ ID NO. 116. The VL region
may further comprise a second CDR (CDR-L2) comprising an amino acid
sequence as set out in SEQ ID NO. 117. The VL region may in
addition comprise a first CDR (CDR-L1) comprising an amino acid
sequence as set out in SEQ ID NO. 118. The VL region of the first
binding domain thus may comprise one, two or all three of the
mentioned CDRs. The above-indicated CDRs are included for example
in the VL regions shown in SEQ ID NOs. 4, 148 and 168.
[0088] Alternatively, it is envisaged that the VL region of the
first binding domain comprises a third CDR (CDR-L3) comprising an
amino acid sequence as set out in SEQ ID NO. 164. Preferably, the
first binding domain additionally comprises a second CDR (CDR-L2)
comprising an amino acid sequence as set out in SEQ ID NO. 165.
Especially preferred, the first binding domain additionally
comprises a first CDR (CDR-L1) comprising an amino acid sequence as
set out in SEQ ID NO. 166. The VL region of the first binding
domain thus may comprise one, two or all three of the mentioned
CDRs. The above-indicated CDRs are included for example in the VL
region shown in SEQ ID NO. 8.
[0089] Preferably, the first binding domain comprises CDR-L1 (SEQ
ID NO. 118), CDR-L2 (SEQ ID NO. 117), and CDR-L3 (SEQ ID NO. 116)
and CDR-H1 (SEQ ID NO. 115), CDR-H2 (SEQ ID NO. 114) and CDR-H3
(SEQ ID NO. 112) or CDR-H3* comprising the amino acid sequence
"VSWFAY" (SEQ ID NO. 113).
[0090] Alternatively, the first binding domain comprises CDR-L1
(SEQ ID NO. 166), CDR-L2 (SEQ ID NO. 165), and CDR-L3 (SEQ ID NO.
164) and CDR-H1 (SEQ ID NO. 121), CDR-H2 (SEQ ID NO. 120) and CDR
H3 (SEQ ID NO. 119).
[0091] Even more preferred, the VH region of the first binding
domain comprises or consists of the amino acid sequence shown in
SEQ ID NO. 2 and the VL region of the first binding domain
comprises or consists of the amino acid sequence shown SEQ ID NO.
4; or the VH region of the first binding domain comprises or
consists of the amino acid sequence shown in SEQ ID NO. 110 and the
VL region of the first binding domain comprises or consists of the
amino acid sequence shown in SEQ ID NO. 148; or the VH region of
the first binding domain comprises or consists of the amino acid
sequence shown in SEQ ID NO. 110 and the VL region of the first
binding domain comprises or consists of the amino acid sequence
shown in SEQ ID NO. 168, or the VH region of the first binding
domain comprises or consists of the amino acid sequence shown in
SEQ ID NO. 6 and the VL region of the first binding domain
comprises or consists of the amino acid sequence shown in SEQ ID
NO. 8. Or the VH region of the first binding domain comprises or
consists of the amino acid sequence shown in SEQ ID NO. 2 and the
VL region of the first binding domain comprises or consists of the
amino acid sequence shown in SEQ ID NO. 148. Or the VH region of
the first binding domain comprises or consists of the amino acid
sequence shown in SEQ ID NO. 110 and the VL region of the first
binding domain comprises or consists of the amino acid sequence
shown in SEQ ID NO. 4. Or the VH region of the first binding domain
comprises or consists of the amino acid sequence shown in SEQ ID
NO. 2 and the VL region of the first binding domain comprises or
consists of the amino acid sequence shown in SEQ ID NO. 168.
[0092] As set forth above, the order of the variable regions of the
first binding domain may be VH-VL or VL-VH. Both arrangements are
within the scope of the invention. For a first binding domain
comprising the VH of SEQ ID NO. 2 and the VL of SEQ ID NO. 4, the
VH-VL arrangement is shown in SEQ ID NOs. 9 and 10, whereas the
VL-VH arrangement is depicted in SEQ ID NOs. 11 and 12.
[0093] For a first binding domain comprising the VH of SEQ ID NO.
110 and the VL of SEQ ID NO. 148, the VH-VL arrangement is shown in
SEQ ID NOs. 146 and 147. For a first binding domain comprising the
VH of SEQ ID NO. 110 and the VL of SEQ ID NO. 168, the VH-VL
arrangement is shown in SEQ ID NOs. 169 and 170, whereas the VL-VH
arrangement is depicted in SEQ ID NOs. 193 and 194. For a first
binding domain comprising the VH of SEQ ID NO. 6 and the VL of SEQ
ID NO. 8, the VH-VL arrangement is shown in SEQ ID NOs. 13 and 14,
whereas the VL-VH arrangement is depicted in SEQ ID NOs. 15 and
16.
[0094] Similarly, the order of the variable regions of the second
binding domain may be VH-VL or VL-VH. Both arrangements are within
the scope of the invention. For example, the VH-VL arrangement of a
second binding domain exhibiting cross-species specificity to human
and cynomolgus EpCAM is shown in SEQ ID NOs. 53 and 54, whereas the
VL-VH arrangement is depicted in SEQ ID NOs. 55 and 56.
[0095] In a particularly preferred embodiment of the pharmaceutical
composition of the invention, the bispecific single chain antibody
as defined herein comprises an amino acid sequence selected from
the group consisting of: [0096] (a) an amino acid sequence as
depicted in any of SEQ ID NOs. 38, 40, 124, 42 or 44; [0097] (b) an
amino acid sequence encoded by a nucleic acid sequence as shown in
SEQ ID NOs. 37, 39, 125, 41 or 43; [0098] (c) an amino acid
sequence encoded by a nucleic acid sequence hybridizing under
stringent conditions to the complementary nucleic acid sequence of
(b); [0099] (d) an amino acid sequence encoded by a nucleic acid
sequence which is degenerate as a result of the genetic code to a
nucleotide sequence of (b); and [0100] (e) an amino acid sequence
at least 85% identical, more preferred at least 90% identical, most
preferred at least 95% identical to the amino acid sequence of (a)
or (b).
[0101] In the above-indicated preferred embodiment, only the first
binding domain (binding to CD3) exhibits cross-species
specificity.
[0102] Most preferably, the bispecific single chain antibody as
defined herein comprises an amino acid sequence selected from the
group consisting of: [0103] (a) an amino acid sequence as depicted
in any of SEQ ID NOs. 66, 68, 74, 76, 122, 70, 72, 78, 80, 172,
174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; [0104] (b) an
amino acid sequence encoded by a nucleic acid sequence as shown in
SEQ ID NOs. 65, 67, 73, 75, 123, 69, 71, 77, 79, 171, 173, 175,
177, 179, 181, 183, 185, 187, 189, or 191; [0105] (c) an amino acid
sequence encoded by a nucleic acid sequence hybridizing under
stringent conditions to the complementary nucleic acid sequence of
(b); [0106] (f) an amino acid sequence encoded by a nucleic acid
sequence which is degenerate as a result of the genetic code to a
nucleotide sequence of (b); and [0107] (g) an amino acid sequence
at least 85% identical, more preferred at least 90% identical, most
preferred at least 95% identical to the amino acid sequence of (a)
or (b).
[0108] In this above-indicated embodiment, both the first and
second binding domains exhibit cross-species specificity.
[0109] In another preferred embodiment of the pharmaceutical
composition, the non-chimpanzee primate is a baboon, marmoset or an
old world monkey.
[0110] In an even more preferred embodiment of the pharmaceutical
composition, the old world monkey is a monkey of the macaque
genus.
[0111] Most preferably, the monkey of the macaque genus is Assamese
macaque (Macaca assamensis), Barbary macaque (Macaca sylvanus),
Bonnet macaque (Macaca radiata), Booted or Sulawesi-Booted macaque
(Macaca ochreata), Sulawesi-crested macaque (Macaca nigra),
Formosan rock macaque (Macaca cyclopsis), Japanese snow macaque or
Japanese macaque (Macaca fuscata), Cynomologus monkey or
crab-eating macaque or long-tailed macaque or Java macaque (Macaca
fascicularis), Lion-tailed macaque (Macaca silenus), Pigtailed
macaque (Macaca nemestrina), Rhesus macaque (Macaca mulatta),
Tibetan macaque (Macaca thibetana), Tonkean macaque (Macaca
tonkeana), Toque macaque (Macaca sinica), Stump-tailed macaque or
Red-faced macaque or Bear monkey (Macaca arctoides), or Moor
macaque (Macaca maurus).
[0112] Preferably, the non-chimpanzee primate CD3 comprises or
consists of an amino acid sequence shown in SEQ ID NOs. 135, 136,
144, or 145.
[0113] According to a further embodiment of the pharmaceutical
composition of the invention, at least one of said first or second
binding domains is human, humanized, CDR-grafted and/or
deimmunized
[0114] The term "human" antibody as used herein is to be understood
as meaning that the bispecific single chain antibody as defined
herein, comprises (an) amino acid sequence(s) contained in the
human germline antibody repertoire. For the purposes of definition
herein, said bispecific single chain antibody may therefore be
considered human if it consists of such (a) human germline amino
acid sequence(s), i.e. if the amino acid sequence(s) of the
bispecific single chain antibody in question is (are) identical to
(an) expressed human germline amino acid sequence(s). A bispecific
single chain antibody as defined herein may also be regarded as
human if it consists of (a) sequence(s) that deviate(s) from its
(their) closest human germline sequence(s) by no more than would be
expected due to the imprint of somatic hypermutation. Additionally,
the antibodies of many non-human mammals, for example rodents such
as mice and rats, comprise VH CDR3 amino acid sequences which one
may expect to exist in the expressed human antibody repertoire as
well. Any such sequence(s) of human or non-human origin which may
be expected to exist in the expressed human repertoire would also
be considered "human" for the purposes of the present
invention.
[0115] As used herein, the term "humanized", "humanization",
"human-like" or grammatically related variants thereof are used
interchangeably to refer to a bispecific single chain antibody
comprising in at least one of its binding domains at least one
complementarity determining region ("CDR") from a non-human
antibody or fragment thereof. Humanization approaches are described
for example in WO 91/09968 and U.S. Pat. No. 6,407,213. As
non-limiting examples, the term encompasses the case in which a
variable region of at least one binding domain comprises a single
CDR region, for example the third CDR region of the VH, from
another non-human animal, for example a rodent, as well as the case
in which a or both variable region/s comprise at each of their
respective first, second and third CDRs the CDRs from said
non-human animal. In the event that all CDRs of a binding domain of
the bispecific single chain antibody have been replaced by their
corresponding equivalents from, for example, a rodent, one
typically speaks of "CDR-grafting", and this term is to be
understood as being encompassed by the term "humanized" or
grammatically related variants thereof as used herein. The term
"humanized" or grammatically related variants thereof also
encompasses cases in which, in addition to replacement of one or
more CDR regions within a VH and/or VL of the first and/or second
binding domain further mutations (e.g. substitutions) of at least
one single amino acid residue/s within the framework ("FR") regions
between the CDRs has/have been effected such that the amino acids
at that/those positions correspond/s to the amino acid/s at
that/those position/s in the animal from which the CDR regions used
for replacement is/are derived. As is known in the art, such
individual mutations are often made in the framework regions
following CDR-grafting in order to restore the original binding
affinity of the non-human antibody used as a CDR-donor for its
target molecule. The term "humanized" may further encompass (an)
amino acid substitution(s) in the CDR regions from a non-human
animal to the amino acid(s) of a corresponding CDR region from a
human antibody, in addition to the amino acid substitutions in the
framework regions as described above.
[0116] As used herein, the term "deimmunized," "deimmunization" or
grammatically related variants thereof denotes modification of the
first and/or second binding domain vis-a-vis an original wild type
construct by rendering said wild type construct non-immunogenic or
less immunogenic in humans. Deimmunization approaches are shown
e.g. in WO 00/34317, WO 98/52976, WO 02/079415 or WO 92/10755. The
term "deimmunized" also relates to constructs, which show reduced
propensity to generate T cell epitopes. In accordance with this
invention, the term "reduced propensity to generate T cell
epitopes" relates to the removal of T-cell epitopes leading to
specific T-cell activation. Furthermore, "reduced propensity to
generate T cell epitopes" means substitution of amino acids
contributing to the formation of T cell epitopes, i.e. substitution
of amino acids, which are essential for formation of a T cell
epitope. In other words, "reduced propensity to generate T cell
epitopes" relates to reduced immunogenicity or reduced capacity to
induce antigen independent T cell proliferation. The term "T cell
epitope" relates to short peptide sequences which can be released
during the degradation of peptides, polypeptides or proteins within
cells and subsequently be presented by molecules of the major
histocompatibility complex (MHC) in order to trigger the activation
of T cells; see inter alia WO 02/066514. For peptides presented by
MHC class II such activation of T cells can then give rise to an
antibody response by direct stimulation of T cells to produce said
antibodies. "Reduced propensity to generate T-cell epitopes" and/or
"deimmunization" may be measured by techniques known in the art.
Preferably, de-immunization of proteins may be tested in vitro by T
cell proliferation assay. In this assay PBMCs from donors
representing >80% of HLA-DR alleles in the world are screened
for proliferation in response to either wild type or de-immunized
peptides. Ideally cell proliferation is only detected upon loading
of the antigen-presenting cells with wild type peptides.
Alternatively, one may test deimmunization by expressing HLA-DR
tetramers representing all haplotypes. These tetramers may be
tested for peptide binding or loaded with peptides substitute for
antigen-presenting cells in proliferation assays. In order to test
whether deimmunized peptides are presented on HLA-DR haplotypes,
binding of e.g. fluorescence-labeled peptides on PBMCs can be
measured. Furthermore, deimmunization can be proven by determining
whether antibodies against the deimmunized molecules have been
formed after administration in patients. Preferably, antibody
derived molecules are deimmunized in the framework regions and most
of the CDR regions are not modified in order to generate reduced
propensity to induce T cell epitope so that the binding affinity of
the CDR regions is not affected. Even elimination of one T cell
epitope results in reduced immunogenicity.
[0117] The invention also provides for a pharmaceutical composition
comprising a nucleic acid sequence encoding a bispecific single
chain antibody as defined herein.
[0118] The invention further relates to a pharmaceutical
composition comprising a vector which comprises a nucleic acid
sequence as defined above. Preferably said vector further comprises
a regulatory sequence which is operably linked to said nucleic acid
sequence defined above. More preferably, said vector is an
expression vector.
[0119] In a further aspect, the invention relates to a
pharmaceutical composition comprising a host transformed or
transfected with a vector defined above.
[0120] A further aspect of the invention relates to a
pharmaceutical composition as defined hereinabove, further
comprising a proteinaceous compound capable of providing an
activation signal for immune effector cells.
[0121] Preferably, the pharmaceutical composition further comprises
suitable formulations of carriers, stabilizers and/or
excipients.
[0122] In another aspect, the invention relates to a process for
the production of a pharmaceutical composition as defined above,
said process comprising culturing a host as defined above under
conditions allowing the expression of the bispecific single chain
antibody as defined hereinabove and recovering the produced
bispecific single chain antibody from the culture.
[0123] A further aspect of the invention relates to a use of a
bispecific single chain antibody as defined hereinabove or as
produced by the process as defined hereinabove, a nucleic acid
molecule as defined hereinabove, a vector as defined hereinabove or
a host as defined hereinabove for the preparation of a
pharmaceutical composition for the prevention, treatment or
amelioration of a disease. Another aspect of the invention relates
to a method for the prevention, treatment or amelioration of a
disease in a subject in the need thereof, said method comprising
the step of administration of an effective amount of a
pharmaceutical composition of the invention or as produced
according by the process set forth above.
[0124] Preferably, said disease is a proliferative disease, a
tumorous disease, or an immunological disorder. Even more
preferred, said tumorous disease is a malignant disease, preferably
cancer. Cross-species specific bispecific single chain antibodies
as defined herein with specificity for EpCAM, EGFR or EGFRvIII can
be used for the therapy of epithelial cancers and tumors.
Cross-species specific bispecific single chain antibody constructs
as defined herein with specificity for CAIX can be used for the
treatment of tumors with hypoxical regions or areas. Moreover, said
CAIX constructs may be used for the treatment of renal or cervical
carcinomas. In another preferred embodiment of the uses or methods
of the invention, said pharmaceutical composition as defined
hereinabove is suitable to be administered in combination with an
additional drug, i.e. as part of a co-therapy. In said co-therapy,
an active agent may be optionally included in the same
pharmaceutical composition as the bispecific single chain antibody,
or may be included in a separate pharmaceutical composition. In
this latter case, said separate pharmaceutical composition is
suitable for administration prior to, simultaneously as or
following administration of said pharmaceutical composition
comprising the bispecific single chain antibody. The additional
drug or pharmaceutical composition may be a non-proteinaceous
compound or a proteinaceous compound. In the case that the
additional drug is a proteinaceous compound, it is advantageous
that the proteinaceous compound be capable of providing an
activation signal for immune effector cells.
[0125] Preferably, said proteinaceous compound or non-proteinaceous
compound may be administered simultaneously or non-simultaneously
with a bispecific single chain antibody as defined hereinabove, a
nucleic acid molecule as defined hereinabove, a vector as defined
as defined hereinabove, or a host as defined as defined
hereinabove. Preferably, said subject to be treated is a human.
[0126] In a further aspect, the invention relates to a kit
comprising a bispecific single chain antibody as defined
hereinabove, a nucleic acid molecule as defined hereinabove, a
vector as defined hereinabove, or a host as defined
hereinabove.
[0127] These and other embodiments are disclosed and encompassed by
the description and Examples of the present invention. Recombinant
techniques and methods in immunology are described e.g. in Sambrook
et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor
Laboratory Press, 3.sup.rd edition 2001; Lefkovits; Immunology
Methods Manual; The Comprehensive Sourcebook of Techniques;
Academic Press, 1997; Golemis; Protein-Protein Interactions: A
Molecular Cloning Manual; Cold Spring Laboratory Press, 2002.
Further literature concerning any one of the antibodies, methods,
uses and compounds to be employed in accordance with the present
invention may be retrieved from public libraries and databases,
using for example electronic devices. For example, the public
database "Medline", available on the Internet, may be utilized, for
example under http://www.ncbi.nlm.nih.gov/PubMed/medline.html.
Further databases and addresses, such as
http://www.ncbi.nim.nih.gov/, http://www.infobioaen.fr/,
http://www.fmi.ch/bioloqv/research tools.html,
http://www.tiqr.orQ/. are known to the person skilled in the art
and can also be obtained using, e.g., http://www.lvcos.com
[0128] The Figures show:
[0129] FIG. 1: Identification of cross-species specific antibodies
to macaque CD3: Cross-species specificity of an anti-CD3 antibody
shown in SEQ ID NO.162 described in WO 99/54440, OKT-3, an Ig
comprising SEQ ID NOs. 6 and 8, an Ig comprising SEQ ID NOs. 2 and
4 and UCHT-1 to macaque (cynomolgus) CD3 were tested with Flow
Cytometry as described in Example 1. An immunoglobulin (Ig)
comprising SEQ ID NOs. 6 and 8 and an Ig comprising SEQ ID NOs. 2
and 4 show cross-species specificity to macaque CD3. In contrast,
the anti-CD3 antibody shown in SEQ ID NO.162, OKT-3 and UCHT-1 fail
to bind to macaque CD3.
[0130] FIG. 2: FACS assay for binding of an Ig comprising SEQ ID
NOs. 2 and 4, an Ig comprising SEQ ID NOs. 6 and 8 and monoclonal
antibody (mAb) FN-18 to HPB-ALL cells and PBMC of Macaca
fascicularis (cynomolgus). HPB-ALL cells express the human CD3
complex. Cells stained with the respective antibodies are shown in
comparison to unstained cells. Strong antigen binding on human as
well as on cynomolgus cells was detected for the Ig comprising SEQ
ID NOs. 2 and 4. For the Ig comprising SEQ ID NOs. 6 and 8, strong
binding to human cells but weaker binding to cynomolgus cells was
observed. For FN-18, strong binding to cynomolgus cells could be
observed, whereas no binding to human cells could be detected.
[0131] FIG. 3: FACS assay for binding of 5-10LHxSEQ ID NO.12,
5-10LHxSEQ ID NO.10, 5-10LHxSEQ ID NO.16 and 5-10LHxSEQ ID NO.14 to
human Kato III cells expressing EpCAM or human EpCAM transfected
CHO cells and to HPB-ALL cells. Cells bound by the respective
constructs (depicted as non-filled curves) are shown in comparison
to cells incubated only with the detection antibodies (depicted as
filled curves). Antigen binding of all bispecific constructs was
clearly detectable for the anti human EpCAM specificity as well as
for the anti CD3 specificities on the HPB-ALL cell line positive
for human CD3.
[0132] FIG. 4: Cytotoxicity assay for 5-10LHxSEQ ID NO.12,
5-10LHxSEQ ID NO.10 and 5-10LHxSEQ ID NO.14 with human Kato III
cells as target cells and human PBMC as effector cells. All
constructs showed cytotoxic activity.
[0133] FIG. 5: Cytotoxicity assay for 5-10LHxSEQ ID NO.12,
5-10LHxSEQ ID NO.10, and 5-10LHxSEQ ID NO.14 with Kato III cells as
target cells and cynomolgus PBMC as effector cells. 5-10LHxSEQ ID
NO.14, 5-10LHxSEQ ID NO.12 and 5-10LHxSEQ ID NO.10 showed cytotoxic
activity. 5-10LHxdi-anti CD3 (deimmunised anti-CD3 antibody shown
in SEQ ID NO.163) which fails to bind to cynomolgus CD3 was used as
a negative control.
[0134] FIG. 6: Amino acid sequence alignment of the extracellular
portion of the cynomolgus EpCAM antigen (also shown in SEQ ID NO.
48) and the human EpCAM antigen.
[0135] FIG. 7: FACS assay for the detection of the cynomolgus EpCAM
antigen on transfected CHO cells. Supernatants of three different
anti human EpCAM hybridomas (M79, 3B10, 2G8) were tested for
binding. Transfectants (depicted as non-filled curves) as compared
to untransfected cells (depicted as filled curves) showed binding
only with the supernatant of the 2G8 hybridoma which is therefore
recognized as antibody cross-species specific for human and
cynomolgus EpCAM.
[0136] FIG. 8: FACS assay for binding of 2G8LHxSEQ ID NO.12,
2G8LHxSEQ ID NO.10, 2G8LHxSEQ ID NO.16, 2G8LHxSEQ ID NO.14,
2G8HLxSEQ ID NO.12, 2G8HLxSEQ ID NO.10, 2G8HLxSEQ ID NO.16 and
2G8HLxSEQ ID NO.14 on Kato III (FIG. 8A) cells or cynomolgus EpCAM
transfected CHO cells (FIG. 8B) and HPB-ALL cells. Antigen binding
was clearly detectable for the anti EpCAM specificities as well as
for the anti CD3 specificities. As a negative control for binding
to cynomolgus EpCAM, the 5-10LHxSEQ ID NO.10 construct was included
which shows binding to human CD3 (on HPB-ALL cells) but no binding
to cynomolgus EpCAM (cynomolgus EpCAM transfected CHO cells).
[0137] FIG. 9: Cytotoxicity assay for 2G8LHxSEQ ID NO.10 and
2G8HLxSEQ ID NO.12 with cynomolgus EpCAM transfected CHO cells as
target cells and human PBMC as effector cells. 2G8LHxSEQ ID NO.10
and 2G8HLxSEQ ID NO.12 showed cytotoxic activity. 5-10LHxdi-anti
CD3 (deimmunised anti-CD3 antibody shown in SEQ ID NO.163) was
included as negative control. 5-10LH fails to bind to cynomolgus
EpCAM.
[0138] FIG. 10: Cytotoxicity assay for 2G8LHxSEQ ID NO.10 and
2G8HLxSEQ ID NO.12 with cynomolgus EpCAM transfected CHO cells as
target cells and cynomolgus PBMC as effector cells. 2G8LHxSEQ ID
NO.10 and 2G8HLxSEQ ID NO.12 showed cytotoxic activity.
5-10LHxdi-anti CD3 (deimmunised anti-CD3 antibody shown in SEQ ID
NO.163) was included as negative control. This construct fails to
bind to cynomolgus CD3 and cynomolgus EpCAM.
[0139] FIG. 11: Amino acid comparison of SEQ ID NO. 2 and human VH
segment (hu)3-73.
[0140] FIG. 12: Amino acid and nucleotide sequences of a
cross-species specific human-like VH region (also shown in SEQ ID
NOs. 110 and 111, respectively).
[0141] FIG. 13: FACS analysis of a scFv comprising the human-like
VH chain shown in SEQ ID NO. 110 and the VL chain shown in SEQ ID
NO: 148. The complete scFv amino acid sequence is shown in SEQ ID
NO.146. The control scFv of SEQ ID NO.10 shows a clear shift on
human CD3 positive HPB-All cells and thus binds to human CD3. The
scFv depicted in SEQ ID NO. 146 also shows clear binding to said
CD3 positive human cells.
[0142] FIG. 14: Binding analysis of the scFv of SEQ ID NO. 146. The
control scFv of SEQ ID NO. 10 shows a clear shift on cynomolgus CD3
positive T cells and thus binds to cynomolgus CD3 positive cells.
Also the scFv of SEQ ID NO: 146 shows clear binding to cynomolgus
CD3 positive cells.
[0143] FIG. 15: Alignment of amino acid sequences of human and
cynomolgus CD3 epsilon.
[0144] FIG. 16: Amino acid sequences of the 13 mer peptides derived
from cynomolgus CD3 epsilon (43 peptide-spots).
[0145] FIG. 17: Amino acid sequences of the 13 mer peptides derived
from human CD3 epsilon (47 peptide-spots).
[0146] FIG. 18: Pepspots developed by enhanced chemiluminescence
(A) Control pepspot with horseradish-peroxidase conjugated
goat-anti-mouse IgG (B) Pepspot with cross-species specific
anti-CD3 antibody I corresponding to an immunoglobulin (Ig)
comprising the VH chain shown in SEQ ID NO. 2 and the VL chain
shown in SEQ ID NO. 4.
[0147] FIG. 19: Pepspot with cross-species specific anti-CD3
antibody II corresponding to an immunoglobulin (Ig) comprising the
VH chain shown in SEQ ID NO. 6 and the VL chain shown in SEQ ID NO.
8.
[0148] FIG. 20: Contact residues of OKT-3 and UCHT-1 and E-F-loop
epitope of cross-species specific anti-CD3 antibodies I and II
referred to in FIGS. 18 and 19, respectively, on cynomolgus and
human CD3 epsilon.
[0149] FIG. 21: Amino acid sequence comparison of the murine VL
shown in SEQ ID NO. 4 to the human germline lambda 7a segment.
[0150] FIG. 22: Binding of the murine scFv shown in SEQ ID NO. 10
and the human-like scFv shown in SEQ ID NO. 170 to human
CD3-positive HPB-ALL cells.
[0151] FIG. 23: Upper Panel: Equal binding of the murine scFv shown
in SEQ ID NO. 10 and the human-like scFv shown in SEQ ID NO. 170 to
human and cynomolgus T cells in PBMCs. Lower Panel When
preincubated with 10 ng/ml of the murine IgG antibody mAb I
described in Example 1 having the same binding specificity as the
scFvs (i.e. for CD3 epsilon), the shifts of cells stained with the
above-mentioned murine scFv or the human-like scFv decrease
significantly, underlining the similar binding region of the scFvs
and the original murine antibody mAb I.
[0152] FIG. 24: Pepspots developed by the alkaline phosphatase
detection system (A) Control pepspot with alkaline phosphatase
conjugated goat-anti-mouse IgG (B) Pepspot with cross-species
specific anti-CD3 antibody comprising the human-like VH shown in
SEQ ID NO. 110 and the human-like VL shown in SEQ ID NO. 168 as
described in Example 18.
[0153] FIG. 25: Dot Blot Assay with the cross-species specific
anti-CD3 antibody comprising the human-like VH of SEQ ID NO. 110
and the human-like VL of SEQ ID NO. 168 as described in Example 19
in (A) and the anti-CD3 murine IgG1 antibody UCHT1 (B) binding to
the blotted peptides "biotin-linker-EFSELEQSGYYVC" (1) and
"EFSELEQSGYYVC-biotin" (2) derived from human CD3 epsilon
[0154] FIG. 26: FACS binding analysis of cross-species specific
bispecific single chain construct CAIX HL.times.SEQ ID NO. 194 to
HPB-ALL (human CD3+), cynomolgus PBMC (cynomolgus CD3+), A549
(human CAIX+) and CYNOM-K1 (cynomolgus CAIX+) cells, respectively.
The FACS staining was performed as described in Example 23. The
thick line represents cells incubated with 1 ng/ml purified
monomeric protein that were subsequently incubated with the
anti-his antibody and the PE labeled detection antibody. The thin
histogram line reflects the negative control: cells only incubated
with the anti-his antibody and the detection antibody.
[0155] FIG. 27: FACS binding analysis of cross-species specific
bispecific single chain construct CAIX HL.times.SEQ ID NO. 170 to
HPB-ALL (human CD3+), cynomolgus PBMC (cynomolgus CD3+), A549
(human CAIX+) and 4 MBr-5 (macaque CAIX+) cells, respectively. The
FACS staining was performed as described in Example 23. The thick
line represents cells incubated with 1 ng/ml purified monomeric
protein that were subsequently incubated with the anti-his antibody
and the PE labeled detection antibody. The thin histogram line
reflects the negative control: cells only incubated with the
anti-his antibody and the detection antibody.
[0156] FIG. 28: FACS binding analysis of cross-species specific
bispecific single chain construct CAIX LH.times.SEQ ID NO. 170 to
HPB-ALL (human CD3+), cynomolgus PBMC (cynomolgus CD3+), A549
(human CAIX+) and 4 MBr-5 (macaque CAIX+) cells, respectively. The
FACS staining was performed as described in Example 23. The thick
line represents cells incubated with 1 ng/ml purified monomeric
protein that were subsequently incubated with the anti-his antibody
and the PE labeled detection antibody. The thin histogram line
reflects the negative control: cells only incubated with the
anti-his antibody and the detection antibody.
[0157] FIG. 29: FACS binding analysis of cross-species specific
bispecific single chain construct EGFR HL.times.SEQ ID NO. 170 to
HPB-ALL (human CD3+), cynomolgus PBMC (cynomolgus CD3+), A431
(human EGFR+) and CHO cells transfected with cynomolgus EGFR
(cynomolgus EGFR+), respectively. The FACS staining was performed
as described in Example 23. The thick line represents cells
incubated with 1 ng/ml purified monomeric protein that were
subsequently incubated with the anti-his antibody and the PE
labeled detection antibody. The thin histogram line reflects the
negative control: cells only incubated with the anti-his antibody
and the detection antibody.
[0158] FIG. 30: FACS binding analysis of cross-species specific
bispecific single chain construct EGFR LH.times.SEQ ID NO. 170 to
HPB-ALL (human CD3+), cynomolgus PBMC (cynomolgus CD3+), A431
(human EGFR+) and CHO cells transfected with cynomolgus EGFR
(cynomolgus EGFR+), respectively. The FACS staining was performed
as described in Example 23. The thick line represents cells
incubated with 1 .mu.g/ml purified monomeric protein that were
subsequently incubated with the anti-his antibody and the PE
labeled detection antibody. The thin histogram line reflects the
negative control: cells only incubated with the anti-his antibody
and the detection antibody.
[0159] FIG. 31: FACS binding analysis of cross-species specific
bispecific single chain construct EGFR HL.times.SEQ ID NO. 194 to
HPB-ALL (human CD3+), cynomolgus PBMC (cynomolgus CD3+), A431
(human EGFR+) and CHO cells transfected with cynomolgus EGFR
(cynomolgus EGFR+), respectively. The FACS staining was performed
as described in Example 23. The thick line represents cells
incubated with 1 .mu.g/ml purified monomeric protein that were
subsequently incubated with the anti-his antibody and the PE
labeled detection antibody. The thin histogram line reflects the
negative control: cells only incubated with the anti-his antibody
and the detection antibody.
[0160] FIG. 32: FACS binding analysis of cross-species specific
bispecific single chain construct EGFR LH.times.SEQ ID NO. 194 to
HPB-ALL (human CD3+), cynomolgus PBMC (cynomolgus CD3+), A431
(human EGFR+) and CHO cells transfected with cynomolgus EGFR
(cynomolgus EGFR+), respectively. The FACS staining was performed
as described in Example 23. The thick line represents cells
incubated with 1 .mu.g/ml purified monomeric protein that were
subsequently incubated with the anti-his antibody and the PE
labeled detection antibody. The thin histogram line reflects the
negative control: cells only incubated with the anti-his antibody
and the detection antibody.
[0161] FIG. 33: FACS binding analysis of cross-species specific
bispecific single chain construct SEQ ID NO. 170.times.EGFR HL to
HPB-ALL (human CD3+), cynomolgus PBMC (cynomolgus CD3+), A431
(human EGFR+) and CHO cells transfected with cynomolgus EGFR
(cynomolgus EGFR+), respectively. The FACS staining was performed
as described in Example 23. The thick line represents cells
incubated with 1 ng/ml purified monomeric protein that were
subsequently incubated with the anti-his antibody and the PE
labeled detection antibody. The thin histogram line reflects the
negative control: cells only incubated with the anti-his antibody
and the detection antibody.
[0162] FIG. 34: FACS binding analysis of cross-species specific
bispecific single chain construct SEQ ID NO. 170.times.EGFR LH to
HPB-ALL (human CD3+), cynomolgus PBMC (cynomolgus CD3+), A431
(human EGFR+) and CHO cells transfected with cynomolgus EGFR
(cynomolgus EGFR+), respectively. The FACS staining was performed
as described in Example 23. The thick line represents cells
incubated with 1 ng/ml purified monomeric protein that were
subsequently incubated with the anti-his antibody and the PE
labeled detection antibody. The thin histogram line reflects the
negative control: cells only incubated with the anti-his antibody
and the detection antibody.
[0163] FIG. 35: FACS binding analysis of cross-species specific
bispecific single chain construct SEQ ID NO. 194.times.EGFR HL to
HPB-ALL (human CD3+), cynomolgus PBMC (cynomolgus CD3+), A431
(human EGFR+) and CHO cells transfected with cynomolgus EGFR
(cynomolgus EGFR+), respectively. The FACS staining was performed
as described in Example 23. The thick line represents cells
incubated with 1 ng/ml purified monomeric protein that were
subsequently incubated with the anti-his antibody and the PE
labeled detection antibody. The thin histogram line reflects the
negative control: cells only incubated with the anti-his antibody
and the detection antibody.
[0164] FIG. 36: FACS binding analysis of cross-species specific
bispecific single chain construct SEQ ID NO. 194.times.EGFR LH to
HPB-ALL (human CD3+), cynomolgus PBMC (cynomolgus CD3+), A431
(human EGFR+) and CHO cells transfected with cynomolgus EGFR
(cynomolgus EGFR+), respectively. The FACS staining was performed
as described in Example 23. The thick line represents cells
incubated with 1 ng/ml purified monomeric protein that were
subsequently incubated with the anti-his antibody and the PE
labeled detection antibody. The thin histogram line reflects the
negative control: cells only incubated with the anti-his antibody
and the detection antibody.
[0165] FIG. 37: Cytotoxic activity induced by CAIX and CD3
cross-species specific bispecific single chain antibody constructs
redirected to indicated target cell lines. Stimulated CD8 positive
T cells from human and cynomolgus origin were used as effector
cells, respectively. The assay was performed as described in
Examples 24 and 25. In the left panel of FIG. 37, a bispecific
single chain antibody with a variable domain reactive with CAIX and
a de-immunized human CD3-specific variable domain has been used as
a positive control. In the right panel, the same construct has been
used as a negative control.
[0166] FIG. 38: Cytotoxic activity induced by the CAIX and CD3
cross-species specific bispecific single chain antibody construct
CAIX HL.times.SEQ ID NO. 194 redirected to target cell line A549.
Stimulated CD8 positive T cells from human and cynomolgus origin
were used as effector cells, respectively. The assay was performed
as described in Examples 24 and 25.
[0167] FIG. 39: Cytotoxic activity induced by EGFR and CD3
cross-species specific bispecific single chain antibody constructs
redirected to CHO cells transfected with cynomolgus EGFR as target
cell line. Stimulated CD8 positive T cells from cynomolgus origin
were used as effector cells. The measurements shown in this figure
were performed in a single assay. The assay was performed as
described in Example 24. A bispecific single chain antibody with a
variable domain reactive with EGFR and a de-immunized human
CD3-specific variable domain (EGFR LH.times.di-anti CD3) has been
used as a negative control.
[0168] FIG. 40: Cytotoxic activity induced by EGFR and CD3
cross-species specific bispecific single chain antibody constructs
redirected to human A431 as target cell line. Stimulated CD8
positive T cells from human origin were used as effector cells. The
measurements shown in this figure were performed in a single assay.
The assay was performed as described in Example 24. A bispecific
single chain antibody with a variable domain reactive with EGFR and
a de-immunized human CD3-specific variable domain (EGFR
LH.times.di-anti CD3) has been used as a positive control. As a
negative control, an irrelevant bispecific single chain antibody
has been used.
[0169] The following Examples illustrate the invention:
EXAMPLE 1
Flow Cytometric Analysis of Cross-Species Specific Antibodies
[0170] Cross-species specificity of anti-human CD3 antibodies to
macaque CD3 (CD3 of Macaca fascicularis, in the following also
named "Cynomolgus") was tested by flow cytometric analysis.
Antibodies tested were an anti-CD3 antibody as described in WO
99/54440 (as shown in SEQ ID NO. 162 of the present application),
monoclonal antibody (mAb) OKT-3 (Jansen-Cilag), UCHT-1-PE (BD
PharMingen, San Diego, Calif.), an immunoglobulin (Ig) comprising
the VH and VL chains shown in SEQ ID NOs. 2 and 4, respectively,
and an Ig comprising the VH and VL chains shown in SEQ ID NOs. 6
and 8, respectively. 2.times.10.sup.5 cells (macaque T cell lines
of Macaca fascicularis and Macaca mulatta, respectively, as kindly
provided by H. Fickenscher, Heidelberg, Germany) per sample were
stained for 30 minutes at 4.degree. C. in 25 of PBS/1% FCS/0.05%
NaN.sub.3 containing working dilutions of monoclonal antibodies (as
determined individually by titration). Cells were washed two times
in PBS/1% FCS/0.05% NaN.sub.3 and a secondary antibody was added
where necessary. After the addition of the secondary antibody,
cells were washed again two times in the same solution and 10.000
living cells were acquired. A FACS Calibur flow cytometer and the
CellQuest software from Becton Dickinson were used to collect and
analyze the data. Non viable cells were excluded using forward and
side scatter electronic gating. Isotype control or secondary
antibody only were used as a negative control. As can be seen from
FIG. 1, only the Ig comprising the VH and VL chains shown in SEQ ID
NOs. 2 and 4, respectively, and the Ig comprising the VH and VL
chains shown in SEQ ID NOs. 6 and 8, respectively, showed
cross-species specificity for a non-chimpanzee primate CD3, i.e.
macaque CD3.
EXAMPLE 2
FACS Assay for Binding of an Ig Comprising SEQ ID NOs. 2 and 4, an
Ig Comprising SEQ ID NOs. 6 and 8 and mAb FN18 to HPB-ALL Cells and
Cynomolgus PBMC
[0171] Binding of an Ig comprising SEQ ID NOs. 2 and 4, an Ig
comprising SEQ ID NOs. 6 and 8 and mAb FN18 to the cynomolgus CD3
antigen on cynomolgus PBMC and to the human CD3 antigen on HPB-ALL
cells (DSMZ No. ACC 483) was tested using an FACS assay. For that
purpose, 2.5.times.10.sup.5 cells were incubated with the
FITC-conjugated Ig comprising SEQ ID NOs. 6 and 8 and the
FITC-conjugated Ig comprising SEQ ID NOs. 2 and 4 diluted 1:25 in
50 nl PBS with 2% FCS, respectively. The incubation with the
FITC-conjugated mAb FN18 antibody (Biosource International) was
performed in 50 nl of undiluted antibody. The samples were measured
on a FACSscan (BD biosciences, Heidelberg, FRG). The results for
the assay are shown in FIG. 2. Strong antigen binding on human as
well as on cynomolgus cells was detected for the Ig comprising SEQ
ID NOs. 2 and 4. For the Ig comprising SEQ ID NOs. 6 and 8, strong
binding to human cells but weaker binding to cynomolgus cells was
observed. For FN18, strong binding to cynomolgus cells could be
observed whereas no binding to human cells could be detected.
EXAMPLE 3
Sequence Determination of the Variable Regions of Two Anti-Human
CD3 Antibodies Exhibiting Species Specificity for Non-Human
Primates
[0172] For the sequence determination of the variable regions of
the cross-species specific anti-CD3 Igs of Examples 1 and 2, PCR
(denaturation at 93.degree. C. for 5 min, annealing at 58.degree.
C. for 1 min, elongation at 72.degree. C. for 1 min for the first
cycle; denaturation at 93.degree. C. for 1 min, annealing at
58.degree. C. for 1 min, elongation at 72.degree. C. for 1 min for
30 cycles; terminal extension at 72.degree. C. for 5 min) was used
to amplify the coding sequences of the variable regions of the
antibodies. As the sequence of the 5' region of the variable
regions is not known, instead of a single primer a set of 5'
primers was used in combination with a constant 3' primer whereby
the 3' primer was chosen according to the isotype of the respective
antibody and there were two different sets of primers for the 5'
region, one for the light chain variable region and the other for
the heavy chain variable region. The primer combinations used in
the PCR reactions are given below.
TABLE-US-00001 Heavy chain variable region: 5' primer: (SEQ ID NO.
81) 5'-SAGGTGCAGCTCGAGGAGTCAGGACCT-3' (SEQ ID NO. 82)
5'-GAGGTCCAGCTCGAGCAGTCTGGACCT-3' (SEQ ID NO. 83)
5'-CAGGTCCAACTCGAGCAGCCTGGGGCT-3' (SEQ ID NO. 84)
5'-GAGGTTCAGCTCGAGCAGTCTGGGGCA-3' (SEQ ID NO. 85)
5'-GARGTGAAGCTCGAGGAGTCTGGAGGA-3' (SEQ ID NO. 86)
5'-GAGGTGAAGCTTCTCGAGTCTGGAGGT-3' (SEQ ID NO. 87)
5'-GAAGTGAAGCTCGAGGAGTCTGGGGGA-3' (SEQ ID NO. 88)
5'-GAGGTTCAGCTCGAGCAGTCTGGAGCT-3' (SEQ ID NO. 89)
5'-GGGCTCGAGCACCATGGRATGSAGCTGKGTMATSCTCTT-3' (SEQ ID NO. 90)
5'-GGGCTCGAGCACCATGRACTTCGGGYTGAGCTKGGTTTT-3' (SEQ ID NO. 91)
5'-GGGCTCGAGCACCATGGCTGTCTTGGGGCTGCTCTTCT-3' 3' primer: (SEQ ID NO.
92) 5'-GAGGAATTCGAACTGGACAGGGATCCAGAGTTCC-3' (SEQ ID NO. 93)
5'-CGGAATTCGAATGACATGGACATCTGGGTCATCC-3' Light chain variable
region: 5' primer: (SEQ ID NO. 94)
5'-CCAGTTCCGAGCTCGTTGTGACTCAGGAATCT-3' (SEQ ID NO. 95)
5'-CCAGTTCCGAGCTCGTGTTGACGCAGCCGCCC-3' (SEQ ID NO. 96)
5'-CCAGTTCCGAGCTCGTGCTCACCCAGTCTCCA-3' (SEQ ID NO. 97)
5'-CCAGTTCCGAGCTCCAGATGACCCAGTCTCCA-3' (SEQ ID NO. 98)
5'-CCAGATGTGAGCTCGTGATGACCCAGACTCCA-3' (SEQ ID NO. 99)
5'-CCAGATGTGAGCTCGTCATGACCCAGTCTCCA-3' (SEQ ID NO. 100)
5'-CCAGTTCCGAGCTCGTGATGACACAGTCTCCA-3' (SEQ ID NO. 101)
5'-GGGGAGCTCCACCATGGAGACAGACACACTCCTGCTAT-3' (SEQ ID NO. 102)
5'-GGGGAGCTCCACCATGGATTTTCAAGTGCAGATTTTCAG-3' (SEQ ID NO. 103)
5'-GGGGAGCTCCACCATGGAGWCACAKWCTCAGGTCTTTRTA-3' (SEQ ID NO. 104)
5'-GGGGAGCTCCACCATGKCCCCWRCTCAGYTYCTKGT-3' 3' primer: (SEQ ID NO.
105) 5'-GAGGAATTCGAACTGCTCACTGGATGGTGGG-3' (SEQ ID NO. 106)
5'-CGGAATTCGAACAAACTCTTCTCCACAGTGTGACC-3'
[0173] All PCR products with a length between 350 and 700 base
pairs were isolated, purified and sequenced with the respective 3'
primer according to standard protocols (Sambrook, Molecular
Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989) (2001)).
[0174] The obtained sequences were examined for functional variable
region coding sequences and for the heavy chain and the light chain
of each antibody a sequence coding for the variable region was
obtained. The nucleotide and amino acid sequences of the heavy and
light chain variable regions of the cross-species specific anti-CD3
antibodies are described in SEQ ID NOs. 1 through 8 in the sequence
listing included in the description, respectively.
EXAMPLE 4
Cloning of Anti Human EpCAM and CD3 Cross-Species Specific
Bispecific Single Chain Antibodies
[0175] To generate bispecific single chain antibodies comprising
the aforementioned CD3 cross-species specificities, the amplified
variable regions had to be modified by PCR to obtain the
corresponding single chain Fv antibody fragments. To determine
suitable arrangements of the light and heavy chain variable regions
in the single chain Fv antibody, two different single chain Fv
antibodies were generated for each antibody. To this end, a
two-step fusion PCR was used to amplify the sequence coding for the
variable regions. A set of appropriate primers was designed to
perform the PCR-based cloning steps, finally resulting in a single
chain antibody connecting the two variable domains with a 15 amino
acid linker ([Gly.sub.4Ser].sub.3) in the order VH-Linker-VL and
VL-Linker-VH. The corresponding nucleotide and amino acid sequences
are described in SEQ ID NO. 9 through 12 and in SEQ ID NO. 13
through 16 of the sequence listing included in the description.
[0176] In short the following primer combinations were used:
[0177] For VL-VH scFv antibody shown in SEQ ID NOs. 11 and 12: SEQ
ID NOs. 17 to 20.
[0178] For VH-VL scFv antibody shown in SEQ ID NOs. 9 and 10: SEQ
ID NOs. 21 to 24.
[0179] For VL-VH scFv antibody shown in SEQ ID NOs. 15 and 16: SEQ
ID NOs. 25 to 28.
[0180] For VH-VL scFv antibody shown in SEQ ID NOs. 13 and 14: SEQ
ID NOs. 29 to 32.
[0181] To generate the single chain antibody, two PCRs with the
respective primer combinations were performed. During this PCR
overlapping complementary sequences were introduced into the
PCR-products stemming from the respective linker primers that
combined to form the coding sequence of the 15 amino acid linker
during the subsequent fusion PCR. The amplified VH and VL domains
were fused in a next PCR in which only the outer primers and both
PCR-products were required. The resulting scFv antibody is flanked
at the 5' end with a small Ser(Gly.sub.4)Ser linker preceded by the
restriction enzyme recognition site for BspEI and at the 3' end
with a 6 histidine affinity tag followed by a stop codon and by the
restriction enzyme recognition site for SalI. The second single
chain Fv antibody was an anti human EpCAM specificity designated
"5-10" which is described in SEQ ID NO. 33 and 34 of the sequence
listing included in the description. To accomplish the fusion of
the single chain Fv antibodies and to allow for eukaryotic
expression, the coding sequence of the single chain Fv antibodies
was then cloned via BspEI (5' to the Ser(Gly.sub.4)Ser linker) and
SalI into the pEFDHFR expression vector (pEFDHFR was described in
Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025)
containing the coding sequence for the human EpCAM specific single
chain Fv antibody 5-10 and the restriction enzyme recognition site
for BspEI. The coding sequence of an murine immunoglobulin leader
peptide is described in SEQ ID NO. 35 and 36 of the sequence
listing included in the description, preceded by a Kozak
translation initiation consensus sequence and the restriction
enzyme recognition site for EcoRI. Single clones of the constructs
were isolated and sequenced with primers complementary to flanking
regions in the vector according to standard protocols (Sambrook,
Molecular Cloning; A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989) (2001)). For
further experiments a clone of each construct was selected. The
nucleotide and amino acid sequences are described for 5-10LHxSEQ ID
NO.12 in SEQ ID NOs. 37 and 38, for 5-10LHxSEQ ID NO.10 in SEQ ID
NOs. 39 and 40, for 5-10LHxSEQ ID NO.16 in SEQ ID NOs. 41 and 42
and for 5-10LHxSEQ ID NOs.14 in SEQ ID NO. 43 and 44 of the
sequence listing included in the description.
EXAMPLE 5
Expression of the 5-10LHxSEQ ID NO.12, 5-10LHxSEQ ID NO.10,
5-10LHxSEQ ID NO.16 and 5-10LHxSEQ ID NO.14 Bispecific Single Chain
Antibodies in CHO Cells
[0182] The plasmids with the sequences coding for the bispecific
single chain antibodies were transfected into DHFR deficient CHO
cells for eukaryotic expression of the construct as described in
Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566). Gene
amplification of the construct was induced by increasing
concentrations of Methotrexat (MTX) to a final concentration of up
to 500 nM MTX. The transfected cells were then expanded and 1 liter
of supernatant produced. The construct was finally purified out of
the culture supernatant as described in Kufer et al. Cancer
immunity Vol. 1, p. 10 (2001).
EXAMPLE 6
FACS Assay for Binding of 5-10LHxSEQ ID NO.12, 5-10LHxSEQ ID NO.10,
5-10LHxSEQ ID NO.16 and 5-10LHxSEQ ID NO.14 to Kato III Cells or
Human EpCAM Transfected CHO Cells and to HPB-ALL Cells
[0183] Binding of the bifunctional constructs to the EpCAM antigen
on human Kato III cells expressing EpCAM (ATCC No. HTB-103) or on
human EpCAM transfected CHO cells and to the human CD3 antigen on
HPB-ALL cells was tested using an FACS assay. For that purpose
2.5.times.10.sup.5 cells were incubated with 50u1 of cell culture
supernatant containing the construct. The binding of the construct
was detected with an anti-His antibody (Penta-His Antibody, BSA
free, obtained from Qiagen GmbH, Hilden, FRG) at 2 .mu.g/ml in 50
ul PBS with 2% FCS. As a second step reagent a
R-Phycoerythrin-conjugated affinity purified F(ab')2 fragment, goat
anti-mouse IgG, Fc-gamma fragment specific antibody, diluted 1:100
in 50 ul PBS with 2% FCS (obtained from Dianova, Hamburg, FRG) was
used. The samples were measured on a FACSscan (BD biosciences,
Heidelberg, FRG). Antigen binding was clearly detectable for the
anti human EpCAM specificity as well as for the anti CD3
specificities on the cell line positive for human CD3 (see FIG.
3).
EXAMPLE 7
Cytotoxicity Assay for 5-10LHxSEQ ID NO. 12, 5-10LHxSEQ ID NO.10,
and 5-10LHxSEQ ID NO.14 with Kato III Cells as Target Cells and
Human PBMC as Effector Cells
[0184] Bioactivity of 5-10LHxSEQ ID NO.12, 5-10LHxSEQ ID NO.10, and
5LH-10.times.SEQ ID NO.14 was analyzed by FACS-based in vitro
cytotoxicity assays using the human EpCAM positive Kato III cells
as target cells and human PBMCs as effector cells. Target cells
were washed twice with PBS and labeled with PKH26 dye
(Sigma-Aldrich, Germany) according to the manufacturer's
instructions. Labeled target cells were washed twice with RPMI/10%
FCS and mixed with freshly isolated effector cells at an E:T ratio
of 10:1. Two times 10.sup.4 target and 2.times.10.sup.5 effector
cells in a volume of 50 .mu.l RPMI/10% FCS were added per well in a
96-well round bottom plate. Ten-fold serial dilutions of different
bispecific single chain constructs were prepared in RPMI/10% FCS to
obtain a starting concentration of 1000 ng/ml in the final reaction
volume. 50 .mu.l of the different solutions were added in
triplicates to the corresponding wells. Individual cytotoxicity
mixtures were incubated for 24 to 48 hours at 37.degree. C., 5%
CO.sub.2.
[0185] Subsequently the measurement of cytotoxic activity was
performed. To this end, Propidium iodide (PI) was added to a final
concentration of 1 .mu.g/ml per well and plates were incubated for
10 minutes at room temperature. The number of PKH and PI positive
and negative target cells was determined by FACS. Cytotoxicity was
measured as the ratio of PKH-positive and PI negative (living
target cells) over the mean of living target cells (PKH-positive
and PI negative) in the control containing no construct according
to the formula: cytotoxicity (%)=[(PI-negative cells/mean of
PI-negative cells in control).times.100]. Sigmoidal dose response
killing curves were analyzed by Prism Software (GraphPad Software
Inc., San Diego, USA) and the BiTE concentration calculated that
induced half maximal killing (EC50 value). The results of this
assay are shown below in FIG. 4. All constructs showed cytotoxic
activity. The resulting EC50 values for 5-10LHxSEQ ID NO.14,
5-10LHxSEQ ID NO.12 and 5-10LHxSEQ ID NO.10 were 1.3 pg/ml, 1.5
pg/ml and 5.8 pg/ml respectively.
EXAMPLE 8
Cytotoxicity Assay for 5-10LHxSEQ ID NO.12, 5-10LHxSEQ ID NO.10,
and 5-10LHxSEQ ID NO.14 with Kato III Cells as Target Cells and
Cynomolgus PBMC as Effector Cells
[0186] Bioactivity of 5-10LHxSEQ ID NO.12, 5-10LHxSEQ ID NO.10, and
5-10LHxSEQ ID NO.14 was analyzed by FACS-based in vitro
cytotoxicity assays using the human EpCAM positive Kato III cells
as target cells and cynomolgus PBMCs as effector cells.
[0187] Target cells were washed twice with PBS and labeled with
PKH26 dye (Sigma-Aldrich, Germany) according to the manufacturer's
instructions. Labeled target cells were washed twice with RPMI/10%
FCS and mixed with freshly isolated effector cells at an E:T ratio
of 10:1. Two times 10.sup.4 target and 2.times.10.sup.5 effector
cells in a volume of 50 .mu.l RPMI/10% FCS were added per well in a
96-well round bottom plate. Ten-fold serial dilutions of different
Bispecific single chain antibodies were prepared in RPMI/10% FCS to
obtain a starting concentration of 1000 ng/ml in the final reaction
volume. 50 .mu.l of the different solutions were added in
triplicates to the corresponding wells. Individual cytotoxicity
mixtures were incubated for 24 to 48 hours at 37.degree. C., 5%
CO.sub.2.
[0188] Subsequently the measurement of cytotoxic activity was
performed. To this end, propidium iodide (PI) was added to a final
concentration of 1 .mu.g/ml per well and plates were incubated for
10 minutes at room temperature. The number of PKH and PI positive
and negative target cells was determined by FACS. Cytotoxicity was
measured as the ratio of PKH-positive and PI negative (living
target cells) over the mean of living target cells (PKH-positive
and PI negative) in the control containing no construct according
to the formula: cytotoxicity (%)=[(PI-negative cells/mean of
PI-negative cells in control).times.100]. Sigmoidal dose response
killing curves were analyzed by Prism Software (GraphPad Software
Inc., San Diego, USA) and the bispecific single chain antibody
concentration calculated that induced half maximal killing (EC50
value). The results of this assay are shown below in FIG. 5.
5-10LHxSEQ ID NO.14, 5-10LHxSEQ ID NO.12 and 5-10LHxSEQ ID NO.10
showed cytotoxic activity. The resulting EC50 values for 5-10LHxSEQ
ID NO.14, 5-10LHxSEQ ID NO.12 and 5-10LHxSEQ ID NO.10 were 87
pg/ml, 69 pg/ml and 52 pg/ml respectively. 5-10LHxdi-anti CD3
(deimmunised anti-CD3 antibody as shown in SEQ ID NO.163) showed no
activity. This is due to the fact that di-anti CD3 antibody only
binds to human CD3, but not to cynomolgus CD3.
EXAMPLE 9
Sequence Determination of the Cynomolgus EpCAM Antigen and
Generation of Cynomolgus EpCAM Transfected CHO Cells
[0189] To obtain the cynomolgus EpCAM antigen for testing of
cross-species specificity of anti human EpCAM antibodies, first the
coding sequence of the cynomolgus EpCAM antigen had to be
determined. To this end, colon tissue samples of 3 animals were
used in parallel for the isolation of total RNA and cDNA synthesis
by random-primed reverse transcription, which were performed
according to standard protocols (Sambrook, Molecular Cloning; A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989) (2001)). A PCR (denaturation at 93.degree. C.
for 5 min, annealing at 58.degree. C. for 1 min, elongation at
72.degree. C. for 1 min for the first cycle; denaturation at
93.degree. C. for 1 min, annealing at 58.degree. C. for 1 min,
elongation at 72.degree. C. for 1 min for 35 cycles; terminal
extension at 72.degree. C. for 5 min) was used to amplify the
coding sequence of the EpCAM antigen. As the coding sequence of the
cynomolgus EpCAM antigen was not known, appropriate primers (5'
primer described in SEQ ID NO. 45, 3' primer described in SEQ ID
NO. 46) for the PCR reaction were designed according to the known
coding sequence of the human EpCAM antigen (Szala S. et al., Proc
Natl Acad Sci USA. 87 (1990); p. 3542-6). Primers were also
designed as to allow for expression of the coding sequence of the
entire antigen. For the 3 samples, PCR of 960 base pairs were
isolated, purified and subcloned via XbaI and SalI, into pEFDHFR.
Multiple clones for each sample were sequenced according to
standard protocols (Sambrook, Molecular Cloning; A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989) (2001)) using appropriate sequencing primers
complementary to flanking sequences in the vector.
[0190] The novel nucleotide and amino acid sequences of the
cynomolgus EpCAM antigen are described in SEQ ID NOs. 47 and 48 in
the sequence listing included in the description, respectively.
[0191] The obtained sequences were examined by comparison with the
coding sequence of the human EpCAM antigen. As shown in FIG. 6,
there is a high degree of sequence homology between the coding
sequence of the human EpCAM antigen and the sequences obtained from
the colon samples of the 3 cynomolgus monkeys.
[0192] To generate a cell line positive for cynomolgus EpCAM, a
clone of the aforementioned coding sequence of the cynomolgus EpCAM
antigen subcloned into pEFDHFR with a verified nucleotide sequence
was transfected into DHFR deficient CHO cells for eukaryotic
expression of the construct as described in Kaufmann R. J. (1990)
Methods Enzymol. 185, 537-566). Gene amplification of the construct
was induced by increasing concentrations of MTX to a final
concentration of up to 500 nM MTX. The transfected cells were then
tested for expression of cynomolgus EpCAM using an FACS assay. For
that purpose, a number of 2.5.times.10.sup.5 cells was incubated
with 50 .mu.l supernatant three different mouse anti human EpCAM
hybridomas (M79-Fogler et al., Cancer Res. 48 (1988); p. 6303-8;
3B10--Passlick et al. Int. J. Cancer 87 (2000), p. 548-552;
2G8--Balzar et al., J. Mol. Med. 77 (1999), p. 699-712). The
binding of the antibodies was detected with a
R-Phycoerythrin-conjugated affinity purified F(ab')2 fragment, goat
anti-mouse IgG, Fc-gamma fragment specific antibody, diluted 1:100
in 50 .mu.l PBS with 2% FCS (obtained from Dianova, Hamburg, FRG)
was used. The samples were measured on a FACSscan (BD biosciences,
Heidelberg, FRG). The anti EpCAM antibody 2G8 was recognized as
cross-species specific and the expression of cynomolgus EpCAM was
confirmed (see FIG. 7). Transfectants (depicted as non-filled
curves) as compared to untransfected cells (depicted as filled
curves) showed binding only with the supernatant of the 2G8
hybridoma which is therefore recognized as antibody species
specific for human and cynomolgus EpCAM.
EXAMPLE 10
Sequence Determination of the Variable Regions of an Anti Human
EpCAM Antibody Cross-Species Specific for Non-Human Primates
[0193] For the sequence determination of the variable regions of
the anti-EpCAM antibody 2G8, the respective hybridoma cell line was
used for isolation of total RNA and cDNA synthesis by random-primed
reverse transcription, which were performed according to standard
protocols (Sambrook, Molecular Cloning; A Laboratory Manual, Cold
Spring Harbour Laboratory Press, Cold Spring Harbour, New York
(1989) (2001)). A PCR (denaturation at 93.degree. C. for 5 min,
annealing at 58.degree. C. for 1 min, elongation at 72.degree. C.
for 1 min for the first cycle; denaturation at 93.degree. C. for 1
min, annealing at 58.degree. C. for 1 min, elongation at 72.degree.
C. for 1 min for 30 cycles; terminal extension at 72.degree. C. for
5 min) was used to amplify the coding sequences of the variable
regions of the antibody. As the sequence of the 5' region of the
variable regions is not known the aforementioned set of 5' primers
was used in combination with a constant 3' primer whereby the 3'
primer was chosen according to the isotype of the antibody.
TABLE-US-00002 Heavy chain variable region: 3' primer: (SEQ ID NO.
107) 5'-TATGCAACTAGTACAACCACAATCCCTGGG-3' Light chain variable
region: 3' primer: (SEQ ID NO. 108)
5'-GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3'
[0194] All PCR products with a length between 350 and 700 base
pairs were isolated, purified and sequenced with the respective 3'
primer according to standard protocols (Molecular Cloning; A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989) (2001)).
[0195] The obtained sequences were examined for functional variable
region coding sequences and for the heavy chain and the light chain
of the antibody a sequence coding for the variable region was
isolated. The nucleotide and amino acid sequences of the variable
regions are described in SEQ ID NOs. 49 through 52 in the sequence
listing included in the description, respectively.
EXAMPLE 11
Cloning of EpCAM and CD3 Cross-Species Specific Bispecific Single
Chain Antibodies
[0196] To generate bispecific single chain antibody molecules
comprising the aforementioned CD3 cross-species specificity and the
aforementioned EpCAM cross-species specificity, the amplified
variable regions of the 2G8 antibody had to be modified by PCR to
obtain the corresponding single chain Fv antibody fragments. Two
single chain Fv antibodies with different arrangements of the light
and heavy chain variable regions were generated. To this end, a
two-step fusion PCR was used to amplify the sequence coding for the
variable regions. A set of appropriate primers was designed to
perform the PCR-based cloning steps, finally resulting in a 2G8
single chain antibody connecting the two variable domains with a 15
amino acid linker ([Gly.sub.4Ser].sub.3) in the order VH-Linker-VL
and VL-Linker-VH. The nucleotide and amino acid sequences are
described in SEQ ID NOs. 53 through 56 of the sequence listing
included in the description, respectively.
[0197] In short the following primer combinations were used:
[0198] For 2G8 VL-VH scFv antibody (hereafter designated as 2G8LH
shown in SEQ ID NOs. 55 and 56): SEQ ID NOs. 57 to 60.
[0199] For 2G8 VH-VL scFv antibody (hereafter designated as 2G8HL
shown in SEQ ID NOs. 53 and 54): SEQ ID NOs. 61 to 64.
[0200] To generate the single chain antibody, two PCRs with the
respective primer combinations were performed. During this PCR,
overlapping complementary sequences were introduced into the
PCR-products (stemming from the respective linker primers that
combined to form the coding sequence of the 15 amino acid linker
during the subsequent fusion PCR). The amplified VH and VL domains
were fused in this fusion PCR in which only the outer primers and
both PCR-products were required. The resulting scFv antibody is
flanked at the 5' end with the restriction enzyme recognition site
for BsrGI and at the 3' end with the restriction enzyme recognition
site for BspEI. The coding sequence of the EpCAM specific single
chain Fv antibodies was then cloned via BsrGI and BspEI into the
pEFDHFR expression vectors described above replacing the 5-10LH
scFv. Single clones of the constructs were isolated and sequenced
with primers complementary to flanking regions in the vector
according to standard protocols (Sambrook, Molecular Cloning; A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989) (2001)). For further experiments a clone of
each construct was selected. The nucleotide and amino acid
sequences are described for 2G8LHxSEQ ID NO.12 in SEQ ID NOs. 65
and 66, for 2G8LHxSEQ ID NO.10 in SEQ ID NOs. 67 and 68, for
2G8LHxSEQ ID NO.16 in SEQ ID NOs. 69 and 70, for 2G8LHxSEQ ID NO.14
in SEQ ID NOs. 71 and 72, for 2G8HLxSEQ ID NO.12 in SEQ ID NOs. 73
and 74, for 2G8HLxSEQ ID NO.10 in SEQ ID NOs. 75 and 76, for
2G8HLxSEQ ID NO.16 in SEQ ID NOs. 77 and 78, and for 2G8HLxSEQ ID
NO.14 in SEQ ID NOs. 79 and 80 of the sequence listing included in
the description.
EXAMPLE 12
Expression of the 2G8LHxSEQ ID NO.12, 2G8LHxSEQ ID NO.10, 2G8LHxSEQ
ID NO.16, 2G8LHxSEQ ID NO.14, 2G8HLxSEQ ID NO.12, 2G8HLxSEQ ID
NO.10, 2G8HLxSEQ ID NO.16 and 2G8HLxSEQ ID NO.14 Bispecific Single
Chain antibodies in CHO cells
[0201] The plasmids with the sequences coding for the bispecific
single chain antibodies were transfected into DHFR deficient CHO
cells for eukaryotic expression of the construct as described in
Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566). Gene
amplification of the construct was induced by increasing
concentrations of MTX to a final concentration of up to 500 nM MTX.
The transfected cells were then expanded and 1 liter of supernatant
produced. The construct was finally purified out of the culture
supernatant as described in Kufer et al. Cancer Immunity Vol. 1, p.
10 (2001).
EXAMPLE 13
FACS assay for binding of 2G8LHxSEQ ID NO.12, 2G8LHxSEQ ID NO.10,
2G8LHxSEQ ID NO.16, 2G8LHxSEQ ID NO.14, 2G8HLxSEQ ID NO.12,
2G8HLxSEQ ID NO.10, 2G8HLxSEQ ID NO.16 and 2G8HLxSEQ ID NO.14 on
Kato III Cells or Cynomolgus EpCAM Transfected CHO Cells and
HPB-ALL Cells
[0202] Binding of the bifunctional constructs from cell culture
supernatants or binding of purified bifunctional constructs to the
human EpCAM antigen on Kato III cells or cynomolgus EpCAM
transfected CHO cells and to the CD3 antigen on HPB-ALL cells was
tested using an FACS assay. For that purpose 2.5.times.10.sup.5
cells were incubated with 50 .mu.l supernatant or with 5 .mu.g/ml
of the purified constructs in 50 .mu.l PBS with 2% FCS. The binding
of the constructs was detected with an anti-His antibody (Penta-His
Antibody, BSA free, obtained from Qiagen GmbH, Hilden, FRG) at 2
.mu.g/ml in 50 .mu.l PBS with 2% FCS. As a second step reagent a
R-Phycoerythrin-conjugated affinity purified F(ab').sub.2 fragment,
goat anti-mouse IgG, Fc-gamma fragment specific antibody, diluted
1:100 in 50 .mu.l PBS with 2% FCS (obtained from Dianova, Hamburg,
FRG) was used. The samples were measured on a FACSscan (BD
biosciences, Heidelberg, FRG). Antigen binding was clearly
detectable for the anti EpCAM specificities as well as for the anti
CD3 specificities (see FIG. 8). As a negative control for binding
to cynomolgus EpCAM, the 5-10LHxSEQ ID NO.10 construct was included
which shows binding on human CD3 (HPB-ALL cells) but no binding to
cynomolgus EpCAM (cynomolgus EpCAM transfected CHO cells). The
5-10LH part only binds to human EpCAM.
EXAMPLE 14
Cytotoxicity Assay for 2G8LHxSEQ ID NO.10 and 2G8HLxSEQ ID NO.12
with Cynomolgus EpCAM Transfected CHO Cells as Target Cells and
Human PBMC as Effector Cells
[0203] Bioactivity of selected bispecific single chain antibodies
was analyzed by FACS-based in vitro cytotoxicity assays using the
cynomolgus EpCAM transfected CHO cells as target cells and human
PBMCs as effector cells.
[0204] Target cells were washed twice with PBS and labeled with
PKH26 dye (Sigma-Aldrich, Germany) according to the manufacturer's
instructions. Labeled target cells were washed twice with RPMI/10%
FCS and mixed with freshly isolated effector cells at an E:T ratio
of 10:1. 2.times.10.sup.4 target and 2.times.10.sup.5 effector
cells in a volume of 50 nl RPMI/10% FCS were added per well in a
96-well round bottom plate. Ten-fold serial dilutions of different
bispecific single chain antibodies were prepared in RPMI/10% FCS to
obtain a starting concentration of 5000 ng/ml in the final reaction
volume. 50 nl of the different solutions were added in triplicates
to the corresponding wells and incubated for 24 to 48 hours at
37.degree. C., 5% CO.sub.2.
[0205] Subsequently, the measurement of cytotoxic activity was
performed. To this end propidium iodide (PI) was added to a final
concentration of 1 ng/ml per well and plates were incubated for 10
minutes at room temperature. The number of PKH and PI positive
target cells was determined by FACS. Cytotoxicity was measured as
the ratio of PI positive (dead cells) over total number of target
cells (PKH-positive) according to the formula: cytotoxicity
(%)=[(PI-positive cells/PKH-positive cells).times.100]. Sigmoidal
dose response killing curves were analyzed by Prism Software
(GraphPad Software Inc., San Diego, USA) and the bispecific single
chain antibody concentration calculated that induced half maximal
killing (EC50 value). The results of this assay are shown below in
FIG. 9. The resulting EC50 values for 2G8LHxSEQ ID NO.10 and
2G8HLxSEQ ID NO.12 were 1103 pg/ml and 3638 pg/ml, respectively.
5-10LHxdi-anti CD3 (deimmunised version of the anti-CD3 antibody as
shown in SEQ ID NO.163 binding to human CD3, but not to cynomolgus
CD3) was included as negative control and showed no activity. This
is due to the fact that 5-10LH only binds to human EpCAM but lacks
cross-species specificity to cynomolgus EpCAM.
EXAMPLE 15
Cytotoxicity Assay for 2G8LHxSEQ ID NO.10 and 2G8HLxSEQ ID NO.12
with Cynomolgus EpCAM Transfected CHO Cells as Target Cells and
Cynomolgus PBMC as Effector Cells
[0206] Bioactivity of selected bispecific single chain antibodies
was analyzed by FACS-based in vitro cytotoxicity assays using the
cynomolgus EpCAM transfected CHO cells as target cells and
cynomolgus PBMCs as effector cells.
[0207] Target cells were washed twice with PBS and labeled with
PKH26 dye (Sigma-Aldrich, Germany) according to the manufacturer's
instructions. Labeled target cells were washed twice with RPMI/10%
FCS and mixed with freshly isolated effector cells at an E:T ratio
of 10:1. 2.times.10.sup.4 target and 2.times.10.sup.5 effector
cells in a volume of 50 nl RPMI/10% FCS were added per well in a
96-well round bottom plate. Ten-fold serial dilutions of different
bispecific single chain antibodies were prepared in RPMI/10% FCS to
obtain a starting concentration of 5000 ng/ml in the final reaction
volume. 50 .mu.l of the different solutions were added in
triplicates to the corresponding wells. Individual cytotoxicity
mixtures were incubated for 24 to 48 hours at 37.degree. C., 5%
CO.sub.2.
[0208] Subsequently the measurement of cytotoxic activity was
performed as described in Example 14. The resulting EC50 values for
2G8LHxSEQ ID NO.10 and 2G8HLxSEQ ID NO.12 were 39810 pg/ml and
60350 pg/ml respectively. 5-10LHxdi-anti CD3 (deimmunised version
of the anti-CD3 antibody as shown in SEQ ID NO.163) was included as
negative control and showed no activity. Di-anti CD3 only binds to
human CD3, but fails to bind to macaque/cynomolgus CD3. 5-10LH only
binds to human EpCAM but lacks cross-species specificity to
cynomolgus EpCAM.
EXAMPLE 16
Generation of a Human-like CD3 Antibody Fragment that Binds to
Human and Cynomolgus CD3
1. Determination of a Correlating Human VH
[0209] The amino acid sequence of the murine VH chain shown in SEQ
ID NO. 2 was aligned to the repertoire of human VH germline
sequences (http://vbase.mrc-cpe.cam.ac.uk) using the Vector NTI DNA
analysis software. On the basis of this analysis, the human VH
segment 3-73 was chosen as a template sequence (see FIG. 11).
Definitions of CDRs and frameworks are according to the Kabat
numbering scheme.
[0210] The corresponding amino acid residues that differ between
the VH chain shown in SEQ ID NO. 2 and the human VH segment 3-73
within the framework regions were mutated on the DNA level towards
the human residues. However, the construct retained potentially
crucial framework residues of the original murine VH sequence
(according to the Kabat numbering scheme): H-30, H-41, H49, H82b,
H-93 (see FIG. 12). In this way, an amino acid sequence was
designed that was identical to the murine VH chain shown in SEQ ID
NO. 2 sequence within its CDRs. The corresponding amino acid
sequence is shown in SEQ ID NO. 110, whereas the corresponding
nucleic acid sequence is shown in SEQ ID NO. 111; see also FIG. 12.
The N-terminal VH sequence was changed to "EVQLLE" to generate a
suitable N-terminal cloning site (see FIG. 12).
2. Gene Synthesis and Cloning of the Human-Like VH Region
[0211] The afore-mentioned human-like VH region was gene
synthesized (Entelechon, Germany) and subcloned via the restriction
sites XhoI and BstEII into a suitable bacterial expression vector.
This vector already contained the sequence coding for a VL chain
(amino acid sequence shown in SEQ ID NO. 148; N-terminus in
comparison to the original VL shown in SEQ ID NO. 4 slightly
changed for cloning reasons) pairing with the human like VH region
followed by a Flag and a His-6 Tag and preceded by a leader
sequence that directs the functional scFv into the periplasma of E.
coli. The functional domain arrangement after cloning was Leader
sequence-VH-(G.sub.4S).sub.3-VL-Flag-His6.
3. Functional Analysis of scFv Constructs Having the Original
Murine VH Shown in SEQ ID NO.2/VL Shown in SEQ ID NO.4 in
Comparison to the Human-Like VH Shown in SEQ ID NO.110/VL Shown in
SEQ ID NO. 148
[0212] Plasmid DNA encoding a) for the original murine VH (SEQ ID
NO. 2) and VL (SEQ ID NO. 4) and b) for the human-like VH (SEQ ID
NO. 110) combined with the VL (SEQ ID NO. 148) was each transformed
into E. coli TG1 according to standard protocols. The nucleotide
and amino acid sequences of the VH-VL scFv comprising the original
murine VH (SEQ ID NO. 2) and VL (SEQ ID NO. 4) are shown in SEQ ID
NOs. 9 and 10, respectively. The nucleotide and amino acid
sequences of the VH-VL scFv comprising the human-like VH (SEQ ID
NO. 110) and the VL (SEQ ID NO. 148) are shown in SEQ ID NOs. 147
and 146, respectively.
[0213] Expression of different clones was performed in E. coli TG-1
in 96-well format. 100 .mu.l LB/0.1% glucose were inoculated with
10 .mu.l of an overnight culture of single clones and grown for 4 h
at 37.degree. C. After addition of IPTG to a final concentration of
1 mM, the culture was grown at 30.degree. C. for another 18-20 h.
Per well, 40 .mu.l of BEL-buffer (400 mM boric acid, 320 mM NaCl, 4
mM EDTA pH 8.0+2.5 mg/ml lysozyme) were added and shaken at room
temperature for 1 h. Cellular debris was eliminated by
centrifugation and supernatants were tested in flow cytometric
experiments.
[0214] The human T cell line HPB-All and T cells in cynomolgus
peripheric blood mononuclear cells (PBMC) were used as human CD3
and cynomolgus CD3 positive cells, respectively. Typically 100,000
cells were incubated with 50 .mu.l of the scFv containing bacterial
supernatants and incubated for 30 min on ice.
[0215] Afterwards the cells were washed three times with PBS and
subsequently resuspended in 50 .mu.l PBS containing anti-His
antibody (Pentahis, Roche) and further incubated on ice for 30 min.
Then the cells were washed three times with PBS and incubated with
a PE labeled anti mouse IgG antibody for 30 more min. on ice (in
this step cynomolgus PBMCs were coincubated with anti-CD2 FITC to
identify the T cells in the PBMC mixture). After washing the cells
for one time the cells were resuspended in a suitable buffer and
positivity of cell bound antibody construct determined in a flow
cytometer (FACScalibur) and analyzed. The control scFv of SEQ ID
NO. 10 shows a clear shift on human CD3 positive cells as well as
on cynomolgus CD3 positive cells indicative of binding to both
human and cynomolgus CD3. The scFv shown in SEQ ID NO. 146
containing the human-like VH also shows clear binding to CD3
positive human (see FIG. 13) and cynomolgus cells (see FIG.
14).
4. Determination of a Correlating Human VL
[0216] The amino acid sequence of the murine VH chain shown in SEQ
ID NO. 2 was aligned to the repertoire of human VL germline
sequences (http://vbase.mrc-cpe.cam.ac.uk) using the Vector NTI DNA
analysis software. On the basis of this analysis, the human Vlambda
segment 7a was chosen as a template sequence (see FIG. 21).
Definitions of CDRs and frameworks are according to the Kabat
numbering scheme. The corresponding amino acid residues that differ
between the murine VL chain shown in SEQ ID NO. 4 and the human
Vlambda segment 7a within the framework regions were mutated on the
DNA level towards the human residues. However, the construct
retained potentially crucial framework residues of the original
murine Vlambda sequence (according to the Kabat numbering scheme):
L 36, L 46, L 49, L 57 (see FIG. 21). In this way, an amino acid
sequence was designed that was identical to the murine VL chain
shown in SEQ ID NO. 4 sequence within its CDRs. The corresponding
amino acid sequence of the generated human-like VL is shown in SEQ
ID NO. 168, whereas the corresponding nucleic acid sequence is
shown in SEQ ID NO. 167. The N-terminal VL sequence was changed to
"EL" to generate a suitable N-terminal cloning site.
5. Gene Synthesis and Cloning of the Human-Like VL Region
[0217] The above-mentioned human-like VL region was gene
synthesized (Entelechon, Germany) and subcloned via the restriction
sites Sad and BsiWI into a suitable bacterial expression vector.
This vector already contained the sequence coding for the
above-mentioned human-like VH chain (amino acid sequence shown in
SEQ ID NO. 110) pairing with the human-like VL region (amino acid
sequence shown in SEQ ID NO. 168) followed by a Flag and a His-6
Tag and preceded by a leader sequence that directs the functional
scFv into the periplasma of E. coli. The functional domain
arrangement after cloning was Leader sequence-VH-(G.sub.4S).sub.3
linker-VL-Flag tag-His6 tag.
6. Functional Analysis of scFv Constructs Having the Human-Like VH
Shown in SEQ ID NO. 110 Combined with the Human-Like VL Shown in
SEQ ID NO. 168
[0218] Plasmid DNA encoding a) for the original murine VH (SEQ ID
NO. 2) and VL (SEQ ID NO. 4) and b) for the human-like VH (SEQ ID
NO. 110) combined with the human-like VL (SEQ ID NO. 168) was each
transformed into E. coli TG1 according to standard protocols. The
nucleotide and amino acid sequences of the VH-VL scFv comprising
the original murine VH (SEQ ID NO. 2) and VL (SEQ ID NO. 4) are
shown in SEQ ID NOs. 9 and 10, respectively. The nucleotide and
amino acid sequences of the VH-VL scFv comprising the human-like VH
(SEQ ID NO. 110) and the human-like VL (SEQ ID NO. 168) are shown
in SEQ ID NOs. 169 and 170, respectively. The nucleotide and amino
acid sequences of the VL-VH scFv comprising the human-like VL (SEQ
ID NO. 168) and the human-like VH (SEQ ID NO. 110) are shown in SEQ
ID NOs. 193 and 194, respectively. Due to different cloning
strategies, the amino acid sequence of the VL-VH scFv of SEQ ID NO.
194 shows three amino acid exchanges in comparison to the one of
the VH-VL scFv of SEQ ID NO. 170, however, without affecting the
binding capacity and specificity of said scFv. Expression of
different clones was performed in E. coli TG-1 in 96-well format.
100 .mu.l LB/0.1% glucose were inoculated with 10 .mu.l of an
overnight culture of single clones and grown for 4 h at 37.degree.
C. After addition of IPTG to a final concentration of 1 mM, the
culture was grown at 30.degree. C. for another 18-20 h. Per well,
40 p. 1 of BEL-buffer (400 mM boric acid, 320 mM NaCl, 4 mM EDTA pH
8.0+2.5 mg/ml lysozyme) were added and shaken at room temperature
for 1 h. Cellular debris was eliminated by centrifugation and
supernatants were tested in flow cytometric experiments.
[0219] The human T cell line HPB-ALL and human and cynomolgus T
cells in peripheric blood mononuclear cells (PBMCs) were used as
human CD3 and cynomolgus CD3 positive cells, respectively.
[0220] Typically 100,000 cells were incubated with 50 .mu.l of the
scFv containing bacterial supernatants and incubated for 30 min on
ice.
a) HPB-ALL cells were washed three times with PBS and subsequently
resuspended in 50 .mu.l PBS containing anti-His antibody (Pentahis,
Roche) and further incubated on ice for 30 mM. Then the cells were
washed three times with PBS and incubated with a PE labeled anti
mouse IgG antibody for 30 more min. on ice. After washing the cells
for one time the cells were resuspended in a suitable buffer and
positivity of cell bound antibody construct determined in a flow
cytometer (FACScalibur) and analyzed. b) Human and cynomolgus PBMCs
(containing T cells) were washed three times with PBS and
subsequently resuspended in 50 .mu.l PBS containing biotinylated
anti-His antibody (biotinylated Pentahis, Roche) and further
incubated on ice for 30 min. Then the cells were washed three times
with PBS and incubated with PE labeled Streptavidin for 30 more
min. on ice. In this step, PBMCs were coincubated with anti-CD2
FITC to identify the T cells in the PBMC mixture.
[0221] After washing the cells from a) or b) for one time the cells
were resuspended in a suitable buffer and positivity of cell bound
antibody construct determined in a flow cytometer (FACScalibur) and
analyzed.
[0222] The control scFv of SEQ ID NO. 10 (murine VH of SEQ ID
NO.4--murine VL of SEQ ID NO. 2) shows a clear shift on human CD3
positive cells as depicted in FIG. 22. The shift on human and
cynomolgus T cells is less pronounced, most probably due to the
less sensitive detection system (FIG. 23).
[0223] The human-like scFv of SEQ ID NO. 170 (human-like VH of SEQ
ID NO.110--human-like VL of SEQ ID NO. 168) shows a positive shift
on HPB-ALL cells (FIG. 22) and clear shifts on human as well as
cynomolgus T cells (FIG. 23, upper panel). When preincubated with
10 .mu.g/ml of the murine IgG antibody mAb I described in Example 1
having the same specificity as the scFvs (i.e. for CD3 epsilon),
the shifts of cells stained with the above-mentioned murine scFv or
the human-like scFv decrease significantly, underlining the similar
binding region of the scFvs and the original murine antibody; see
FIG. 23 lower panel.
EXAMPLE 17
Determination of an Epitope for Cross-Species Specific Anti-CD3
Antibodies Binding Both Human and Cynomolgus CD3 Epsilon
[0224] In order to determine the epitope of human and cynomolgus
CD3 epsilon bound by cross-species specific anti-CD3 antibodies,
epitope mapping was carried out with antibody I (Ig comprising the
VH chain shown in SEQ ID NO. 2 and the VL chain shown in SEQ ID NO.
4) and antibody II (Ig comprising the VH chain shown in SEQ ID NO.
6 and the VL chain shown in SEQ ID NO. 8), both binding to human
and cynomolgus CD3 epsilon; see also FIG. 1. For the
peptide-spotting ("pepspot") analysis, overlapping 13 mer peptides
derived from the amino acid sequences of human and cynomolgus CD3
epsilon (see FIG. 15) were covalently linked to a Whatman 50
cellulose-B-alanine-membrane via the C-terminus while the
acetylated N-terminus remained free. In the peptides, the amino
acid cystein--wherever occurring in the corresponding CD3 epsilon
sequence--was exchanged by the amino acid serin. The individual 13
mer peptides generated (by JPT Peptide Technologies GmbH) are shown
in FIGS. 16 and 17. For cynomolgus CD3 epsilon, 43 spots have been
tested, whereas for the human CD3 epsilon 47 spots have been
tested. The length of the overlapping sequence of two adjacent
peptides was set to be 11 amino acids. The pepspot experiments were
performed as follows. According to the manufacturer's protocol, the
membrane was rinsed with methanol for 1 min, washed with
1.times.TBS and blocked with 1.times.TBS/1% (w/v) blocking reagent
(BM Chemiluminescence Blotting Substrate (POD) of Roche Diagnostics
GmbH) for 3 h. All incubation and washing steps were performed on
an orbital shaker at room temperature, except for the overnight
incubation of the primary antibody. Directly after discarding the
blocking solution, the membranes were incubated overnight with 5 or
3 .mu.g/ml of cross-species specific anti-CD3 antibodies as set
forth above in 1.times.TBS/0.5% (w/v) blocking reagent at 4.degree.
C. on an orbital shaker. After washing 4 times with
1.times.TBS/0.05% Tween for 15 min, detection of bound anti-CD3
antibody was accomplished by incubation for 2 h with a commercially
available horseradish-peroxidase-conjugated anti-IgG (F(ab).sub.2
specific) antibody or an alkaline phosphatase-labeled anti-IgG
antibody (diluted according to the manufacturer's recommendation in
1.times.TBS/0.5% blocking reagent, respectively). Subsequently, the
membranes were washed 6 times with 1.times.TBS/0.05% Tween for 15
min. Horseradish-peroxidase was visualized by enhanced
chemiluminescence (luminescence substrate solution A and starting
solution B mixed 100:1; BM Chemiluminescence Blotting Substrate
(POD) of Roche Diagnostics GmbH) and a BioMax Film (Kodak).
Alkaline phosphatase was visualized using 5-bromo-4-chloro-indolyl
phosphate/nitro blue tetrazolium liquid substrate system (Sigma).
To exclude unspecific binding of horseradish-peroxidase-conjugated
secondary antibody, the membrane was incubated with secondary
antibody only. All other steps were performed as in the experiment
above.
[0225] The control pepspot assay (see FIG. 18(A)) showed signals on
spots 33 and 42 of cynomolgus CD3 epsilon and on spots 37, 39 and
46 of human CD3 epsilon. These signals are regarded as unspecific
and will not be mentioned further.
1. Anti-CD3 Antibody I (Ig Comprising the VH Chain Shown in SEQ ID
NO. 2 and the VL Chain Shown in SEQ ID NO. 4)
(i) Binding on Cynomolgus CD3 Epsilon
[0226] Strong binding signals of cross-species specific anti-CD3
antibody I (Ig comprising the VH chain shown in SEQ ID NO. 2 and
the VL chain shown in SEQ ID NO. 4) to peptides derived from
cynomolgus CD3 epsilon were detected on spot 1 as well as on the
stretch of peptide-spots 24-29 (FIG. 18(B)). The latter corresponds
to amino acid residues 47-69 of cynomolgus CD3 epsilon (see FIG.
15). All 13 mer peptides covering this region contain one minimal
amino acid motif 56-59 (EFSE). Spot 1 corresponds to amino acid
residues 1-13 (QDGNEEMGSITQT) of cynomolgus CD3 epsilon.
(ii) Binding on Human CD3 Epsilon
[0227] Cross-species specific anti-CD3 antibody I bound to
peptide-spots 15, 28, 32, 33 and 40 derived from human CD3 epsilon
(see FIG. 18(B)). The stretch of peptide-spots 28 to 33 corresponds
to the amino acid residues 47-69 of human CD3 epsilon and comprises
the minimal amino acid motif 57-59 (FSE). Spots 15 and 40
correspond to amino acid residues 30-42 (QYPGSEILWQHND) and 71-83
(RGSKPEDANFYLY), respectively.
2. Anti-CD3 Antibody II (Ig Comprising the VH Chain Shown in SEQ ID
NO. 6 and the VL Chain Shown in SEQ ID NO. 8)
(i) Binding on Cynomolgus CD3 Epsilon
[0228] The pepspot analysis with cross-species specific anti-CD3
antibody II (Ig comprising the VH chain shown in SEQ ID NO. 6 and
the VL chain shown in SEQ ID NO. 8) showed strong signals to
cynomolgus CD3 epsilon on the stretch of peptide-spots 27-29 as
well as on spot 33 (see FIG. 19). The stretch spanning spots 27 and
29 corresponds to the amino acid residues 53-69 of cynomolgus CD3
epsilon (see FIG. 15), wherein the 13 mer peptides have the minimal
stretch of amino acids 57-61 (FSEME) in common Spot 33 correlates
with amino acid residues 65-77 (YYVSYPRGSNPED).
(ii) Binding on Human CD3 Epsilon
[0229] Cross-reactive anti-CD3 antibody II bound the peptide-spots
15, 19, 32 and 33, 37, 39 and 40 of human CD3 epsilon (see FIG.
19). Spot 19 corresponds to amino acid residues 38-46d
(WQHNDKNIGGDED) of human CD3 epsilon (see FIG. 15). The small
stretch of spots 32 to 33 corresponds to amino acid residues 55-69
containing the minimal peptide FSELE (amino acids 57-61). The spots
37 and 39 match amino acid residues 65-77 (YYVSYPRGSKPED) and 69-81
(YPRGSKPEDANFY) of human CD3 epsilon, respectively. The
correlations of spots 15 and 40 are already mentioned above.
[0230] In summary, both cross-species specific anti-CD3 antibodies
recognize discontinous epitopes on human and cynomolgus CD3
epsilon. Regarding cynomolgus CD3 epsilon both cross-species
specific anti-CD3 antibodies recognized a clear overlapping stretch
of peptide-spots 27-29 (see FIG. 16). All 13 mer peptides covering
this region contain one minimal peptide FSEME (amino acid residues
57-61 of cynomolgus CD3 epsilon). The peptide-intersection on human
CD3 epsilon bound by both antibodies can be determined for spots 32
and 33 (see FIG. 17). This section contains the minimal peptide
FSELE corresponding to residues 57-61 of human CD3 epsilon.
[0231] Based on these results it is concluded that cross-species
specific CD3 antibody fragments contact CD3 epsilon in the area of
amino acid residues 57-61 of both cynomolgus and human CD3 epsilon
comprising, the amino acid stretches FSEME and FSELE of cynomolgus
and human CD3 epsilon, respectively, with the motif FSE forming the
epitope core. This result--although plausible because of the
accessibility of the E-F-loop (amino acids 56-62; see FIG. 15) of
human CD3 epsilon (Kjer-Nielsen et al., PNAS 101 (2004), p.
7675-80) comprising the amino acids FSELE or FSEME--is nevertheless
surprising since there is no overlap of this newly defined epitope
with the known epitope on the CD3 epsilon-chain of anti-CD3
antibodies OKT3 and UCHT1 (see FIG. 17; Kjer-Nielsen et al.,
loc.cit; Arnett et al., PNAS 101 (2004), p. 16268-73) which have so
far been regarded as representative of all anti-CD3 antibodies
thought to form a single family with the same or a very similar
epitope.
EXAMPLE 18
Determination of the Epitope for the Human-Like Cross-Species
Specific Anti-CD3 Antibody Binding Both to Human and Cynomolgus CD3
Epsilon
[0232] The epitope mapping of the human-like cross-species specific
anti-CD3 antibody fragment described in Example 16 (SEQ ID NO. 170)
was carried out by peptide-spotting ("pepspot") analysis as
described in Example 17. For this purpose, said single chain Fv
fragment shown in SEQ ID NO. 170 was converted into a full IgG
antibody with a murine gammal heavy chain comprising the VH region
as shown in SEQ ID NO. 110 and a kappa light chain comprising the
VL region as shown in SEQ ID NO. 168. The procedure of the pepspot
experiment was identical to the protocol used in Example 17.
[0233] The pepspot membrane was incubated with 4 ng/ml of the
mentioned IgG1 antibody, and an alkaline phosphatase-labeled
goat-anti-mouse IgG antibody detecting bound CD3 antibody. A second
membrane was incubated with alkaline phosphatase-labeled
goat-anti-mouse IgG antibody alone to reveal unspecific binding of
the detection antibody.
[0234] The following signals detected in the control pepspot assay
(see FIG. 24(A)) have been regarded as unspecific and will not be
mentioned further: the stained spot-stretches 10-13, 15-19, 30-32,
35-41 of cynomolgus CD3 epsilon and 2-6, 14-19, 26, 34-39 and 46 of
human CD3 epsilon.
(i) Binding on Cynomolgus CD3 Epsilon
[0235] The cross-species specific anti-CD3 antibody (murine IgG1
comprising the VH chain shown in SEQ ID NO. 110 and the VL chain
shown in SEQ ID NO. 168) bound to the peptide-spots 1 and 33 as
well as to the amino acid stretch of peptide-spots 24-29 (FIG.
24(B)) derived from cynomolgus CD3 epsilon. The stretch spanning
spots 24 and 29 corresponds to the amino acid residues 47-69 of
cynomolgus CD3 epsilon (see FIGS. 15 and 16), wherein the 13 mer
peptides have the minimal stretch of amino acids 56-59 (EFSE) in
common. Spot 1 and spot 33 correspond to amino acid residues 1-13
("QDGNEEMGSITQT"; SEQ ID NO. 199) and 65-77 ("YYVSYPRGSNPED"; SEQ
ID NO. 200) of cynomolgus CD3 epsilon, respectively.
(ii) Binding on Human CD3 Epsilon
[0236] Binding signals of the mentioned cross-species specific
anti-CD3 IgG1 antibody to peptides derived from human CD3 epsilon
(see FIG. 24(B)) were found on spots 28 and 33, which correspond to
the amino acid residues 47-59 and 57-69 of human CD3 epsilon (see
FIG. 17), respectively. The two stained spots comprise the minimal
amino acid motif 57-59 (FSE).
[0237] The human-like cross-species specific anti-CD3 antibody
recognizes the same discontinuous epitopes on human and cynomolgus
CD3 epsilon as antibody I and II described in Examples 1 and 17.
Binding signals of said human-like antibody on the peptide membrane
reveal the peptide-intersections corresponding to the amino acid
sequence "FSEME" (amino acid residues 57-61) of cynomolgus CD3
epsilon and those corresponding to amino acid sequence "FSELE"
(amino acid residues 57-61) of human CD3 epsilon as core region.
This is in line with the epitope determined for the cross-species
specific anti-CD3 antibodies I and II on both cynomolgus and human
CD3 epsilon (see Example 17).
EXAMPLE 19
Verification of the Identified Epitope on Human CD3 Epsilon for the
Human-Like Cross-Species Specific Anti-CD3 Antibody
[0238] To verify the epitope of the human-like cross-species
specific anti-CD3 antibody fragment described in Example 16 on
human CD3 epsilon, the identified binding region as determined in
Experiment 18 was further analyzed by a dot-blotting assay using a
13 mer peptide covering the defined binding area of amino acid
residues "FSELE" on human CD3 epsilon. This peptide comprises the
amino acid sequence "EFSELEQSGYYVC" (SEQ ID NO. 195) of human CD3
epsilon. The peptide exists in two forms and is either biotinylated
N- or C-terminally. In case of the N-terminal labelling, a short
linker connects the peptide with the biotin. As described in
Example 18, the antibody fragment was converted to a murine IgG
format with a murine gammal heavy chain comprising the VH region as
shown in SEQ ID NO. 110 and a kappa light chain comprising the VL
region as shown in SEQ ID NO. 168. The dot blotting was performed
as follows. The Minifold I Spot Blot System from Schleicher &
Schuell was used for immobilizing the peptides on a nitrocellulose
membrane (Protran BA 85, 0.45 .mu.m). 75 .mu.g of each peptide in
100 p. 1 TBS were filtered through the membrane using vacuum. After
the filtration step the membrane was blocked with 1.times.TBS/1%
(w/v) blocking reagent (BM Chemiluminescence Blotting Substrate
(POD) of Roche Diagnostics GmbH) for 2 h. All incubation and
washing steps were performed on an orbital shaker at room
temperature, except for the overnight incubation of the primary
antibody. Directly after discarding the blocking solution, the
membrane was incubated overnight with 3 .mu.g/ml of the
above-mentioned anti-CD3 antibody in 1.times.TBS/0.5% (w/v)
blocking reagent at 4.degree. C. on an orbital shaker. As a
control, the anti-CD3 murine IgG1 antibody UCHT1 (BD Biosciences)
binding to human CD3 epsilon was applied to a second membrane
blotted with the same amounts of the two peptides. After washing
three times with 1.times.TBS/0.05% Tween for 10 min, detection of
bound anti-CD3 antibody was accomplished by incubation for 2 h with
a commercially available alkaline phosphatase-conjugated anti-IgG
antibody (diluted according to the manufacturer's recommendation in
1.times.TBS/0.5% blocking reagent). Subsequently, the membranes
were washed three times with 1.times.TBS/0.05% Tween for 10 min.
Alkaline phosphatase was visualized using 5-bromo-4-chloro-indolyl
phosphate/nitro blue tetrazolium liquid substrate system
(Sigma).
[0239] The mentioned CD3 specific antibody comprising the VH region
shown in SEQ ID NO. 110 and the VL region shown in SEQ ID NO. 168
bound to both forms of the peptide "EFSELEQSGYYVC" (SEQ ID NO. 195)
blotted to the membrane (see FIGS. 25 (A)(1) and (2)), whereas no
binding could be obtained for the anti-CD3 murine IgG antibody
UCHT1 (see FIGS. 25 (B) (1) and (2)). The epitope recognized by
anti-CD3 antibody UCHT1 is described e.g. in Kjer-Nielsen et al.,
loc.cit; Arnett et al., PNAS (2204), p. 16268-73. These results
support the identification of the newly defined epitope of the
herein-described anti-CD3 antibody (with the VH region shown in SEQ
ID NO. 110 and the VL region shown in SEQ ID NO. 168). Said epitope
corresponds to the amino acid residues "EFSELEQSGYYVC" (SEQ ID NO.
195) on the human CD3 epsilon chain and comprises the amino acid
stretch "FSELE".
EXAMPLE 20
Generation of CHO Cells Transfected with Cynomolgus EGFR
[0240] A shock frozen piece of EGFR positive cynomolgus colon was
used to obtain the total RNA that was isolated according to the
instructions of the kit manual (Qiagen, RNeasy Mini Kit). The
obtained RNA was used for cDNA synthesis by random-primed reverse
transcription. For cloning of the full length sequence of the EGFR
antigen the following oligonucleotides were used:
TABLE-US-00003 5' EGFR AG XbaI (SEQ ID NO. 197)
5'-GGTCTAGAGCATGCGACCCTCCGGGACGGCCGGG-3' 3' EGFR AG SalI (SEQ ID
NO. 199) 5'-TTTTAAGTCGACTCATGCTCCAATAAATTCACTGCT-3'.
[0241] A PCR (denaturation at 93.degree. C. for 5 min, annealing at
58.degree. C. for 1 min, elongation at 72.degree. C. for 2 min for
the first cycle; denaturation at 93.degree. C. for 1 min, annealing
at 58.degree. C. for 1 min, elongation at 72.degree. C. for 2 min
for 30 cycles; terminal extension at 72.degree. C. for 5 min) was
used to amplify the coding sequence. The PCR product was
subsequently digested with XbaI and SalI, ligated into the
appropriately digested expression vector pEF-DHFR, and transformed
into E. coli. The afore-mentioned procedures were carried out
according to standard protocols (Sambrook, Molecular Cloning; A
Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory
Press, Cold Spring Harbour, New York (2001)). A clone with
sequence-verified nucleotide sequence was transfected into DHFR
deficient CHO cells for eukaryotic expression of the construct.
Eukaryotic protein expression in DHFR deficient CHO cells was
performed as described in Kaufmann R.J. (1990) Methods Enzymol.
185, 537-566. Gene amplification of the construct was induced by
increasing concentrations of MTX to a final concentration of up to
20 nM MTX.
EXAMPLE 21
Generation of EGFR and CD3 Cross-Species Specific Bispecific Single
Chain Antibodies
[0242] Generally, bispecific single chain antibody molecules, each
comprising a domain with a binding specificity for the human and
the cynomolgus CD3 antigen as well as a domain with a binding
specificity for the human and the cynomolgus EGFR antigen, were
designed as set out in the following Table 1:
TABLE-US-00004 TABLE 1 Formats of anti-CD3 and anti-EGFR
cross-species specific bispecific single chain antibody molecules
SEQ ID Formats of protein constructs (nucl/prot) (N .fwdarw. C)
171/172 EGFR HL .times. SEQ ID NO. 170 173/174 EGFR LH .times. SEQ
ID NO. 170 175/176 EGFR HL .times. SEQ ID NO. 194 177/178 EGFR LH
.times. SEQ ID NO. 194 179/180 SEQ ID NO. 170 .times. EGFR HL
181/182 SEQ ID NO. 194 .times. EGFR HL 183/184 SEQ ID NO. 170
.times. EGFR LH 185/186 SEQ ID NO. 194 .times. EGFR LH
[0243] The afore-mentioned constructs containing the variable
light-chain (L) and variable heavy-chain (H) domains reactive with
the human and cynomolgus EGFR derived from murine hybridomas were
obtained by gene synthesis and subsequent cloning into an
expression vector comprising the CD3 specific VH and VL
combinations reactive with the human and cynomolgus CD3. Herein,
SEQ ID NO. 170 corresponds to amino acid sequence of the anti-CD3
VH-VL scFv comprising the human-like VH (SEQ ID NO. 110) and the
human-like VL (SEQ ID NO. 168). SEQ ID NO. 194 corresponds to the
amino acid sequence of the anti-CD3 VL-VH scFv comprising the
human-like VL (SEQ ID NO. 168) and the human-like VH (SEQ ID NO.
110). The constructs were then transfected into DHFR-deficient
CHO-cells by electroporation.
EXAMPLE 22
Expression and Purification of the EGFR and CD3 Cross-Species
Specific Bispecific Single Chain Antibodies
[0244] The bispecific single chain antibodies were expressed in
chinese hamster ovary cells (CHO). Eukaryotic protein expression in
DHFR deficient CHO cells was performed as described in Kaufmann R.
J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the
constructs were induced by increasing concentrations of MTX to a
final concentration of up to 20 nM MTX. After two passages of
stationary culture the cells were grown in roller bottles with CHO
modified MEM medium for 7 days before harvest. The cells were
removed by centrifugation and the supernatant containing the
expressed protein was stored at -20.degree. C.
[0245] Akta.RTM. FPLC System (Pharmacia) and Unicorn.RTM. Software
were used for chromatography. All chemicals were of research grade
and purchased from Sigma (Deisenhofen) or Merck (Darmstadt)
Immobilized metal affinity chromatography ("IMAC") was performed
using a Fractogel.RTM. column (Merck) which was loaded with
ZnCl.sub.2 according to the protocol provided by the manufacturer.
The column was equilibrated with buffer A2 (20 mM sodium phosphate
buffer pH 7.5, 0.4 M NaCl) and the cell culture supernatant (500
ml) was applied to the column (10 ml) at a flow rate of 3 ml/min.
The column was washed with buffer A2 to remove unbound sample.
Bound protein was eluted using a two step gradient of buffer B2 (20
mM sodium phosphate buffer pH 7.5, 0.4 M NaCl, 0.5 M Imidazol)
according to the following:
[0246] Step 1: 20% buffer B2 in 6 column volumes;
[0247] Step 2: 100% buffer B2 in 6 column volumes.
[0248] Eluted protein fractions from step 2 were pooled for further
purification.
[0249] Gel filtration chromatography was performed on a Sephadex
S200 HiPrep column
[0250] (Pharmacia) equilibrated with PBS (Gibco). Eluted protein
samples (flow rate 1 ml/min) were subjected to standard SDS-PAGE
and Western Blot for detection. Prior to purification, the column
was calibrated for molecular weight determination (molecular weight
marker kit, Sigma MW GF-200). Protein concentrations were
determined using protein assay dye (MicroBCA, Pierce) and IgG
(Biorad) as standard protein.
[0251] The bispecific single chain antibodies were isolated in a
two step purification process of IMAC and gel filtration. The main
product had a molecular weight of about 52 kDa under native
conditions as determined by gel filtration in PBS. This molecular
weight corresponds to the bispecific single chain antibody. All
constructs were purified according to this method.
[0252] Purified bispecific single chain antibody protein was
analyzed in SDS PAGE under reducing conditions performed with
pre-cast 4-12% Bis Tris gels (Invitrogen). Sample preparation and
application were performed according to the protocol provided by
the manufacturer. The molecular weight was determined with
MultiMark protein standard (Invitrogen). The gel was stained with
colloidal Coomassie (Invitrogen protocol). The purity of the
isolated protein was >95% as determined by SDS-PAGE.
[0253] Western Blot was performed using an Optitran.RTM. BA-S83
membrane and the Invitrogen Blot Module according to the protocol
provided by the manufacturer. The antibodies used were directed
against the H is Tag (Penta H is, Qiagen) and Goat-anti-mouse Ig
labeled with alkaline phosphatase (AP) (Sigma), and BCIP/NBT
(Sigma) as substrate. The bispecific single chain antibody could be
specifically detected by Western Blot. A single band was detected
at 52 kD corresponding to the purified bispecific molecule.
EXAMPLE 23
Flow Cytometric Binding Analysis of the EGFR and CD3 Cross-Species
Specific Bispecific Antibodies
[0254] In order to test the functionality of the cross-species
specific bispecific antibody constructs with regard to binding
capability to human and cynomolgus EGFR and CD3, respectively, a
FACS analysis was performed. For this purpose the EGFR positive
epidermoid carcinoma A431 cells (ATCC, CRL-1555) and CD3 positive
human T cell leukemia cell line HPB-ALL (DSMZ, Braunschweig,
ACC483) were used to check the binding to human antigens. The
binding reactivity to cynomolgus antigens was tested by using the
generated cynomolgus EGFR transfectants described in Example 20 and
cynomolgus PBMCs which were obtained by Ficoll density gradient
centrifugation. 200,000 cells of the respective cell population
were incubated for 30 min on ice with 50 nl of the purified protein
of the cross-species specific bispecific antibody constructs (1
ng/ml). The cells were washed twice in PBS and binding of the
construct was detected with an unlabeled murine Penta H is antibody
(diluted 1:20 in 50 nl PBS with 2% FCS; Qiagen; Order No. 34660).
After washing, bound anti H is antibodies were detected with an Fc
gamma-specific antibody (Dianova) conjugated to phycoerythrin,
diluted 1:100 in 50 nl PBS with 2% FCS. Fresh culture medium was
used as a negative control.
[0255] Cells were analyzed by flow cytometry on a FACS-Calibur
apparatus (Becton Dickinson, Heidelberg). FACS staining and
measuring of the fluorescence intensity were performed as described
in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies,
Shevach and Strober, Wiley-Interscience, 2002).
[0256] The binding ability of several domain arrangements were
clearly detectable as shown in FIGS. 29 to 36. In FACS analysis,
all constructs with different arrangement of VH and VL domains
specific for EGFR and CD3 showed binding to CD3 and EGFR compared
to the negative control using culture medium and 1. and 2.
detection antibody. In summary, the cross-species specificity of
the bispecific antibody to human and cynomolgus CD3 and EGFR
antigens could clearly be demonstrated.
EXAMPLE 24
Bioactivity of EGFR and CD3 Cross-Species Specific Bispecific
Single Chain Antibodies
[0257] Bioactivity of the generated bispecific single chain
antibodies was analyzed by chromium 51 release in vitro
cytotoxicity assays using the EGFR positive cell lines described in
Example 23; see also FIGS. 39 and 40. As effector cells stimulated
human CD8 positive T cells or stimulated cynomolgus PBMCs were
used, respectively.
[0258] The generation of the stimulated CD8+T cells was performed
as follows:
[0259] A Petri dish (85 mm diameter, Nunc) was pre-coated with a
commercially available anti-CD3 specific antibody in a final
concentration of 1 ng/ml for 1 hour at 37.degree. C. Unbound
protein was removed by one washing step with PBS. The fresh PBMC's
were isolated from peripheral blood (30-50 ml human blood or 10 ml
cynomolgus blood) by Ficoll gradient centrifugation according to
standard protocols. 3-5.times.10.sup.7 PBMCs were added to the
precoated petri dish in 50 ml of RPMI 1640/10% FCS/IL-2 20 U/ml
(Proleukin, Chiron) and stimulated for 2 days. At the third day the
cells were collected, washed once with RPMI 1640. IL-2 was added to
a final concentration of 20 U/ml and cultivated again for one day.
The CD8+CTLs were isolated by depleting CD4+T cells an CD56+NK
cells.
[0260] Target cells were washed twice with PBS and labeled with
11.1 MBq .sup.51Cr in a final volume of 100 nl RPMI with 50% FCS
for 45 minutes at 37.degree. C. Subsequently the labeled target
cells were washed 3 times with 5 ml RPMI and then used in the
cytotoxicity assay. The assay was performed in a 96 well plate in a
total volume of 250 nl supplemented RPMI (as above) with an E:T
ratio of 10:1 corresponding to 1000 target cells and 10000 effector
cells per well. 1 ng/ml of the cross-species specific bispecific
single chain antibody molecules and 20 threefold dilutions thereof
were applied. The assay time was 18 hours and cytotoxicity was
measured as relative values of released chromium in the supernatant
related to the difference of maximum lysis (addition of Triton-X)
and spontaneous lysis (without effector cells). All measurements
were done in quadruplicates. Measurement of chromium activity in
the supernatants was performed with a Wizard 3 gammacounter (Perkin
Elmer Life Sciences GmbH, Koln, Germany). Analysis of the
experimental data was performed with Prism 4 for Windows (version
4.02, GraphPad Software Inc., San Diego, Calif., USA). Sigmoidal
dose response curves typically had R.sup.2 values >0.90 as
determined by the software. EC.sub.50 values calculated by the
analysis program were used for comparison of bioactivity.
[0261] As shown in FIGS. 39 and 40, all of the generated
cross-species specific bispecific single chain antibody constructs
revealed cytotoxic activity against human EGFR positive target
cells elicited by human CD8+ cells and cynomolgus EGFR positive
target cells elicited by cynomolgus CD8+ cells. In FIG. 39, a
bispecific single chain antibody with a variable domain reactive
with EGFR and a de-immunized human CD3-specific variable domain
(EGFR LH.times.di-anti CD3) has been used as a negative control. In
FIG. 40, the same construct (EGFR LH.times.di-anti CD3) has been
used as a positive control. As a negative control, an irrelevant
bispecific single chain antibody has been used.
EXAMPLE 25
Generation and Characterization of Carboanhydrase IX (CAIX) and CD3
Cross-Species Specific Bispecific Single Chain Antibodies
TABLE-US-00005 [0262] TABLE 2 Formats of CAIX and CD3 cross-species
specific bispecific single chain antibodies SEQ ID Formats of
protein constructs (nucl/prot) (N .fwdarw. C) 189/190 CAIX HL
.times. SEQ ID NO. 170 191/192 CAIX LH .times. SEQ ID NO. 170
187/188 CAIX HL .times. SEQ ID NO. 194
[0263] In analogy to the afore-mentioned Examples, Carboanhydrase
IX (CAIX/MN) and CD3 cross-species specific bispecific single chain
antibodies containing the variable light-chain (L) and variable
heavy-chain (H) domains reactive with the human and cynomolgus CAIX
antigen were created and subsequently cloned into an expression
vector comprising the CD3 specific VH and VL combinations reactive
with the human and cynomolgus CD3. The experiments were carried out
in essence as described in Examples 20 to 24, with the following
exceptions:
[0264] The FACS binding experiments were performed with the CAIX
positive human lung carcinoma cell line A549 (ATCC, CCL-185) to
assess the binding capability to the human CAIX antigen. The
cross-species specificity to cynomolgus tissue was tested by
deploying the cynomolgus skin cell line CYNOM-K1 (National
Institute for Cancer Research (IST) of Genova, Italy, ECACC
90071809) or the rhesus monkey epithelial cell line 4 MBr-5 (ATCC,
CCL-208). The same changes in cell lines apply to the cytotoxicity
assays performed with the CAIX and CD3 cross-species specific
bispecific single chain antibodies.
[0265] As depicted in FIGS. 26 to 28, the generated CAIX and CD3
cross-species specific bispecific single chain antibodies
demonstrated binding to both the human and cynomolgus antigens and
proved to be fully cross-species specific. The cytolytic
bioactivity of the analysed constructs is shown in FIGS. 37 and 38.
In the left panel of FIG. 37, a bispecific single chain antibody
with a variable domain reactive with CAIX and a de-immunized human
CD3-specific variable domain has been used as a positive control.
In the right panel, the same construct has been used as a negative
control.
TABLE-US-00006 APPENDIX SEQ ID NO. DESIGNATION SOURCE TYPE SEQUENCE
1 VH Murine NA
gaggtgaagcttctcgagtctggaggaggattggtgcagcctaaagggtcattgaaactctcatgtgcagcct-
ctggattca
ccttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgcat-
aagaagtaaat
ataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagagatgattcaca-
aagcattctctatct
acaaatgaacaacttgaaaactgaggacacagccatgtactactgtgtgagacatgggaacttcggtaat-
agctacgtttcct ggtttgcttactggggccaagggactctggtcactgtctctgca 2 VH
Murine AA EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI
RSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHG
NFGNSYVSWFAYWGQGTLVTVSA 3 VL Murine NA
caggctgttgtgactcaggaatctgcactcaccacatcacctggtgaaacagtcacactcacttgtcgctcaa-
gtactgggg
ctgttacaactagtaactatgccaactgggtccaagaaaaaccagatcatttattcactggtctaatagg-
tggtaccaacaag
cgagctccaggtgtgcctgccagattctcaggctccctgattggagacaaggctgccctcaccatcacag-
gggcacagac
tgaggatgaggcaatatatttctgtgctctatggtacagcaacctctgggtgttcggtggaggaaccaaa-
ctgactgtccta 4 VL Murine AA
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGG
TNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGT KLTVL 5 VH
Murine NA
caggtccagctgcagcagtctggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaaggctt-
ctggcta
cacctttactagatctacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatac-
attaatcctagc
agtgcttatactaattacaatcagaaattcaaggacaaggccacattgactgcagacaaatcctccagta-
cagcctacatgc
aactgagtagcctgacatctgaggactctgcagtctattactgtgcaagtccgcaagtccactatgatta-
caacgggtttcctt actggggccaagggactctggtcactgtctctgca 6 VH Murine AA
QVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHWVKQRPGQGLEWIGYI
NPSSAYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHY
DYNGFPYWGQGTLVTVSA 7 VL Murine NA
caagttgttctcacccagtctccagcaatcatgtctgcatttccaggggagaaggtcaccatgacctgcagtg-
ccagctcaa
gtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgactcatc-
caaactggcttct
ggagtccctgctcgcttcagtggcagtgggtctgggacctcttattctctcacaatcagcagcatggaga-
ctgaagatgctg
ccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattac-
a 8 VL Murine AA
QVVLTQSPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSK
LASGVPARFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQI T 9 VH-VL
scFv artificial NA
gaggtgaagcttctcgagtctggaggaggattggtgcagcctaaagggtcattgaaactctcatgtgcagcct-
ctggattca
ccttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgcat-
aagaagtaaat
ataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagagatgattcaca-
aagcattctctatct
acaaatgaacaacttgaaaactgaggacacagccatgtactactgtgtgagacatgggaacttcggtaat-
agctacgtttcct
ggtttgcttactggggccaagggactctggtcactgtctctgcaggtggtggtggttctggcggcggcgg-
ctccggtggtg
gtggttctcaggctgttgtgactcaggaatctgcactcaccacatcacctggtgaaacagtcacactcac-
ttgtcgctcaagt
actggggctgttacaactagtaactatgccaactgggtccaagaaaaaccagatcatttattcactggtc-
taataggtggtac
caacaagcgagctccaggtgtgcctgccagattctcaggctccctgattggagacaaggctgccctcacc-
atcacagggg
cacagactgaggatgaggcaatatatttctgtgctctatggtacagcaacctctgggtgttcggtggagg-
aaccaaactgac tgtccta 10 VH-VL scFv artificial AA
EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI
RSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHG
NFGNSYVSWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSQAVVTQESALTT
SPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARF
SGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL 11 VL-VH scFv
artificial NA
caggctgttgtgactcaggaatctgcactcaccacatcacctggtgaaacagtcacactcacttgtcgctcaa-
gtactgggg
ctgttacaactagtaactatgccaactgggtccaagaaaaaccagatcatttattcactggtctaatagg-
tggtaccaacaag
cgagctccaggtgtgcctgccagattctcaggctccctgattggagacaaggctgccctcaccatcacag-
gggcacagac
tgaggatgaggcaatatatttctgtgctctatggtacagcaacctctgggtgttcggtggaggaaccaaa-
ctgactgtcctag
gtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgaagcttctcgagtctggagg-
aggattggtgc
agcctaaagggtcattgaaactctcatgtgcagcctctggattcaccttcaatacctacgccatgaactg-
ggtccgccaggct
ccaggaaagggtttggaatgggttgctcgcataagaagtaaatataataattatgcaacatattatgccg-
attcagtgaaaga
caggttcaccatctccagagatgattcacaaagcattctctatctacaaatgaacaacttgaaaactgag-
gacacagccatgt
actactgtgtgagacatgggaacttcggtaatagctacgtttcctggtttgcttactggggccaagggac-
tctggtcactgtct ctgca 12 VL-VH scFv artificial AA
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGG
TNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGT
KLTVLGGGGSGGGGSGGGGSEVKLLESGGGLVQPKGSLKLSCAASGFTFNTY
AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYL
QMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA 13 VH-VL scFv artificial
NA
caggtccagctgcagcagtctggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaaggctt-
ctggcta
cacctttactagatctacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatac-
attaatcctagc
agtgcttatactaattacaatcagaaattcaaggacaaggccacattgactgcagacaaatcctccagta-
cagcctacatgc
aactgagtagcctgacatctgaggactctgcagtctattactgtgcaagtccgcaagtccactatgatta-
caacgggtttcctt
actggggccaagggactctggtcactgtctctgcaggtggtggtggttctggcggcggcggctccggtgg-
tggtggttctc
aagttgttctcacccagtctccagcaatcatgtctgcatttccaggggagaaggtcaccatgacctgcag-
tgccagctcaag
tgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgactcatcc-
aaactggcttctg
gagtccctgctcgcttcagtggcagtgggtctgggacctcttattctctcacaatcagcagcatggagac-
tgaagatgctgc
cacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattaca
14 VH-VL scFv artificial AA
QVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHWVKQRPGQGLEWIGYI
NPSSAYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHY
DYNGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSQVVLTQSPAIMSAFPGEK
VTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGVPARFSGSGSGTSY
SLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQIT 15 VL-VH scFv artificial NA
caagttgttctcacccagtctccagcaatcatgtctgcatttccaggggagaaggtcaccatgacctgcagtg-
ccagctcaa
gtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgactcatc-
caaactggcttct
ggagtccctgctcgcttcagtggcagtgggtctgggacctcttattctctcacaatcagcagcatggaga-
ctgaagatgctg
ccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattac-
aggtggtggt
ggttctggcggcggcggctccggtggtggtggttctcaggtccagctgcagcagtctggggctgaactgg-
caagacctg
gggcctcagtgaagatgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaa-
acagaggcctgg
acagggtctggaatggattggatacattaatcctagcagtgcttatactaattacaatcagaaattcaag-
gacaaggccacat
tgactgcagacaaatcctccagtacagcctacatgcaactgagtagcctgacatctgaggactctgcagt-
ctattactgtgca
agtccgcaagtccactatgattacaacgggtttccttactggggccaagggactctggtcactgtctctg-
ca 16 VL-VH scFv artificial AA
QVVLTQSPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSK
LASGVPARFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQI
TGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRSTM
HWVKQRPGQGLEWIGYINPSSAYTNYNQKFKDKATLTADKSSSTAYMQLSS
LTSEDSAVYYCASPQVHYDYNGFPYWGQGTLVTVSA 17 5' LH artificial NA
acatccggaggtggtggatcccaggctgttgtgactcaggaatctgc 18 3' VL Linker
artificial NA ggagccgccgccgccagaaccaccaccacctaggacagtcagtttggttcc
19 5' VH Linker artificial NA
tctggcggcggcggctccggtggtggtggttctgaggtgaagcttctcgagtctggaggaggattggtgc
20 3' LH artificial NA
agtgggtcgacctaatgatgatggtgatgatgtgcagagacagtgaccagagtccc 21 5' HL
artificial NA
acatccggaggtggtggatccgaggtgaagcttctcgagtctggaggaggattggtgc 22 3' VH
Linker artificial NA
ggagccgccgccgccagaaccaccaccacctgcagagacagtgaccagagtccc 23 5' VL
Linker artificial NA
tctggcggcggcggctccggtggtggtggttctcaggctgttgtgactcaggaatctgc 24 3'
HL artificial NA
agtgggtcgacctaatgatgatggtgatgatgtaggacagtcagtttggttcctcc 25 5' LH
artificial NA acatccggaggtggtggatcccaagttgttctcacccagtctcc 26 3' VL
Linker artificial NA
ggagccgccgccgccagaaccaccaccacctgtaatttgtagcttggtccccc 27 5' VH
Linker artificial NA
tctggcggcggcggctccggtggtggtggttctcaggtccagctgcagcagtctgg 28 3' LH
artificial NA
agtgggtcgacctaatgatgatggtgatgatgtgcagagacagtgaccagagtcc 29 5' HL
artificial NA acatccggaggtggtggatcccaggtccagctgcagcagtctgg 30 3' VH
Linker artificial NA
ggagccgccgccgccagaaccaccaccacctgcagagacagtgaccagagtcc 31 5' VL
Linker artificial NA
tctggcggcggcggctccggtggtggtggttctcaagttgttctcacccagtctcc 32 3' HL
artificial NA
agtgggtcgacctaatgatgatggtgatgatgtgtaatttgtagcttggtccccc 33 5-10 LH
scFv artificial NA
gagctcgtgatgacacagtctccatcctccctgactgtgacagcaggagagaaggtcactatgagctgcaagt-
ccagtcag
agtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcctccta-
aactgttgatct
actgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctggaacagatttcac-
tctcaccatcag
cagtgtgcaggctgaagacctggcagtttattactgtcagaatgattatagttatccgctcacgttcggt-
gctgggaccaagc
ttgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagctgct-
cgagcagtctg
gagctgagctggtaaggcctgggacttcagtgaagatatcctgcaaggcttctggatacgccttcactaa-
ctactggctagg
ttgggtaaagcagaggcctggacatggacttgagtggattggagatattttccctggaagtggtaatatc-
cactacaatgag
aagttcaagggcaaagccacactgactgcagacaaatcttcgagcacagcctatatgcagctcagtagcc-
tgacatttgag
gactctgctgtctatttctgtgcaagactgaggaactgggacgagcctatggactactggggccaaggga-
ccacggtcacc gtctcctcc 34 5-10 LH scFv artificial AA
ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPK
LLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTF
GAGTKLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPGTSVKISCKASGY
AFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTA
YMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSS 35 Leader peptide mouse NA
atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtacactcc cDNA
36 Leader peptide mouse AA MGWSCIILFLVATATGVHS cDNA 37 5-10 LHx
artificial NA
gagctcgtgatgacacagtctccatcctccctgactgtgacagcaggagagaaggtcactatgagctgcaagt-
ccagtcag SEQ ID NO. 12
agtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcctcctaaac-
tgttgatct
actgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctggaacagatttcac-
tctcaccatcag
cagtgtgcaggctgaagacctggcagtttattactgtcagaatgattatagttatccgctcacgttcggt-
gctgggaccaagc
ttgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagctgct-
cgagcagtctg
gagctgagctggtaaggcctgggacttcagtgaagatatcctgcaaggcttctggatacgccttcactaa-
ctactggctagg
ttgggtaaagcagaggcctggacatggacttgagtggattggagatattttccctggaagtggtaatatc-
cactacaatgag
aagttcaagggcaaagccacactgactgcagacaaatcttcgagcacagcctatatgcagctcagtagcc-
tgacatttgag
gactctgctgtctatttctgtgcaagactgaggaactgggacgagcctatggactactggggccaaggga-
ccacggtcacc
gtctcctccggaggtggtggatcccaggctgttgtgactcaggaatctgcactcaccacatcacctggtg-
aaacagtcaca
ctcacttgtcgctcaagtactggggctgttacaactagtaactatgccaactgggtccaagaaaaaccag-
atcatttattcact
ggtctaataggtggtaccaacaagcgagctccaggtgtgcctgccagattctcaggctccctgattggag-
acaaggctgcc
ctcaccatcacaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaacctct-
gggtgttcggtg
gaggaaccaaactgactgtcctaggtggtggtggttctggcggcggcggctccggtggtggtggttctga-
ggtgaagctt
ctcgagtctggaggaggattggtgcagcctaaagggtcattgaaactctcatgtgcagcctctggattca-
ccttcaataccta
cgccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgcataagaagtaaatat-
aataattatgca
acatattatgccgattcagtgaaagacaggttcaccatctccagagatgattcacaaagcattctctatc-
tacaaatgaacaac
ttgaaaactgaggacacagccatgtactactgtgtgagacatgggaacttcggtaatagctacgtttcct-
ggtttgcttactgg ggccaagggactctggtcactgtctctgca 38 5-10 LHx
artificial AA ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPK
SEQ ID NO. 12 LLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTF
GAGTKLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPGTSVKISCKASGY
AFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTA
YMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSGGGGSQAVVT
QESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRA
PGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL
GGGGSGGGGSGGGGSEVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNW
VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNN
LKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA 39 5-10 LHx artificial NA
gagctcgtgatgacacagtctccatcctccctgactgtgacagcaggagagaaggtcactatgagctgcaagt-
ccagtcag SEQ ID NO. 10
agtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcctcctaaac-
tgttgatct
actgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctggaacagatttcac-
tctcaccatcag
cagtgtgcaggctgaagacctggcagtttattactgtcagaatgattatagttatccgctcacgttcggt-
gctgggaccaagc
ttgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagctgct-
cgagcagtctg
gagctgagctggtaaggcctgggacttcagtgaagatatcctgcaaggcttctggatacgccttcactaa-
ctactggctagg
ttgggtaaagcagaggcctggacatggacttgagtggattggagatattttccctggaagtggtaatatc-
cactacaatgag
aagttcaagggcaaagccacactgactgcagacaaatcttcgagcacagcctatatgcagctcagtagcc-
tgacatttgag
gactctgctgtctatttctgtgcaagactgaggaactgggacgagcctatggactactggggccaaggga-
ccacggtcacc
gtctcctccggaggtggtggatccgaggtgaagcttctcgagtctggaggaggattggtgcagcctaaag-
ggtcattgaaa
ctctcatgtgcagcctctggattcaccttcaatacctacgccatgaactgggtccgccaggctccaggaa-
agggtttggaat
gggttgctcgcataagaagtaaatataataattatgcaacatattatgccgattcagtgaaagacaggtt-
caccatctccaga
gatgattcacaaagcattctctatctacaaatgaacaacttgaaaactgaggacacagccatgtactact-
gtgtgagacatgg
gaacttcggtaatagctacgtttcctggtttgcttactggggccaagggactctggtcactgtctctgca-
ggtggtggtggttc
tggcggcggcggctccggtggtggtggttctcaggctgttgtgactcaggaatctgcactcaccacatca-
cctggtgaaac
agtcacactcacttgtcgctcaagtactggggctgttacaactagtaactatgccaactgggtccaagaa-
aaaccagatcatt
tattcactggtctaataggtggtaccaacaagcgagctccaggtgtgcctgccagattctcaggctccct-
gattggagacaa
ggctgccctcaccatcacaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacagc-
aacctctgggtg ttcggtggaggaaccaaactgactgtccta 40 5-10 LHx artificial
AA ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPK SEQ ID NO.
10 LLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTF
GAGTKLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPGTSVKISCKASGY
AFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTA
YMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSGGGGSEVKLLE
SGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYN
NYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNS
YVSWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSQAVVTQESALTTSPGET
VTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIG
DKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL 41 5-10 LHx artificial NA
gagctcgtgatgacacagtctccatcctccctgactgtgacagcaggagagaaggtcactatgagctgcaagt-
ccagtcag SEQ ID NO. 16
agtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcctcctaaac-
tgttgatct
actgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctggaacagatttcac-
tctcaccatcag
cagtgtgcaggctgaagacctggcagtttattactgtcagaatgattatagttatccgctcacgttcggt-
gctgggaccaagc
ttgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagctgct-
cgagcagtctg
gagctgagctggtaaggcctgggacttcagtgaagatatcctgcaaggcttctggatacgccttcactaa-
ctactggctagg
ttgggtaaagcagaggcctggacatggacttgagtggattggagatattttccctggaagtggtaatatc-
cactacaatgag
aagttcaagggcaaagccacactgactgcagacaaatcttcgagcacagcctatatgcagctcagtagcc-
tgacatttgag
gactctgctgtctatttctgtgcaagactgaggaactgggacgagcctatggactactggggccaaggga-
ccacggtcacc
gtctcctccggaggtggtggatcccaagttgttctcacccagtctccagcaatcatgtctgcatttccag-
gggagaaggtca
ccatgacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccc-
caaaagatgg
atttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctctt-
attctctcacaatca
gcagcatggagactgaagatgctgccacttattactgccagcagtggagtcgtaacccacccacgttcgg-
aggggggac
caagctacaaattacaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtccag-
ctgcagcagt
ctggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaaggcttctggctacacctttac-
tagatctacgat
gcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagcagtgcttat-
actaattacaatc
agaaattcaaggacaaggccacattgactgcagacaaatcctccagtacagcctacatgcaactgagtag-
cctgacatctg
aggactctgcagtctattactgtgcaagtccgcaagtccactatgattacaacgggtttccttactgggg-
ccaagggactctg gtcactgtctctgca 42 5-10 LHx artificial AA
ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPK SEQ ID NO. 16
LLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTF
GAGTKLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPGTSVKISCKASGY
AFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTA
YMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSGGGGSQVVLT
QSPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGV
PARFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQITGGGG
SGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHWVKQ
RPGQGLEWIGYINPSSAYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDS
AVYYCASPQVHYDYNGFPYWGQGTLVTVSA 43 5-10 LHx artificial NA
gagctcgtgatgacacagtctccatcctccctgactgtgacagcaggagagaaggtcactatgagctgcaagt-
ccagtcag SEQ ID NO.14
agtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcctcctaaac-
tgttgatct
actgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctggaacagatttcac-
tctcaccatcag
cagtgtgcaggctgaagacctggcagtttattactgtcagaatgattatagttatccgctcacgttcggt-
gctgggaccaagc
ttgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagctgct-
cgagcagtctg
gagctgagctggtaaggcctgggacttcagtgaagatatcctgcaaggcttctggatacgccttcactaa-
ctactggctagg
ttgggtaaagcagaggcctggacatggacttgagtggattggagatattttccctggaagtggtaatatc-
cactacaatgag
aagttcaagggcaaagccacactgactgcagacaaatcttcgagcacagcctatatgcagctcagtagcc-
tgacatttgag
gactctgctgtctatttctgtgcaagactgaggaactgggacgagcctatggactactggggccaaggga-
ccacggtcacc
gtctcctccggaggtggtggatcccaggtccagctgcagcagtctggggctgaactggcaagacctgggg-
cctcagtga
agatgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaaacagaggcctgg-
acagggtctgga
atggattggatacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaaggccaca-
ttgactgcagaca
aatcctccagtacagcctacatgcaactgagtagcctgacatctgaggactctgcagtctattactgtgc-
aagtccgcaagtc
cactatgattacaacgggtttccttactggggccaagggactctggtcactgtctctgcaggtggtggtg-
gttctggcggcg
gcggctccggtggtggtggttctcaagttgttctcacccagtctccagcaatcatgtctgcatttccagg-
ggagaaggtcacc
atgacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctccccca-
aaagatggatt
tatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttatt-
ctctcacaatcagc
agcatggagactgaagatgctgccacttattactgccagcagtggagtcgtaacccacccacgttcggag-
gggggaccaa gctacaaattaca 44 5-10 LHx artificial AA
ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPK SEQ ID NO. 14
LLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTF
GAGTKLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPGTSVKISCKASGY
AFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTA
YMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSGGGGSQVQLQ
QSGAELARPGASVKMSCKASGYTFTRSTMHWVKQRPGQGLEWIGYINPSSA
YTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHYDYNG
FPYWGQGTLVTVSAGGGGSGGGGSGGGGSQVVLTQSPAIMSAFPGEKVTMT
CSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGVPARFSGSGSGTSYSLTIS
SMETEDAATYYCQQWSRNPPTFGGGTKLQIT 45 5'EpCAM artificial NA
ggttctagaccaccatggcgcccccgcaggtcctcgcgttcgg 46 3'EpCAM artificial
NA agtgggtcgacttatgcattgagttccctatgcatctcaccc 47 cynomolgus Cyno-
NA
cagaaagaatgtgtctgtgaaaactacaagctggccgtaaactgctttttgaatgacaatggtcaatgccagt-
gtacttcgatt EpCAM molgus
ggtgcacaaaatactgtcctttgctcaaagctggctgccaaatgtttggtgatgaaggcagaaatgaacggct-
caaaacttg extracellular cDNA
ggagaagagcgaaacctgaaggggctctccagaacaatgatggcctttacgatcctgactgcgatgagagcgg-
gctcttt portion
aaggccaagcagtgcaacggcacctccacgtgctggtgtgtgaacactgctggggtcagaaga-
actgacaaggacactg
aaataacctgctctgagcgagtgagaacctactggatcatcattgaattaaaacacaaagcaagagaaaa-
accttatgatgtt
caaagtttgcggactgcacttgaggaggcgatcaaaacgcgttatcaactggatccaaaatttatcacaa-
atattttgtatgag
gataatgttatcactattgatctggttcaaaattcttctcagaaaactcagaatgatgtggacatagctg-
atgtggcttattattttg
aaaaagatgttaaaggtgaatccttgtttcattctaagaaaatggacctgagagtaaatggggaacaact-
ggatctggatcct
ggtcaaactttaatttattatgtcgatgaaaaagcacctgaattctcaatgcagggtctaaaa 48
cynomolgus Cyno- AA
QKECVCENYKLAVNCFLNDNGQCQCTSIGAQNTVLCSKLAAKCLVMKAEM EpCAM molgus
NGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSTCWCVNTAG extracellular
cDNA VRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDVQSLRTALEEAIKTRYQL portion
DPKFITNILYEDNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSK
KMDLRVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLK 49 2G8 VH Hybri- NA
gaggttcagctgcagcagtctggggcagagcttgtgaggtcaggggcctcagtcaagttgtcctgcacagctt-
ctggcttc doma
aacattaaagactactatttgcactgggtgaagcagaggcctgaacagggcctggagtggattgcc-
tggattgatcttgaga
atggtgatattaaatatgccccgaagtttcagggcaaggccactataactgcagacacatcctccaacac-
agcctacctgca
gctcagcagcctgacatctgaggacactgccgtctattactgtaatccctattactacggtagtaactac-
gactatgctatgga ctactggggtcaaggaacctcagtcaccgtctcctca 50 2G8 VH
Hybri- AA EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIAWI doma
DLENGDIKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCNPYYYGS
NYDYAMDYWGQGTSVTVSS 51 2G8 VL Hybri- NA
gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagt-
caagtcagagc doma
ctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtctccaaag-
cgcctaatctatctggtg
tctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaaa-
tcagcagagtgg
aggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctgggac-
caagctggagctg aaa 52 2G8 VL Hybri- AA
DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIY doma
LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGT KLELK 53
2G8VH-VL scFv artificial NA
gaggttcagctgcagcagtctggggcagagcttgtgaggtcaggggcctcagtcaagttgtcctgcacagctt-
ctggcttc
aacattaaagactactatttgcactgggtgaagcagaggcctgaacagggcctggagtggattgcctgga-
ttgatcttgaga
atggtgatattaaatatgccccgaagtttcagggcaaggccactataactgcagacacatcctccaacac-
agcctacctgca
gctcagcagcctgacatctgaggacactgccgtctattactgtaatccctattactacggtagtaactac-
gactatgctatgga
ctactggggtcaaggaacctcagtcaccgtctcctcaggtggtggtggttctggcggcggcggctccggt-
ggtggtggttc
tgatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgc-
aagtcaagtcagag
cctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtctccaaagcgc-
ctaatctatctggt
gtctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaa-
atcagcagagtg
gaggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctggga-
ccaagctggagct gaaa 54 2G8VH-VL scFv artificial AA
EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIAWI
DLENGDIKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCNPYYYGS
NYDYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIG
QPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIYLVSKLDSGVPDRFTGS
GSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGTKLELK 55 2G8VL-VH scFv
artificial NA
gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagt-
caagtcagagc
ctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtctccaaagcgcc-
taatctatctggtg
tctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaaa-
tcagcagagtgg
aggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctgggac-
caagctggagctg
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggttcagctgcagcagtctg-
gggcagagct
tgtgaggtcaggggcctcagtcaagttgtcctgcacagcttctggcttcaacattaaagactactatttg-
cactgggtgaagc
agaggcctgaacagggcctggagtggattgcctggattgatcttgagaatggtgatattaaatatgcccc-
gaagtttcaggg
caaggccactataactgcagacacatcctccaacacagcctacctgcagctcagcagcctgacatctgag-
gacactgccg
tctattactgtaatccctattactacggtagtaactacgactatgctatggactactggggtcaaggaac-
ctcagtcaccgtctc ctcc 56 2G8VL-VH scFv artificial AA
DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIY
LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGT
KLELKGGGGSGGGGSGGGGSEVQLQQSGAELVRSGASVKLSCTASGFNIKD
YYLHWVKQRPEQGLEWIAWIDLENGDIKYAPKFQGKATITADTSSNTAYLQL
SSLTSEDTAVYYCNPYYYGSNYDYAMDYWGQGTSVTVSS 57 5'2G8 LH artificial NA
aggtgtacactccgatgttgtgatgacccagactccactcactttgtcg 58 3'2G8 VL
Linker artificial NA
ggagccgccgccgccagaaccaccaccacctttcagctccagcttggtcccagc 59 5'2G8 VH
Linker artificial NA
tctggcggcggcggctccggtggtggtggttctgaggttcagctgcagcagtctgg 60 3'2G8
LH artificial NA acatccggaggagacggtgactgaggttcc 61 5'2G8 HL
artificial NA aggtgtacactccgaggttcagctgcagcagtctggg 62 3'2G8 VH
Linker artificial NA
ggagccgccgccgccagaaccaccaccacctgaggagacggtgactgaggttcc 63 5'2G8 VL
Linker artificial NA
tctggcggcggcggctccggtggtggtggttctgatgttgtgatgacccagactccactcactttgtcg
64 3'2G8 HL artificial NA acatccggatttcagctccagcttggtcccagc 65
2G8LHx artificial NA
gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagt-
caagtcagagc SEQ ID NO. 12
ctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtctccaaagcgcctaa-
tctatctggtg
tctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaaa-
tcagcagagtgg
aggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctgggac-
caagctggagctg
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggttcagctgcagcagtctg-
gggcagagct
tgtgaggtcaggggcctcagtcaagttgtcctgcacagcttctggcttcaacattaaagactactatttg-
cactgggtgaagc
agaggcctgaacagggcctggagtggattgcctggattgatcttgagaatggtgatattaaatatgcccc-
gaagtttcaggg
caaggccactataactgcagacacatcctccaacacagcctacctgcagctcagcagcctgacatctgag-
gacactgccg
tctattactgtaatccctattactacggtagtaactacgactatgctatggactactggggtcaaggaac-
ctcagtcaccgtctc
ctccggaggtggtggatcccaggctgttgtgactcaggaatctgcactcaccacatcacctggtgaaaca-
gtcacactcact
tgtcgctcaagtactggggctgttacaactagtaactatgccaactgggtccaagaaaaaccagatcatt-
tattcactggtcta
ataggtggtaccaacaagcgagctccaggtgtgcctgccagattctcaggctccctgattggagacaagg-
ctgccctcacc
atcacaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaacctctgggtgt-
tcggtggaggaa
ccaaactgactgtcctaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgaa-
gcttctcgagt
ctggaggaggattggtgcagcctaaagggtcattgaaactctcatgtgcagcctctggattcaccttcaa-
tacctacgccatg
aactgggtccgccaggctccaggaaagggtttggaatgggttgctcgcataagaagtaaatataataatt-
atgcaacatatta
tgccgattcagtgaaagacaggttcaccatctccagagatgattcacaaagcattctctatctacaaatg-
aacaacttgaaaa
ctgaggacacagccatgtactactgtgtgagacatgggaacttcggtaatagctacgtttcctggtttgc-
ttactggggccaa gggactctggtcactgtctctgca 66 2G8LHx artificial AA
DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIY SEQ ID NO.
12 LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGT
KLELKGGGGSGGGGSGGGGSEVQLQQSGAELVRSGASVKLSCTASGFNIKD
YYLHWVKQRPEQGLEWIAWIDLENGDIKYAPKFQGKATITADTSSNTAYLQL
SSLTSEDTAVYYCNPYYYGSNYDYAMDYWGQGTSVTVSSGGGGSQAVVTQ
ESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAP
GVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLG
GGGSGGGGSGGGGSEVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWV
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNL
KTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA 67 2G8LHx artificial NA
gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagt-
caagtcagagc SEQ ID NO. 10
ctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtctccaaagcgcctaa-
tctatctggtg
tctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaaa-
tcagcagagtgg
aggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctgggac-
caagctggagctg
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggttcagctgcagcagtctg-
gggcagagct
tgtgaggtcaggggcctcagtcaagttgtcctgcacagcttctggcttcaacattaaagactactatttg-
cactgggtgaagc
agaggcctgaacagggcctggagtggattgcctggattgatcttgagaatggtgatattaaatatgcccc-
gaagtttcaggg
caaggccactataactgcagacacatcctccaacacagcctacctgcagctcagcagcctgacatctgag-
gacactgccg
tctattactgtaatccctattactacggtagtaactacgactatgctatggactactggggtcaaggaac-
ctcagtcaccgtctc
ctccggaggtggtggatccgaggtgaagcttctcgagtctggaggaggattggtgcagcctaaagggtca-
ttgaaactctc
atgtgcagcctctggattcaccttcaatacctacgccatgaactgggtccgccaggctccaggaaagggt-
ttggaatgggtt
gctcgcataagaagtaaatataataattatgcaacatattatgccgattcagtgaaagacaggttcacca-
tctccagagatgat
tcacaaagcattctctatctacaaatgaacaacttgaaaactgaggacacagccatgtactactgtgtga-
gacatgggaactt
cggtaatagctacgtttcctggtttgcttactggggccaagggactctggtcactgtctctgcaggtggt-
ggtggttctggcg
gcggcggctccggtggtggtggttctcaggctgttgtgactcaggaatctgcactcaccacatcacctgg-
tgaaacagtca
cactcacttgtcgctcaagtactggggctgttacaactagtaactatgccaactgggtccaagaaaaacc-
agatcatttattca
ctggtctaataggtggtaccaacaagcgagctccaggtgtgcctgccagattctcaggctccctgattgg-
agacaaggctg
ccctcaccatcacaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaacct-
ctgggtgttcgg tggaggaaccaaactgactgtccta 68 2G8LHx artificial AA
DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIY SEQ ID NO.
10 LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGT
KLELKGGGGSGGGGSGGGGSEVQLQQSGAELVRSGASVKLSCTASGFNIKD
YYLHWVKQRPEQGLEWIAWIDLENGDIKYAPKFQGKATITADTSSNTAYLQL
SSLTSEDTAVYYCNPYYYGSNYDYAMDYWGQGTSVTVSSGGGGSEVKLLES
GGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN
YATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSY
VSWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSQAVVTQESALTTSPGETV
TLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGD
KAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL 69 2G8LHx artificial NA
gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagt-
caagtcagagc SEQ ID NO. 16
ctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtctccaaagcgcctaa-
tctatctggtg
tctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaaa-
tcagcagagtgg
aggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctgggac-
caagctggagctg
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggttcagctgcagcagtctg-
gggcagagct
tgtgaggtcaggggcctcagtcaagttgtcctgcacagcttctggcttcaacattaaagactactatttg-
cactgggtgaagc
agaggcctgaacagggcctggagtggattgcctggattgatcttgagaatggtgatattaaatatgcccc-
gaagtttcaggg
caaggccactataactgcagacacatcctccaacacagcctacctgcagctcagcagcctgacatctgag-
gacactgccg
tctattactgtaatccctattactacggtagtaactacgactatgctatggactactggggtcaaggaac-
ctcagtcaccgtctc
ctccggaggtggtggatcccaagttgttctcacccagtctccagcaatcatgtctgcatttccaggggag-
aaggtcaccatg
acctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaa-
gatggatttat
gactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttattctc-
tcacaatcagcag
catggagactgaagatgctgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggg-
gggaccaagc
tacaaattacaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtccagctgca-
gcagtctggg
gctgaactggcaagacctggggcctcagtgaagatgtcctgcaaggcttctggctacacctttactagat-
ctacgatgcact
gggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagcagtgcttatactaa-
ttacaatcagaa
attcaaggacaaggccacattgactgcagacaaatcctccagtacagcctacatgcaactgagtagcctg-
acatctgagga
ctctgcagtctattactgtgcaagtccgcaagtccactatgattacaacgggtttccttactggggccaa-
gggactctggtca ctgtctctgca 70 2G8LHx artificial AA
DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIY SEQ ID NO.
16 LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGT
KLELKGGGGSGGGGSGGGGSEVQLQQSGAELVRSGASVKLSCTASGFNIKD
YYLHWVKQRPEQGLEWIAWIDLENGDIKYAPKFQGKATITADTSSNTAYLQL
SSLTSEDTAVYYCNPYYYGSNYDYAMDYWGQGTSVTVSSGGGGSQVVLTQ
SPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGVP
ARFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQITGGGGS
GGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHWVKQR
PGQGLEWIGYINPSSAYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSA
VYYCASPQVHYDYNGFPYWGQGTLVTVSA 71 2G8LHx artificial NA
gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagt-
caagtcagagc SEQ ID NO. 14
ctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtctccaaagcgcctaa-
tctatctggtg
tctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaaa-
tcagcagagtgg
aggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctgggac-
caagctggagctg
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggttcagctgcagcagtctg-
gggcagagct
tgtgaggtcaggggcctcagtcaagttgtcctgcacagcttctggcttcaacattaaagactactatttg-
cactgggtgaagc
agaggcctgaacagggcctggagtggattgcctggattgatcttgagaatggtgatattaaatatgcccc-
gaagtttcaggg
caaggccactataactgcagacacatcctccaacacagcctacctgcagctcagcagcctgacatctgag-
gacactgccg
tctattactgtaatccctattactacggtagtaactacgactatgctatggactactggggtcaaggaac-
ctcagtcaccgtctc
ctccggaggtggtggatcccaggtccagctgcagcagtctggggctgaactggcaagacctggggcctca-
gtgaagatg
tcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaaacagaggcctggacagg-
gtctggaatgga
ttggatacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaaggccacattgac-
tgcagacaaatcct
ccagtacagcctacatgcaactgagtagcctgacatctgaggactctgcagtctattactgtgcaagtcc-
gcaagtccactat
gattacaacgggtttccttactggggccaagggactctggtcactgtctctgcaggtggtggtggttctg-
gcggcggcggct
ccggtggtggtggttctcaagttgttctcacccagtctccagcaatcatgtctgcatttccaggggagaa-
ggtcaccatgacc
tgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagat-
ggatttatgact
catccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttattctctcac-
aatcagcagcatg
gagactgaagatgctgccacttattactgccagcagtggagtcgtaacccacccacgttcggagggggga-
ccaagctaca aattaca 72 2G8LHx artificial AA
DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIY SEQ ID NO.
14 LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGT
KLELKGGGGSGGGGSGGGGSEVQLQQSGAELVRSGASVKLSCTASGFNIKD
YYLHWVKQRPEQGLEWIAWIDLENGDIKYAPKFQGKATITADTSSNTAYLQL
SSLTSEDTAVYYCNPYYYGSNYDYAMDYWGQGTSVTVSSGGGGSQVQLQQ
SGAELARPGASVKMSCKASGYTFTRSTMHWVKQRPGQGLEWIGYINPSSAY
TNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHYDYNGF
PYWGQGTLVTVSAGGGGSGGGGSGGGGSQVVLTQSPAIMSAFPGEKVTMTC
SASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGVPARFSGSGSGTSYSLTISS
METEDAATYYCQQWSRNPPTFGGGTKLQIT 73 2G8HLx artificial NA
gaggttcagctgcagcagtctggggcagagcttgtgaggtcaggggcctcagtcaagttgtcctgcacagctt-
ctggcttc SEQ ID NO. 12
aacattaaagactactatttgcactgggtgaagcagaggcctgaacagggcctggagtggattgcctggattg-
atcttgaga
atggtgatattaaatatgccccgaagtttcagggcaaggccactataactgcagacacatcctccaacac-
agcctacctgca
gctcagcagcctgacatctgaggacactgccgtctattactgtaatccctattactacggtagtaactac-
gactatgctatgga
ctactggggtcaaggaacctcagtcaccgtctcctcaggtggtggtggttctggcggcggcggctccggt-
ggtggtggttc
tgatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgc-
aagtcaagtcagag
cctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtaccaaagcgcc-
taatctatctggt
gtctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaa-
atcagcagagtg
gaggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctggga-
ccaagctggagct
gaaatccggaggtggtggatcccaggctgttgtgactcaggaatctgcactcaccacatcacctggtgaa-
acagtcacact
cacttgtcgctcaagtactggggctgttacaactagtaactatgccaactgggtccaagaaaaaccagat-
catttattcactg
gtctaataggtggtaccaacaagcgagctccaggtgtgcctgccagattctcaggctccctgattggaga-
caaggctgccc
tcaccatcacaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaacctctg-
ggtgttcggtgg
aggaaccaaactgactgtcctaggtggtggtggttctggcggcggcggctccggtggtggtggttctgag-
gtgaagcttct
cgagtctggaggaggattggtgcagcctaaagggtcattgaaactctcatgtgcagcctaggattcacct-
tcaatacctac
gccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgcataagaagtaaatata-
ataattatgcaa
catattatgccgattcagtgaaagacaggttcaccatctccagagatgattcacaaagcattctctatct-
acaaatgaacaact
tgaaaactgaggacacagccatgtactactgtgtgagacatgggaacttcggtaatagctacgtttcctg-
gtttgcttactgg ggccaagggactctggtcactgtctctgca 74 2G8HLx artificial
AA EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIAWI SEQ ID NO.
12 DLENGDIKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCNPYYYGS
NYDYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIG
QPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIYLVSKLDSGVPDRFTGS
GSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGTKLELKSGGGGSQAVV
TQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKR
APGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTV
LGGGGSGGGGSGGGGSEVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMN
WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMN
NLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA 75 2G8HLx artificial NA
gaggttcagctgcagcagtctggggcagagcttgtgaggtcaggggcctcagtcaagttgtcctgcacagctt-
ctggcttc SEQ ID NO. 10
aacattaaagactactatttgcactgggtgaagcagaggcctgaacagggcctggagtggattgcctggattg-
atcttgaga
atggtgatattaaatatgccccgaagtttcagggcaaggccactataactgcagacacatcctccaacac-
agcctacctgca
gctcagcagcctgacatctgaggacactgccgtctattactgtaatccctattactacggtagtaactac-
gactatgctatgga
ctactggggtcaaggaacctcagtcaccgtctcctcaggtggtggtggttctggcggcggcggctccggt-
ggtggtggttc
tgatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgc-
aagtcaagtcagag
cctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtctccaaagcgc-
ctaatctatctggt
gtctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaa-
atcagcagagtg
gaggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctggga-
ccaagctggagct
gaaatccggaggtggtggatccgaggtgaagcttctcgagtctggaggaggattggtgcagcctaaaggg-
tcattgaaac
tctcatgtgcagcctctggattcaccttcaatacctacgccatgaactgggtccgccaggctccaggaaa-
gggtttggaatg
ggttgctcgcataagaagtaaatataataattatgcaacatattatgccgattcagtgaaagacaggttc-
accatctccagaga
tgattcacaaagcattctctatctacaaatgaacaacttgaaaactgaggacacagccatgtactactgt-
gtgagacatggga
acttcggtaatagctacgtttcctggtttgcttactggggccaagggactctggtcactgtctctgcagg-
tggtggtggttctg
gcggcggcggctccggtggtggtggttctcaggctgttgtgactcaggaatctgcactcaccacatcacc-
tggtgaaacag
tcacactcacttgtcgctcaagtactggggctgttacaactagtaactatgccaactgggtccaagaaaa-
accagatcatttat
tcactggtctaataggtggtaccaacaagcgagctccaggtgtgcctgccagattctcaggctccctgat-
tggagacaagg
ctgccctcaccatcacaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaa-
cctctgggtgttc ggtggaggaaccaaactgactgtccta 76 2G8HLx artificial AA
EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIAWI SEQ ID NO. 10
DLENGDIKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCNPYYYGS
NYDYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIG
QPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIYLVSKLDSGVPDRFTGS
GSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGTKLELKSGGGGSEVKL
LESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKY
NNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGN
SYVSWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSQAVVTQESALTTSPGET
VTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIG
DKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL 77 2G8HLx artificial NA
gaggttcagctgcagcagtctggggcagagcttgtgaggtcaggggcctcagtcaagttgtcctgcacagctt-
ctggcttc SEQ ID NO. 16
aacattaaagactactatttgcactgggtgaagcagaggcctgaacagggcctggagtggattgcctggattg-
atcttgaga
atggtgatattaaatatgccccgaagtttcagggcaaggccactataactgcagacacatcctccaacac-
agcctacctgca
gctcagcagcctgacatctgaggacactgccgtctattactgtaatccctattactacggtagtaactac-
gactatgctatgga
ctactggggtcaaggaacctcagtcaccgtctcctcaggtggtggtggttctggcggcggcggctccggt-
ggtggtggttc
tgatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgc-
aagtcaagtcagag
cctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtctccaaagcgc-
ctaatctatctggt
gtctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaa-
atcagcagagtg
gaggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctggga-
ccaagctggagct
gaaatccggaggtggtggatcccaagttgttctcacccagtctccagcaatcatgtctgcatttccaggg-
gagaaggtcacc
atgacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctccccca-
aaagatggatt
tatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttatt-
ctctcacaatcagc
agcatggagactgaagatgctgccacttattactgccagcagtggagtcgtaacccacccacgttcggag-
gggggaccaa
gctacaaattacaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtccagctg-
cagcagtctgg
ggctgaactggcaagacctggggcctcagtgaagatgtcctgcaaggcttctggctacacctttactaga-
tctacgatgcac
tgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagcagtgcttatacta-
attacaatcagaa
attcaaggacaaggccacattgactgcagacaaatcctccagtacagcctacatgcaactgagtagcctg-
acatctgagga
ctctgcagtctattactgtgcaagtccgcaagtccactatgattacaacgggtttccttactggggccaa-
gggactctggtca ctgtctctgca 78 2G8HLx artificial AA
EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIAWI SEQ ID NO. 16
DLENGDIKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCNPYYYGS
NYDYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIG
QPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIYLVSKLDSGVPDRFTGS
GSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGTKLELKSGGGGSQVVL
TQSPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASG
VPARFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQITGGG
GSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHWVK
QRPGQGLEWIGYINPSSAYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSED
SAVYYCASPQVHYDYNGFPYWGQGTLVTVSA 79 2G8HLx artificial NA
gaggttcagctgcagcagtctggggcagagcttgtgaggtcaggggcctcagtcaagttgtcctgcacagatc-
tggcttc SEQ ID NO. 14
aacattaaagactactatttgcactgggtgaagcagaggcctgaacagggcctggagtggattgcctggattg-
atcttgaga
atggtgatattaaatatgccccgaagtttcagggcaaggccactataactgcagacacatcctccaacac-
agcctacctgca
gctcagcagcctgacatctgaggacactgccgtctattactgtaatccctattactacggtagtaactac-
gactatgctatgga
ctactggggtcaaggaacctcagtcaccgtctcctcaggtggtggtggttctggcggcggcggctccggt-
ggtggtggttc
tgatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgc-
aagtcaagtcagag
cctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtctccaaagcgc-
ctaatctatctggt
gtctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaa-
atcagcagagtg
gaggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctggga-
ccaagctggagct
gaaatccggaggtggtggatcccaggtccagctgcagcagtctggggctgaactggcaagacctggggcc-
tcagtgaa
gatgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaaacagaggcctgga-
cagggtctggaa
tggattggatacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaaggccacat-
tgactgcagacaa
atcctccagtacagcctacatgcaactgagtagcctgacatctgaggactctgcagtctattactgtgca-
agtccgcaagtcc
actatgattacaacgggtttccttactggggccaagggactctggtcactgtctctgcaggtggtggtgg-
ttctggcggcgg
cggctccggtggtggtggttctcaagttgttctcacccagtctccagcaatcatgtctgcatttccaggg-
gagaaggtcacca
tgacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaa-
aagatggattt
atgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttattc-
tctcacaatcagca
gcatggagactgaagatgctgccacttattactgccagcagtggagtcgtaacccacccacgttcggagg-
ggggaccaa gctacaaattaca 80 2G8HLx artificial AA
EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIAWI SEQ ID NO. 14
DLENGDIKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCNPYYYGS
NYDYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIG
QPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIYLVSKLDSGVPDRFTGS
GSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGTKLELKSGGGGSQVQL
QQSGAELARPGASVKMSCKASGYTFTRSTMHWVKQRPGQGLEWIGYINPSS
AYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHYDYN
GFPYWGQGTLVTVSAGGGGSGGGGSGGGGSQVVLTQSPAIMSAFPGEKVTM
TCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGVPARFSGSGSGTSYSLTI
SSMETEDAATYYCQQWSRNPPTFGGGTKLQIT 81 5' primer VH artificial NA
5'-SAGGTGCAGCTCGAGGAGTCAGGACCT-3' 82 5' primer VH artificial NA
5'-GAGGTCCAGCTCGAGCAGTCTGGACCT-3' 83 5' primer VH artificial NA
5'-CAGGTCCAACTCGAGCAGCCTGGGGCT-3' 84 5' primer VH artificial NA
5'-GAGGTTCAGCTCGAGCAGTCTGGGGCA-3' 85 5' primer VH artificial NA
5'-GARGTGAAGCTCGAGGAGTCTGGAGGA-3' 86 5' primer VH artificial NA
5'-GAGGTGAAGCTTCTCGAGTCTGGAGGT-3' 87 5' primer VH artificial NA
5'-GAAGTGAAGCTCGAGGAGTCTGGGGGA-3' 88 5' primer VH artificial NA
5'-GAGGTTCAGCTCGAGCAGTCTGGAGCT-3' 89 5' primer VH artificial NA
5'-GGGCTCGAGCACCATGGRATGSAGCTGKGTMATSCTCTT-3' 90 5' primer VH
artificial NA 5'-GGGCTCGAGCACCATGRACTTCGGGYTGAGCTKGGTTTT-3' 91 5'
primer VH artificial NA
5'-GGGCTCGAGCACCATGGCTGTCTTGGGGCTGCTCTTCT-3' 92 3' primer VH
artificial NA 5'-GAGGAATTCGAACTGGACAGGGATCCAGAGTTCC-3' 93 3' primer
VH artificial NA 5'-CGGAATTCGAATGACATGGACATCTGGGTCATCC-3' 94 5'
primer VL artificial NA 5'-CCAGTTCCGAGCTCGTTGTGACTCAGGAATCT-3' 95
5' primer VL artificial NA 5'-CCAGTTCCGAGCTCGTGTTGACGCAGCCGCCC-3'
96 5' primer VL artificial NA
5'-CCAGTTCCGAGCTCGTGCTCACCCAGTCTCCA-3' 97 5' primer VL artificial
NA 5'-CCAGTTCCGAGCTCCAGATGACCCAGTCTCCA-3' 98 5' primer VL
artificial NA 5'-CCAGATGTGAGCTCGTGATGACCCAGACTCCA-3' 99 5' primer
VL artificial NA 5'-CCAGATGTGAGCTCGTCATGACCCAGTCTCCA-3' 100 5'
primer VL artificial NA 5'-CCAGTTCCGAGCTCGTGATGACACAGTCTCCA-3' 101
5' primer VL artificial NA
5'-GGGGAGCTCCACCATGGAGACAGACACACTCCTGCTAT-3' 102 5' primer VL
artificial NA 5'-GGGGAGCTCCACCATGGATTTTCAAGTGCAGATTTTCAG-3' 103 5'
primer VL artificial NA
5'-GGGGAGCTCCACCATGGAGWCACAKWCTCAGGTCTTTRTA-3' 104 5' primer VL
artificial NA 5'-GGGGAGCTCCACCATGKCCCCWRCTCAGYTYCTKGT-3' 105 3'
primer VL artificial NA 5'-GAGGAATTCGAACTGCTCACTGGATGGTGGG-3' 106
3' primer VL artificial NA
5'-CGGAATTCGAACAAACTCTTCTCCACAGTGTGACC-3' 107 3' primer VH
artificial NA 5'-TATGCAACTAGTACAACCACAATCCCTGGG-3' 108 3' primer VL
artificial NA 5'-GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3' 109 5-10 LH
x artificial AA ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPK
deimmunised (di)
LLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTF anti-CD3
GAGTKLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPGTSVKISCKASGY
AFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTA
YMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSGGGGSDVQLV
QSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRG
YTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDY
WGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCR
ASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINS
LEAEDAATYYCQQWSSNPLTFGGGTKVEIK 110 Human-like VH artificial AA
EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI
RSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 111 Human-like VH artificial NA
gaggtgcagctgctcgagtctggaggaggattggtgcagcctggagggtcattgaaactc
tcatgtgcagcctctggattcaccttcaatacctacgccatgaactgggtccgccaggct
ccaggaaagggtttggaatgggttgctcgcataagaagtaaatataataattatgcaaca
tattatgccgattcagtgaaagacaggttcaccatctccagagatgattcaaaaaacact
gcctatctacaaatgaacaacttgaaaactgaggacactgccgtgtactactgtgtgaga
catgggaacttcggtaatagctacgtttcctggtttgcttactggggccaagggactctg
gtcaccgtctcctca 112 VH: CDR3 murine AA HGNFGNSYVSWFAY 113 VH: short
CDR3 murine AA VSWFAY 114 VH: CDR2 murine AA RIRSKYNNYATYYADSVKD
115 VH: CDR1 murine AA TYAMN 116 VL: CDR3 murine AA ALWYSNLWV 117
VL: CDR2 murine AA GTNKRAP 118 VL: CDR1 murine AA RSSTGAVTTSNYAN
119 VH: CDR3 murine AA PQVHYDYNGFPY 120 VH: CDR2 murine AA
YINPSSAYTNYNQKFKD 121 VH: CDR1 murine AA GYTFTRSTMH 122 2G8 LHx
artificial AA
DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWILQRPGQSPKRLIY SEQ ID NO.
146 LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPLTFGAGT
KLELKGGGGSGGGGSGGGGSEVQLQQSGAELVRSGASVKLSCTASGFNIKD
YYLHWVKQRPEQGLEWIAWIDLENGDIKYAPKFQGKATITADTSSNTAYLQL
SSLTSEDTAVYYCNPYYYGSNYDYAMDYWGQGTSVTVSSGGGGSEVKLLES
GGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN
YATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY
VSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQESALTTSPGETVT
LTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGD
KAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLHHHHHH 123 2G8 LHx artificial
NA
gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagt-
caagtcagagc SEQ ID NO. 146
ctcttatatagtaatggaaaaacctatttgaactggatattacagaggccaggccagtctccaaagcgcctaa-
tctatctggtg
tctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacgctgaaaa-
tcagcagagtgg
aggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctctcacgttcggtgctgggac-
caagctggagctg
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggttcagctgcagcagtctg-
gggcagagct
tgtgaggtcaggggcctcagtcaagttgtcctgcacagcttctggcttcaacattaaagactactatttg-
cactgggtgaagc
agaggcctgaacagggcctggagtggattgcctggattgatcttgagaatggtgatattaaatatgcccc-
gaagtttcaggg
caaggccactataactgcagacacatcctccaacacagcctacctgcagctcagcagcctgacatctgag-
gacactgccg
tctattactgtaatccctattactacggtagtaactacgactatgctatggactactggggtcaaggaac-
ctcagtcaccgtctc
ctccggaggtggtggatccgaggtgaagcttctcgagtctggaggaggattggtgcagcctggagggtca-
ttgaaactct
catgtgcagcctctggattcaccttcaatacctacgccatgaactgggtccgccaggctccaggaaaggg-
tttggaatgggt
tgctcgcataagaagtaaatataataattatgcaacatattatgccgattcagtgaaagacaggttcacc-
atctccagagatga
ttcaaaaaacactgcctatctacaaatgaacaacttgaaaactgaggacactgccgtgtactactgtgtg-
agacatgggaac
ttcggtaatagctacgtttcctggtttgcttactggggccaagggactctggtcaccgtctcctcaggtg-
gtggtggttctggc
ggcggcggctccggtggtggtggttctgagctcgttgtgactcaggaatctgcactcaccacatcacctg-
gtgaaacagtc
acactcacttgtcgctcaagtactggggctgttacaactagtaactatgccaactgggtccaagaaaaac-
cagatcatttattc
actggtctaataggtggtaccaacaagcgagcaccaggtgtgcctgccagattctcaggctccctgattg-
gagacaaggct
gccctcaccatcacaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaacc-
tctgggtgttcg gtggaggaaccaaactgactgtcctacatcatcaccatcatcat 124 5-10
LHx artificial AA
ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPK SEQ ID NO. 146
LLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTF
GAGTKLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPGTSVKISCKASGY
AFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTA
YMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSGGGGSEVKLLE
SGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYN
NYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNS
YVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQESALTTSPGETV
TLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGD
KAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLHHHHHH 125 5-10 LHx
artificial NA
gagctcgtgatgacacagtctccatcctccctgactgtgacagcaggagagaaggtcactatgagctgcaagt-
ccagtcag SEQ ID NO. 146
agtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcctcctaaac-
tgttgatct
actgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctggaacagatttcac-
tctcaccatcag
cagtgtgcaggctgaagacctggcagtttattactgtcagaatgattatagttatccgctcacgttcggt-
gctgggaccaagc
ttgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagctgct-
cgagcagtctg
gagctgagctggtaaggcctgggacttcagtgaagatatcctgcaaggcttctggatacgccttcactaa-
ctactggctagg
ttgggtaaagcagaggcctggacatggacttgagtggattggagatattttccctggaagtggtaatatc-
cactacaatgag
aagttcaagggcaaagccacactgactgcagacaaatcttcgagcacagcctatatgcagctcagtagcc-
tgacatttgag
gactctgctgtctatttctgtgcaagactgaggaactgggacgagcctatggactactggggccaaggga-
ccacggtcacc
gtctcctccggaggtggtggatccgaggtgaagcttctcgagtctggaggaggattggtgcagcctggag-
ggtcattgaa
actctcatgtgcagcctctggattcaccttcaatacctacgccatgaactgggtccgccaggctccagga-
aagggtttggaa
tgggttgctcgcataagaagtaaatataataattatgcaacatattatgccgattcagtgaaagacaggt-
tcaccatctccaga
gatgattcaaaaaacactgcctatctacaaatgaacaacttgaaaactgaggacactgccgtgtactact-
gtgtgagacatg
ggaacttcggtaatagctacgtttcctggtttgcttactggggccaagggactctggtcaccgtctcctc-
aggtggtggtggtt
ctggcggcggcggctccggtggtggtggttctgagctcgttgtgactcaggaatctgcactcaccacatc-
acctggtgaaa
cagtcacactcacttgtcgctcaagtactggggctgttacaactagtaactatgccaactgggtccaaga-
aaaaccagatca
tttattcactggtctaataggtggtaccaacaagcgagcaccaggtgtgcctgccagattctcaggctcc-
ctgattggagaca
aggctgccctcaccatcacaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacag-
caacctctgggt gttcggtggaggaaccaaactgactgtcctacatcatcaccatcatcat 126
FN18 VH hybridoma NA
caggtccagctgcagcagtctgaagctgaactggcaagacctggggcctcagtgaagatgtcctgcaaggctt-
ctggcta
cacctttactgactacacgatacactggttaaaacagaggcctggacagggtctggactggattggatat-
tttaatcctagca
gtgaatctactgaatacaatcggaaattcaaggacaggaccatattgactgcagacagatcctcaaccac-
agcctacatgc
aactgagcagcctgacatctgaggactctgcggtctattactgttcaaggaaaggggagaaactacttgg-
taaccgttactg gtacttcgatgtctggggcgcagggacctcggtcaccgtctcctca 127
FN18 VH hybridoma AA
QVQLQQSEAELARPGASVKMSCKASGYTFTDYTIHWLKQRPGQGLDWIGYF
NPSSESTEYNRKFKDRTILTADRSSTTAYMQLSSLTSEDSAVYYCSRKGEKLL
GNRYWYFDVWGAGTSVTVSS 128 FN18 VL hybridoma NA
gacattgtgatgtcacagtctccatcctccctagctgtgtcagttggagagaaggttactatgagctgcaagt-
ccagtcagag
ccttttatatagtagcaatcaaaagaactacttggcctggtaccagcagaagccagggcagtctcctaaa-
ttgctgattaact
gggcatccaccagggaatctggggtccctgatcgcttcacaggcagtggatctaggacagatttcactct-
caccatcagca
gtgtgaaggctgaagacctggcagtttatttctgtcagcaattttatagttatcctccgacgttcggtgg-
aggcaccaagctgg aaatcaaa 129 FN18 VL hybridoma AA
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKL
LINWASTRESGVPDRFTGSGSRTDFTLTISSVKAEDLAVYFCQQFYSYPPTFGG GTKLEIK 130
FN18 VH-VL scFv artificial NA
caggtccagctgcagcagtctgaagctgaactggcaagacctggggcctcagtgaagatgtcctgcaaggctt-
ctggcta
cacctttactgactacacgatacactggttaaaacagaggcctggacagggtctggactggattggatat-
tttaatcctagca
gtgaatctactgaatacaatcggaaattcaaggacaggaccatattgactgcagacagatcctcaaccac-
agcctacatgc
aactgagcagcctgacatctgaggactctgcggtctattactgttcaaggaaaggggagaaactacttgg-
taaccgttactg
gtacttcgatgtctggggcgcagggacctcggtcaccgtctcctcaggtggtggtggttctggcggcggc-
ggctccggtg
gtggtggttctgacattgtgatgtcacagtctccatcctccctagctgtgtcagttggagagaaggttac-
tatgagctgcaagt
ccagtcagagccttttatatagtagcaatcaaaagaactacttggcctggtaccagcagaagccagggca-
gtctcctaaatt
gctgattaactgggcatccaccagggaatctggggtccctgatcgcttcacaggcagtggatctaggaca-
gatttcactctc
accatcagcagtgtgaaggctgaagacctggcagtttatttctgtcagcaattttatagttatcctccga-
cgttcggtggaggc accaagctggaaatcaaa 131 FN18 VH-VL scFv artificial AA
QVQLQQSEAELARPGASVKMSCKASGYTFTDYTIHWLKQRPGQGLDWIGYF
NPSSESTEYNRKFKDRTILTADRSSTTAYMQLSSLTSEDSAVYYCSRKGEKLL
GNRYWYFDVWGAGTSVTVSSGGGGSGGGGSGGGGSDIVMSQSPSSLAVSVG
EKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLINWASTRESGVPDRF
TGSGSRTDFTLTISSVKAEDLAVYFCQQFYSYPPTFGGGTKLEIK 132 FN18 VL-VH scFv
artificial NA
gacattgtgatgtcacagtctccatcctccctagctgtgtcagttggagagaaggttactatgagctgcaagt-
ccagtcagag
ccttttatatagtagcaatcaaaagaactacttggcctggtaccagcagaagccagggcagtctcctaaa-
ttgctgattaact
gggcatccaccagggaatctggggtccctgatcgcttcacaggcagtggatctaggacagatttcactct-
caccatcagca
gtgtgaaggctgaagacctggcagtttatttctgtcagcaattttatagttatcctccgacgttcggtgg-
aggcaccaagctgg
aaatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtccagctgcagca-
gtctgaagct
gaactggcaagacctggggcctcagtgaagatgtcctgcaaggcttctggctacacctttactgactaca-
cgatacactggt
taaaacagaggcctggacagggtctggactggattggatattttaatcctagcagtgaatctactgaata-
caatcggaaattc
aaggacaggaccatattgactgcagacagatcctcaaccacagcctacatgcaactgagcagcctgacat-
ctgaggactc
tgcggtctattactgttcaaggaaaggggagaaactacttggtaaccgttactggtacttcgatgtctgg-
ggcgcagggacc tcggtcaccgtctcctca 133 FN18 VL-VH scFv artificial AA
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKL
LINWASTRESGVPDRFTGSGSRTDFTLTISSVKAEDLAVYFCQQFYSYPPTFGG
GTKLEIKGGGGSGGGGSGGGGSQVQLQQSEAELARPGASVKMSCKASGYTF
TDYTIHWLKQRPGQGLDWIGYFNPSSESTEYNRKFKDRTILTADRSSTTAYM
QLSSLTSEDSAVYYCSRKGEKLLGNRYWYFDVWGAGTSVTVSS 134 CD3 epsilon human
AA QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKN
extracellular cDNA;
IGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD portion
NM_0007 33 135 CD3 epsilon Cyno- AA
QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRL extracellular
molgus FLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMD portion FN18+
cDNA; AB 073993 136 CD3 epsilon Cyno- AA
QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKGDSGDQL extracellular
molgus FLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMD portion FN18-
cDNA; AB 073993 137 EpCAM human AA
QEECVCENYKLAVNCFVNNNRQCQCTSVGAQNTVICSKLAAKCLVMKAEM extracellular
cDNA NGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSTCWCVNTAG portion
VRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDSKSLRTALQKEITTRYQLD
PKFITSILYENNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKK
MDLTVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLK 138 EpCAM chimp AA
QEECVCENYKLAVNCFVNNNHQCQCTSIGAQNTVICSKLAAKCLVMKAEMN extracellular
cDNA GSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSTCWCVNTAGV portion
RRTDKDTEITCSERVRTYWIIIELKHKAREKPYDGKSLRTALQKEITTRYQLDP
KFITNILYENNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKM
DLTVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLK 139 EpCAM rhesus AA IDENTICAL
WITH CYNOMOLGUS EPCAM SHOWN extracellular cDNA IN FIG. 6 AND SEQ ID
NO. 48 portion 142 human CD3 human AA
QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWN gamma cDNA;
LGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELN extracellular NM_0000
portion 73 143 human CD3 delta human AA
FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGT
extracellular cDNA; DIYKDKESTVQVHYRMCQSCVELDPAT portion NM_0007 32
144 cynomolgus CD3 cyno- AA
QSFEENRKLNVYNQEDGSVLLTCHVKNTNITWFKEGKMIDILTAHKNKWNL gamma molgus
GSNTKDPRGVYQCKGSKDKSKTLQVYYRMCQNCIELN extracellular cDNA; portion
AB 073992 145 cynomolgus CD3 cyno- AA
FKIPVEELEDRVFVKCNTSVTWVEGTVGTLLTNNTRLDLGKRILDPRGIYRCN delta molgus
GTDIYKDKESAVQVHYRMCQNCVELDPAT extracellular cDNA; portion AB 073991
146 Human-like VH artificial AA
EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI (SEQ ID NO.
110) RSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRH x murine VL
GNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQESALTT (SEQ ID NO.
148) SPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARF scFv
SGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL 147 Human-like VH
artificial NA
gaggtgcagctgctcgagtctggaggaggattggtgcagcctggagggtcattgaaactctcatgtgcagcct-
ctggattc (SEQ ID NO. 110)
accttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgcataa-
gaagtaaa x murine VL
tataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagagatgattcaaaaa-
acactgcctat (SEQ ID NO. 148)
ctacaaatgaacaacttgaaaactgaggacactgccgtgtactactgtgtgagacatgggaacttcggtaata-
gctacgtttc scFv
ctggtttgcttactggggccaagggactctggtcaccgtctcctcaggtggtggtggttctggcgg-
cggcggctccggtgg
tggtggttctgagctcgttgtgactcaggaatctgcactcaccacatcacctggtgaaacagtcacactc-
acttgtcgctcaa
gtactggggctgttacaactagtaactatgccaactgggtccaagaaaaaccagatcatttattcactgg-
tctaataggtggta
ccaacaagcgagcaccaggtgtgcctgccagattctcaggctccctgattggagacaaggctgccctcac-
catcacaggg
gcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaacctctgggtgttcggtggag-
gaaccaaactga ctgtccta 148 murine VL artificial AA
ELVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGG [identical
TNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGT to VL of KLTVL
SEQ ID NO. 4, with exception of the first two amino acid residues]
149 murine VL artificial NA
Gagctcgttgtgactcaggaatctgcactcaccacatcacctggtgaaacagtcacactcacttgtcgctcaa-
gtactgggg [identical
ctgttacaactagtaactatgccaactgggtccaagaaaaaccagatcatttattcactggtctaataggtgg-
taccaacaag to VL of
cgagcaccaggtgtgcctgccagattctcaggctccctgattggagacaaggctgccctcaccatcacagggg-
cacaga SEQ ID NO. 4,
ctgaggatgaggcaatatatttctgtgctctatggtacagcaacctctgggtgttcggtggaggaaccaaact-
gactgtccta with exception of nucleotides encoding the first two
amino acid residues] 150 CAIX LH scFv artificial NA
gacattgtgatgacccagtctcaaagattcatgtccacaacagtaggagacagggtcagcatcacctgcaagg-
ccagtca
gaatgtggtttctgctgttgcctggtatcaacagaaaccaggacaatctcctaaactactgatttactca-
gcatccaatcggta
cactggagtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccattagcaatatg-
cagtctgaagac
ctggctgattttttctgtcaacaatatagcaactatccgtggacgttcggtggaggcaccaagctggaaa-
tcaaaggtggtgg
tggttctggcggcggcggctccggtggtggtggttctgacgtgaagctcgtggagtctgggggaggctta-
gtgaagcttg
gagggtccctgaaactctcctgtgcagcctctggattcactttcagtaactattacatgtcttgggttcg-
ccagactccagaga
agaggctggagttggtcgcagccattaatagtgatggtggtatcacctactatctagacactgtgaaggg-
ccgattcaccatt
tcaagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccttgt-
tttactgtgcaa
gacaccgctcgggctacttttctatggactactggggtcaaggaacctcagtcaccgtctcctcc
151 CAIX LH scFv artificial AA
DIVMTQSQRFMSTTVGDRVSITCKASQNVVSAVAWYQQKPGQSPKLLIYSAS
NRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADFFCQQYSNYPWTFGGGTKL
EIKGGGGSGGGGSGGGGSDVKLVESGGGLVKLGGSLKLSCAASGFTFSNYY
MSWVRQTPEKRLELVAAINSDGGITYYLDTVKGRFTISRDNAKNTLYLQMSS
LKSEDTALFYCARHRSGYFSMDYWGQGTSVTVSS 152 EGFR21 LH scFv artificial NA
gacattgtgctgacacagtctcctgcttccttacctgtgtctctggggcagagggccaccatctcatgcaggg-
ccagccaaa
gtgtcagttcatctacttatagttatatacactggtaccaacagaaaccaggacagccacccaaactcct-
catcacgtatgcat
ccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcgacat-
ccatcctgtgg
aggaggatgattcttcaacatattactgtcagcacagttgggagattccatttacgttcggctcggggac-
aaagttggaaata
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggttcagctgcagcagtctg-
gacctgatctg
gtgaagcctggggcctcagtgaagatgtcctgcaaggcttctggacacactttcactgactgtgttataa-
tctgggtgaaaca
gagagctggacagggccttgagtggattggacagatttatccagggactggtcgttcttactacaatgag-
attttcaagggc
aaggccacactgactgcagacaaatcctccaacacagtccacattcaactcagcagcctgacatctgagg-
actctgcggtc
tatttctgtgccctatctactatattcacgggacctggttttcttattggggccaagggactctggtcac-
tgtctcttcc 153 EGFR21 LH scFv artificial AA
DIVLTQSPASLPVSLGQRATISCRASQSVSSSTYSYIHWYQQKPGQPPKLLITY
ASNLESGVPARFSGSGSGTDFTLDIHPVEEDDSSTYYCQHSWEIPFTFGSGTKL
EIKGGGGSGGGGSGGGGSQVQLQQSGPDLVKPGASVKMSCKASGHTFTDCV
IIWVKQRAGQGLEWIGQIYPGTGRSYYNEIFKGKATLTADKSSNTVHIQLSSL
TSEDSAVYFCALSTLIHGTWFSYWGQGTLVTVSS 154 EGFRvIII-LH artificial NA
gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagt-
caagtcagagc scFv
ctatatatagtaatggaaaaacctatttgaattggttattacagaggccaggccagtctccaaagc-
gcctaatctatctggtat
ctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacactgaaaat-
cagcagagtgg
aggctgaggatttgggaatttattactgcgtgcaagatacacattttcctcagacattcggtggaggcac-
caagctggaaatc
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtccagctgcaacagtctg-
gacctgagct
gctgaagcctggggcttcagtgaagatatcctgcaagacttctggatacacattcactgaatacaccata-
cactgggtgaag
cagagccatggaaagagccttgagtggattggaggtattgatcctaacaatggtggtactatgtataacc-
aaaaattcaagg
gcaaggccacattgactgtagacaagtcttccagcacagcctacacggacctccgcagcctgacgtctga-
ggattctgca
gtctattactgcacaagagcagaggctatggactactggggtcaaggaacctcagtcaccgtctcctcc
155 EGFRvIIILH- artificial AA
DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLI scFv
YLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCVQDTHFPQTFGGG
TKLEIKGGGGSGGGGSGGGGSEVQLQQSGPELLKPGASVKISCKTSGYTFTEY
TIHWVKQSHGKSLEWIGGIDPNNGGTMYNQKFKGKATLTVDKSSSTAYTDL
RSLTSEDSAVYYCTRAEAMDYWGQGTSVTVSS 156 CAIX LHx artificial NA
gacattgtgatgacccagtctcaaagattcatgtccacaacagtaggagacagggtcagcatcacctgcaagg-
ccagtca SEQ ID NO. 10
gaatgtggtttctgctgttgcctggtatcaacagaaaccaggacaatctcctaaactactgatttactcagca-
tccaatcggta
cactggagtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccattagcaatatg-
cagtctgaagac
ctggctgattttttctgtcaacaatatagcaactatccgtggacgttcggtggaggcaccaagctggaaa-
tcaaaggtggtgg
tggttctggcggcggcggctccggtggtggtggttctgacgtgaagctcgtggagtctgggggaggctta-
gtgaagcttg
gagggtccctgaaactctcctgtgcagcctctggattcactttcagtaactattacatgtcttgggttcg-
ccagactccagaga
agaggctggagttggtcgcagccattaatagtgatggtggtatcacctactatctagacactgtgaaggg-
ccgattcaccatt
tcaagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccttgt-
tttactgtgcaa
gacaccgctcgggctacttttctatggactactggggtcaaggaacctcagtcaccgtctcctccggagg-
tggtggatccg
aggtgaagcttctcgagtctggaggaggattggtgcagcctaaagggtcattgaaactctcatgtgcagc-
ctctggattcac
cttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgcata-
agaagtaaatat
aataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagagatgattcacaaa-
gcattctctatctac
aaatgaacaacttgaaaactgaggacacagccatgtactactgtgtgagacatgggaacttcggtaatag-
ctacgtttcctg
gtttgcttactggggccaagggactctggtcactgtctctgcaggtggtggtggttctggcggcggcggc-
tccggtggtgg
tggttctcaggctgttgtgactcaggaatctgcactcaccacatcacctggtgaaacagtcacactcact-
tgtcgctcaagta
ctggggctgttacaactagtaactatgccaactgggtccaagaaaaaccagatcatttattcactggtct-
aataggtggtacc
aacaagcgagctccaggtgtgcctgccagattctcaggctccctgattggagacaaggctgccctcacca-
tcacaggggc
acagactgaggatgaggcaatatatttctgtgctctatggtacagcaacctctgggtgttcggtggagga-
accaaactgact gtccta 157 CAIX LHx artificial AA
DIVMTQSQRFMSTTVGDRVSITCKASQNVVSAVAWYQQKPGQSPKLLIYSAS SEQ ID NO. 10
NRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADFFCQQYSNYPWTFGGGTKL
EIKGGGGSGGGGSGGGGSDVKLVESGGGLVKLGGSLKLSCAASGFTFSNYY
MSWVRQTPEKRLELVAAINSDGGITYYLDTVKGRFTISRDNAKNTLYLQMSS
LKSEDTALFYCARHRSGYFSMDYWGQGTSVTVSSGGGGSEVKLLESGGGLV
QPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYY
ADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFA
YWGQGTLVTVSAGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRS
STGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTI
TGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL 158 EGFR21 LHx artificial NA
gacattgtgctgacacagtctcctgcttccttacctgtgtctctggggcagagggccaccatctcatgcaggg-
ccagccaaa SEQ ID O. 10
gtgtcagttcatctacttatagttatatacactggtaccaacagaaaccaggacagccacccaaactcctcat-
cacgtatgcat N
ccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcgaca-
tccatcctgtgg
aggaggatgattcttcaacatattactgtcagcacagttgggagattccatttacgttcggctcggggac-
aaagttggaaata
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggttcagctgcagcagtctg-
gacctgatctg
gtgaagcctggggcctcagtgaagatgtcctgcaaggatctggacacactttcactgactgtgttataat-
ctgggtgaaaca
gagagctggacagggccttgagtggattggacagatttatccagggactggtcgttcttactacaatgag-
attttcaagggc
aaggccacactgactgcagacaaatcctccaacacagtccacattcaactcagcagcctgacatctgagg-
actctgcggtc
tatttctgtgccctatctactatattcacgggacctggttttcttattggggccaagggactctggtcac-
tgtctcttccggaggt
ggtggatccgaggtgaagatctcgagtctggaggaggattggtgcagcctaaagggtcattgaaactctc-
atgtgcagcc
tctggattcaccttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatggg-
ttgctcgcataa
gaagtaaatataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagaga-
tgattcacaaagc
attctctatctacaaatgaacaacttgaaaactgaggacacagccatgtactactgtgtgagacatggga-
acttcggtaatag
ctacgtttcctggtttgcttactggggccaagggactctggtcactgtctctgcaggtggtggtggttct-
ggcggcggcggct
ccggtggtggtggttctcaggctgttgtgactcaggaatctgcactcaccacatcacctggtgaaacagt-
cacactcacttgt
cgctcaagtactggggctgttacaactagtaactatgccaactgggtccaagaaaaaccagatcatttat-
tcactggtctaata
ggtggtaccaacaagcgagctccaggtgtgcctgccagattctcaggctccctgattggagacaaggctg-
ccctcaccatc
acaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaacctctgggtgttcg-
gtggaggaacc aaactgactgtccta 159 EGFR21 LHx artificial AA
DIVLTQSPASLPVSLGQRATISCRASQSVSSSTYSYIHWYQQKPGQPPKLLITY SEQ ID NO.
10 ASNLESGVPARFSGSGSGTDFTLDIHPVEEDDSSTYYCQHSWEIPFTFGSGTKL
EIKGGGGSGGGGSGGGGSQVQLQQSGPDLVKPGASVKMSCKASGHTFTDCV
IIWVKQRAGQGLEWIGQIYPGTGRSYYNEIFKGKATLTADKSSNTVHIQLSSL
TSEDSAVYFCALSTLIHGTWFSYWGQGTLVTVSSGGGGSEVKLLESGGGLVQ
PKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAY
WGQGTLVTVSAGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSS
TGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTI
TGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL 160 EGFRvIII-LHx artificial NA
gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagt-
caagtcagagc SEQ ID NO. 10
ctcttatatagtaatggaaaaacctatttgaattggttattacagaggccaggccagtctccaaagcgcctaa-
tctatctggtat
ctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacactgaaaat-
cagcagagtgg
aggctgaggatttgggaatttattactgcgtgcaagatacacattttcctcagacattcggtggaggcac-
caagctggaaatc
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtccagctgcaacagtctg-
gacctgagct
gctgaagcctggggcttcagtgaagatatcctgcaagacttctggatacacattcactgaatacaccata-
cactgggtgaag
cagagccatggaaagagccttgagtggattggaggtattgatcctaacaatggtggtactatgtataacc-
aaaaattcaagg
gcaaggccacattgactgtagacaagtcttccagcacagcctacacggacctccgcagcctgacgtctga-
ggattctgca
gtctattactgcacaagagcagaggctatggactactggggtcaaggaacctcagtcaccgtctcctccg-
gaggtggtgga
tccgaggtgaagcttctcgagtctggaggaggattggtgcagcctaaagggtcattgaaactctcatgtg-
cagcctctggat
tcaccttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcg-
cataagaagta
aatataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagagatgattc-
acaaagcattctcta
tctacaaatgaacaacttgaaaactgaggacacagccatgtactactgtgtgagacatgggaacttcggt-
aatagctacgttt
cctggtttgcttactggggccaagggactctggtcactgtctctgcaggtggtggtggttctggcggcgg-
cggctccggtg
gtggtggttctcaggctgttgtgactcaggaatctgcactcaccacatcacctggtgaaacagtcacact-
cacttgtcgctca
agtactggggctgttacaactagtaactatgccaactgggtccaagaaaaaccagatcatttattcactg-
gtctaataggtggt
accaacaagcgagaccaggtgtgcctgccagattctcaggctccctgattggagacaaggctgccctcac-
catcacagg
ggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaacctctgggtgttcggtgga-
ggaaccaaactg actgtccta 161 EGFRvIII-LHx artificial AA
DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLI SEQ ID NO. 10
YLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCVQDTHFPQTFGGG
TKLEIKGGGGSGGGGSGGGGSEVQLQQSGPELLKPGASVKISCKTSGYTFTEY
TIHWVKQSHGKSLEWIGGIDPNNGGTMYNQKFKGKATLTVDKSSSTAYTDL
RSLTSEDSAVYYCTRAEAMDYWGQGTSVTVSSGGGGSEVKLLESGGGLVQP
KGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
SVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYW
GQGTLVTVSAGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTG
AVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITG
AQTEDEAIYFCALWYSNLWVFGGGTKLTVL 162 anti CD3 (as artificial AA
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYI used in WO
NPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD 99/54440
HYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGE
KVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGT
SYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK 163 deimmunised(di)-
artificial AA DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGY
anti CD3 INPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDD
HYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGE
RATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGT
DYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIK 164 VL: CDR3 murine AA
QQWSRNPPT 165 VL: CDR2 murine AA DSSKLAS 166 VL: CDR1 murine AA
SASSSVSYMN 167 Human-like VL artificial NA
gagctcgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcacactcacttgtcgctcgt-
cgactgggg
ctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccccgtggtctaatagg-
tggtaccaaca
agcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggctgccctcaccactca-
ggggtacag
ccagaggatgaggcagaatattactgtgactatggtacagcaacctctgggtgttcggtggaggaaccaa-
actgactgtc cta 168 Human-like VL artificial AA
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG
TNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGG TKLTVL 169
Human-like VH artificial NA
gaggtgcagctgctcgagtctggaggaggattggtgcagcctggagggtcattgaaactctcatgtgcagcct-
ctggattc (SEQ ID NO. 110)
accttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgcataa-
gaagtaaa x Human-like VL
tataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagagatgattcaaaaa-
acactgcctat (SEQ ID NO. 168)
ctacaaatgaacaacttgaaaactgaggacactgccgtgtactactgtgtgagacatgggaacttcggtaata-
gctacgtttc scFv
ctggtttgcttactggggccaagggactctggtcaccgtctcctcaggtggtggtggttctggcgg-
cggcggctccggtgg
tggtggttctgagctcgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcacactc-
acttgtcgctcgtc
gactggggctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccccgtggt-
ctaataggtg
gtaccaacaagcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggctgccct-
caccctctcag
gggtacagccagaggatgaggcagaatattactgtgctctatggtacagcaacctctgggtgttcggtgg-
aggaaccaaa ctgactgtccta 170 Human-like VH artificial AA
EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI (SEQ ID NO.
110) RSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRH x
Human-like VL GNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTV
(SEQ ID NO. 168)
SPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF scFv
SGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL 171 EGFR HL x
artificial NA
caggtgcagctgcagcagtctgggcctgatctggtgaagcctggggcctcagtgaagatgtcctgcaaggctt-
ctggaca SEQ ID NO. 170
cactttcactgactgtgttataatctgggtgaaacagagagctggacagggccttgagtggattggacagatt-
tatccaggg
actggtcgttcttactacaatgagattttcaagggcaaggccacactgactgcagacaaatcctccaaca-
cagtccacattca
actcagcagcctgacatctgaggactctgcggtctatttctgtgccctatctactcttattcacgggacc-
tggttttcttattggg
gccaagggactctggtcactgtctcttccggtggtggtggttctggcggcggcggctccggtggtggtgg-
ttctgacattgt
actgacccagtctccagcttccttacctgtgtctctggggcagagggccaccatctcatgcagggccagc-
caaagtgtcag
ttcatctacttatagttatatacactggtaccaacagaaaccaggacagccacccaaactcctcatcacg-
tatgcatccaacct
agaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcgacatccatcct-
gtggaggagg
atgattcttcaacatattactgtcagcacagttgggagattccatttacgttcggctcggggacaaagtt-
ggaaataaaatccg
gaggtggtggctccgaggtgcagctggtggagtctggaggaggattggtgcagcctggagggtcattgaa-
actctcatgt
gcagcctctggattcaccttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttgg-
aatgggttgctc
gcataagaagtaaatataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctc-
cagagatgattcaa
aaaacactgcctatctacaaatgaacaacttgaaaactgaggacactgccgtgtactactgtgtgagaca-
tgggaacttcgg
taatagctacgtttcctggtttgatactggggccaagggactctggtcaccgtctcctcaggtggtggtg-
gttctggcggcg
gcggctccggtggtggtggttctcagaccgttgtgactcaggaaccttcactcaccgtatcacctggtgg-
aacagtcacact
cacttgtcgctcgtccactggggctgttacaactagcaactatgccaactgggtccaacaaaaaccaggt-
caggcaccccg
tggtctaataggtggtaccaacaagcgcgcaccaggtactcctgccagattctcaggctccctgcttgga-
ggcaaggctgc
cctcaccctctcaggggtacagccagaggatgaggcagaatattactgtgctctatggtacagcaacctc-
tgggtgttcggt ggaggaaccaaactgactgtcctacatcatcaccatcatcattaggtcgac
172 EGFR HL x artificial AA
QVQLQQSGPDLVKPGASVKMSCKASGHTFTDCVIIWVKQRAGQGLEWIGQI SEQ ID NO. 170
YPGTGRSYYNEIFKGKATLTADKSSNTVHIQLSSLTSEDSAVYFCALSTLIHGT
WFSYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLPVSLGQRATIS
CRASQSVSSSTYSYIHWYQQKPGQPPKLLITYASNLESGVPARFSGSGSGTDF
TLDIHPVEEDDSSTYYCQHSWEIPFTFGSGTKLEIKSGGGGSEVQLVESGGGL
VQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY
YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWF
AYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCR
SSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAA
LTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLHHHHHH*VD 173 EGFR LH x
artificial NA
gacattgtgctgacacagtctcctgcttccttacctgtgtctctggggcagagggccaccatctcatgcaggg-
ccagccaaa SEQ ID NO. 170
gtgtcagttcatctacttatagttatatacactggtaccaacagaaaccaggacagccacccaaactcctcat-
cacgtatgcat
ccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcgacat-
ccatcctgtgg
aggaggatgattcttcaacatattactgtcagcacagttgggagattccatttacgttcggctcggggac-
aaagttggaaata
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggttcagctgcagcagtctg-
gacctgatctg
gtgaagcctggggcctcagtgaagatgtcctgcaaggcttctggacacactttcactgactgtgttataa-
tctgggtgaaaca
gagagctggacagggccttgagtggattggacagatttatccagggactggtcgttcttactacaatgag-
attttcaagggc
aaggccacactgactgcagacaaatcctccaacacagtccacattcaactcagcagcctgacatctgagg-
actctgcggtc
tatttctgtgccctatctactatattcacgggacctggttttcttattggggccaagggactctggtcac-
tgtctcttccggaggt
ggtggctccgaggtgcagctggtggagtctggaggaggattggtgcagcctggagggtcattgaaactct-
catgtgcagc
ctctggattcaccttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatgg-
gttgctcgcata
agaagtaaatataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagag-
atgattcaaaaaac
actgcctatctacaaatgaacaacttgaaaactgaggacactgccgtgtactactgtgtgagacatggga-
acttcggtaata
gctacgtttcctggtttgatactggggccaagggactctggtcaccgtctcctcaggtggtggtggttct-
ggcggcggcgg
ctccggtggtggtggttctcagaccgttgtgactcaggaaccttcactcaccgtatcacctggtggaaca-
gtcacactcactt
gtcgctcgtccactggggctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggc-
accccgtggt
ctaataggtggtaccaacaagcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggca-
aggctgccctc
accctctcaggggtacagccagaggatgaggcagaatattactgtgctctatggtacagcaacctctggg-
tgttcggtgga ggaaccaaactgactgtcctacatcatcaccatcatcattaggtcgac 174
EGFR LH x artificial AA
DIVLTQSPASLPVSLGQRATISCRASQSVSSSTYSYIHWYQQKPGQPPKLLITY SEQ ID NO.
170 ASNLESGVPARFSGSGSGTDFTLDIHPVEEDDSSTYYCQHSWEIPFTFGSGTKL
EIKGGGGSGGGGSGGGGSQVQLQQSGPDLVKPGASVKMSCKASGHTFTDCV
IIWVKQRAGQGLEWIGQIYPGTGRSYYNEIFKGKATLTADKSSNTVHIQLSSL
TSEDSAVYFCALSTLIHGTWFSYWGQGTLVTVSSGGGGSEVQLVESGGGLVQ
PGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYV
SWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL
TCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGK
AALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLHHHHHH*VD 175 EGFR HL x
artificial NA
caggtgcagctgcagcagtctgggcctgatctggtgaagcctggggcctcagtgaagatgtcctgcaaggctt-
ctggaca SEQ ID NO. 194
cactttcactgactgtgttataatctgggtgaaacagagagctggacagggccttgagtggattggacagatt-
tatccaggg
actggtcgttcttactacaatgagattttcaagggcaaggccacactgactgcagacaaatcctccaaca-
cagtccacattca
actcagcagcctgacatctgaggactctgcggtctatttctgtgccctatctactcttattcacgggacc-
tggttttcttattggg
gccaagggactctggtcactgtctcttccggtggtggtggttctggcggcggcggctccggtggtggtgg-
ttctgacattgt
actgacccagtctccagcttccttacctgtgtctctggggcagagggccaccatctcatgcagggccagc-
caaagtgtcag
ttcatctacttatagttatatacactggtaccaacagaaaccaggacagccacccaaactcctcatcacg-
tatgcatccaacct
agaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcgacatccatcct-
gtggaggagg
atgattcttcaacatattactgtcagcacagttgggagattccatttacgttcggctcggggacaaagtt-
ggaaataaaatccg
gaggtggtggctcccagaccgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcac-
actcacttgtcg
ctcgtccactggggctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccc-
cgtggtctaat
aggtggtaccaacaagcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggct-
gccctcaccc
tctcaggggtacagccagaggatgaggcagaatattactgtgctctatggtacagcaacctctgggtgtt-
cggtggaggaa
ccaaactgactgtcctaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgca-
gctggtggagt
ctggaggaggattggtgcagcctggagggtcattgaaactctcatgtgcagcctctggattcaccttcaa-
tacctacgccat
gaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgcataagaagtaaatataataat-
tatgcaacatat
tatgccgattcagtgaaagacaggttcaccatctccagagatgattcaaaaaacactgcctatctacaaa-
tgaacaacttgaa
aactgaggacactgccgtgtactactgtgtgagacatgggaacttcggtaatagctacgtttcctggttt-
gcttactggggcc aagggactctggtcaccgtctcctcacatcatcaccatcatcattaggtcgac
176 EGFR HL x artificial AA
QVQLQQSGPDLVKPGASVKMSCKASGHTFTDCVIIWVKQRAGQGLEWIGQI SEQ ID NO. 194
YPGTGRSYYNEIFKGKATLTADKSSNTVHIQLSSLTSEDSAVYFCALSTLIHGT
WFSYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLPVSLGQRATIS
CRASQSVSSSTYSYIHWYQQKPGQPPKLLITYASNLESGVPARFSGSGSGTDF
TLDIHPVEEDDSSTYYCQHSWEIPFTFGSGTKLEIKSGGGGSQTVVTQEPSLTV
SPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF
SGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTV
LGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMN
WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQM
NNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSHHHHHH*VD 177 EGFR LH x
artificial NA
gacattgtgctgacacagtctcctgcttccttacctgtgtctctggggcagagggccaccatctcatgcaggg-
ccagccaaa SEQ ID NO. 194
gtgtcagttcatctacttatagttatatacactggtaccaacagaaaccaggacagccacccaaactcctcat-
cacgtatgcat
ccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcgacat-
ccatcctgtgg
aggaggatgattcttcaacatattactgtcagcacagttgggagattccatttacgttcggctcggggac-
aaagttggaaata
aaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggttcagctgcagcagtctg-
gacctgatctg
gtgaagcctggggcctcagtgaagatgtcctgcaaggcttctggacacactttcactgactgtgttataa-
tctgggtgaaaca
gagagctggacagggccttgagtggattggacagatttatccagggactggtcgttcttactacaatgag-
attttcaagggc
aaggccacactgactgcagacaaatcctccaacacagtccacattcaactcagcagcctgacatctgagg-
actctgcggtc
tatttctgtgccctatctactatattcacgggacctggttttcttattggggccaagggactctggtcac-
tgtctcttcctccgga
ggtggtggctcccagaccgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcacac-
tcacttgtcgct
cgtccactggggctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccccg-
tggtctaatag
gtggtaccaacaagcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggctgc-
cctcaccctct
caggggtacagccagaggatgaggcagaatattactgtgctctatggtacagcaacctctgggtgttcgg-
tggaggaacc
aaactgactgtcctaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagc-
tggtggagtct
ggaggaggattggtgcagcctggagggtcattgaaactctcatgtgcagcctctggattcaccttcaata-
cctacgccatga
actgggtccgccaggctccaggaaagggtttggaatgggttgctcgcataagaagtaaatataataatta-
tgcaacatattat
gccgattcagtgaaagacaggttcaccatctccagagatgattcaaaaaacactgcctatctacaaatga-
acaacttgaaaa
ctgaggacactgccgtgtactactgtgtgagacatgggaacttcggtaatagctacgtttcctggtttgc-
ttactggggccaa gggactctggtcaccgtctcctcacatcatcaccatcatcattaggtcgac
178 EGFR LH x artificial AA
DIVLTQSPASLPVSLGQRATISCRASQSVSSSTYSYIHWYQQKPGQPPKLLITY SEQ ID NO.
194 ASNLESGVPARFSGSGSGTDFTLDIHPVEEDDSSTYYCQHSWEIPFTFGSGTKL
EIKGGGGSGGGGSGGGGSQVQLQQSGPDLVKPGASVKMSCKASGHTFTDCV
IIWVKQRAGQGLEWIGQIYPGTGRSYYNEIFKGKATLTADKSSNTVHIQLSSL
TSEDSAVYFCALSTLIHGTWFSYWGQGTLVTVSSSGGGGSQTVVTQEPSLTV
SPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF
SGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTV
LGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMN
WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQM
NNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSHHHHHH*VD 179 SEQ ID NO.
170 artificial NA
gaggtgcagctggtggagtctggaggaggattggtgcagcctggagggtcattgaaactctcatgtgcagcct-
ctggattc x EGFR HL
accttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgcataa-
gaagtaaa
tataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagagatgattcaa-
aaaacactgcctat
ctacaaatgaacaacttgaaaactgaggacactgccgtgtactactgtgtgagacatgggaacttcggta-
atagctacgtttc
ctggtttgcttactggggccaagggactctggtcaccgtctcctcaggtggtggtggttctggcggcggc-
ggctccggtgg
tggtggttctcagaccgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcacactc-
acttgtcgctcgtc
cactggggctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccccgtggt-
ctaataggtg
gtaccaacaagcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggctgccct-
caccctctcag
gggtacagccagaggatgaggcagaatattactgtgctctatggtacagcaacctctgggtgttcggtgg-
aggaaccaaa
ctgactgtcctatccggaggtggtggctcccaggtgcagctgcagcagtctgggcctgatctggtgaagc-
ctggggcctc
agtgaagatgtcctgcaaggatctggacacactttcactgactgtgttataatctgggtgaaacagagag-
ctggacagggc
cttgagtggattggacagatttatccagggactggtcgttcttactacaatgagattttcaagggcaagg-
ccacactgactgc
agacaaatcctccaacacagtccacattcaactcagcagcctgacatctgaggactctgcggtctatttc-
tgtgccctatctac
tcttattcacgggacctggttttcttattggggccaagggactctggtcactgtctcttccggtggtggt-
ggttctggcggcgg
cggctccggtggtggtggttctgacattgtactgacccagtctccagcttccttacctgtgtctctgggg-
cagagggccacc
atctcatgcagggccagccaaagtgtcagttcatctacttatagttatatacactggtaccaacagaaac-
caggacagccac
ccaaactcctcatcacgtatgcatccaacctagaatctggggtccctgccaggttcagtggcagtgggtc-
tgggacagactt
caccctcgacatccatcctgtggaggaggatgattcttcaacatattactgtcagcacagttgggagatt-
ccatttacgttcgg ctcggggacaaagttggaaataaaacatcatcaccatcatcattaggtcgac
180 SEQ ID NO. 170 artificial AA
EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI x EGFR HL
RSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLT
VSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPAR
FSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLSGGGG
SQVQLQQSGPDLVKPGASVKMSCKASGHTFTDCVIIWVKQRAGQGL
EWIGQIYPGTGRSYYNEIFKGKATLTADKSSNTVHIQLSSLTSEDSAVYFCALS
TLIHGTWFSYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLPVSLG
QRATISCRASQSVSSSTYSYIHWYQQKPGQPPKLLITYASNLESGVPARFSGSG
SGTDFTLDIHPVEEDDSSTYYCQHSWEIPFTFGSGTKLEIKHHHHHH*VD 181 SEQ ID NO.
194 artificial NA
cagaccgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcacactcacttgtcgctcgt-
ccactgggg x EGFR HL
ctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccccgtggtctaataggtgg-
taccaaca
agcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggctgccctcaccctctc-
aggggtacag
ccagaggatgaggcagaatattactgtgctctatggtacagcaacctctgggtgttcggtggaggaacca-
aactgactgtc
ctaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagctggtggagtctg-
gaggaggatt
ggtgcagcctggagggtcattgaaactctcatgtgcagcctctggattcaccttcaatacctacgccatg-
aactgggtccgc
caggctccaggaaagggtttggaatgggttgctcgcataagaagtaaatataataattatgcaacatatt-
atgccgattcagt
gaaagacaggttcaccatctccagagatgattcaaaaaacactgcctatctacaaatgaacaacttgaaa-
actgaggacact
gccgtgtactactgtgtgagacatgggaacttcggtaatagctacgtttcctggtttgcttactggggcc-
aagggactctggt
caccgtctcctcatccggaggtggtggctcccaggtgcagctgcagcagtctgggcctgatctggtgaag-
cctggggcct
cagtgaagatgtcctgcaaggcttctggacacactttcactgactgtgttataatctgggtgaaacagag-
agctggacaggg
ccttgagtggattggacagatttatccagggactggtcgttcttactacaatgagattttcaagggcaag-
gccacactgactg
cagacaaatcctccaacacagtccacattcaactcagcagcctgacatctgaggactctgcggtctattt-
ctgtgccctatcta
ctcttattcacgggacctggrtttcttattggggccaagggactctggtcactgtctcttccggtggtgg-
tggttctggcggcg
gcggctccggtggtggtggttctgacattgtactgacccagtctccagcttccttacctgtgtctctggg-
gcagagggccac
catctcatgcagggccagccaaagtgtcagttcatctacttatagttatatacactggtaccaacagaaa-
ccaggacagcca
cccaaactcctcatcacgtatgcatccaacctagaatctggggtccctgccaggttcagtggcagtgggt-
ctgggacagac
ttcaccctcgacatccatcctgtggaggaggatgattcttcaacatattactgtcagcacagttgggaga-
ttccatttacgttcg
gctcggggacaaagttggaaataaaacatcatcaccatcatcattaggtcgac 182 SEQ ID
NO. 194 artificial AA
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG x EGFR HL
TNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGG
TKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNT
YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTA
YLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSSGGGGS
QVQLQQSGPDLVKPGASVKMSCKASGHTFTDCVIIWVKQRAGQGL
EWIGQIYPGTGRSYYNEIFKGKATLTADKSSNTVHIQLSSLTSEDSAVYFCALS
TLIHGTWFSYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLPVSLG
QRATISCRASQSVSSSTYSYIHWYQQKPGQPPKLLITYASNLESGVPARFSGSG
SGTDFTLDIHPVEEDDSSTYYCQHSWEIPFTFGSGTKLEIKHHHHHH*VD 183 SEQ ID NO.
170 artificial NA
gaggtgcagctggtggagtctggaggaggattggtgcagcctggagggtcattgaaactctcatgtgcagcct-
ctggattc x EGFR LH
accttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgcataa-
gaagtaaa
tataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagagatgattcaa-
aaaacactgcctat
ctacaaatgaacaacttgaaaactgaggacactgccgtgtactactgtgtgagacatgggaacttcggta-
atagctacgtttc
ctggtttgcttactggggccaagggactctggtcaccgtctcctcaggtggtggtggttctggcggcggc-
ggctccggtgg
tggtggttctcagaccgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcacactc-
acttgtcgctcgtc
cactggggctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccccgtggt-
ctaataggtg
gtaccaacaagcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggctgccct-
caccctctcag
gggtacagccagaggatgaggcagaatattactgtgctctatggtacagcaacctctgggtgttcggtgg-
aggaaccaaa
ctgactgtcctatccggaggtggtggctccgacattgtgctgacacagtctcctgcttccttacctgtgt-
ctctggggcagag
ggccaccatctcatgcagggccagccaaagtgtcagttcatctacttatagttatatacactggtaccaa-
cagaaaccagga
cagccacccaaactcctcatcacgtatgcatccaacctagaatctggggtccctgccaggttcagtggca-
gtgggtctggg
acagacttcaccctcgacatccatcctgtggaggaggatgattcttcaacatattactgtcagcacagtt-
gggagattccattt
acgttcggctcggggacaaagttggaaataaaaggtggtggtggttctggcggcggcggctccggtggtg-
gtggttctca
ggttcagctgcagcagtctggacctgatctggtgaagcctggggcctcagtgaagatgtcctgcaaggct-
tctggacacac
tttcactgactgtgttataatctgggtgaaacagagagctggacagggccttgagtggattggacagatt-
tatccagggactg
gtcgttcttactacaatgagattttcaagggcaaggccacactgactgcagacaaatcctccaacacagt-
ccacattcaactc
agcagcctgacatctgaggactctgcggtctatttctgtgccctatctactcttattcacgggacctggt-
tttcttattggggcca
agggactctggtcactgtctcttcccatcatcaccatcatcattaggtcgac 184 SEQ ID NO.
170 artificial AA
EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI x EGFR LH
RSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLT
VSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPAR
FSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLSGGGG
SDIVLTQSPASLPVSLGQRATISCRASQSVSSSTYSYIHWYQQKPGQPPKLLIT
YASNLESGVPARFSGSGSGTDFTLDIHPVEEDDSSTYYCQHSWEIPFTFGSG
TKLEIKGGGGSGGGGSGGGGSQVQLQQSGPDLVKPGASVKMSCKASGHTFT
DCVIIWVKQRAGQGLEWIGQIYPGTGRSYYNEIFKGKATLTADKSSNTVHIQL
SSLTSEDSAVYFCALSTLIHGTWFSYWGQGTLVTVSSHHHHHH*VD 185 SEQ ID NO. 194
artificial NA
cagaccgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcacactcacttgtcgctcgt-
ccactgggg x EGFR LH
ctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccccgtggtctaataggtgg-
taccaaca
agcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggctgccctcaccctctc-
aggggtacag
ccagaggatgaggcagaatattactgtgctctatggtacagcaacctctgggtgttcggtggaggaacca-
aactgactgtc
ctaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagctggtggagtctg-
gaggaggatt
ggtgcagcctggagggtcattgaaactctcatgtgcagcctctggattcaccttcaatacctacgccatg-
aactgggtccgc
caggctccaggaaagggtttggaatgggttgctcgcataagaagtaaatataataattatgcaacatatt-
atgccgattcagt
gaaagacaggttcaccatctccagagatgattcaaaaaacactgcctatctacaaatgaacaacttgaaa-
actgaggacact
gccgtgtactactgtgtgagacatgggaacttcggtaatagctacgtttcctggtttgcttactggggcc-
aagggactctggt
caccgtctcctcatccggaggtggtggctccgacattgtgctgacacagtctcctgcttccttacctgtg-
tctctggggcaga
gggccaccatctcatgcagggccagccaaagtgtcagttcatctacttatagttatatacactggtacca-
acagaaaccagg
acagccacccaaactcctcatcacgtatgcatccaacctagaatctggggtccctgccaggttcagtggc-
agtgggtctgg
gacagacttcaccctcgacatccatcctgtggaggaggatgattcttcaacatattactgtcagcacagt-
tgggagattccatt
tacgttcggctcggggacaaagttggaaataaaaggtggtggtggttctggcggcggcggctccggtggt-
ggtggttctc
aggttcagctgcagcagtctggacctgatctggtgaagcctggggcctcagtgaagatgtcctgcaaggc-
ttctggacaca
ctttcactgactgtgttataatctgggtgaaacagagagctggacagggccttgagtggattggacagat-
ttatccagggact
ggtcgttcttactacaatgagattttcaagggcaaggccacactgactgcagacaaatcctccaacacag-
tccacattcaact
cagcagcctgacatctgaggactctgcggtctatttctgtgccctatctactcttattcacgggacctgg-
ttttcttattggggcc
aagggactctggtcactgtctcttcccatcatcaccatcatcattaggtcgac 186 SEQ ID
NO. 194 artificial AA
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG x EGFR LH
TNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGG
TKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNT
YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTA
YLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSSGGGGS
DIVLTQSPASLPVSLGQRATISCRASQSVSSSTYSYIHWYQQKPGQPPKLLITY
ASNLESGVPARFSGSGSGTDFTLDIHPVEEDDSSTYYCQHSWEIPFTFGSG
TKLEIKGGGGSGGGGSGGGGSQVQLQQSGPDLVKPGASVKMSCKASGHTFT
DCVIIWVKQRAGQGLEWIGQIYPGTGRSYYNEIFKGKATLTADKSSNTVHIQL
SSLTSEDSAVYFCALSTLIHGTWFSYWGQGTLVTVSSHHHHHH*VD 187 CAIX HL x
artificial NA
gacgtgaagctcgtggagtctgggggaggcttagtgaagcttggagggtccctgaaactctcctgtgcagcct-
ctggattc SEQ ID NO. 194
actttcagtaactattacatgtcttgggttcgccagactccagagaagaggctggagttggtcgcagccatta-
atagtgatgg
tggtatcacctactatctagacactgtgaagggccgattcaccatttcaagagacaatgccaagaacacc-
ctgtacctgcaa
atgagcagtctgaagtctgaggacacagccttgttttactgtgcaagacaccgctcgggctacttttcta-
tggactactgggg
tcaaggaacctcagtcaccgtctcctcaggtggtggtggttctggcggcggcggctccggtggtggtggt-
tctgacattgtg
atgacccagtctcaaagattcatgtccacaacagtaggagacagggtcagcatcacctgcaaggccagtc-
agaatgtggtt
tctgctgttgcctggtatcaacagaaaccaggacaatctcctaaactactgatttactcagcatccaatc-
ggtacactggagtc
cctgatcgcttcacaggcagtggatctgggacagatttcactctcaccattagcaatatgcagtctgaag-
acctggctgatttt
ttctgtcaacaatatagcaactatccgtggacgttcggtggaggcaccaagctggaaatcaaatccggag-
gtggtggctcc
cagaccgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcacactcacttgtcgct-
cgtccactgggg
ctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccccgtggtctaatagg-
tggtaccaaca
agcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggctgccctcaccctctc-
aggggtacag
ccagaggatgaggcagaatattactgtgctctatggtacagcaacctctgggtgttcggtggaggaacca-
aactgactgtc
ctaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagctggtggagtctg-
gaggaggatt
ggtgcagcctggagggtcattgaaactctcatgtgcagcctctggattcaccttcaatacctacgccatg-
aactgggtccgc
caggctccaggaaagggtttggaatgggttgctcgcataagaagtaaatataataattatgcaacatatt-
atgccgattcagt
gaaagacaggttcaccatctccagagatgattcaaaaaacactgcctatctacaaatgaacaacttgaaa-
actgaggacact
gccgtgtactactgtgtgagacatgggaacttcggtaatagctacgtttcctggtttgcttactggggcc-
aagggactctggt caccgtctcctcacatcatcaccatcatcattaggtcgac 188 CAIX HL
x artificial AA DVKLVESGGGLVKLGGSLKLSCAASGFTFSNYYMSWVRQTPEKRLELVAAI
SEQ ID NO. 194 NSDGGITYYLDTVKGRFTISRDNAKNTLYLQMSSLKSEDTALFYCARHRSGY
FSMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSQRFMSTTVGDRVS
ITCKASQNVVSAVAWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFT
LTISNMQSEDLADFFCQQYSNYPWTFGGGTKLEIKSGGGGSQTVVTQEPSLT
VSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPAR
FSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGG
SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQ
APGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKT
EDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSHHHHHH*VD 189 CAIX HL x
artificial NA
gacgtgaagctcgtggagtctgggggaggcttagtgaagcttggagggtccctgaaactctcctgtgcagcct-
ctggattc SEQ ID NO. 170
actttcagtaactattacatgtcttgggttcgccagactccagagaagaggctggagttggtcgcagccatta-
atagtgatgg
tggtatcacctactatctagacactgtgaagggccgattcaccatttcaagagacaatgccaagaacacc-
ctgtacctgcaa
atgagcagtctgaagtctgaggacacagccttgttttactgtgcaagacaccgctcgggctacttttcta-
tggactactgggg
tcaaggaacctcagtcaccgtctcctcaggtggtggtggttctggcggcggcggctccggtggtggtggt-
tctgacattgtg
atgacccagtctcaaagattcatgtccacaacagtaggagacagggtcagcatcacctgcaaggccagtc-
agaatgtggtt
tctgctgttgcctggtatcaacagaaaccaggacaatctcctaaactactgatttactcagcatccaatc-
ggtacactggagtc
cctgatcgcttcacaggcagtggatctgggacagatttcactctcaccattagcaatatgcagtctgaag-
acctggctgatttt
ttctgtcaacaatatagcaactatccgtggacgttcggtggaggcaccaagctggaaatcaaatccggag-
gtggtggctcc
gaggtgcagctggtggagtctggaggaggattggtgcagcctggagggtcattgaaactctcatgtgcag-
cctctggattc
accttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgca-
taagaagtaaa
tataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagagatgattcaa-
aaaacactgcctat
ctacaaatgaacaacttgaaaactgaggacactgccgtgtactactgtgtgagacatgggaacttcggta-
atagctacgtttc
ctggtttgcttactggggccaagggactctggtcaccgtctcctcaggtggtggtggttctggcggcggc-
ggctccggtgg
tggtggttctcagaccgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcacactc-
acttgtcgctcgtc
cactggggctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccccgtggt-
ctaataggtg
gtaccaacaagcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggctgccct-
caccctctcag
gggtacagccagaggatgaggcagaatattactgtgctctatggtacagcaacctctgggtgttcggtgg-
aggaaccaaa ctgactgtcctacatcatcaccatcatcattaggtcgac 190 CAIX HL x
artificial AA DVKLVESGGGLVKLGGSLKLSCAASGFTFSNYYMSWVRQTPEKRLELVAAI
SEQ ID NO. 170 NSDGGITYYLDTVKGRFTISRDNAKNTLYLQMSSLKSEDTALFYCARHRSGY
FSMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSQRFMSTTVGDRVS
ITCKASQNVVSAVAWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFT
LTISNMQSEDLADFFCQQYSNYPWTFGGGTKLEIKSGGGGSEVQLVESGGGL
VQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY
YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWF
AYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCR
SSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAA
LTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLHHHHHH*VD 191 CAIX LH x
artificial NA
gacattgtgatgacccagtctcaaagattcatgtccacaacagtaggagacagggtcagcatcacctgcaagg-
ccagtca SEQ ID NO. 170
gaatgtggtttctgctgttgcctggtatcaacagaaaccaggacaatctcctaaactactgatttactcagca-
tccaatcggta
cactggagtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccattagcaatatg-
cagtctgaagac
ctggctgattttttctgtcaacaatatagcaactatccgtggacgttcggtggaggcaccaagctggaaa-
tcaaaggtggtgg
tggttctggcggcggcggctccggtggtggtggttctgacgtgaagctcgtggagtctgggggaggctta-
gtgaagcttg
gagggtccctgaaactctcctgtgcagcctctggattcactttcagtaactattacatgtcttgggttcg-
ccagactccagaga
agaggctggagttggtcgcagccattaatagtgatggtggtatcacctactatctagacactgtgaaggg-
ccgattcaccatt
tcaagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccttgt-
tttactgtgcaa
gacaccgctcgggctacttttctatggactactggggtcaaggaacctcagtcaccgtctcctcctccgg-
aggtggtggctc
cgaggtgcagctggtggagtctggaggaggattggtgcagcctggagggtcattgaaactctcatgtgca-
gcctctggatt
caccttcaatacctacgccatgaactgggtccgccaggctccaggaaagggtttggaatgggttgctcgc-
ataagaagtaa
atataataattatgcaacatattatgccgattcagtgaaagacaggttcaccatctccagagatgattca-
aaaaacactgccta
tctacaaatgaacaacttgaaaactgaggacactgccgtgtactactgtgtgagacatgggaacttcggt-
aatagctacgttt
cctggtttgcttactggggccaagggactctggtcaccgtctcctcaggtggtggtggttctggcggcgg-
cggctccggtg
gtggtggttctcagaccgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcacact-
cacttgtcgctcg
tccactggggctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccccgtg-
gtctaataggt
ggtaccaacaagcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggctgccc-
tcaccctctca
ggggtacagccagaggatgaggcagaatattactgtgctctatggtacagcaacctctgggtgttcggtg-
gaggaaccaa actgactgtcctacatcatcaccatcatcattaggtcgac 192 CAIX LH x
artificial AA DIVMTQSQRFMSTTVGDRVSITCKASQNVVSAVAWYQQKPGQSPKLLIYSAS
SEQ ID NO. 170 NRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADFFCQQYSNYPWTFGGGTKL
EIKGGGGSGGGGSGGGGSDVKLVESGGGLVKLGGSLKLSCAASGFTFSNYY
MSWVRQTPEKRLELVAAINSDGGITYYLDTVKGRFTISRDNAKNTLYLQMSS
LKSEDTALFYCARHRSGYFSMDYWGQGTSVTVSSSGGGGSEVQLVESGGGL
VQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY
YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWF
AYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCR
SSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAA
LTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLHHHHHH*VD 193 Human-like VL
artificial NA
cagaccgttgtgactcaggaaccttcactcaccgtatcacctggtggaacagtcacactcacttgtcgctcgt-
ccactgggg (SEQ ID NO. 168)
ctgttacaactagcaactatgccaactgggtccaacaaaaaccaggtcaggcaccccgtggtctaataggtgg-
taccaaca x Human-like VH
agcgcgcaccaggtactcctgccagattctcaggctccctgcttggaggcaaggctgccctcaccctctcagg-
ggtacag (SEQ ID NO. 110)
ccagaggatgaggcagaatattactgtgctctatggtacagcaacctctgggtgttcggtggaggaaccaaac-
tgactgtc scFv
ctaggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagctggtggag-
tctggaggaggatt
ggtgcagcctggagggtcattgaaactctcatgtgcagcctctggattcaccttcaatacctacgccatg-
aactgggtccgc
caggctccaggaaagggtttggaatgggttgctcgcataagaagtaaatataataattatgcaacatatt-
atgccgattcagt
gaaagacaggttcaccatctccagagatgattcaaaaaacactgcctatctacaaatgaacaacttgaaa-
actgaggacact
gccgtgtactactgtgtgagacatgggaacttcggtaatagctacgtttcctggtttgcttactggggcc-
aagggactctggt caccgtctcctca 194 Human-like VL artificial AA
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG (SEQ ID NO.
168) TNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGG x
Human-like VH TKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNT
(SEQ ID NO. 110) YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTA
scFv YLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 195 epitope
artificial AA EFSELEQSGYYVC 196 epitope artificial AA EFSELEQSGYYVK
197 5' EGFR XbaI artificial NA GGTCTAGAGCATGCGACCCTCCGGGACGGCCGGG
198 3' EGFR SalI artificial NA TTTTAAGTCGACTCATGCTCCAATAAATTCACTGCT
199 epitope artificial AA QDGNEEMGSITQT 200 epitope artificial AA
YYVSYPRGSNPED 201 epitope artificial AA EFSEMEQSGYYVC 202 epitope
artificial AA FSEXE; X as in SEQ ID NO: 204 203 epitope artificial
AA QYPGSEILWQHND 204 epitope artificial AA FSEXE, wherein X
represents L (Leucine) or M (Methionine) 205 epitope artificial AA
FSELE 206 epitope artificial AA FSEME 207 epitope artificial AA
EFSEXEQSGYYVC, wherein X represents L (Leucine) or M (Methionine)
Abbreviations: scFv = single chain Fv AA = amino acid sequence NA =
nuclei acid sequence L = VL region H = VH region Single letter code
as used in the sequence listing: B = C or G or T D = A or G or T H
= A or C or T K = G or T M = A or C N = A or C or G or T R = A or G
S = C or G V = A or C or G W = A or T Y = C or T
Sequence CWU 1
1
2071375DNAmus musculusmisc_feature(1)..(375)VH region 1gaggtgaagc
ttctcgagtc tggaggagga ttggtgcagc ctaaagggtc attgaaactc 60tcatgtgcag
cctctggatt caccttcaat acctacgcca tgaactgggt ccgccaggct
120ccaggaaagg gtttggaatg ggttgctcgc ataagaagta aatataataa
ttatgcaaca 180tattatgccg attcagtgaa agacaggttc accatctcca
gagatgattc acaaagcatt 240ctctatctac aaatgaacaa cttgaaaact
gaggacacag ccatgtacta ctgtgtgaga 300catgggaact tcggtaatag
ctacgtttcc tggtttgctt actggggcca agggactctg 360gtcactgtct ctgca
3752125PRTmus musculusmisc_feature(1)..(125)VH region 2Glu Val Lys
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25
30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr
Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp
Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr
Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe
Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ala 115 120 125 3327DNAMUS
MUSCULUSmisc_feature(1)..(327)VL region 3caggctgttg tgactcagga
atctgcactc accacatcac ctggtgaaac agtcacactc 60acttgtcgct caagtactgg
ggctgttaca actagtaact atgccaactg ggtccaagaa 120aaaccagatc
atttattcac tggtctaata ggtggtacca acaagcgagc tccaggtgtg
180cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat
cacaggggca 240cagactgagg atgaggcaat atatttctgt gctctatggt
acagcaacct ctgggtgttc 300ggtggaggaa ccaaactgac tgtccta
3274109PRTmus musculusmisc_feature(1)..(109)VL region 4Gln Ala Val
Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu 1 5 10 15 Thr
Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25
30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly
35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala
Arg Phe 50 55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr
Ile Thr Gly Ala 65 70 75 80 Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys
Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 5363DNAMUS
MUSCULUSmisc_feature(1)..(363)VH region 5caggtccagc tgcagcagtc
tggggctgaa ctggcaagac ctggggcctc agtgaagatg 60tcctgcaagg cttctggcta
cacctttact agatctacga tgcactgggt aaaacagagg 120cctggacagg
gtctggaatg gattggatac attaatccta gcagtgctta tactaattac
180aatcagaaat tcaaggacaa ggccacattg actgcagaca aatcctccag
tacagcctac 240atgcaactga gtagcctgac atctgaggac tctgcagtct
attactgtgc aagtccgcaa 300gtccactatg attacaacgg gtttccttac
tggggccaag ggactctggt cactgtctct 360gca 3636121PRTmus
musculusmisc_feature(1)..(121)VH region 6Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Ser 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 Ser Ala Tyr Thr Asn Tyr Asn Gln Lys Phe 50
55 60 Lys Asp Lys Ala Thr Leu Thr Ala 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 Ser Pro Gln Val His Tyr Asp Tyr Asn Gly
Phe Pro Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ala
115 120 7318DNAMUS MUSCULUSmisc_feature(1)..(318)VL region
7caagttgttc tcacccagtc tccagcaatc atgtctgcat ttccagggga gaaggtcacc
60atgacctgca gtgccagctc aagtgtaagt tacatgaact ggtaccagca gaagtcaggc
120acctccccca aaagatggat ttatgactca tccaaactgg cttctggagt
ccctgctcgc 180ttcagtggca gtgggtctgg gacctcttat tctctcacaa
tcagcagcat ggagactgaa 240gatgctgcca cttattactg ccagcagtgg
agtcgtaacc cacccacgtt cggagggggg 300accaagctac aaattaca
3188106PRTmus musculusmisc_feature(1)..(106)VL region 8Gln Val Val
Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Phe Pro Gly 1 5 10 15 Glu
Lys Val Thr Met Thr Cys Ser 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 Ser Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser
Met Glu Thr Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
Ser Arg Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Gln
Ile Thr 100 105 9747DNAARTIFICIAL SEQUENCEVH-VL scFV single chain
Fv 9gaggtgaagc ttctcgagtc tggaggagga ttggtgcagc ctaaagggtc
attgaaactc 60tcatgtgcag cctctggatt caccttcaat acctacgcca tgaactgggt
ccgccaggct 120ccaggaaagg gtttggaatg ggttgctcgc ataagaagta
aatataataa ttatgcaaca 180tattatgccg attcagtgaa agacaggttc
accatctcca gagatgattc acaaagcatt 240ctctatctac aaatgaacaa
cttgaaaact gaggacacag ccatgtacta ctgtgtgaga 300catgggaact
tcggtaatag ctacgtttcc tggtttgctt actggggcca agggactctg
360gtcactgtct ctgcaggtgg tggtggttct ggcggcggcg gctccggtgg
tggtggttct 420caggctgttg tgactcagga atctgcactc accacatcac
ctggtgaaac agtcacactc 480acttgtcgct caagtactgg ggctgttaca
actagtaact atgccaactg ggtccaagaa 540aaaccagatc atttattcac
tggtctaata ggtggtacca acaagcgagc tccaggtgtg 600cctgccagat
tctcaggctc cctgattgga gacaaggctg ccctcaccat cacaggggca
660cagactgagg atgaggcaat atatttctgt gctctatggt acagcaacct
ctgggtgttc 720ggtggaggaa ccaaactgac tgtccta 74710249PRTartificial
sequenceVH-VL scFv single chain Fv 10Glu Val Lys Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile
65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala
Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ala Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gln Ala Val Val 130 135 140 Thr Gln Glu Ser Ala Leu
Thr Thr Ser Pro Gly Glu Thr Val Thr Leu 145 150 155 160 Thr Cys Arg
Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 165 170 175 Trp
Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly 180 185
190 Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu
195 200 205 Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr
Glu Asp 210 215 220 Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
Leu Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Thr Val Leu
245 11747DNAARTIFICIAL SEQUENCEVL-VH scFv single chain Fv
11caggctgttg tgactcagga atctgcactc accacatcac ctggtgaaac agtcacactc
60acttgtcgct caagtactgg ggctgttaca actagtaact atgccaactg ggtccaagaa
120aaaccagatc atttattcac tggtctaata ggtggtacca acaagcgagc
tccaggtgtg 180cctgccagat tctcaggctc cctgattgga gacaaggctg
ccctcaccat cacaggggca 240cagactgagg atgaggcaat atatttctgt
gctctatggt acagcaacct ctgggtgttc 300ggtggaggaa ccaaactgac
tgtcctaggt ggtggtggtt ctggcggcgg cggctccggt 360ggtggtggtt
ctgaggtgaa gcttctcgag tctggaggag gattggtgca gcctaaaggg
420tcattgaaac tctcatgtgc agcctctgga ttcaccttca atacctacgc
catgaactgg 480gtccgccagg ctccaggaaa gggtttggaa tgggttgctc
gcataagaag taaatataat 540aattatgcaa catattatgc cgattcagtg
aaagacaggt tcaccatctc cagagatgat 600tcacaaagca ttctctatct
acaaatgaac aacttgaaaa ctgaggacac agccatgtac 660tactgtgtga
gacatgggaa cttcggtaat agctacgttt cctggtttgc ttactggggc
720caagggactc tggtcactgt ctctgca 74712249PRTartificial
sequenceVL-VH scFv single chain Fv 12Gln Ala Val Val Thr Gln Glu
Ser Ala Leu Thr Thr Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr
Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala
Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45 Leu
Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55
60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala
65 70 75 80 Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr
Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Lys Leu 115 120 125 Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Lys Gly Ser Leu Lys Leu 130 135 140 Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp 145 150 155 160 Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg 165 170 175 Ser
Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp 180 185
190 Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln
195 200 205 Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys
Val Arg 210 215 220 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
Ala Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ala
245 13726DNAARTIFICIAL SEQUENCEVH-VL scFv single chain Fv
13caggtccagc tgcagcagtc tggggctgaa ctggcaagac ctggggcctc agtgaagatg
60tcctgcaagg cttctggcta cacctttact agatctacga tgcactgggt aaaacagagg
120cctggacagg gtctggaatg gattggatac attaatccta gcagtgctta
tactaattac 180aatcagaaat tcaaggacaa ggccacattg actgcagaca
aatcctccag tacagcctac 240atgcaactga gtagcctgac atctgaggac
tctgcagtct attactgtgc aagtccgcaa 300gtccactatg attacaacgg
gtttccttac tggggccaag ggactctggt cactgtctct 360gcaggtggtg
gtggttctgg cggcggcggc tccggtggtg gtggttctca agttgttctc
420acccagtctc cagcaatcat gtctgcattt ccaggggaga aggtcaccat
gacctgcagt 480gccagctcaa gtgtaagtta catgaactgg taccagcaga
agtcaggcac ctcccccaaa 540agatggattt atgactcatc caaactggct
tctggagtcc ctgctcgctt cagtggcagt 600gggtctggga cctcttattc
tctcacaatc agcagcatgg agactgaaga tgctgccact 660tattactgcc
agcagtggag tcgtaaccca cccacgttcg gaggggggac caagctacaa 720attaca
72614242PRTartificial sequenceVH-VL scFv single chain Fv 14Gln Val
Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Ser 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 Ser Ala Tyr Thr Asn Tyr Asn
Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala 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 Ser Pro Gln Val His Tyr
Asp Tyr Asn Gly Phe Pro Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val
Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser
Gly Gly Gly Gly Ser Gln Val Val Leu Thr Gln Ser Pro 130 135 140 Ala
Ile Met Ser Ala Phe Pro Gly Glu Lys Val Thr Met Thr Cys Ser 145 150
155 160 Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
Gly 165 170 175 Thr Ser Pro Lys Arg Trp Ile Tyr Asp Ser Ser Lys Leu
Ala Ser Gly 180 185 190 Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly
Thr Ser Tyr Ser Leu 195 200 205 Thr Ile Ser Ser Met Glu Thr Glu Asp
Ala Ala Thr Tyr Tyr Cys Gln 210 215 220 Gln Trp Ser Arg Asn Pro Pro
Thr Phe Gly Gly Gly Thr Lys Leu Gln 225 230 235 240 Ile Thr
15726DNAARTIFICIAL SEQUENCEVL-VH scFv single chain Fv 15caagttgttc
tcacccagtc tccagcaatc atgtctgcat ttccagggga gaaggtcacc 60atgacctgca
gtgccagctc aagtgtaagt tacatgaact ggtaccagca gaagtcaggc
120acctccccca aaagatggat ttatgactca tccaaactgg cttctggagt
ccctgctcgc 180ttcagtggca gtgggtctgg gacctcttat tctctcacaa
tcagcagcat ggagactgaa 240gatgctgcca cttattactg ccagcagtgg
agtcgtaacc cacccacgtt cggagggggg 300accaagctac aaattacagg
tggtggtggt tctggcggcg gcggctccgg tggtggtggt 360tctcaggtcc
agctgcagca gtctggggct gaactggcaa gacctggggc ctcagtgaag
420atgtcctgca aggcttctgg ctacaccttt actagatcta cgatgcactg
ggtaaaacag 480aggcctggac agggtctgga atggattgga tacattaatc
ctagcagtgc ttatactaat 540tacaatcaga aattcaagga caaggccaca
ttgactgcag acaaatcctc cagtacagcc 600tacatgcaac tgagtagcct
gacatctgag gactctgcag tctattactg tgcaagtccg 660caagtccact
atgattacaa cgggtttcct tactggggcc aagggactct ggtcactgtc 720tctgca
72616242PRTartificial sequenceVL-VH scFv single chain Fv 16Gln Val
Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Phe Pro Gly 1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser 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 Ser Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser
Ser Met Glu Thr Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
Trp Ser Arg Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu
Gln Ile Thr Gly Gly Gly Gly Ser Gly 100 105 110 Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser 115 120 125 Gly Ala Glu
Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys 130 135 140 Ala
Ser Gly Tyr Thr Phe Thr Arg Ser Thr Met His Trp Val Lys Gln 145 150
155 160 Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser
Ser 165 170 175 Ala Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala
Thr Leu Thr 180 185 190 Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln
Leu Ser Ser Leu Thr 195 200 205 Ser Glu Asp Ser Ala Val Tyr Tyr Cys
Ala Ser Pro Gln Val His Tyr 210 215 220 Asp Tyr Asn Gly Phe Pro Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val 225 230 235 240 Ser Ala
1747DNAARTIFICIAL SEQUENCE5' LH oligonucleotide 17acatccggag
gtggtggatc ccaggctgtt gtgactcagg aatctgc
471851DNAartificial sequence3' VL Linker oligonucleotide
18ggagccgccg ccgccagaac caccaccacc taggacagtc agtttggttc c
511970DNAartificial sequence5' VH Linker oligonucleotide
19tctggcggcg gcggctccgg tggtggtggt tctgaggtga agcttctcga gtctggagga
60ggattggtgc 702056DNAartificial sequence3' LH oligonucleotide
20agtgggtcga cctaatgatg atggtgatga tgtgcagaga cagtgaccag agtccc
562158DNAartificial sequence5' HL oligonucleotide 21acatccggag
gtggtggatc cgaggtgaag cttctcgagt ctggaggagg attggtgc
582254DNAartificial sequence3' VH Linker oligonucleotide
22ggagccgccg ccgccagaac caccaccacc tgcagagaca gtgaccagag tccc
542359DNAartificial sequence5' VL Linker oligonucleotide
23tctggcggcg gcggctccgg tggtggtggt tctcaggctg ttgtgactca ggaatctgc
592456DNAartificial sequence3' HL oligonucleotide 24agtgggtcga
cctaatgatg atggtgatga tgtaggacag tcagtttggt tcctcc
562544DNAartificial sequence5' LH oligonucleotide 25acatccggag
gtggtggatc ccaagttgtt ctcacccagt ctcc 442653DNAartificial
sequence3' VL Linker oligonucleotide 26ggagccgccg ccgccagaac
caccaccacc tgtaatttgt agcttggtcc ccc 532756DNAartificial sequence5'
VH Linker oligonucleotide 27tctggcggcg gcggctccgg tggtggtggt
tctcaggtcc agctgcagca gtctgg 562855DNAartificial sequence3' LH
oligonucleotide 28agtgggtcga cctaatgatg atggtgatga tgtgcagaga
cagtgaccag agtcc 552944DNAartificial sequence5' HL oligonucleotide
29acatccggag gtggtggatc ccaggtccag ctgcagcagt ctgg
443053DNAartificial sequence3' VH Linker oligonucleotide
30ggagccgccg ccgccagaac caccaccacc tgcagagaca gtgaccagag tcc
533156DNAartificial sequence5' VL Linker oligonucleotide
31tctggcggcg gcggctccgg tggtggtggt tctcaagttg ttctcaccca gtctcc
563255DNAartificial sequence3' HL oligonucleotide 32agtgggtcga
cctaatgatg atggtgatga tgtgtaattt gtagcttggt ccccc
5533744DNAartificial sequence5-10 LH scFv single chain Fv
33gagctcgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact
60atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc
120tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc
atccactagg 180gaatctgggg tccctgatcg cttcacaggc agtggatctg
gaacagattt cactctcacc 240atcagcagtg tgcaggctga agacctggca
gtttattact gtcagaatga ttatagttat 300ccgctcacgt tcggtgctgg
gaccaagctt gagatcaaag gtggtggtgg ttctggcggc 360ggcggctccg
gtggtggtgg ttctgaggtg cagctgctcg agcagtctgg agctgagctg
420gtaaggcctg ggacttcagt gaagatatcc tgcaaggctt ctggatacgc
cttcactaac 480tactggctag gttgggtaaa gcagaggcct ggacatggac
ttgagtggat tggagatatt 540ttccctggaa gtggtaatat ccactacaat
gagaagttca agggcaaagc cacactgact 600gcagacaaat cttcgagcac
agcctatatg cagctcagta gcctgacatt tgaggactct 660gctgtctatt
tctgtgcaag actgaggaac tgggacgagc ctatggacta ctggggccaa
720gggaccacgg tcaccgtctc ctcc 74434248PRTartificial sequence5-10 LH
scFv single chain Fv 34Glu 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 115 120 125 Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu
Val Arg Pro Gly 130 135 140 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Thr Asn 145 150 155 160 Tyr Trp Leu Gly Trp Val Lys
Gln Arg Pro Gly His Gly Leu Glu Trp 165 170 175 Ile Gly Asp Ile Phe
Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys 180 185 190 Phe Lys Gly
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 195 200 205 Tyr
Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe 210 215
220 Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln
225 230 235 240 Gly Thr Thr Val Thr Val Ser Ser 245 3557DNAMUS
MUSCULUSmisc_feature(1)..(57)leader peptide 35atgggatgga gctgtatcat
cctcttcttg gtagcaacag ctacaggtgt acactcc 573619PRTmus
musculusmisc_feature(1)..(19)leader peptide 36Met Gly Trp Ser Cys
Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser
371506DNAARTIFICIAL SEQUENCE5-10 LHxSEQ ID NO 12 ; Bispecific
single chain antibody 37gagctcgtga tgacacagtc tccatcctcc ctgactgtga
cagcaggaga gaaggtcact 60atgagctgca agtccagtca gagtctgtta aacagtggaa
atcaaaagaa ctacttgacc 120tggtaccagc agaaaccagg gcagcctcct
aaactgttga tctactgggc atccactagg 180gaatctgggg tccctgatcg
cttcacaggc agtggatctg gaacagattt cactctcacc 240atcagcagtg
tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat
300ccgctcacgt tcggtgctgg gaccaagctt gagatcaaag gtggtggtgg
ttctggcggc 360ggcggctccg gtggtggtgg ttctgaggtg cagctgctcg
agcagtctgg agctgagctg 420gtaaggcctg ggacttcagt gaagatatcc
tgcaaggctt ctggatacgc cttcactaac 480tactggctag gttgggtaaa
gcagaggcct ggacatggac ttgagtggat tggagatatt 540ttccctggaa
gtggtaatat ccactacaat gagaagttca agggcaaagc cacactgact
600gcagacaaat cttcgagcac agcctatatg cagctcagta gcctgacatt
tgaggactct 660gctgtctatt tctgtgcaag actgaggaac tgggacgagc
ctatggacta ctggggccaa 720gggaccacgg tcaccgtctc ctccggaggt
ggtggatccc aggctgttgt gactcaggaa 780tctgcactca ccacatcacc
tggtgaaaca gtcacactca cttgtcgctc aagtactggg 840gctgttacaa
ctagtaacta tgccaactgg gtccaagaaa aaccagatca tttattcact
900ggtctaatag gtggtaccaa caagcgagct ccaggtgtgc ctgccagatt
ctcaggctcc 960ctgattggag acaaggctgc cctcaccatc acaggggcac
agactgagga tgaggcaata 1020tatttctgtg ctctatggta cagcaacctc
tgggtgttcg gtggaggaac caaactgact 1080gtcctaggtg gtggtggttc
tggcggcggc ggctccggtg gtggtggttc tgaggtgaag 1140cttctcgagt
ctggaggagg attggtgcag cctaaagggt cattgaaact ctcatgtgca
1200gcctctggat tcaccttcaa tacctacgcc atgaactggg tccgccaggc
tccaggaaag 1260ggtttggaat gggttgctcg cataagaagt aaatataata
attatgcaac atattatgcc 1320gattcagtga aagacaggtt caccatctcc
agagatgatt cacaaagcat tctctatcta 1380caaatgaaca acttgaaaac
tgaggacaca gccatgtact actgtgtgag acatgggaac 1440ttcggtaata
gctacgtttc ctggtttgct tactggggcc aagggactct ggtcactgtc 1500tctgca
150638502PRTartificial sequence5-10 LHxSEQ ID NO 12 ; Bispecific
single chain antibody 38Glu 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 115 120 125 Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu
Val Arg Pro Gly 130 135 140 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Thr Asn 145 150 155 160 Tyr Trp Leu Gly Trp Val Lys
Gln Arg Pro Gly His Gly Leu Glu Trp 165 170 175 Ile Gly Asp Ile Phe
Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys 180 185 190 Phe Lys Gly
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 195 200 205 Tyr
Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe 210 215
220 Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln
225 230 235 240 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gln Ala Val 245 250 255 Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro
Gly Glu Thr Val Thr 260 265 270 Leu Thr Cys Arg Ser Ser Thr Gly Ala
Val Thr Thr Ser Asn Tyr Ala 275 280 285 Asn Trp Val Gln Glu Lys Pro
Asp His Leu Phe Thr Gly Leu Ile Gly 290 295 300 Gly Thr Asn Lys Arg
Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser 305 310 315 320 Leu Ile
Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu 325 330 335
Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val 340
345 350 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser
Gly 355 360 365 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu
Leu Glu Ser 370 375 380 Gly Gly Gly Leu Val Gln Pro Lys Gly Ser Leu
Lys Leu Ser Cys Ala 385 390 395 400 Ala Ser Gly Phe Thr Phe Asn Thr
Tyr Ala Met Asn Trp Val Arg Gln 405 410 415 Ala Pro Gly Lys Gly Leu
Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr 420 425 430 Asn Asn Tyr Ala
Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr 435 440 445 Ile Ser
Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn 450 455 460
Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His Gly Asn 465
470 475 480 Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln
Gly Thr 485 490 495 Leu Val Thr Val Ser Ala 500 391506DNAARTIFICIAL
SEQUENCE5-10 LHx SEQ ID NO 10 ; bispecific single chain antibdoy
39gagctcgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact
60atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc
120tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc
atccactagg 180gaatctgggg tccctgatcg cttcacaggc agtggatctg
gaacagattt cactctcacc 240atcagcagtg tgcaggctga agacctggca
gtttattact gtcagaatga ttatagttat 300ccgctcacgt tcggtgctgg
gaccaagctt gagatcaaag gtggtggtgg ttctggcggc 360ggcggctccg
gtggtggtgg ttctgaggtg cagctgctcg agcagtctgg agctgagctg
420gtaaggcctg ggacttcagt gaagatatcc tgcaaggctt ctggatacgc
cttcactaac 480tactggctag gttgggtaaa gcagaggcct ggacatggac
ttgagtggat tggagatatt 540ttccctggaa gtggtaatat ccactacaat
gagaagttca agggcaaagc cacactgact 600gcagacaaat cttcgagcac
agcctatatg cagctcagta gcctgacatt tgaggactct 660gctgtctatt
tctgtgcaag actgaggaac tgggacgagc ctatggacta ctggggccaa
720gggaccacgg tcaccgtctc ctccggaggt ggtggatccg aggtgaagct
tctcgagtct 780ggaggaggat tggtgcagcc taaagggtca ttgaaactct
catgtgcagc ctctggattc 840accttcaata cctacgccat gaactgggtc
cgccaggctc caggaaaggg tttggaatgg 900gttgctcgca taagaagtaa
atataataat tatgcaacat attatgccga ttcagtgaaa 960gacaggttca
ccatctccag agatgattca caaagcattc tctatctaca aatgaacaac
1020ttgaaaactg aggacacagc catgtactac tgtgtgagac atgggaactt
cggtaatagc 1080tacgtttcct ggtttgctta ctggggccaa gggactctgg
tcactgtctc tgcaggtggt 1140ggtggttctg gcggcggcgg ctccggtggt
ggtggttctc aggctgttgt gactcaggaa 1200tctgcactca ccacatcacc
tggtgaaaca gtcacactca cttgtcgctc aagtactggg 1260gctgttacaa
ctagtaacta tgccaactgg gtccaagaaa aaccagatca tttattcact
1320ggtctaatag gtggtaccaa caagcgagct ccaggtgtgc ctgccagatt
ctcaggctcc 1380ctgattggag acaaggctgc cctcaccatc acaggggcac
agactgagga tgaggcaata 1440tatttctgtg ctctatggta cagcaacctc
tgggtgttcg gtggaggaac caaactgact 1500gtccta 150640502PRTartificial
sequence5-10 LHx SEQ ID NO 10 ; bispecific single chain antibody
40Glu 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 Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Glu
Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 130 135
140 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn
145 150 155 160 Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
Leu Glu Trp 165 170 175 Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile
His Tyr Asn Glu Lys 180 185 190 Phe Lys Gly Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser Thr Ala 195 200 205 Tyr Met Gln Leu Ser Ser Leu
Thr Phe Glu Asp Ser Ala Val Tyr Phe 210 215 220 Cys Ala Arg Leu Arg
Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln 225 230 235 240 Gly Thr
Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Val Lys 245 250 255
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser Leu Lys 260
265 270 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met
Asn 275 280 285 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
Ala Arg Ile 290 295 300 Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr
Ala Asp Ser Val Lys 305 310 315 320 Asp Arg Phe Thr Ile Ser Arg Asp
Asp Ser Gln Ser Ile Leu Tyr Leu 325 330 335 Gln Met Asn Asn Leu Lys
Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val 340 345 350 Arg His Gly Asn
Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp 355 360 365 Gly Gln
Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly 370 375 380
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu 385
390 395 400 Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr
Cys Arg 405 410 415 Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala
Asn Trp Val Gln 420 425 430 Glu Lys Pro Asp His Leu Phe Thr Gly Leu
Ile Gly Gly Thr Asn Lys 435 440 445 Arg Ala Pro Gly Val Pro Ala Arg
Phe Ser Gly Ser Leu Ile Gly Asp 450 455 460 Lys Ala Ala Leu Thr Ile
Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile 465 470 475 480 Tyr Phe Cys
Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly 485 490 495 Thr
Lys Leu Thr Val Leu 500 411485DNAARTIFICIAL SEQUENCE5-10 LHx SEQ ID
NO 16 ; bispecific single chain antibody 41gagctcgtga tgacacagtc
tccatcctcc ctgactgtga cagcaggaga gaaggtcact 60atgagctgca agtccagtca
gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 120tggtaccagc
agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg
180gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt
cactctcacc 240atcagcagtg tgcaggctga agacctggca gtttattact
gtcagaatga ttatagttat 300ccgctcacgt tcggtgctgg gaccaagctt
gagatcaaag gtggtggtgg ttctggcggc 360ggcggctccg gtggtggtgg
ttctgaggtg cagctgctcg agcagtctgg agctgagctg 420gtaaggcctg
ggacttcagt gaagatatcc tgcaaggctt ctggatacgc cttcactaac
480tactggctag gttgggtaaa
gcagaggcct ggacatggac ttgagtggat tggagatatt 540ttccctggaa
gtggtaatat ccactacaat gagaagttca agggcaaagc cacactgact
600gcagacaaat cttcgagcac agcctatatg cagctcagta gcctgacatt
tgaggactct 660gctgtctatt tctgtgcaag actgaggaac tgggacgagc
ctatggacta ctggggccaa 720gggaccacgg tcaccgtctc ctccggaggt
ggtggatccc aagttgttct cacccagtct 780ccagcaatca tgtctgcatt
tccaggggag aaggtcacca tgacctgcag tgccagctca 840agtgtaagtt
acatgaactg gtaccagcag aagtcaggca cctcccccaa aagatggatt
900tatgactcat ccaaactggc ttctggagtc cctgctcgct tcagtggcag
tgggtctggg 960acctcttatt ctctcacaat cagcagcatg gagactgaag
atgctgccac ttattactgc 1020cagcagtgga gtcgtaaccc acccacgttc
ggagggggga ccaagctaca aattacaggt 1080ggtggtggtt ctggcggcgg
cggctccggt ggtggtggtt ctcaggtcca gctgcagcag 1140tctggggctg
aactggcaag acctggggcc tcagtgaaga tgtcctgcaa ggcttctggc
1200tacaccttta ctagatctac gatgcactgg gtaaaacaga ggcctggaca
gggtctggaa 1260tggattggat acattaatcc tagcagtgct tatactaatt
acaatcagaa attcaaggac 1320aaggccacat tgactgcaga caaatcctcc
agtacagcct acatgcaact gagtagcctg 1380acatctgagg actctgcagt
ctattactgt gcaagtccgc aagtccacta tgattacaac 1440gggtttcctt
actggggcca agggactctg gtcactgtct ctgca 148542495PRTartificial
sequence5-10 LHxSEQ ID NO 16 ; bispecific single chain antibody
42Glu 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 Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Glu
Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 130 135
140 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn
145 150 155 160 Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
Leu Glu Trp 165 170 175 Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile
His Tyr Asn Glu Lys 180 185 190 Phe Lys Gly Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser Thr Ala 195 200 205 Tyr Met Gln Leu Ser Ser Leu
Thr Phe Glu Asp Ser Ala Val Tyr Phe 210 215 220 Cys Ala Arg Leu Arg
Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln 225 230 235 240 Gly Thr
Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Val Val 245 250 255
Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Phe Pro Gly Glu Lys Val 260
265 270 Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp
Tyr 275 280 285 Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
Asp Ser Ser 290 295 300 Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
Gly Ser Gly Ser Gly 305 310 315 320 Thr Ser Tyr Ser Leu Thr Ile Ser
Ser Met Glu Thr Glu Asp Ala Ala 325 330 335 Thr Tyr Tyr Cys Gln Gln
Trp Ser Arg Asn Pro Pro Thr Phe Gly Gly 340 345 350 Gly Thr Lys Leu
Gln Ile Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly 355 360 365 Ser Gly
Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu 370 375 380
Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly 385
390 395 400 Tyr Thr Phe Thr Arg Ser Thr Met His Trp Val Lys Gln Arg
Pro Gly 405 410 415 Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser
Ser Ala Tyr Thr 420 425 430 Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala
Thr Leu Thr Ala Asp Lys 435 440 445 Ser Ser Ser Thr Ala Tyr Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp 450 455 460 Ser Ala Val Tyr Tyr Cys
Ala Ser Pro Gln Val His Tyr Asp Tyr Asn 465 470 475 480 Gly Phe Pro
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 485 490 495
431485DNAARTIFICIAL SEQUENCE5-10 LHxSEQ ID NO 14 ; bispecific
single chain antibody 43gagctcgtga tgacacagtc tccatcctcc ctgactgtga
cagcaggaga gaaggtcact 60atgagctgca agtccagtca gagtctgtta aacagtggaa
atcaaaagaa ctacttgacc 120tggtaccagc agaaaccagg gcagcctcct
aaactgttga tctactgggc atccactagg 180gaatctgggg tccctgatcg
cttcacaggc agtggatctg gaacagattt cactctcacc 240atcagcagtg
tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat
300ccgctcacgt tcggtgctgg gaccaagctt gagatcaaag gtggtggtgg
ttctggcggc 360ggcggctccg gtggtggtgg ttctgaggtg cagctgctcg
agcagtctgg agctgagctg 420gtaaggcctg ggacttcagt gaagatatcc
tgcaaggctt ctggatacgc cttcactaac 480tactggctag gttgggtaaa
gcagaggcct ggacatggac ttgagtggat tggagatatt 540ttccctggaa
gtggtaatat ccactacaat gagaagttca agggcaaagc cacactgact
600gcagacaaat cttcgagcac agcctatatg cagctcagta gcctgacatt
tgaggactct 660gctgtctatt tctgtgcaag actgaggaac tgggacgagc
ctatggacta ctggggccaa 720gggaccacgg tcaccgtctc ctccggaggt
ggtggatccc aggtccagct gcagcagtct 780ggggctgaac tggcaagacc
tggggcctca gtgaagatgt cctgcaaggc ttctggctac 840acctttacta
gatctacgat gcactgggta aaacagaggc ctggacaggg tctggaatgg
900attggataca ttaatcctag cagtgcttat actaattaca atcagaaatt
caaggacaag 960gccacattga ctgcagacaa atcctccagt acagcctaca
tgcaactgag tagcctgaca 1020tctgaggact ctgcagtcta ttactgtgca
agtccgcaag tccactatga ttacaacggg 1080tttccttact ggggccaagg
gactctggtc actgtctctg caggtggtgg tggttctggc 1140ggcggcggct
ccggtggtgg tggttctcaa gttgttctca cccagtctcc agcaatcatg
1200tctgcatttc caggggagaa ggtcaccatg acctgcagtg ccagctcaag
tgtaagttac 1260atgaactggt accagcagaa gtcaggcacc tcccccaaaa
gatggattta tgactcatcc 1320aaactggctt ctggagtccc tgctcgcttc
agtggcagtg ggtctgggac ctcttattct 1380ctcacaatca gcagcatgga
gactgaagat gctgccactt attactgcca gcagtggagt 1440cgtaacccac
ccacgttcgg aggggggacc aagctacaaa ttaca 148544495PRTartificial
sequence5-10 LHxSEQ ID NO 14 ; bispecific single chain antibody
44Glu 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 Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Glu
Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 130 135
140 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn
145 150 155 160 Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
Leu Glu Trp 165 170 175 Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile
His Tyr Asn Glu Lys 180 185 190 Phe Lys Gly Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser Thr Ala 195 200 205 Tyr Met Gln Leu Ser Ser Leu
Thr Phe Glu Asp Ser Ala Val Tyr Phe 210 215 220 Cys Ala Arg Leu Arg
Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln 225 230 235 240 Gly Thr
Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Val Gln 245 250 255
Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys 260
265 270 Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Ser Thr Met
His 275 280 285 Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
Gly Tyr Ile 290 295 300 Asn Pro Ser Ser Ala Tyr Thr Asn Tyr Asn Gln
Lys Phe Lys Asp Lys 305 310 315 320 Ala Thr Leu Thr Ala Asp Lys Ser
Ser Ser Thr Ala Tyr Met Gln Leu 325 330 335 Ser Ser Leu Thr Ser Glu
Asp Ser Ala Val Tyr Tyr Cys Ala Ser Pro 340 345 350 Gln Val His Tyr
Asp Tyr Asn Gly Phe Pro Tyr Trp Gly Gln Gly Thr 355 360 365 Leu Val
Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 370 375 380
Gly Gly Gly Gly Ser Gln Val Val Leu Thr Gln Ser Pro Ala Ile Met 385
390 395 400 Ser Ala Phe Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala
Ser Ser 405 410 415 Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
Gly Thr Ser Pro 420 425 430 Lys Arg Trp Ile Tyr Asp Ser Ser Lys Leu
Ala Ser Gly Val Pro Ala 435 440 445 Arg Phe Ser Gly Ser Gly Ser Gly
Thr Ser Tyr Ser Leu Thr Ile Ser 450 455 460 Ser Met Glu Thr Glu Asp
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser 465 470 475 480 Arg Asn Pro
Pro Thr Phe Gly Gly Gly Thr Lys Leu Gln Ile Thr 485 490 495
4543DNAARTIFICIAL SEQUENCE5' EpCAM oligonucleotide 45ggttctagac
caccatggcg cccccgcagg tcctcgcgtt cgg 434642DNAartificial sequence3'
EpCAM oligonucleotide 46agtgggtcga cttatgcatt gagttcccta tgcatctcac
cc 4247726DNACynomolgusmisc_feature(1)..(726)cynomolgus EpCAM
extracellular portion 47cagaaagaat gtgtctgtga aaactacaag ctggccgtaa
actgcttttt gaatgacaat 60ggtcaatgcc agtgtacttc gattggtgca caaaatactg
tcctttgctc aaagctggct 120gccaaatgtt tggtgatgaa ggcagaaatg
aacggctcaa aacttgggag aagagcgaaa 180cctgaagggg ctctccagaa
caatgatggc ctttacgatc ctgactgcga tgagagcggg 240ctctttaagg
ccaagcagtg caacggcacc tccacgtgct ggtgtgtgaa cactgctggg
300gtcagaagaa ctgacaagga cactgaaata acctgctctg agcgagtgag
aacctactgg 360atcatcattg aattaaaaca caaagcaaga gaaaaacctt
atgatgttca aagtttgcgg 420actgcacttg aggaggcgat caaaacgcgt
tatcaactgg atccaaaatt tatcacaaat 480attttgtatg aggataatgt
tatcactatt gatctggttc aaaattcttc tcagaaaact 540cagaatgatg
tggacatagc tgatgtggct tattattttg aaaaagatgt taaaggtgaa
600tccttgtttc attctaagaa aatggacctg agagtaaatg gggaacaact
ggatctggat 660cctggtcaaa ctttaattta ttatgtcgat gaaaaagcac
ctgaattctc aatgcagggt 720ctaaaa
72648242PRTCynomolgusmisc_feature(1)..(242)cynomolgus EpCAM
extracellular portion 48Gln Lys Glu Cys Val Cys Glu Asn Tyr Lys Leu
Ala Val Asn Cys Phe 1 5 10 15 Leu Asn Asp Asn Gly Gln Cys Gln Cys
Thr Ser Ile Gly Ala Gln Asn 20 25 30 Thr Val Leu Cys Ser Lys Leu
Ala Ala Lys Cys Leu Val Met Lys Ala 35 40 45 Glu Met Asn Gly Ser
Lys Leu Gly Arg Arg Ala Lys Pro Glu Gly Ala 50 55 60 Leu Gln Asn
Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Glu Ser Gly 65 70 75 80 Leu
Phe Lys Ala Lys Gln Cys Asn Gly Thr Ser Thr Cys Trp Cys Val 85 90
95 Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile Thr Cys
100 105 110 Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu Lys
His Lys 115 120 125 Ala Arg Glu Lys Pro Tyr Asp Val Gln Ser Leu Arg
Thr Ala Leu Glu 130 135 140 Glu Ala Ile Lys Thr Arg Tyr Gln Leu Asp
Pro Lys Phe Ile Thr Asn 145 150 155 160 Ile Leu Tyr Glu Asp Asn Val
Ile Thr Ile Asp Leu Val Gln Asn Ser 165 170 175 Ser Gln Lys Thr Gln
Asn Asp Val Asp Ile Ala Asp Val Ala Tyr Tyr 180 185 190 Phe Glu Lys
Asp Val Lys Gly Glu Ser Leu Phe His Ser Lys Lys Met 195 200 205 Asp
Leu Arg Val Asn Gly Glu Gln Leu Asp Leu Asp Pro Gly Gln Thr 210 215
220 Leu Ile Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met Gln Gly
225 230 235 240 Leu Lys 49366DNAHYBRIDOMAmisc_feature(1)..(366)2G8
VH VH region of monoclonal antibody 2G8 49gaggttcagc tgcagcagtc
tggggcagag cttgtgaggt caggggcctc agtcaagttg 60tcctgcacag cttctggctt
caacattaaa gactactatt tgcactgggt gaagcagagg 120cctgaacagg
gcctggagtg gattgcctgg attgatcttg agaatggtga tattaaatat
180gccccgaagt ttcagggcaa ggccactata actgcagaca catcctccaa
cacagcctac 240ctgcagctca gcagcctgac atctgaggac actgccgtct
attactgtaa tccctattac 300tacggtagta actacgacta tgctatggac
tactggggtc aaggaacctc agtcaccgtc 360tcctca
36650122PRThybridomamisc_feature(1)..(122)2G8 VH VH region of
monoclonal antibody 2G8 50Glu Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Val Arg Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala
Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Leu His Trp Val Lys
Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Ala Trp Ile Asp
Leu Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly
Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Asn Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr Ala Met Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120
51336DNAHYBRIDOMAmisc_feature(1)..(336)2G8 VL VL region of
monoclonal antibody 2G8 51gatgttgtga tgacccagac tccactcact
ttgtcggtta ccattggaca accagcctct 60atctcttgca agtcaagtca gagcctctta
tatagtaatg gaaaaaccta tttgaactgg 120atattacaga ggccaggcca
gtctccaaag cgcctaatct atctggtgtc taaactggac 180tctggagtcc
ctgacaggtt cactggcagt ggatcaggaa cagattttac gctgaaaatc
240agcagagtgg aggctgagga tttgggagtt tattactgcg tgcaaggtac
acattttcct 300ctcacgttcg gtgctgggac caagctggag ctgaaa
33652112PRThybridomamisc_feature(1)..(112)2G8 VL VL region of
monoclonal antibody 2G8 52Asp Val Val Met Thr Gln Thr Pro Leu Thr
Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys
Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Lys Thr Tyr Leu
Asn Trp Ile Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu
Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val Gln Gly 85
90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
Lys 100 105 110 53747DNAartificial sequence2G8VH-VL scFv single
chain Fv 53gaggttcagc tgcagcagtc tggggcagag cttgtgaggt caggggcctc
agtcaagttg 60tcctgcacag cttctggctt caacattaaa gactactatt tgcactgggt
gaagcagagg 120cctgaacagg gcctggagtg gattgcctgg attgatcttg
agaatggtga tattaaatat 180gccccgaagt ttcagggcaa ggccactata
actgcagaca catcctccaa cacagcctac 240ctgcagctca gcagcctgac
atctgaggac actgccgtct attactgtaa tccctattac 300tacggtagta
actacgacta tgctatggac tactggggtc aaggaacctc agtcaccgtc
360tcctcaggtg gtggtggttc tggcggcggc ggctccggtg gtggtggttc
tgatgttgtg 420atgacccaga ctccactcac tttgtcggtt accattggac
aaccagcctc tatctcttgc 480aagtcaagtc agagcctctt atatagtaat
ggaaaaacct atttgaactg gatattacag 540aggccaggcc agtctccaaa
gcgcctaatc tatctggtgt ctaaactgga ctctggagtc 600cctgacaggt
tcactggcag tggatcagga acagatttta cgctgaaaat cagcagagtg
660gaggctgagg atttgggagt ttattactgc gtgcaaggta cacattttcc
tctcacgttc 720ggtgctggga ccaagctgga gctgaaa 74754249PRTartificial
sequence2G8VH-VL scFv single chain Fv 54Glu Val Gln Leu Gln Gln Ser
Gly Ala Glu Leu Val Arg Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser
Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Leu His
Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Ala
Trp Ile Asp Leu Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe 50 55
60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Asn Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr Ala
Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly
Ser Asp Val Val Met Thr Gln Thr 130 135 140 Pro Leu Thr Leu Ser Val
Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys 145 150 155 160 Lys Ser Ser
Gln Ser Leu Leu Tyr Ser Asn Gly Lys Thr Tyr Leu Asn 165 170 175 Trp
Ile Leu Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu 180 185
190 Val Ser Lys Leu Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
195 200 205 Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
Glu Asp 210 215 220 Leu Gly Val Tyr Tyr Cys Val Gln Gly Thr His Phe
Pro Leu Thr Phe 225 230 235 240 Gly Ala Gly Thr Lys Leu Glu Leu Lys
245 55747DNAARTIFICIAL SEQUENCE2G8VL-VH scFv single chain Fv
55gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcctct
60atctcttgca agtcaagtca gagcctctta tatagtaatg gaaaaaccta tttgaactgg
120atattacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc
taaactggac 180tctggagtcc ctgacaggtt cactggcagt ggatcaggaa
cagattttac gctgaaaatc 240agcagagtgg aggctgagga tttgggagtt
tattactgcg tgcaaggtac acattttcct 300ctcacgttcg gtgctgggac
caagctggag ctgaaaggtg gtggtggttc tggcggcggc 360ggctccggtg
gtggtggttc tgaggttcag ctgcagcagt ctggggcaga gcttgtgagg
420tcaggggcct cagtcaagtt gtcctgcaca gcttctggct tcaacattaa
agactactat 480ttgcactggg tgaagcagag gcctgaacag ggcctggagt
ggattgcctg gattgatctt 540gagaatggtg atattaaata tgccccgaag
tttcagggca aggccactat aactgcagac 600acatcctcca acacagccta
cctgcagctc agcagcctga catctgagga cactgccgtc 660tattactgta
atccctatta ctacggtagt aactacgact atgctatgga ctactggggt
720caaggaacct cagtcaccgt ctcctcc 74756249PRTartificial
sequence2G8VL-VH scFv single chain Fv 56Asp Val Val Met Thr Gln Thr
Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile
Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Lys
Thr Tyr Leu Asn Trp Ile Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro
Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55
60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val
Gln Gly 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys
Leu Glu Leu Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu 115 120 125 Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Val Arg Ser Gly Ala Ser 130 135 140 Val Lys Leu Ser Cys Thr
Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr 145 150 155 160 Leu His Trp
Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Ala 165 170 175 Trp
Ile Asp Leu Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe Gln 180 185
190 Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu
195 200 205 Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr
Cys Asn 210 215 220 Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr Ala Met
Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Ser Val Thr Val Ser Ser
245 5749DNAARTIFICIAL SEQUENCE5'2G8 LH oligonucleotide 57aggtgtacac
tccgatgttg tgatgaccca gactccactc actttgtcg 495854DNAartificial
sequence3'2G8 VL Linker oligonucleotide 58ggagccgccg ccgccagaac
caccaccacc tttcagctcc agcttggtcc cagc 545956DNAartificial
sequence5'2G8 VH Linker oligonucleotide 59tctggcggcg gcggctccgg
tggtggtggt tctgaggttc agctgcagca gtctgg 566030DNAartificial
sequence3'2G8 LH oligonucleotide 60acatccggag gagacggtga ctgaggttcc
306137DNAartificial sequence5' 2G8 HL oligonucleotide 61aggtgtacac
tccgaggttc agctgcagca gtctggg 376254DNAartificial sequence3' 2G8 VH
Linker oligonucleotide 62ggagccgccg ccgccagaac caccaccacc
tgaggagacg gtgactgagg ttcc 546369DNAartificial sequence5' 2G8 VL
Linker oligonucleotide 63tctggcggcg gcggctccgg tggtggtggt
tctgatgttg tgatgaccca gactccactc 60actttgtcg 696433DNAartificial
sequence3' 2G8 HL oligonucleotide 64acatccggat ttcagctcca
gcttggtccc agc 33651509DNAartificial sequence2G8LHxSEQ ID NO 12 ;
bispecific single chain antibody 65gatgttgtga tgacccagac tccactcact
ttgtcggtta ccattggaca accagcctct 60atctcttgca agtcaagtca gagcctctta
tatagtaatg gaaaaaccta tttgaactgg 120atattacaga ggccaggcca
gtctccaaag cgcctaatct atctggtgtc taaactggac 180tctggagtcc
ctgacaggtt cactggcagt ggatcaggaa cagattttac gctgaaaatc
240agcagagtgg aggctgagga tttgggagtt tattactgcg tgcaaggtac
acattttcct 300ctcacgttcg gtgctgggac caagctggag ctgaaaggtg
gtggtggttc tggcggcggc 360ggctccggtg gtggtggttc tgaggttcag
ctgcagcagt ctggggcaga gcttgtgagg 420tcaggggcct cagtcaagtt
gtcctgcaca gcttctggct tcaacattaa agactactat 480ttgcactggg
tgaagcagag gcctgaacag ggcctggagt ggattgcctg gattgatctt
540gagaatggtg atattaaata tgccccgaag tttcagggca aggccactat
aactgcagac 600acatcctcca acacagccta cctgcagctc agcagcctga
catctgagga cactgccgtc 660tattactgta atccctatta ctacggtagt
aactacgact atgctatgga ctactggggt 720caaggaacct cagtcaccgt
ctcctccgga ggtggtggat cccaggctgt tgtgactcag 780gaatctgcac
tcaccacatc acctggtgaa acagtcacac tcacttgtcg ctcaagtact
840ggggctgtta caactagtaa ctatgccaac tgggtccaag aaaaaccaga
tcatttattc 900actggtctaa taggtggtac caacaagcga gctccaggtg
tgcctgccag attctcaggc 960tccctgattg gagacaaggc tgccctcacc
atcacagggg cacagactga ggatgaggca 1020atatatttct gtgctctatg
gtacagcaac ctctgggtgt tcggtggagg aaccaaactg 1080actgtcctag
gtggtggtgg ttctggcggc ggcggctccg gtggtggtgg ttctgaggtg
1140aagcttctcg agtctggagg aggattggtg cagcctaaag ggtcattgaa
actctcatgt 1200gcagcctctg gattcacctt caatacctac gccatgaact
gggtccgcca ggctccagga 1260aagggtttgg aatgggttgc tcgcataaga
agtaaatata ataattatgc aacatattat 1320gccgattcag tgaaagacag
gttcaccatc tccagagatg attcacaaag cattctctat 1380ctacaaatga
acaacttgaa aactgaggac acagccatgt actactgtgt gagacatggg
1440aacttcggta atagctacgt ttcctggttt gcttactggg gccaagggac
tctggtcact 1500gtctctgca 150966503PRTartificial sequence2G8LHxSEQ
ID NO 12 ; bispecific single chain antibody 66Asp Val Val Met Thr
Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala
Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn
Gly Lys Thr Tyr Leu Asn Trp Ile Leu Gln Arg Pro Gly Gln Ser 35 40
45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
Cys Val Gln Gly 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu 115 120 125 Val Gln Leu Gln Gln Ser
Gly Ala Glu Leu Val Arg Ser Gly Ala Ser 130 135 140 Val Lys Leu Ser
Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr 145 150 155 160 Leu
His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Ala 165 170
175 Trp Ile Asp Leu Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe Gln
180 185 190 Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala
Tyr Leu 195 200 205 Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 210 215 220 Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr
Ala Met Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Ser Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gln Ala 245 250 255 Val Val Thr Gln Glu
Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val 260 265 270 Thr Leu Thr
Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr 275 280 285 Ala
Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile 290 295
300 Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly
305 310 315 320 Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly
Ala Gln Thr 325 330 335 Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp
Tyr Ser Asn Leu Trp 340 345 350 Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gly Gly Gly Ser 355 360 365 Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Lys Leu Leu Glu 370 375 380 Ser Gly Gly Gly Leu
Val Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys 385 390 395 400 Ala Ala
Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg 405 410 415
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys 420
425 430 Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg
Phe 435 440 445 Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu
Gln Met Asn 450 455 460 Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr
Cys Val Arg His Gly 465 470 475 480 Asn Phe Gly Asn Ser Tyr Val Ser
Trp Phe Ala Tyr Trp Gly Gln Gly 485 490 495 Thr Leu Val Thr Val Ser
Ala 500 671509DNAARTIFICIAL SEQUENCE2G8LHx SEQ ID NO 10 ;
bispecific single chain antibody 67gatgttgtga tgacccagac tccactcact
ttgtcggtta ccattggaca accagcctct 60atctcttgca agtcaagtca gagcctctta
tatagtaatg gaaaaaccta tttgaactgg 120atattacaga ggccaggcca
gtctccaaag cgcctaatct atctggtgtc taaactggac 180tctggagtcc
ctgacaggtt cactggcagt ggatcaggaa cagattttac gctgaaaatc
240agcagagtgg aggctgagga tttgggagtt tattactgcg tgcaaggtac
acattttcct 300ctcacgttcg gtgctgggac caagctggag ctgaaaggtg
gtggtggttc tggcggcggc 360ggctccggtg gtggtggttc tgaggttcag
ctgcagcagt ctggggcaga gcttgtgagg 420tcaggggcct cagtcaagtt
gtcctgcaca gcttctggct tcaacattaa agactactat 480ttgcactggg
tgaagcagag gcctgaacag ggcctggagt ggattgcctg gattgatctt
540gagaatggtg atattaaata tgccccgaag tttcagggca aggccactat
aactgcagac 600acatcctcca acacagccta cctgcagctc agcagcctga
catctgagga cactgccgtc 660tattactgta atccctatta ctacggtagt
aactacgact atgctatgga ctactggggt 720caaggaacct cagtcaccgt
ctcctccgga ggtggtggat ccgaggtgaa gcttctcgag 780tctggaggag
gattggtgca gcctaaaggg tcattgaaac tctcatgtgc agcctctgga
840ttcaccttca atacctacgc catgaactgg gtccgccagg ctccaggaaa
gggtttggaa 900tgggttgctc gcataagaag taaatataat aattatgcaa
catattatgc cgattcagtg 960aaagacaggt tcaccatctc cagagatgat
tcacaaagca ttctctatct acaaatgaac 1020aacttgaaaa ctgaggacac
agccatgtac tactgtgtga gacatgggaa cttcggtaat 1080agctacgttt
cctggtttgc ttactggggc caagggactc tggtcactgt ctctgcaggt
1140ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctcaggctgt
tgtgactcag 1200gaatctgcac tcaccacatc acctggtgaa acagtcacac
tcacttgtcg ctcaagtact 1260ggggctgtta caactagtaa ctatgccaac
tgggtccaag aaaaaccaga tcatttattc 1320actggtctaa taggtggtac
caacaagcga gctccaggtg tgcctgccag attctcaggc 1380tccctgattg
gagacaaggc tgccctcacc atcacagggg cacagactga ggatgaggca
1440atatatttct gtgctctatg gtacagcaac ctctgggtgt tcggtggagg
aaccaaactg 1500actgtccta 150968503PRTartificial sequence2G8LHxSEQ
ID NO 10 ; bispecific single chain antibody 68Asp Val Val Met Thr
Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala
Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn
Gly Lys Thr Tyr Leu Asn Trp Ile Leu Gln Arg Pro Gly Gln Ser 35 40
45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
Cys Val Gln Gly 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu 115 120 125 Val Gln Leu Gln Gln Ser
Gly Ala Glu Leu Val Arg Ser Gly Ala Ser 130 135 140 Val Lys Leu Ser
Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr 145 150 155 160 Leu
His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Ala 165 170
175 Trp Ile Asp Leu Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe Gln
180 185 190 Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala
Tyr Leu 195 200 205 Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 210 215 220 Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr
Ala Met Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Ser Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Glu Val 245 250 255 Lys Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Lys Gly Ser Leu 260 265 270 Lys Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met 275 280 285 Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg 290 295
300 Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val
305 310 315 320 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser
Ile Leu Tyr 325 330 335 Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Met Tyr Tyr Cys 340 345 350 Val Arg His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe Ala Tyr 355 360 365 Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ala Gly Gly Gly Gly Ser 370 375 380 Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln 385 390 395 400 Glu Ser
Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys 405 410 415
Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val 420
425 430 Gln Glu Lys Pro Asp His Leu
Phe Thr Gly Leu Ile Gly Gly Thr Asn 435 440 445 Lys Arg Ala Pro Gly
Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly 450 455 460 Asp Lys Ala
Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala 465 470 475 480
Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly 485
490 495 Gly Thr Lys Leu Thr Val Leu 500 691488DNAARTIFICIAL
SEQUENCE2G8LHxSEQ ID NO 16 ; bispecific single chain antibody
69gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcctct
60atctcttgca agtcaagtca gagcctctta tatagtaatg gaaaaaccta tttgaactgg
120atattacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc
taaactggac 180tctggagtcc ctgacaggtt cactggcagt ggatcaggaa
cagattttac gctgaaaatc 240agcagagtgg aggctgagga tttgggagtt
tattactgcg tgcaaggtac acattttcct 300ctcacgttcg gtgctgggac
caagctggag ctgaaaggtg gtggtggttc tggcggcggc 360ggctccggtg
gtggtggttc tgaggttcag ctgcagcagt ctggggcaga gcttgtgagg
420tcaggggcct cagtcaagtt gtcctgcaca gcttctggct tcaacattaa
agactactat 480ttgcactggg tgaagcagag gcctgaacag ggcctggagt
ggattgcctg gattgatctt 540gagaatggtg atattaaata tgccccgaag
tttcagggca aggccactat aactgcagac 600acatcctcca acacagccta
cctgcagctc agcagcctga catctgagga cactgccgtc 660tattactgta
atccctatta ctacggtagt aactacgact atgctatgga ctactggggt
720caaggaacct cagtcaccgt ctcctccgga ggtggtggat cccaagttgt
tctcacccag 780tctccagcaa tcatgtctgc atttccaggg gagaaggtca
ccatgacctg cagtgccagc 840tcaagtgtaa gttacatgaa ctggtaccag
cagaagtcag gcacctcccc caaaagatgg 900atttatgact catccaaact
ggcttctgga gtccctgctc gcttcagtgg cagtgggtct 960gggacctctt
attctctcac aatcagcagc atggagactg aagatgctgc cacttattac
1020tgccagcagt ggagtcgtaa cccacccacg ttcggagggg ggaccaagct
acaaattaca 1080ggtggtggtg gttctggcgg cggcggctcc ggtggtggtg
gttctcaggt ccagctgcag 1140cagtctgggg ctgaactggc aagacctggg
gcctcagtga agatgtcctg caaggcttct 1200ggctacacct ttactagatc
tacgatgcac tgggtaaaac agaggcctgg acagggtctg 1260gaatggattg
gatacattaa tcctagcagt gcttatacta attacaatca gaaattcaag
1320gacaaggcca cattgactgc agacaaatcc tccagtacag cctacatgca
actgagtagc 1380ctgacatctg aggactctgc agtctattac tgtgcaagtc
cgcaagtcca ctatgattac 1440aacgggtttc cttactgggg ccaagggact
ctggtcactg tctctgca 148870496PRTartificial sequence2G8LHxSEQ ID NO
16 ; bispecific single chain antibody 70Asp Val Val Met Thr Gln Thr
Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile
Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Lys
Thr Tyr Leu Asn Trp Ile Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro
Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55
60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val
Gln Gly 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys
Leu Glu Leu Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu 115 120 125 Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Val Arg Ser Gly Ala Ser 130 135 140 Val Lys Leu Ser Cys Thr
Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr 145 150 155 160 Leu His Trp
Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Ala 165 170 175 Trp
Ile Asp Leu Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe Gln 180 185
190 Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu
195 200 205 Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr
Cys Asn 210 215 220 Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr Ala Met
Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Ser Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gln Val 245 250 255 Val Leu Thr Gln Ser Pro Ala
Ile Met Ser Ala Phe Pro Gly Glu Lys 260 265 270 Val Thr Met Thr Cys
Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp 275 280 285 Tyr Gln Gln
Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Ser 290 295 300 Ser
Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser 305 310
315 320 Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Thr Glu Asp
Ala 325 330 335 Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Arg Asn Pro Pro
Thr Phe Gly 340 345 350 Gly Gly Thr Lys Leu Gln Ile Thr Gly Gly Gly
Gly Ser Gly Gly Gly 355 360 365 Gly Ser Gly Gly Gly Gly Ser Gln Val
Gln Leu Gln Gln Ser Gly Ala 370 375 380 Glu Leu Ala Arg Pro Gly Ala
Ser Val Lys Met Ser Cys Lys Ala Ser 385 390 395 400 Gly Tyr Thr Phe
Thr Arg Ser Thr Met His Trp Val Lys Gln Arg Pro 405 410 415 Gly Gln
Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Ser Ala Tyr 420 425 430
Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp 435
440 445 Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser
Glu 450 455 460 Asp Ser Ala Val Tyr Tyr Cys Ala Ser Pro Gln Val His
Tyr Asp Tyr 465 470 475 480 Asn Gly Phe Pro Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ala 485 490 495 711488DNAARTIFICIAL
SEQUENCE2G8LHxSEQ ID NO 14 ; bispecific single chain antibody
71gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcctct
60atctcttgca agtcaagtca gagcctctta tatagtaatg gaaaaaccta tttgaactgg
120atattacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc
taaactggac 180tctggagtcc ctgacaggtt cactggcagt ggatcaggaa
cagattttac gctgaaaatc 240agcagagtgg aggctgagga tttgggagtt
tattactgcg tgcaaggtac acattttcct 300ctcacgttcg gtgctgggac
caagctggag ctgaaaggtg gtggtggttc tggcggcggc 360ggctccggtg
gtggtggttc tgaggttcag ctgcagcagt ctggggcaga gcttgtgagg
420tcaggggcct cagtcaagtt gtcctgcaca gcttctggct tcaacattaa
agactactat 480ttgcactggg tgaagcagag gcctgaacag ggcctggagt
ggattgcctg gattgatctt 540gagaatggtg atattaaata tgccccgaag
tttcagggca aggccactat aactgcagac 600acatcctcca acacagccta
cctgcagctc agcagcctga catctgagga cactgccgtc 660tattactgta
atccctatta ctacggtagt aactacgact atgctatgga ctactggggt
720caaggaacct cagtcaccgt ctcctccgga ggtggtggat cccaggtcca
gctgcagcag 780tctggggctg aactggcaag acctggggcc tcagtgaaga
tgtcctgcaa ggcttctggc 840tacaccttta ctagatctac gatgcactgg
gtaaaacaga ggcctggaca gggtctggaa 900tggattggat acattaatcc
tagcagtgct tatactaatt acaatcagaa attcaaggac 960aaggccacat
tgactgcaga caaatcctcc agtacagcct acatgcaact gagtagcctg
1020acatctgagg actctgcagt ctattactgt gcaagtccgc aagtccacta
tgattacaac 1080gggtttcctt actggggcca agggactctg gtcactgtct
ctgcaggtgg tggtggttct 1140ggcggcggcg gctccggtgg tggtggttct
caagttgttc tcacccagtc tccagcaatc 1200atgtctgcat ttccagggga
gaaggtcacc atgacctgca gtgccagctc aagtgtaagt 1260tacatgaact
ggtaccagca gaagtcaggc acctccccca aaagatggat ttatgactca
1320tccaaactgg cttctggagt ccctgctcgc ttcagtggca gtgggtctgg
gacctcttat 1380tctctcacaa tcagcagcat ggagactgaa gatgctgcca
cttattactg ccagcagtgg 1440agtcgtaacc cacccacgtt cggagggggg
accaagctac aaattaca 148872496PRTartificial sequence2G8LHxSEQ ID NO
14 ; bispecific single chain antibody 72Asp Val Val Met Thr Gln Thr
Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile
Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Lys
Thr Tyr Leu Asn Trp Ile Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro
Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55
60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val
Gln Gly 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys
Leu Glu Leu Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu 115 120 125 Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Val Arg Ser Gly Ala Ser 130 135 140 Val Lys Leu Ser Cys Thr
Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr 145 150 155 160 Leu His Trp
Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Ala 165 170 175 Trp
Ile Asp Leu Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe Gln 180 185
190 Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu
195 200 205 Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr
Cys Asn 210 215 220 Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr Ala Met
Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Ser Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gln Val 245 250 255 Gln Leu Gln Gln Ser Gly Ala
Glu Leu Ala Arg Pro Gly Ala Ser Val 260 265 270 Lys Met Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Arg Ser Thr Met 275 280 285 His Trp Val
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr 290 295 300 Ile
Asn Pro Ser Ser Ala Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp 305 310
315 320 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met
Gln 325 330 335 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys Ala Ser 340 345 350 Pro Gln Val His Tyr Asp Tyr Asn Gly Phe Pro
Tyr Trp Gly Gln Gly 355 360 365 Thr Leu Val Thr Val Ser Ala Gly Gly
Gly Gly Ser Gly Gly Gly Gly 370 375 380 Ser Gly Gly Gly Gly Ser Gln
Val Val Leu Thr Gln Ser Pro Ala Ile 385 390 395 400 Met Ser Ala Phe
Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 405 410 415 Ser Ser
Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser 420 425 430
Pro Lys Arg Trp Ile Tyr Asp Ser Ser Lys Leu Ala Ser Gly Val Pro 435
440 445 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile 450 455 460 Ser Ser Met Glu Thr Glu Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Trp 465 470 475 480 Ser Arg Asn Pro Pro Thr Phe Gly Gly Gly
Thr Lys Leu Gln Ile Thr 485 490 495 731512DNAARTIFICIAL
SEQUENCE2G8HLxSEQ ID NO 12 ; bispecific single chain antibody
73gaggttcagc tgcagcagtc tggggcagag cttgtgaggt caggggcctc agtcaagttg
60tcctgcacag cttctggctt caacattaaa gactactatt tgcactgggt gaagcagagg
120cctgaacagg gcctggagtg gattgcctgg attgatcttg agaatggtga
tattaaatat 180gccccgaagt ttcagggcaa ggccactata actgcagaca
catcctccaa cacagcctac 240ctgcagctca gcagcctgac atctgaggac
actgccgtct attactgtaa tccctattac 300tacggtagta actacgacta
tgctatggac tactggggtc aaggaacctc agtcaccgtc 360tcctcaggtg
gtggtggttc tggcggcggc ggctccggtg gtggtggttc tgatgttgtg
420atgacccaga ctccactcac tttgtcggtt accattggac aaccagcctc
tatctcttgc 480aagtcaagtc agagcctctt atatagtaat ggaaaaacct
atttgaactg gatattacag 540aggccaggcc agtctccaaa gcgcctaatc
tatctggtgt ctaaactgga ctctggagtc 600cctgacaggt tcactggcag
tggatcagga acagatttta cgctgaaaat cagcagagtg 660gaggctgagg
atttgggagt ttattactgc gtgcaaggta cacattttcc tctcacgttc
720ggtgctggga ccaagctgga gctgaaatcc ggaggtggtg gatcccaggc
tgttgtgact 780caggaatctg cactcaccac atcacctggt gaaacagtca
cactcacttg tcgctcaagt 840actggggctg ttacaactag taactatgcc
aactgggtcc aagaaaaacc agatcattta 900ttcactggtc taataggtgg
taccaacaag cgagctccag gtgtgcctgc cagattctca 960ggctccctga
ttggagacaa ggctgccctc accatcacag gggcacagac tgaggatgag
1020gcaatatatt tctgtgctct atggtacagc aacctctggg tgttcggtgg
aggaaccaaa 1080ctgactgtcc taggtggtgg tggttctggc ggcggcggct
ccggtggtgg tggttctgag 1140gtgaagcttc tcgagtctgg aggaggattg
gtgcagccta aagggtcatt gaaactctca 1200tgtgcagcct ctggattcac
cttcaatacc tacgccatga actgggtccg ccaggctcca 1260ggaaagggtt
tggaatgggt tgctcgcata agaagtaaat ataataatta tgcaacatat
1320tatgccgatt cagtgaaaga caggttcacc atctccagag atgattcaca
aagcattctc 1380tatctacaaa tgaacaactt gaaaactgag gacacagcca
tgtactactg tgtgagacat 1440gggaacttcg gtaatagcta cgtttcctgg
tttgcttact ggggccaagg gactctggtc 1500actgtctctg ca
151274504PRTartificial sequence2G8HLxSEQ ID NO 12 ; bispecific
single chain antibody 74Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser
Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Leu His Trp Val Lys Gln
Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Ala Trp Ile Asp Leu
Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys
Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu
Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Asn Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr Ala Met Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val
Met Thr Gln Thr 130 135 140 Pro Leu Thr Leu Ser Val Thr Ile Gly Gln
Pro Ala Ser Ile Ser Cys 145 150 155 160 Lys Ser Ser Gln Ser Leu Leu
Tyr Ser Asn Gly Lys Thr Tyr Leu Asn 165 170 175 Trp Ile Leu Gln Arg
Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu 180 185 190 Val Ser Lys
Leu Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 195 200 205 Ser
Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp 210 215
220 Leu Gly Val Tyr Tyr Cys Val Gln Gly Thr His Phe Pro Leu Thr Phe
225 230 235 240 Gly Ala Gly Thr Lys Leu Glu Leu Lys Ser Gly Gly Gly
Gly Ser Gln 245 250 255 Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr
Ser Pro Gly Glu Thr 260 265 270 Val Thr Leu Thr Cys Arg Ser Ser Thr
Gly Ala Val Thr Thr Ser Asn 275 280 285 Tyr Ala Asn Trp Val Gln Glu
Lys Pro Asp His Leu Phe Thr Gly Leu 290 295 300 Ile Gly Gly Thr Asn
Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser 305 310 315 320 Gly Ser
Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln 325 330 335
Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu 340
345 350 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly
Gly 355 360 365 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
Lys Leu Leu 370 375 380 Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly
Ser Leu Lys Leu Ser 385 390 395 400 Cys Ala Ala Ser Gly Phe Thr Phe
Asn Thr Tyr Ala Met Asn Trp Val 405 410 415 Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 420 425 430 Lys Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg 435 440 445 Phe Thr
Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met 450 455 460
Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His 465
470 475 480 Gly Asn Phe
Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln 485 490 495 Gly
Thr Leu Val Thr Val Ser Ala 500 751512DNAARTIFICIAL
SEQUENCE2G8HLxSEQ ID NO 10 ; bispecific single chain antibody
75gaggttcagc tgcagcagtc tggggcagag cttgtgaggt caggggcctc agtcaagttg
60tcctgcacag cttctggctt caacattaaa gactactatt tgcactgggt gaagcagagg
120cctgaacagg gcctggagtg gattgcctgg attgatcttg agaatggtga
tattaaatat 180gccccgaagt ttcagggcaa ggccactata actgcagaca
catcctccaa cacagcctac 240ctgcagctca gcagcctgac atctgaggac
actgccgtct attactgtaa tccctattac 300tacggtagta actacgacta
tgctatggac tactggggtc aaggaacctc agtcaccgtc 360tcctcaggtg
gtggtggttc tggcggcggc ggctccggtg gtggtggttc tgatgttgtg
420atgacccaga ctccactcac tttgtcggtt accattggac aaccagcctc
tatctcttgc 480aagtcaagtc agagcctctt atatagtaat ggaaaaacct
atttgaactg gatattacag 540aggccaggcc agtctccaaa gcgcctaatc
tatctggtgt ctaaactgga ctctggagtc 600cctgacaggt tcactggcag
tggatcagga acagatttta cgctgaaaat cagcagagtg 660gaggctgagg
atttgggagt ttattactgc gtgcaaggta cacattttcc tctcacgttc
720ggtgctggga ccaagctgga gctgaaatcc ggaggtggtg gatccgaggt
gaagcttctc 780gagtctggag gaggattggt gcagcctaaa gggtcattga
aactctcatg tgcagcctct 840ggattcacct tcaataccta cgccatgaac
tgggtccgcc aggctccagg aaagggtttg 900gaatgggttg ctcgcataag
aagtaaatat aataattatg caacatatta tgccgattca 960gtgaaagaca
ggttcaccat ctccagagat gattcacaaa gcattctcta tctacaaatg
1020aacaacttga aaactgagga cacagccatg tactactgtg tgagacatgg
gaacttcggt 1080aatagctacg tttcctggtt tgcttactgg ggccaaggga
ctctggtcac tgtctctgca 1140ggtggtggtg gttctggcgg cggcggctcc
ggtggtggtg gttctcaggc tgttgtgact 1200caggaatctg cactcaccac
atcacctggt gaaacagtca cactcacttg tcgctcaagt 1260actggggctg
ttacaactag taactatgcc aactgggtcc aagaaaaacc agatcattta
1320ttcactggtc taataggtgg taccaacaag cgagctccag gtgtgcctgc
cagattctca 1380ggctccctga ttggagacaa ggctgccctc accatcacag
gggcacagac tgaggatgag 1440gcaatatatt tctgtgctct atggtacagc
aacctctggg tgttcggtgg aggaaccaaa 1500ctgactgtcc ta
151276504PRTartificial sequence2G8HLxSEQ ID NO 10 ; bispecific
single chain antibody 76Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser
Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Leu His Trp Val Lys Gln
Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Ala Trp Ile Asp Leu
Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys
Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu
Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Asn Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr Ala Met Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val
Met Thr Gln Thr 130 135 140 Pro Leu Thr Leu Ser Val Thr Ile Gly Gln
Pro Ala Ser Ile Ser Cys 145 150 155 160 Lys Ser Ser Gln Ser Leu Leu
Tyr Ser Asn Gly Lys Thr Tyr Leu Asn 165 170 175 Trp Ile Leu Gln Arg
Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu 180 185 190 Val Ser Lys
Leu Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 195 200 205 Ser
Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp 210 215
220 Leu Gly Val Tyr Tyr Cys Val Gln Gly Thr His Phe Pro Leu Thr Phe
225 230 235 240 Gly Ala Gly Thr Lys Leu Glu Leu Lys Ser Gly Gly Gly
Gly Ser Glu 245 250 255 Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Lys Gly Ser 260 265 270 Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Thr Tyr Ala 275 280 285 Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Ala 290 295 300 Arg Ile Arg Ser Lys
Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 305 310 315 320 Val Lys
Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu 325 330 335
Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr 340
345 350 Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
Ala 355 360 365 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly
Gly Gly Gly 370 375 380 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gln Ala Val Val Thr 385 390 395 400 Gln Glu Ser Ala Leu Thr Thr Ser
Pro Gly Glu Thr Val Thr Leu Thr 405 410 415 Cys Arg Ser Ser Thr Gly
Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp 420 425 430 Val Gln Glu Lys
Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr 435 440 445 Asn Lys
Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile 450 455 460
Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu 465
470 475 480 Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val
Phe Gly 485 490 495 Gly Gly Thr Lys Leu Thr Val Leu 500
771491DNAARTIFICIAL SEQUENCE2G8HLxSEQ ID NO 16 ; bispecific single
chain antibody 77gaggttcagc tgcagcagtc tggggcagag cttgtgaggt
caggggcctc agtcaagttg 60tcctgcacag cttctggctt caacattaaa gactactatt
tgcactgggt gaagcagagg 120cctgaacagg gcctggagtg gattgcctgg
attgatcttg agaatggtga tattaaatat 180gccccgaagt ttcagggcaa
ggccactata actgcagaca catcctccaa cacagcctac 240ctgcagctca
gcagcctgac atctgaggac actgccgtct attactgtaa tccctattac
300tacggtagta actacgacta tgctatggac tactggggtc aaggaacctc
agtcaccgtc 360tcctcaggtg gtggtggttc tggcggcggc ggctccggtg
gtggtggttc tgatgttgtg 420atgacccaga ctccactcac tttgtcggtt
accattggac aaccagcctc tatctcttgc 480aagtcaagtc agagcctctt
atatagtaat ggaaaaacct atttgaactg gatattacag 540aggccaggcc
agtctccaaa gcgcctaatc tatctggtgt ctaaactgga ctctggagtc
600cctgacaggt tcactggcag tggatcagga acagatttta cgctgaaaat
cagcagagtg 660gaggctgagg atttgggagt ttattactgc gtgcaaggta
cacattttcc tctcacgttc 720ggtgctggga ccaagctgga gctgaaatcc
ggaggtggtg gatcccaagt tgttctcacc 780cagtctccag caatcatgtc
tgcatttcca ggggagaagg tcaccatgac ctgcagtgcc 840agctcaagtg
taagttacat gaactggtac cagcagaagt caggcacctc ccccaaaaga
900tggatttatg actcatccaa actggcttct ggagtccctg ctcgcttcag
tggcagtggg 960tctgggacct cttattctct cacaatcagc agcatggaga
ctgaagatgc tgccacttat 1020tactgccagc agtggagtcg taacccaccc
acgttcggag gggggaccaa gctacaaatt 1080acaggtggtg gtggttctgg
cggcggcggc tccggtggtg gtggttctca ggtccagctg 1140cagcagtctg
gggctgaact ggcaagacct ggggcctcag tgaagatgtc ctgcaaggct
1200tctggctaca cctttactag atctacgatg cactgggtaa aacagaggcc
tggacagggt 1260ctggaatgga ttggatacat taatcctagc agtgcttata
ctaattacaa tcagaaattc 1320aaggacaagg ccacattgac tgcagacaaa
tcctccagta cagcctacat gcaactgagt 1380agcctgacat ctgaggactc
tgcagtctat tactgtgcaa gtccgcaagt ccactatgat 1440tacaacgggt
ttccttactg gggccaaggg actctggtca ctgtctctgc a
149178497PRTartificial sequence2G8HLxSEQ ID NO 16 ; bispecific
single chain antibody 78Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser
Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Leu His Trp Val Lys Gln
Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Ala Trp Ile Asp Leu
Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys
Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu
Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Asn Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr Ala Met Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val
Met Thr Gln Thr 130 135 140 Pro Leu Thr Leu Ser Val Thr Ile Gly Gln
Pro Ala Ser Ile Ser Cys 145 150 155 160 Lys Ser Ser Gln Ser Leu Leu
Tyr Ser Asn Gly Lys Thr Tyr Leu Asn 165 170 175 Trp Ile Leu Gln Arg
Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu 180 185 190 Val Ser Lys
Leu Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 195 200 205 Ser
Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp 210 215
220 Leu Gly Val Tyr Tyr Cys Val Gln Gly Thr His Phe Pro Leu Thr Phe
225 230 235 240 Gly Ala Gly Thr Lys Leu Glu Leu Lys Ser Gly Gly Gly
Gly Ser Gln 245 250 255 Val Val Leu Thr Gln Ser Pro Ala Ile Met Ser
Ala Phe Pro Gly Glu 260 265 270 Lys Val Thr Met Thr Cys Ser Ala Ser
Ser Ser Val Ser Tyr Met Asn 275 280 285 Trp Tyr Gln Gln Lys Ser Gly
Thr Ser Pro Lys Arg Trp Ile Tyr Asp 290 295 300 Ser Ser Lys Leu Ala
Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 305 310 315 320 Ser Gly
Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Thr Glu Asp 325 330 335
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Arg Asn Pro Pro Thr Phe 340
345 350 Gly Gly Gly Thr Lys Leu Gln Ile Thr Gly Gly Gly Gly Ser Gly
Gly 355 360 365 Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln
Gln Ser Gly 370 375 380 Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys
Met Ser Cys Lys Ala 385 390 395 400 Ser Gly Tyr Thr Phe Thr Arg Ser
Thr Met His Trp Val Lys Gln Arg 405 410 415 Pro Gly Gln Gly Leu Glu
Trp Ile Gly Tyr Ile Asn Pro Ser Ser Ala 420 425 430 Tyr Thr Asn Tyr
Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala 435 440 445 Asp Lys
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser 450 455 460
Glu Asp Ser Ala Val Tyr Tyr Cys Ala Ser Pro Gln Val His Tyr Asp 465
470 475 480 Tyr Asn Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser 485 490 495 Ala 791491DNAARTIFICIAL SEQUENCE2G8HLxSEQ ID NO
14 ; bispecific single chain antibody 79gaggttcagc tgcagcagtc
tggggcagag cttgtgaggt caggggcctc agtcaagttg 60tcctgcacag cttctggctt
caacattaaa gactactatt tgcactgggt gaagcagagg 120cctgaacagg
gcctggagtg gattgcctgg attgatcttg agaatggtga tattaaatat
180gccccgaagt ttcagggcaa ggccactata actgcagaca catcctccaa
cacagcctac 240ctgcagctca gcagcctgac atctgaggac actgccgtct
attactgtaa tccctattac 300tacggtagta actacgacta tgctatggac
tactggggtc aaggaacctc agtcaccgtc 360tcctcaggtg gtggtggttc
tggcggcggc ggctccggtg gtggtggttc tgatgttgtg 420atgacccaga
ctccactcac tttgtcggtt accattggac aaccagcctc tatctcttgc
480aagtcaagtc agagcctctt atatagtaat ggaaaaacct atttgaactg
gatattacag 540aggccaggcc agtctccaaa gcgcctaatc tatctggtgt
ctaaactgga ctctggagtc 600cctgacaggt tcactggcag tggatcagga
acagatttta cgctgaaaat cagcagagtg 660gaggctgagg atttgggagt
ttattactgc gtgcaaggta cacattttcc tctcacgttc 720ggtgctggga
ccaagctgga gctgaaatcc ggaggtggtg gatcccaggt ccagctgcag
780cagtctgggg ctgaactggc aagacctggg gcctcagtga agatgtcctg
caaggcttct 840ggctacacct ttactagatc tacgatgcac tgggtaaaac
agaggcctgg acagggtctg 900gaatggattg gatacattaa tcctagcagt
gcttatacta attacaatca gaaattcaag 960gacaaggcca cattgactgc
agacaaatcc tccagtacag cctacatgca actgagtagc 1020ctgacatctg
aggactctgc agtctattac tgtgcaagtc cgcaagtcca ctatgattac
1080aacgggtttc cttactgggg ccaagggact ctggtcactg tctctgcagg
tggtggtggt 1140tctggcggcg gcggctccgg tggtggtggt tctcaagttg
ttctcaccca gtctccagca 1200atcatgtctg catttccagg ggagaaggtc
accatgacct gcagtgccag ctcaagtgta 1260agttacatga actggtacca
gcagaagtca ggcacctccc ccaaaagatg gatttatgac 1320tcatccaaac
tggcttctgg agtccctgct cgcttcagtg gcagtgggtc tgggacctct
1380tattctctca caatcagcag catggagact gaagatgctg ccacttatta
ctgccagcag 1440tggagtcgta acccacccac gttcggaggg gggaccaagc
tacaaattac a 149180497PRTArtificial sequence2G8HLxSEQ ID NO 14 ;
bispecific single chain antibody 80Glu Val Gln Leu Gln Gln Ser Gly
Ala Glu Leu Val Arg Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys
Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Leu His Trp
Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Ala Trp
Ile Asp Leu Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe 50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65
70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Asn Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr Ala
Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly
Ser Asp Val Val Met Thr Gln Thr 130 135 140 Pro Leu Thr Leu Ser Val
Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys 145 150 155 160 Lys Ser Ser
Gln Ser Leu Leu Tyr Ser Asn Gly Lys Thr Tyr Leu Asn 165 170 175 Trp
Ile Leu Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu 180 185
190 Val Ser Lys Leu Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
195 200 205 Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
Glu Asp 210 215 220 Leu Gly Val Tyr Tyr Cys Val Gln Gly Thr His Phe
Pro Leu Thr Phe 225 230 235 240 Gly Ala Gly Thr Lys Leu Glu Leu Lys
Ser Gly Gly Gly Gly Ser Gln 245 250 255 Val Gln Leu Gln Gln Ser Gly
Ala Glu Leu Ala Arg Pro Gly Ala Ser 260 265 270 Val Lys Met Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Arg Ser Thr 275 280 285 Met His Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 290 295 300 Tyr
Ile Asn Pro Ser Ser Ala Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 305 310
315 320 Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
Met 325 330 335 Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys Ala 340 345 350 Ser Pro Gln Val His Tyr Asp Tyr Asn Gly Phe
Pro Tyr Trp Gly Gln 355 360 365 Gly Thr Leu Val Thr Val Ser Ala Gly
Gly Gly Gly Ser Gly Gly Gly 370 375 380 Gly Ser Gly Gly Gly Gly Ser
Gln Val Val Leu Thr Gln Ser Pro Ala 385 390 395 400 Ile Met Ser Ala
Phe Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala 405 410 415 Ser Ser
Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr 420 425 430
Ser Pro Lys Arg Trp Ile Tyr Asp Ser Ser Lys Leu Ala Ser Gly Val 435
440 445 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
Thr 450 455 460 Ile Ser Ser Met Glu Thr Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln 465 470 475 480 Trp Ser Arg Asn Pro Pro Thr Phe Gly Gly
Gly Thr Lys Leu Gln Ile 485 490 495 Thr 8127DNAARTIFICIAL
SEQUENCE5' primer VH ; oligonucleotide 81saggtgcagc tcgaggagtc
aggacct
278227DNAartificial sequence5' primer VH ; oligonucleotide
82gaggtccagc tcgagcagtc tggacct 278327DNAartificial sequence5'
primer VH ; oligonucleotide 83caggtccaac tcgagcagcc tggggct
278427DNAartificial sequence5' primer VH ; oligonucleotide
84gaggttcagc tcgagcagtc tggggca 278527DNAartificial sequence5'
primer VH ; oligonucleotide 85gargtgaagc tcgaggagtc tggagga
278627DNAartificial sequence5' primer VH ; oligonucleotide
86gaggtgaagc ttctcgagtc tggaggt 278727DNAartificial sequence5'
primer VH ; oligonucleotide 87gaagtgaagc tcgaggagtc tggggga
278827DNAartificial sequence5' primer VH ; oligonucleotide
88gaggttcagc tcgagcagtc tggagct 278939DNAartificial sequence5'
primer VH ; oligonucleotide 89gggctcgagc accatggrat gsagctgkgt
matsctctt 399039DNAartificial sequence5' primer VH ;
oligonucleotide 90gggctcgagc accatgract tcgggytgag ctkggtttt
399138DNAartificial sequence5' primer VH ; oligonucleotide
91gggctcgagc accatggctg tcttggggct gctcttct 389234DNAartificial
sequence3' primer VH ; oligonucleotide 92gaggaattcg aactggacag
ggatccagag ttcc 349334DNAARTIFICIAL SEQUENCE3' primer VH ;
oligonucleotide 93cggaattcga atgacatgga catctgggtc atcc
349432DNAARTIFICIAL SEQUENCE5' primer VL ; oligonucleotide
94ccagttccga gctcgttgtg actcaggaat ct 329532DNAartificial
sequence5' primer VL ; oligonucleotide 95ccagttccga gctcgtgttg
acgcagccgc cc 329632DNAartificial sequence5' primer VL ;
oligonucleotide 96ccagttccga gctcgtgctc acccagtctc ca
329732DNAartificial sequence5' primer VL ; oligonucleotide
97ccagttccga gctccagatg acccagtctc ca 329832DNAartificial
sequence5' primer VL ; oligonucleotide 98ccagatgtga gctcgtgatg
acccagactc ca 329932DNAartificial sequence5' primer VL ;
oligonucleotide 99ccagatgtga gctcgtcatg acccagtctc ca
3210032DNAartificial sequence5' primer VL ; oligonucleotide
100ccagttccga gctcgtgatg acacagtctc ca 3210138DNAartificial
sequence5' primer VL ; oligonucleotide 101ggggagctcc accatggaga
cagacacact cctgctat 3810239DNAartificial sequence5' primer VL ;
oligonucleotide 102ggggagctcc accatggatt ttcaagtgca gattttcag
3910340DNAartificial sequence5' primer VL ; oligonucleotide
103ggggagctcc accatggagw cacakwctca ggtctttrta 4010436DNAartificial
sequence5' primer VL ; oligonucleotide 104ggggagctcc accatgkccc
cwrctcagyt yctkgt 3610531DNAartificial sequence3' primer VL ;
oligonucleotide 105gaggaattcg aactgctcac tggatggtgg g
3110635DNAARTIFICIAL SEQUENCE3' primer VL ; oligonucleotide
106cggaattcga acaaactctt ctccacagtg tgacc 3510730DNAARTIFICIAL
SEQUENCE3' primer VH ; oligonucleotide 107tatgcaacta gtacaaccac
aatccctggg 3010834DNAartificial sequence3' primer VL ;
oligonucleotide 108gcgccgtcta gaattaacac tcattcctgt tgaa
34109496PRTartificial sequence5-10 LH x deimmunised (di) anti-CD3 ;
bispecific single chain antibody 109Glu 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 115 120 125 Glu Val Gln Leu Leu Glu Gln Ser
Gly Ala Glu Leu Val Arg Pro Gly 130 135 140 Thr Ser Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn 145 150 155 160 Tyr Trp Leu
Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp 165 170 175 Ile
Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys 180 185
190 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
195 200 205 Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val
Tyr Phe 210 215 220 Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp
Tyr Trp Gly Gln 225 230 235 240 Gly Thr Thr Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Asp Val Gln 245 250 255 Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala Ser Val Lys 260 265 270 Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His 275 280 285 Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile 290 295 300 Asn
Pro Ser Arg Gly Tyr Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg 305 310
315 320 Phe Thr Ile Thr Thr Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu
Leu 325 330 335 Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
Ala Arg Tyr 340 345 350 Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly
Gln Gly Thr Thr Val 355 360 365 Thr Val Ser Ser Gly Glu Gly Thr Ser
Thr Gly Ser Gly Gly Ser Gly 370 375 380 Gly Ser Gly Gly Ala Asp Asp
Ile Val Leu Thr Gln Ser Pro Ala Thr 385 390 395 400 Leu Ser Leu Ser
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 405 410 415 Gln Ser
Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala 420 425 430
Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro 435
440 445 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
Ile 450 455 460 Asn Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Trp 465 470 475 480 Ser Ser Asn Pro Leu Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 485 490 495 110125PRTARTIFICIAL
SEQUENCEHuman-like VH VH region 110Glu Val Gln Leu Leu 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 Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65
70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 125 111375DNAartificial sequenceHuman-like VH
VH region 111gaggtgcagc tgctcgagtc tggaggagga ttggtgcagc ctggagggtc
attgaaactc 60tcatgtgcag cctctggatt caccttcaat acctacgcca tgaactgggt
ccgccaggct 120ccaggaaagg gtttggaatg ggttgctcgc ataagaagta
aatataataa ttatgcaaca 180tattatgccg attcagtgaa agacaggttc
accatctcca gagatgattc aaaaaacact 240gcctatctac aaatgaacaa
cttgaaaact gaggacactg ccgtgtacta ctgtgtgaga 300catgggaact
tcggtaatag ctacgtttcc tggtttgctt actggggcca agggactctg
360gtcaccgtct cctca 37511214PRTMUS MUSCULUSmisc_feature(1)..()CDR3
of VH 112His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1
5 10 1136PRTmus musculusmisc_feature(1)..(6)VH short CDR3 short
CDR3 of SEQ ID NO 112 113Val Ser Trp Phe Ala Tyr 1 5 11419PRTmus
musculusmisc_feature(1)..(19)CDR2 of VH 114Arg Ile Arg Ser Lys Tyr
Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp
1155PRTmus musculusmisc_feature(1)..(5)CDR1 of VH 115Thr Tyr Ala
Met Asn 1 5 1169PRTmus musculusmisc_feature(1)..(9)CDR3 of VL
116Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 1177PRTmus
musculusmisc_feature(1)..(7)CDR2 of VL 117Gly Thr Asn Lys Arg Ala
Pro 1 5 11814PRTmus musculusmisc_feature(1)..(14)CDR1 of VL 118Arg
Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10
11912PRTmus musculusmisc_feature(1)..(12)CDR3 of VH 119Pro Gln Val
His Tyr Asp Tyr Asn Gly Phe Pro Tyr 1 5 10 12017PRTmus
musculusmisc_feature(1)..(17)CDR2 of VH 120Tyr Ile Asn Pro Ser Ser
Ala Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp 12110PRTmus
musculusmisc_feature(1)..(10)CDR1 of VH 121Gly Tyr Thr Phe Thr Arg
Ser Thr Met His 1 5 10 122509PRTartificial sequence2G8 LHx SEQ ID
NO 146 ; bispecific single chain antibody 122Asp Val Val Met Thr
Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala
Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn
Gly Lys Thr Tyr Leu Asn Trp Ile Leu Gln Arg Pro Gly Gln Ser 35 40
45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
Cys Val Gln Gly 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu 115 120 125 Val Gln Leu Gln Gln Ser
Gly Ala Glu Leu Val Arg Ser Gly Ala Ser 130 135 140 Val Lys Leu Ser
Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr 145 150 155 160 Leu
His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Ala 165 170
175 Trp Ile Asp Leu Glu Asn Gly Asp Ile Lys Tyr Ala Pro Lys Phe Gln
180 185 190 Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala
Tyr Leu 195 200 205 Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 210 215 220 Pro Tyr Tyr Tyr Gly Ser Asn Tyr Asp Tyr
Ala Met Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Ser Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Glu Val 245 250 255 Lys Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 260 265 270 Lys Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met 275 280 285 Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg 290 295
300 Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val
305 310 315 320 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Thr Ala Tyr 325 330 335 Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Val Tyr Tyr Cys 340 345 350 Val Arg His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe Ala Tyr 355 360 365 Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Gly Gly Gly Gly Ser 370 375 380 Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Leu Val Val Thr Gln 385 390 395 400 Glu Ser
Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys 405 410 415
Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val 420
425 430 Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr
Asn 435 440 445 Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser
Leu Ile Gly 450 455 460 Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln
Thr Glu Asp Glu Ala 465 470 475 480 Ile Tyr Phe Cys Ala Leu Trp Tyr
Ser Asn Leu Trp Val Phe Gly Gly 485 490 495 Gly Thr Lys Leu Thr Val
Leu His His His His His His 500 505 1231527DNAARTIFICIAL
SEQUENCE2G8 LHxSEQ ID NO 146 ; bispecific single chain antibody
123gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca
accagcctct 60atctcttgca agtcaagtca gagcctctta tatagtaatg gaaaaaccta
tttgaactgg 120atattacaga ggccaggcca gtctccaaag cgcctaatct
atctggtgtc taaactggac 180tctggagtcc ctgacaggtt cactggcagt
ggatcaggaa cagattttac gctgaaaatc 240agcagagtgg aggctgagga
tttgggagtt tattactgcg tgcaaggtac acattttcct 300ctcacgttcg
gtgctgggac caagctggag ctgaaaggtg gtggtggttc tggcggcggc
360ggctccggtg gtggtggttc tgaggttcag ctgcagcagt ctggggcaga
gcttgtgagg 420tcaggggcct cagtcaagtt gtcctgcaca gcttctggct
tcaacattaa agactactat 480ttgcactggg tgaagcagag gcctgaacag
ggcctggagt ggattgcctg gattgatctt 540gagaatggtg atattaaata
tgccccgaag tttcagggca aggccactat aactgcagac 600acatcctcca
acacagccta cctgcagctc agcagcctga catctgagga cactgccgtc
660tattactgta atccctatta ctacggtagt aactacgact atgctatgga
ctactggggt 720caaggaacct cagtcaccgt ctcctccgga ggtggtggat
ccgaggtgaa gcttctcgag 780tctggaggag gattggtgca gcctggaggg
tcattgaaac tctcatgtgc agcctctgga 840ttcaccttca atacctacgc
catgaactgg gtccgccagg ctccaggaaa gggtttggaa 900tgggttgctc
gcataagaag taaatataat aattatgcaa catattatgc cgattcagtg
960aaagacaggt tcaccatctc cagagatgat tcaaaaaaca ctgcctatct
acaaatgaac 1020aacttgaaaa ctgaggacac tgccgtgtac tactgtgtga
gacatgggaa cttcggtaat 1080agctacgttt cctggtttgc ttactggggc
caagggactc tggtcaccgt ctcctcaggt 1140ggtggtggtt ctggcggcgg
cggctccggt ggtggtggtt ctgagctcgt tgtgactcag 1200gaatctgcac
tcaccacatc acctggtgaa acagtcacac tcacttgtcg ctcaagtact
1260ggggctgtta caactagtaa ctatgccaac tgggtccaag aaaaaccaga
tcatttattc 1320actggtctaa taggtggtac caacaagcga gcaccaggtg
tgcctgccag attctcaggc 1380tccctgattg gagacaaggc tgccctcacc
atcacagggg cacagactga ggatgaggca 1440atatatttct gtgctctatg
gtacagcaac ctctgggtgt tcggtggagg aaccaaactg 1500actgtcctac
atcatcacca tcatcat 1527124508PRTartificial sequence5-10 LHxSEQ ID
NO 146 ; bispecific single chain antibody 124Glu 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 Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125 Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg
Pro Gly 130 135 140 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
Ala Phe Thr Asn 145 150 155 160 Tyr Trp Leu Gly Trp Val Lys Gln Arg
Pro Gly His Gly Leu Glu Trp 165 170 175 Ile Gly Asp Ile Phe Pro Gly
Ser Gly Asn Ile His Tyr Asn Glu Lys 180 185 190 Phe Lys Gly Lys Ala
Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 195 200 205 Tyr Met Gln
Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe 210 215 220 Cys
Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln 225 230
235 240 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Val
Lys 245 250 255 Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
Ser Leu Lys 260 265 270 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn
Thr Tyr Ala Met Asn 275 280 285 Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ala Arg Ile 290 295 300 Arg Ser Lys Tyr Asn Asn Tyr
Ala Thr Tyr Tyr Ala Asp Ser Val Lys 305 310 315 320 Asp Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu 325 330 335 Gln Met
Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val 340 345 350
Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp 355
360 365 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly 370 375 380 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Val
Thr Gln Glu 385 390 395 400 Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr
Val Thr Leu Thr Cys Arg 405 410 415 Ser Ser Thr Gly Ala Val Thr Thr
Ser Asn Tyr Ala Asn Trp Val Gln 420 425 430 Glu Lys Pro Asp His Leu
Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys 435 440 445 Arg Ala Pro Gly
Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp 450 455 460 Lys Ala
Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile 465 470 475
480 Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly
485 490 495 Thr Lys Leu Thr Val Leu His His His His His His 500 505
1251524DNAARTIFICIAL SEQUENCE5-10 LHxSEQ ID NO 146 ; bispecific
single chain antibody 125gagctcgtga tgacacagtc tccatcctcc
ctgactgtga cagcaggaga gaaggtcact 60atgagctgca agtccagtca gagtctgtta
aacagtggaa atcaaaagaa ctacttgacc 120tggtaccagc agaaaccagg
gcagcctcct aaactgttga tctactgggc atccactagg 180gaatctgggg
tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc
240atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga
ttatagttat 300ccgctcacgt tcggtgctgg gaccaagctt gagatcaaag
gtggtggtgg ttctggcggc 360ggcggctccg gtggtggtgg ttctgaggtg
cagctgctcg agcagtctgg agctgagctg 420gtaaggcctg ggacttcagt
gaagatatcc tgcaaggctt ctggatacgc cttcactaac 480tactggctag
gttgggtaaa gcagaggcct ggacatggac ttgagtggat tggagatatt
540ttccctggaa gtggtaatat ccactacaat gagaagttca agggcaaagc
cacactgact 600gcagacaaat cttcgagcac agcctatatg cagctcagta
gcctgacatt tgaggactct 660gctgtctatt tctgtgcaag actgaggaac
tgggacgagc ctatggacta ctggggccaa 720gggaccacgg tcaccgtctc
ctccggaggt ggtggatccg aggtgaagct tctcgagtct 780ggaggaggat
tggtgcagcc tggagggtca ttgaaactct catgtgcagc ctctggattc
840accttcaata cctacgccat gaactgggtc cgccaggctc caggaaaggg
tttggaatgg 900gttgctcgca taagaagtaa atataataat tatgcaacat
attatgccga ttcagtgaaa 960gacaggttca ccatctccag agatgattca
aaaaacactg cctatctaca aatgaacaac 1020ttgaaaactg aggacactgc
cgtgtactac tgtgtgagac atgggaactt cggtaatagc 1080tacgtttcct
ggtttgctta ctggggccaa gggactctgg tcaccgtctc ctcaggtggt
1140ggtggttctg gcggcggcgg ctccggtggt ggtggttctg agctcgttgt
gactcaggaa 1200tctgcactca ccacatcacc tggtgaaaca gtcacactca
cttgtcgctc aagtactggg 1260gctgttacaa ctagtaacta tgccaactgg
gtccaagaaa aaccagatca tttattcact 1320ggtctaatag gtggtaccaa
caagcgagca ccaggtgtgc ctgccagatt ctcaggctcc 1380ctgattggag
acaaggctgc cctcaccatc acaggggcac agactgagga tgaggcaata
1440tatttctgtg ctctatggta cagcaacctc tgggtgttcg gtggaggaac
caaactgact 1500gtcctacatc atcaccatca tcat
1524126372DNAhybridomamisc_feature(1)..(372)FN18 VH VH region of
monoclonal antibody FN18 126caggtccagc tgcagcagtc tgaagctgaa
ctggcaagac ctggggcctc agtgaagatg 60tcctgcaagg cttctggcta cacctttact
gactacacga tacactggtt aaaacagagg 120cctggacagg gtctggactg
gattggatat tttaatccta gcagtgaatc tactgaatac 180aatcggaaat
tcaaggacag gaccatattg actgcagaca gatcctcaac cacagcctac
240atgcaactga gcagcctgac atctgaggac tctgcggtct attactgttc
aaggaaaggg 300gagaaactac ttggtaaccg ttactggtac ttcgatgtct
ggggcgcagg gacctcggtc 360accgtctcct ca
372127124PRThybridomamisc_feature(1)..(124)FN18 VH VH region of
monoclonal antibody FN18 127Gln Val Gln Leu Gln Gln Ser Glu Ala Glu
Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Thr Ile His Trp Leu Lys
Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile 35 40 45 Gly Tyr Phe Asn
Pro Ser Ser Glu Ser Thr Glu Tyr Asn Arg Lys Phe 50 55 60 Lys Asp
Arg Thr Ile Leu Thr Ala Asp Arg Ser Ser Thr 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 Ser Arg Lys Gly Glu Lys Leu Leu Gly Asn Arg Tyr Trp Tyr Phe
Asp 100 105 110 Val Trp Gly Ala Gly Thr Ser Val Thr Val Ser Ser 115
120 128339DNAHYBRIDOMAmisc_feature(1)..(339)FN18 VL VL region of
monoclonal antibody FN18 128gacattgtga tgtcacagtc tccatcctcc
ctagctgtgt cagttggaga gaaggttact 60atgagctgca agtccagtca gagcctttta
tatagtagca atcaaaagaa ctacttggcc 120tggtaccagc agaagccagg
gcagtctcct aaattgctga ttaactgggc atccaccagg 180gaatctgggg
tccctgatcg cttcacaggc agtggatcta ggacagattt cactctcacc
240atcagcagtg tgaaggctga agacctggca gtttatttct gtcagcaatt
ttatagttat 300cctccgacgt tcggtggagg caccaagctg gaaatcaaa
339129113PRThybridomamisc_feature(1)..(113)FN18 VL VL region of
monoclonal antibody FN18 129Asp Ile Val Met Ser Gln Ser Pro Ser Ser
Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys
Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu
Leu Ile Asn Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp
Arg Phe Thr Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr 65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln 85
90 95 Phe Tyr Ser Tyr Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile 100 105 110 Lys 130756DNAARTIFICIAL SEQUENCEFN18 VH-VL scFv ;
single chain Fv 130caggtccagc tgcagcagtc tgaagctgaa ctggcaagac
ctggggcctc agtgaagatg 60tcctgcaagg cttctggcta cacctttact gactacacga
tacactggtt aaaacagagg 120cctggacagg gtctggactg gattggatat
tttaatccta gcagtgaatc tactgaatac 180aatcggaaat tcaaggacag
gaccatattg actgcagaca gatcctcaac cacagcctac 240atgcaactga
gcagcctgac atctgaggac tctgcggtct attactgttc aaggaaaggg
300gagaaactac ttggtaaccg ttactggtac ttcgatgtct ggggcgcagg
gacctcggtc 360accgtctcct caggtggtgg tggttctggc ggcggcggct
ccggtggtgg tggttctgac 420attgtgatgt cacagtctcc atcctcccta
gctgtgtcag ttggagagaa ggttactatg 480agctgcaagt ccagtcagag
ccttttatat agtagcaatc aaaagaacta cttggcctgg 540taccagcaga
agccagggca gtctcctaaa ttgctgatta actgggcatc caccagggaa
600tctggggtcc ctgatcgctt cacaggcagt ggatctagga cagatttcac
tctcaccatc 660agcagtgtga aggctgaaga cctggcagtt tatttctgtc
agcaatttta tagttatcct 720ccgacgttcg gtggaggcac caagctggaa atcaaa
756131252PRTartificial sequenceFN18 VH-VL scFv ; single chain Fv
131Gln Val Gln Leu Gln Gln Ser Glu Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Asp Tyr 20 25 30 Thr Ile His Trp Leu Lys Gln Arg Pro Gly Gln Gly
Leu Asp Trp Ile 35 40 45 Gly Tyr Phe Asn Pro Ser Ser Glu Ser Thr
Glu Tyr Asn Arg Lys Phe 50 55 60 Lys Asp Arg Thr Ile Leu Thr Ala
Asp Arg Ser Ser Thr 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 Ser Arg Lys Gly
Glu Lys Leu Leu Gly Asn Arg Tyr Trp Tyr Phe Asp 100 105 110 Val Trp
Gly Ala Gly Thr Ser 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 Val Met Ser 130
135 140 Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly Glu Lys Val Thr
Met 145 150 155 160 Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser
Asn Gln Lys Asn 165 170 175 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ser Pro Lys Leu Leu 180 185 190 Ile Asn Trp Ala Ser Thr Arg Glu
Ser Gly Val Pro Asp Arg Phe Thr 195 200 205 Gly Ser Gly Ser Arg Thr
Asp Phe Thr Leu Thr Ile Ser Ser Val Lys 210 215 220 Ala Glu Asp Leu
Ala Val Tyr Phe Cys Gln Gln Phe Tyr Ser Tyr Pro 225 230 235 240 Pro
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 245 250
132756DNAARTIFICIAL SEQUENCEFN18 VL-VH scFv ; single chain Fv
132gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga
gaaggttact 60atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa
ctacttggcc 120tggtaccagc agaagccagg gcagtctcct aaattgctga
ttaactgggc atccaccagg 180gaatctgggg tccctgatcg cttcacaggc
agtggatcta ggacagattt cactctcacc 240atcagcagtg tgaaggctga
agacctggca gtttatttct gtcagcaatt ttatagttat 300cctccgacgt
tcggtggagg caccaagctg gaaatcaaag gtggtggtgg ttctggcggc
360ggcggctccg gtggtggtgg ttctcaggtc cagctgcagc agtctgaagc
tgaactggca 420agacctgggg cctcagtgaa gatgtcctgc aaggcttctg
gctacacctt tactgactac 480acgatacact ggttaaaaca gaggcctgga
cagggtctgg actggattgg atattttaat 540cctagcagtg aatctactga
atacaatcgg aaattcaagg acaggaccat attgactgca 600gacagatcct
caaccacagc ctacatgcaa ctgagcagcc tgacatctga ggactctgcg
660gtctattact gttcaaggaa aggggagaaa ctacttggta accgttactg
gtacttcgat 720gtctggggcg cagggacctc ggtcaccgtc tcctca
756133252PRTartificial sequenceFN18 VL-VH scFv ; single chain Fv
133Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu
Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Asn Trp Ala Ser
Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly
Ser Arg Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala
Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln 85 90 95 Phe Tyr Ser Tyr
Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125
Gln Val Gln Leu Gln Gln Ser Glu Ala Glu Leu Ala Arg Pro Gly Ala 130
135 140 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 145 150 155 160 Thr Ile His Trp Leu Lys Gln Arg Pro Gly Gln Gly
Leu Asp Trp Ile 165 170 175 Gly Tyr Phe Asn Pro Ser Ser Glu Ser Thr
Glu Tyr Asn Arg Lys Phe 180 185 190 Lys Asp Arg Thr Ile Leu Thr Ala
Asp Arg Ser Ser Thr Thr Ala Tyr 195 200 205 Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 210 215 220 Ser Arg Lys Gly
Glu Lys Leu Leu Gly Asn Arg Tyr Trp Tyr Phe Asp 225 230 235 240 Val
Trp Gly Ala Gly Thr Ser Val Thr Val Ser Ser 245 250 134105PRTHOMO
SAPIENSmisc_feature(1)..(105)CD3 epsilon extracellular portion
134Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys
1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln
Tyr Pro 20 25 30 Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn
Ile Gly Gly Asp 35 40 45 Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu
Asp His Leu Ser Leu Lys 50 55 60 Glu Phe Ser Glu Leu Glu Gln Ser
Gly Tyr Tyr Val Cys Tyr Pro Arg 65 70 75 80 Gly Ser Lys Pro Glu Asp
Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg 85 90 95 Val Cys Glu Asn
Cys Met Glu Met Asp 100 105
13596PRTCYNOMOLGUSmisc_feature(1)..(96)CD3 epsilon extracellular
portion FN18+ 135Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln
Thr Pro Tyr Gln 1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu
Thr Cys Ser Gln His Leu 20 25 30 Gly Ser Glu Ala Gln Trp Gln His
Asn Gly Lys Asn Lys Glu Asp Ser 35 40 45 Gly Asp Arg Leu Phe Leu
Pro Glu Phe Ser Glu Met Glu Gln Ser Gly 50 55 60 Tyr Tyr Val Cys
Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His 65 70 75 80 His Leu
Tyr Leu Lys Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp 85 90 95
13696PRTCYNOMOLGUSmisc_feature(1)..(96)CD3 epsilon extracellular
portion FN18- 136Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln
Thr Pro Tyr Gln 1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu
Thr Cys Ser Gln His Leu 20 25 30 Gly Ser Glu Ala Gln Trp Gln His
Asn Gly Lys Asn Lys Gly Asp Ser 35 40 45 Gly Asp Gln Leu Phe Leu
Pro Glu Phe Ser Glu Met Glu Gln Ser Gly 50 55 60 Tyr Tyr Val Cys
Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His 65 70 75 80 His Leu
Tyr Leu Lys Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp 85 90 95
137242PRTHOMO SAPIENSmisc_feature(1)..(242)EpCAM extracellular
portion 137Gln Glu Glu Cys Val Cys Glu Asn Tyr Lys Leu Ala Val Asn
Cys Phe 1 5 10 15 Val Asn Asn Asn Arg Gln Cys Gln Cys Thr Ser Val
Gly Ala Gln Asn 20 25 30 Thr Val Ile Cys Ser Lys Leu Ala Ala Lys
Cys Leu Val Met Lys Ala 35 40 45 Glu Met Asn Gly Ser Lys Leu Gly
Arg Arg Ala Lys Pro Glu Gly Ala 50 55 60 Leu Gln Asn Asn Asp Gly
Leu Tyr Asp Pro Asp Cys Asp Glu Ser Gly 65 70 75 80 Leu Phe Lys Ala
Lys Gln Cys Asn Gly Thr Ser Thr Cys Trp Cys Val 85 90 95 Asn Thr
Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile Thr Cys 100 105 110
Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu Lys His Lys 115
120 125 Ala Arg Glu Lys Pro Tyr Asp Ser Lys Ser Leu Arg Thr Ala Leu
Gln 130
135 140 Lys Glu Ile Thr Thr Arg Tyr Gln Leu Asp Pro Lys Phe Ile Thr
Ser 145 150 155 160 Ile Leu Tyr Glu Asn Asn Val Ile Thr Ile Asp Leu
Val Gln Asn Ser 165 170 175 Ser Gln Lys Thr Gln Asn Asp Val Asp Ile
Ala Asp Val Ala Tyr Tyr 180 185 190 Phe Glu Lys Asp Val Lys Gly Glu
Ser Leu Phe His Ser Lys Lys Met 195 200 205 Asp Leu Thr Val Asn Gly
Glu Gln Leu Asp Leu Asp Pro Gly Gln Thr 210 215 220 Leu Ile Tyr Tyr
Val Asp Glu Lys Ala Pro Glu Phe Ser Met Gln Gly 225 230 235 240 Leu
Lys 138242PRTChimpanzeemisc_feature(1)..(242)EpCAM extracellular
portion 138Gln Glu Glu Cys Val Cys Glu Asn Tyr Lys Leu Ala Val Asn
Cys Phe 1 5 10 15 Val Asn Asn Asn His Gln Cys Gln Cys Thr Ser Ile
Gly Ala Gln Asn 20 25 30 Thr Val Ile Cys Ser Lys Leu Ala Ala Lys
Cys Leu Val Met Lys Ala 35 40 45 Glu Met Asn Gly Ser Lys Leu Gly
Arg Arg Ala Lys Pro Glu Gly Ala 50 55 60 Leu Gln Asn Asn Asp Gly
Leu Tyr Asp Pro Asp Cys Asp Glu Ser Gly 65 70 75 80 Leu Phe Lys Ala
Lys Gln Cys Asn Gly Thr Ser Thr Cys Trp Cys Val 85 90 95 Asn Thr
Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile Thr Cys 100 105 110
Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu Lys His Lys 115
120 125 Ala Arg Glu Lys Pro Tyr Asp Gly Lys Ser Leu Arg Thr Ala Leu
Gln 130 135 140 Lys Glu Ile Thr Thr Arg Tyr Gln Leu Asp Pro Lys Phe
Ile Thr Asn 145 150 155 160 Ile Leu Tyr Glu Asn Asn Val Ile Thr Ile
Asp Leu Val Gln Asn Ser 165 170 175 Ser Gln Lys Thr Gln Asn Asp Val
Asp Ile Ala Asp Val Ala Tyr Tyr 180 185 190 Phe Glu Lys Asp Val Lys
Gly Glu Ser Leu Phe His Ser Lys Lys Met 195 200 205 Asp Leu Thr Val
Asn Gly Glu Gln Leu Asp Leu Asp Pro Gly Gln Thr 210 215 220 Leu Ile
Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met Gln Gly 225 230 235
240 Leu Lys 139242PRTRhesus monkeymisc_feature(1)..(242)EpCAM
extracellular portion 139Gln Lys Glu Cys Val Cys Glu Asn Tyr Lys
Leu Ala Val Asn Cys Phe 1 5 10 15 Leu Asn Asp Asn Gly Gln Cys Gln
Cys Thr Ser Ile Gly Ala Gln Asn 20 25 30 Thr Val Leu Cys Ser Lys
Leu Ala Ala Lys Cys Leu Val Met Lys Ala 35 40 45 Glu Met Asn Gly
Ser Lys Leu Gly Arg Arg Ala Lys Pro Glu Gly Ala 50 55 60 Leu Gln
Asn Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Glu Ser Gly 65 70 75 80
Leu Phe Lys Ala Lys Gln Cys Asn Gly Thr Ser Thr Cys Trp Cys Val 85
90 95 Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile Thr
Cys 100 105 110 Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu
Lys His Lys 115 120 125 Ala Arg Glu Lys Pro Tyr Asp Val Gln Ser Leu
Arg Thr Ala Leu Glu 130 135 140 Glu Ala Ile Lys Thr Arg Tyr Gln Leu
Asp Pro Lys Phe Ile Thr Asn 145 150 155 160 Ile Leu Tyr Glu Asp Asn
Val Ile Thr Ile Asp Leu Val Gln Asn Ser 165 170 175 Ser Gln Lys Thr
Gln Asn Asp Val Asp Ile Ala Asp Val Ala Tyr Tyr 180 185 190 Phe Glu
Lys Asp Val Lys Gly Glu Ser Leu Phe His Ser Lys Lys Met 195 200 205
Asp Leu Arg Val Asn Gly Glu Gln Leu Asp Leu Asp Pro Gly Gln Thr 210
215 220 Leu Ile Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met Gln
Gly 225 230 235 240 Leu Lys
140242PRTCHIMPANZEEmisc_feature(1)..(242)EpCAM extracellular
portion 140Gln Glu Glu Cys Val Cys Glu Asn Tyr Lys Leu Ala Val Asn
Cys Phe 1 5 10 15 Val Asn Asn Asn His Gln Cys Gln Cys Thr Ser Ile
Gly Ala Gln Asn 20 25 30 Thr Val Ile Cys Ser Lys Leu Ala Ala Lys
Cys Leu Val Met Lys Ala 35 40 45 Glu Met Asn Gly Ser Lys Leu Gly
Arg Arg Ala Lys Pro Glu Gly Ala 50 55 60 Leu Gln Asn Asn Asp Gly
Leu Tyr Asp Pro Asp Cys Asp Glu Ser Gly 65 70 75 80 Leu Phe Lys Ala
Lys Gln Cys Asn Gly Thr Ser Thr Cys Trp Cys Val 85 90 95 Asn Thr
Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile Thr Cys 100 105 110
Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu Lys His Lys 115
120 125 Ala Arg Glu Lys Pro Tyr Asp Gly Lys Ser Leu Arg Thr Ala Leu
Gln 130 135 140 Lys Glu Ile Thr Thr Arg Tyr Gln Leu Asp Pro Lys Phe
Ile Thr Asn 145 150 155 160 Ile Leu Tyr Glu Asn Asn Val Ile Thr Ile
Asp Leu Val Gln Asn Ser 165 170 175 Ser Gln Lys Thr Gln Asn Asp Val
Asp Ile Ala Asp Val Ala Tyr Tyr 180 185 190 Phe Glu Lys Asp Val Lys
Gly Glu Ser Leu Phe His Ser Lys Lys Met 195 200 205 Asp Leu Thr Val
Asn Gly Glu Gln Leu Asp Leu Asp Pro Gly Gln Thr 210 215 220 Leu Ile
Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met Gln Gly 225 230 235
240 Leu Lys 141242PRTRhesus monkeymisc_feat(1)..(242)EpCAM
extracellular portion 141Gln Lys Glu Cys Val Cys Glu Asn Tyr Lys
Leu Ala Val Asn Cys Phe 1 5 10 15 Leu Asn Asp Asn Gly Gln Cys Gln
Cys Thr Ser Ile Gly Ala Gln Asn 20 25 30 Thr Val Leu Cys Ser Lys
Leu Ala Ala Lys Cys Leu Val Met Lys Ala 35 40 45 Glu Met Asn Gly
Ser Lys Leu Gly Arg Arg Ala Lys Pro Glu Gly Ala 50 55 60 Leu Gln
Asn Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Glu Ser Gly 65 70 75 80
Leu Phe Lys Ala Lys Gln Cys Asn Gly Thr Ser Thr Cys Trp Cys Val 85
90 95 Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile Thr
Cys 100 105 110 Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu
Lys His Lys 115 120 125 Ala Arg Glu Lys Pro Tyr Asp Val Gln Ser Leu
Arg Thr Ala Leu Glu 130 135 140 Glu Ala Ile Lys Thr Arg Tyr Gln Leu
Asp Pro Lys Phe Ile Thr Asn 145 150 155 160 Ile Leu Tyr Glu Asp Asn
Val Ile Thr Ile Asp Leu Val Gln Asn Ser 165 170 175 Ser Gln Lys Thr
Gln Asn Asp Val Asp Ile Ala Asp Val Ala Tyr Tyr 180 185 190 Phe Glu
Lys Asp Val Lys Gly Glu Ser Leu Phe His Ser Lys Lys Met 195 200 205
Asp Leu Arg Val Asn Gly Glu Gln Leu Asp Leu Asp Pro Gly Gln Thr 210
215 220 Leu Ile Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met Gln
Gly 225 230 235 240 Leu Lys 14289PRTHOMO
SAPIENSmisc_feature(1)..(89)human CD3 gamma extracellular portion
142Gln Ser Ile Lys Gly Asn His Leu Val Lys Val Tyr Asp Tyr Gln Glu
1 5 10 15 Asp Gly Ser Val Leu Leu Thr Cys Asp Ala Glu Ala Lys Asn
Ile Thr 20 25 30 Trp Phe Lys Asp Gly Lys Met Ile Gly Phe Leu Thr
Glu Asp Lys Lys 35 40 45 Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp
Pro Arg Gly Met Tyr Gln 50 55 60 Cys Lys Gly Ser Gln Asn Lys Ser
Lys Pro Leu Gln Val Tyr Tyr Arg 65 70 75 80 Met Cys Gln Asn Cys Ile
Glu Leu Asn 85 14382PRTHOMO SAPIENSmisc_feature(1)..(82)human CD3
delta extracellular portion 143Phe Lys Ile Pro Ile Glu Glu Leu Glu
Asp Arg Val Phe Val Asn Cys 1 5 10 15 Asn Thr Ser Ile Thr Trp Val
Glu Gly Thr Val Gly Thr Leu Leu Ser 20 25 30 Asp Ile Thr Arg Leu
Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly 35 40 45 Ile Tyr Arg
Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Thr 50 55 60 Val
Gln Val His Tyr Arg Met Cys Gln Ser Cys Val Glu Leu Asp Pro 65 70
75 80 Ala Thr 14488PRTCYNOMOLGUSmisc_feature(1)..(88)cynomolgus CD3
gamma extracellular portion 144Gln Ser Phe Glu Glu Asn Arg Lys Leu
Asn Val Tyr Asn Gln Glu Asp 1 5 10 15 Gly Ser Val Leu Leu Thr Cys
His Val Lys Asn Thr Asn Ile Thr Trp 20 25 30 Phe Lys Glu Gly Lys
Met Ile Asp Ile Leu Thr Ala His Lys Asn Lys 35 40 45 Trp Asn Leu
Gly Ser Asn Thr Lys Asp Pro Arg Gly Val Tyr Gln Cys 50 55 60 Lys
Gly Ser Lys Asp Lys Ser Lys Thr Leu Gln Val Tyr Tyr Arg Met 65 70
75 80 Cys Gln Asn Cys Ile Glu Leu Asn 85
14582PRTCYNOMOLGUSmisc_feature(1)..(82)cynomolgus CD3 delta
extracellular portion 145Phe Lys Ile Pro Val Glu Glu Leu Glu Asp
Arg Val Phe Val Lys Cys 1 5 10 15 Asn Thr Ser Val Thr Trp Val Glu
Gly Thr Val Gly Thr Leu Leu Thr 20 25 30 Asn Asn Thr Arg Leu Asp
Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly 35 40 45 Ile Tyr Arg Cys
Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Ala 50 55 60 Val Gln
Val His Tyr Arg Met Cys Gln Asn Cys Val Glu Leu Asp Pro 65 70 75 80
Ala Thr 146249PRTARTIFICIAL SEQUENCEHuman-like VH (SEQ ID NO 110) x
murine VL (SEQ ID NO 148) scFv ; single chain Fv 146Glu Val Gln Leu
Leu 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 Thr Phe Asn Thr Tyr 20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala
Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu
Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly
Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Val 130 135 140 Thr Gln Glu
Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu 145 150 155 160
Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 165
170 175 Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly
Gly 180 185 190 Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser
Gly Ser Leu 195 200 205 Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly
Ala Gln Thr Glu Asp 210 215 220 Glu Ala Ile Tyr Phe Cys Ala Leu Trp
Tyr Ser Asn Leu Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu
Thr Val Leu 245 147747DNAARTIFICIAL SEQUENCEHuman-like VH (SEQ ID
NO 110) x murine VL (SEQ ID NO 148) scFv ; single chain Fv
147gaggtgcagc tgctcgagtc tggaggagga ttggtgcagc ctggagggtc
attgaaactc 60tcatgtgcag cctctggatt caccttcaat acctacgcca tgaactgggt
ccgccaggct 120ccaggaaagg gtttggaatg ggttgctcgc ataagaagta
aatataataa ttatgcaaca 180tattatgccg attcagtgaa agacaggttc
accatctcca gagatgattc aaaaaacact 240gcctatctac aaatgaacaa
cttgaaaact gaggacactg ccgtgtacta ctgtgtgaga 300catgggaact
tcggtaatag ctacgtttcc tggtttgctt actggggcca agggactctg
360gtcaccgtct cctcaggtgg tggtggttct ggcggcggcg gctccggtgg
tggtggttct 420gagctcgttg tgactcagga atctgcactc accacatcac
ctggtgaaac agtcacactc 480acttgtcgct caagtactgg ggctgttaca
actagtaact atgccaactg ggtccaagaa 540aaaccagatc atttattcac
tggtctaata ggtggtacca acaagcgagc accaggtgtg 600cctgccagat
tctcaggctc cctgattgga gacaaggctg ccctcaccat cacaggggca
660cagactgagg atgaggcaat atatttctgt gctctatggt acagcaacct
ctgggtgttc 720ggtggaggaa ccaaactgac tgtccta 747148109PRTartificial
sequencemurine VL 148Glu Leu Val Val Thr Gln Glu Ser Ala Leu Thr
Thr Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser
Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln
Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45 Leu Ile Gly Gly Thr
Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser
Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln
Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90
95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
149327DNAARTIFICIAL SEQUENCEmurine VL 149gagctcgttg tgactcagga
atctgcactc accacatcac ctggtgaaac agtcacactc 60acttgtcgct caagtactgg
ggctgttaca actagtaact atgccaactg ggtccaagaa 120aaaccagatc
atttattcac tggtctaata ggtggtacca acaagcgagc accaggtgtg
180cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat
cacaggggca 240cagactgagg atgaggcaat atatttctgt gctctatggt
acagcaacct ctgggtgttc 300ggtggaggaa ccaaactgac tgtccta
327150723DNAartificial sequenceCAIX LH scFv , single chain Fv
150gacattgtga tgacccagtc tcaaagattc atgtccacaa cagtaggaga
cagggtcagc 60atcacctgca aggccagtca gaatgtggtt tctgctgttg cctggtatca
acagaaacca 120ggacaatctc ctaaactact gatttactca gcatccaatc
ggtacactgg agtccctgat 180cgcttcacag gcagtggatc tgggacagat
ttcactctca ccattagcaa tatgcagtct 240gaagacctgg ctgatttttt
ctgtcaacaa tatagcaact atccgtggac gttcggtgga 300ggcaccaagc
tggaaatcaa aggtggtggt ggttctggcg gcggcggctc cggtggtggt
360ggttctgacg tgaagctcgt ggagtctggg ggaggcttag tgaagcttgg
agggtccctg 420aaactctcct gtgcagcctc tggattcact ttcagtaact
attacatgtc ttgggttcgc 480cagactccag agaagaggct ggagttggtc
gcagccatta atagtgatgg tggtatcacc 540tactatctag acactgtgaa
gggccgattc accatttcaa gagacaatgc caagaacacc 600ctgtacctgc
aaatgagcag tctgaagtct gaggacacag ccttgtttta ctgtgcaaga
660caccgctcgg gctacttttc tatggactac tggggtcaag gaacctcagt
caccgtctcc 720tcc 723151241PRTartificial sequenceCAIX LH scFv ;
single chain Fv 151Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Ser
Thr Thr Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser
Gln Asn Val Val Ser Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg
Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser 65 70 75 80 Glu Asp
Leu Ala Asp Phe Phe Cys Gln Gln Tyr Ser Asn Tyr Pro Trp 85
90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly
Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Lys
Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Lys Leu Gly Gly Ser
Leu Lys Leu Ser Cys 130 135 140 Ala Ala Ser Gly Phe Thr Phe Ser Asn
Tyr Tyr Met Ser Trp Val Arg 145 150 155 160 Gln Thr Pro Glu Lys Arg
Leu Glu Leu Val Ala Ala Ile Asn Ser Asp 165 170 175 Gly Gly Ile Thr
Tyr Tyr Leu Asp Thr Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg
Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu 195 200 205
Lys Ser Glu Asp Thr Ala Leu Phe Tyr Cys Ala Arg His Arg Ser Gly 210
215 220 Tyr Phe Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
Ser 225 230 235 240 Ser 152738DNAARTIFICIAL SEQUENCEEGFR21 LH scFv
; single chain Fv 152gacattgtgc tgacacagtc tcctgcttcc ttacctgtgt
ctctggggca gagggccacc 60atctcatgca gggccagcca aagtgtcagt tcatctactt
atagttatat acactggtac 120caacagaaac caggacagcc acccaaactc
ctcatcacgt atgcatccaa cctagaatct 180ggggtccctg ccaggttcag
tggcagtggg tctgggacag acttcaccct cgacatccat 240cctgtggagg
aggatgattc ttcaacatat tactgtcagc acagttggga gattccattt
300acgttcggct cggggacaaa gttggaaata aaaggtggtg gtggttctgg
cggcggcggc 360tccggtggtg gtggttctca ggttcagctg cagcagtctg
gacctgatct ggtgaagcct 420ggggcctcag tgaagatgtc ctgcaaggct
tctggacaca ctttcactga ctgtgttata 480atctgggtga aacagagagc
tggacagggc cttgagtgga ttggacagat ttatccaggg 540actggtcgtt
cttactacaa tgagattttc aagggcaagg ccacactgac tgcagacaaa
600tcctccaaca cagtccacat tcaactcagc agcctgacat ctgaggactc
tgcggtctat 660ttctgtgccc tatctactct tattcacggg acctggtttt
cttattgggg ccaagggact 720ctggtcactg tctcttcc 738153246PRTartificial
sequenceEGFR21 LH scFv ; single chain Fv 153Asp Ile Val Leu Thr Gln
Ser Pro Ala Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr
Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Thr Tyr
Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45
Lys Leu Leu Ile Thr Tyr 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 Asp Ile
His 65 70 75 80 Pro Val Glu Glu Asp Asp Ser Ser Thr Tyr Tyr Cys Gln
His Ser Trp 85 90 95 Glu Ile Pro Phe Thr Phe Gly Ser Gly Thr Lys
Leu Glu Ile Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gln Val 115 120 125 Gln Leu Gln Gln Ser Gly Pro
Asp Leu Val Lys Pro Gly Ala Ser Val 130 135 140 Lys Met Ser Cys Lys
Ala Ser Gly His Thr Phe Thr Asp Cys Val Ile 145 150 155 160 Ile Trp
Val Lys Gln Arg Ala Gly Gln Gly Leu Glu Trp Ile Gly Gln 165 170 175
Ile Tyr Pro Gly Thr Gly Arg Ser Tyr Tyr Asn Glu Ile Phe Lys Gly 180
185 190 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Val His Ile
Gln 195 200 205 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe
Cys Ala Leu 210 215 220 Ser Thr Leu Ile His Gly Thr Trp Phe Ser Tyr
Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser 245
154726DNAARTIFICIAL SEQUENCEEGFRvIII-LH scFv ; single chain Fv
154gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca
accagcctct 60atctcttgca agtcaagtca gagcctctta tatagtaatg gaaaaaccta
tttgaattgg 120ttattacaga ggccaggcca gtctccaaag cgcctaatct
atctggtatc taaactggac 180tctggagtcc ctgacaggtt cactggcagt
ggatcaggaa cagattttac actgaaaatc 240agcagagtgg aggctgagga
tttgggaatt tattactgcg tgcaagatac acattttcct 300cagacattcg
gtggaggcac caagctggaa atcaaaggtg gtggtggttc tggcggcggc
360ggctccggtg gtggtggttc tgaggtccag ctgcaacagt ctggacctga
gctgctgaag 420cctggggctt cagtgaagat atcctgcaag acttctggat
acacattcac tgaatacacc 480atacactggg tgaagcagag ccatggaaag
agccttgagt ggattggagg tattgatcct 540aacaatggtg gtactatgta
taaccaaaaa ttcaagggca aggccacatt gactgtagac 600aagtcttcca
gcacagccta cacggacctc cgcagcctga cgtctgagga ttctgcagtc
660tattactgca caagagcaga ggctatggac tactggggtc aaggaacctc
agtcaccgtc 720tcctcc 726155242PRTartificial sequenceEGFRvIII-LH
scFv ; single chain Fv 155Asp Val Val Met Thr Gln Thr Pro Leu Thr
Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys
Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Lys Thr Tyr Leu
Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu
Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Val Gln Asp 85
90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu 115 120 125 Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Leu
Lys Pro Gly Ala Ser 130 135 140 Val Lys Ile Ser Cys Lys Thr Ser Gly
Tyr Thr Phe Thr Glu Tyr Thr 145 150 155 160 Ile His Trp Val Lys Gln
Ser His Gly Lys Ser Leu Glu Trp Ile Gly 165 170 175 Gly Ile Asp Pro
Asn Asn Gly Gly Thr Met Tyr Asn Gln Lys Phe Lys 180 185 190 Gly Lys
Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Thr 195 200 205
Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr 210
215 220 Arg Ala Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
Val 225 230 235 240 Ser Ser 1561485DNAARTIFICIAL SEQUENCECAIX
LHxSEQ ID NO 10 ; bispecific single chain antibody 156gacattgtga
tgacccagtc tcaaagattc atgtccacaa cagtaggaga cagggtcagc 60atcacctgca
aggccagtca gaatgtggtt tctgctgttg cctggtatca acagaaacca
120ggacaatctc ctaaactact gatttactca gcatccaatc ggtacactgg
agtccctgat 180cgcttcacag gcagtggatc tgggacagat ttcactctca
ccattagcaa tatgcagtct 240gaagacctgg ctgatttttt ctgtcaacaa
tatagcaact atccgtggac gttcggtgga 300ggcaccaagc tggaaatcaa
aggtggtggt ggttctggcg gcggcggctc cggtggtggt 360ggttctgacg
tgaagctcgt ggagtctggg ggaggcttag tgaagcttgg agggtccctg
420aaactctcct gtgcagcctc tggattcact ttcagtaact attacatgtc
ttgggttcgc 480cagactccag agaagaggct ggagttggtc gcagccatta
atagtgatgg tggtatcacc 540tactatctag acactgtgaa gggccgattc
accatttcaa gagacaatgc caagaacacc 600ctgtacctgc aaatgagcag
tctgaagtct gaggacacag ccttgtttta ctgtgcaaga 660caccgctcgg
gctacttttc tatggactac tggggtcaag gaacctcagt caccgtctcc
720tccggaggtg gtggatccga ggtgaagctt ctcgagtctg gaggaggatt
ggtgcagcct 780aaagggtcat tgaaactctc atgtgcagcc tctggattca
ccttcaatac ctacgccatg 840aactgggtcc gccaggctcc aggaaagggt
ttggaatggg ttgctcgcat aagaagtaaa 900tataataatt atgcaacata
ttatgccgat tcagtgaaag acaggttcac catctccaga 960gatgattcac
aaagcattct ctatctacaa atgaacaact tgaaaactga ggacacagcc
1020atgtactact gtgtgagaca tgggaacttc ggtaatagct acgtttcctg
gtttgcttac 1080tggggccaag ggactctggt cactgtctct gcaggtggtg
gtggttctgg cggcggcggc 1140tccggtggtg gtggttctca ggctgttgtg
actcaggaat ctgcactcac cacatcacct 1200ggtgaaacag tcacactcac
ttgtcgctca agtactgggg ctgttacaac tagtaactat 1260gccaactggg
tccaagaaaa accagatcat ttattcactg gtctaatagg tggtaccaac
1320aagcgagctc caggtgtgcc tgccagattc tcaggctccc tgattggaga
caaggctgcc 1380ctcaccatca caggggcaca gactgaggat gaggcaatat
atttctgtgc tctatggtac 1440agcaacctct gggtgttcgg tggaggaacc
aaactgactg tccta 1485157495PRTartificial sequenceCAIX LHxSEQ ID NO
10 ; bispecific single chain antibody 157Asp Ile Val Met Thr Gln
Ser Gln Arg Phe Met Ser Thr Thr Val Gly 1 5 10 15 Asp Arg Val Ser
Ile Thr Cys Lys Ala Ser Gln Asn Val Val Ser Ala 20 25 30 Val Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45
Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln
Ser 65 70 75 80 Glu Asp Leu Ala Asp Phe Phe Cys Gln Gln Tyr Ser Asn
Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Asp Val Lys Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Lys
Leu Gly Gly Ser Leu Lys Leu Ser Cys 130 135 140 Ala Ala Ser Gly Phe
Thr Phe Ser Asn Tyr Tyr Met Ser Trp Val Arg 145 150 155 160 Gln Thr
Pro Glu Lys Arg Leu Glu Leu Val Ala Ala Ile Asn Ser Asp 165 170 175
Gly Gly Ile Thr Tyr Tyr Leu Asp Thr Val Lys Gly Arg Phe Thr Ile 180
185 190 Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser
Leu 195 200 205 Lys Ser Glu Asp Thr Ala Leu Phe Tyr Cys Ala Arg His
Arg Ser Gly 210 215 220 Tyr Phe Ser Met Asp Tyr Trp Gly Gln Gly Thr
Ser Val Thr Val Ser 225 230 235 240 Ser Gly Gly Gly Gly Ser Glu Val
Lys Leu Leu Glu Ser Gly Gly Gly 245 250 255 Leu Val Gln Pro Lys Gly
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly 260 265 270 Phe Thr Phe Asn
Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly 275 280 285 Lys Gly
Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr 290 295 300
Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg 305
310 315 320 Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu
Lys Thr 325 330 335 Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His Gly
Asn Phe Gly Asn 340 345 350 Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly
Gln Gly Thr Leu Val Thr 355 360 365 Val Ser Ala Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly 370 375 380 Gly Ser Gln Ala Val Val
Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro 385 390 395 400 Gly Glu Thr
Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr 405 410 415 Thr
Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe 420 425
430 Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala
435 440 445 Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr
Ile Thr 450 455 460 Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys
Ala Leu Trp Tyr 465 470 475 480 Ser Asn Leu Trp Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu 485 490 495 1581500DNAARTIFICIAL
SEQUENCEEGFR21 LHxSEQ ID NO 10 ; bispecific single chain antibody
158gacattgtgc tgacacagtc tcctgcttcc ttacctgtgt ctctggggca
gagggccacc 60atctcatgca gggccagcca aagtgtcagt tcatctactt atagttatat
acactggtac 120caacagaaac caggacagcc acccaaactc ctcatcacgt
atgcatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg
tctgggacag acttcaccct cgacatccat 240cctgtggagg aggatgattc
ttcaacatat tactgtcagc acagttggga gattccattt 300acgttcggct
cggggacaaa gttggaaata aaaggtggtg gtggttctgg cggcggcggc
360tccggtggtg gtggttctca ggttcagctg cagcagtctg gacctgatct
ggtgaagcct 420ggggcctcag tgaagatgtc ctgcaaggct tctggacaca
ctttcactga ctgtgttata 480atctgggtga aacagagagc tggacagggc
cttgagtgga ttggacagat ttatccaggg 540actggtcgtt cttactacaa
tgagattttc aagggcaagg ccacactgac tgcagacaaa 600tcctccaaca
cagtccacat tcaactcagc agcctgacat ctgaggactc tgcggtctat
660ttctgtgccc tatctactct tattcacggg acctggtttt cttattgggg
ccaagggact 720ctggtcactg tctcttccgg aggtggtgga tccgaggtga
agcttctcga gtctggagga 780ggattggtgc agcctaaagg gtcattgaaa
ctctcatgtg cagcctctgg attcaccttc 840aatacctacg ccatgaactg
ggtccgccag gctccaggaa agggtttgga atgggttgct 900cgcataagaa
gtaaatataa taattatgca acatattatg ccgattcagt gaaagacagg
960ttcaccatct ccagagatga ttcacaaagc attctctatc tacaaatgaa
caacttgaaa 1020actgaggaca cagccatgta ctactgtgtg agacatggga
acttcggtaa tagctacgtt 1080tcctggtttg cttactgggg ccaagggact
ctggtcactg tctctgcagg tggtggtggt 1140tctggcggcg gcggctccgg
tggtggtggt tctcaggctg ttgtgactca ggaatctgca 1200ctcaccacat
cacctggtga aacagtcaca ctcacttgtc gctcaagtac tggggctgtt
1260acaactagta actatgccaa ctgggtccaa gaaaaaccag atcatttatt
cactggtcta 1320ataggtggta ccaacaagcg agctccaggt gtgcctgcca
gattctcagg ctccctgatt 1380ggagacaagg ctgccctcac catcacaggg
gcacagactg aggatgaggc aatatatttc 1440tgtgctctat ggtacagcaa
cctctgggtg ttcggtggag gaaccaaact gactgtccta 1500159500PRTartificial
sequenceEGFR21 LHxSEQ ID NO 10 ; bispecific single chain antibody
159Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Pro Val Ser Leu Gly
1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Ser 20 25 30 Thr Tyr Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro
Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Thr Tyr 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 Asp Ile His 65 70 75 80 Pro Val Glu Glu Asp Asp
Ser Ser Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Phe
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110 Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 115 120 125
Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala Ser Val 130
135 140 Lys Met Ser Cys Lys Ala Ser Gly His Thr Phe Thr Asp Cys Val
Ile 145 150 155 160 Ile Trp Val Lys Gln Arg Ala Gly Gln Gly Leu Glu
Trp Ile Gly Gln 165 170 175 Ile Tyr Pro Gly Thr Gly Arg Ser Tyr Tyr
Asn Glu Ile Phe Lys Gly 180 185 190 Lys Ala Thr Leu Thr Ala Asp Lys
Ser Ser Asn Thr Val His Ile Gln 195 200 205 Leu Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Phe Cys Ala Leu 210 215 220 Ser Thr Leu Ile
His Gly Thr Trp Phe Ser Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Leu 245 250
255 Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser Leu Lys Leu Ser
260 265 270 Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn
Trp Val 275 280 285 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
Arg Ile Arg Ser 290 295 300 Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala
Asp Ser Val Lys Asp Arg 305 310 315 320 Phe Thr Ile Ser Arg Asp Asp
Ser Gln Ser Ile Leu Tyr Leu Gln Met 325 330 335 Asn Asn Leu Lys Thr
Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His 340 345 350 Gly Asn Phe
Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln 355 360 365 Gly
Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly 370 375
380 Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Ser Ala
385
390 395 400 Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg
Ser Ser 405 410 415 Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp
Val Gln Glu Lys 420 425 430 Pro Asp His Leu Phe Thr Gly Leu Ile Gly
Gly Thr Asn Lys Arg Ala 435 440 445 Pro Gly Val Pro Ala Arg Phe Ser
Gly Ser Leu Ile Gly Asp Lys Ala 450 455 460 Ala Leu Thr Ile Thr Gly
Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe 465 470 475 480 Cys Ala Leu
Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys 485 490 495 Leu
Thr Val Leu 500 1601488DNAARTIFICIAL SEQUENCEEGFRvIII-LHxSEQ ID NO
10 ; bispecific single chain antibody 160gatgttgtga tgacccagac
tccactcact ttgtcggtta ccattggaca accagcctct 60atctcttgca agtcaagtca
gagcctctta tatagtaatg gaaaaaccta tttgaattgg 120ttattacaga
ggccaggcca gtctccaaag cgcctaatct atctggtatc taaactggac
180tctggagtcc ctgacaggtt cactggcagt ggatcaggaa cagattttac
actgaaaatc 240agcagagtgg aggctgagga tttgggaatt tattactgcg
tgcaagatac acattttcct 300cagacattcg gtggaggcac caagctggaa
atcaaaggtg gtggtggttc tggcggcggc 360ggctccggtg gtggtggttc
tgaggtccag ctgcaacagt ctggacctga gctgctgaag 420cctggggctt
cagtgaagat atcctgcaag acttctggat acacattcac tgaatacacc
480atacactggg tgaagcagag ccatggaaag agccttgagt ggattggagg
tattgatcct 540aacaatggtg gtactatgta taaccaaaaa ttcaagggca
aggccacatt gactgtagac 600aagtcttcca gcacagccta cacggacctc
cgcagcctga cgtctgagga ttctgcagtc 660tattactgca caagagcaga
ggctatggac tactggggtc aaggaacctc agtcaccgtc 720tcctccggag
gtggtggatc cgaggtgaag cttctcgagt ctggaggagg attggtgcag
780cctaaagggt cattgaaact ctcatgtgca gcctctggat tcaccttcaa
tacctacgcc 840atgaactggg tccgccaggc tccaggaaag ggtttggaat
gggttgctcg cataagaagt 900aaatataata attatgcaac atattatgcc
gattcagtga aagacaggtt caccatctcc 960agagatgatt cacaaagcat
tctctatcta caaatgaaca acttgaaaac tgaggacaca 1020gccatgtact
actgtgtgag acatgggaac ttcggtaata gctacgtttc ctggtttgct
1080tactggggcc aagggactct ggtcactgtc tctgcaggtg gtggtggttc
tggcggcggc 1140ggctccggtg gtggtggttc tcaggctgtt gtgactcagg
aatctgcact caccacatca 1200cctggtgaaa cagtcacact cacttgtcgc
tcaagtactg gggctgttac aactagtaac 1260tatgccaact gggtccaaga
aaaaccagat catttattca ctggtctaat aggtggtacc 1320aacaagcgag
ctccaggtgt gcctgccaga ttctcaggct ccctgattgg agacaaggct
1380gccctcacca tcacaggggc acagactgag gatgaggcaa tatatttctg
tgctctatgg 1440tacagcaacc tctgggtgtt cggtggagga accaaactga ctgtccta
1488161496PRTartificial sequenceEGFRvIII-LHxSEQ ID NO 10 ;
bispecific single chain antibody 161Asp Val Val Met Thr Gln Thr Pro
Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser
Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Lys Thr
Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys
Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Val
Gln Asp 85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu 115 120 125 Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Leu Lys Pro Gly Ala Ser 130 135 140 Val Lys Ile Ser Cys Lys
Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr 145 150 155 160 Ile His Trp
Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly 165 170 175 Gly
Ile Asp Pro Asn Asn Gly Gly Thr Met Tyr Asn Gln Lys Phe Lys 180 185
190 Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Thr
195 200 205 Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys Thr 210 215 220 Arg Ala Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr
Ser Val Thr Val 225 230 235 240 Ser Ser Gly Gly Gly Gly Ser Glu Val
Lys Leu Leu Glu Ser Gly Gly 245 250 255 Gly Leu Val Gln Pro Lys Gly
Ser Leu Lys Leu Ser Cys Ala Ala Ser 260 265 270 Gly Phe Thr Phe Asn
Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro 275 280 285 Gly Lys Gly
Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn 290 295 300 Tyr
Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser 305 310
315 320 Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu
Lys 325 330 335 Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His Gly
Asn Phe Gly 340 345 350 Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly
Gln Gly Thr Leu Val 355 360 365 Thr Val Ser Ala Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly 370 375 380 Gly Gly Ser Gln Ala Val Val
Thr Gln Glu Ser Ala Leu Thr Thr Ser 385 390 395 400 Pro Gly Glu Thr
Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val 405 410 415 Thr Thr
Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu 420 425 430
Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro 435
440 445 Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr
Ile 450 455 460 Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys
Ala Leu Trp 465 470 475 480 Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu 485 490 495 162243PRTARTIFICIAL
SEQUENCEanti CD3 162Asp 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 Val Glu Gly Gly Ser Gly Gly Ser
Gly 115 120 125 Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu
Thr Gln Ser 130 135 140 Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys
Val Thr Met Thr Cys 145 150 155 160 Arg Ala Ser Ser Ser Val Ser Tyr
Met Asn Trp Tyr Gln Gln Lys Ser 165 170 175 Gly Thr Ser Pro Lys Arg
Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser 180 185 190 Gly Val Pro Tyr
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 195 200 205 Leu Thr
Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 210 215 220
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 225
230 235 240 Glu Leu Lys 163243PRTartificial
sequencedeimmunised(di)-anti CD3 deimmunised single chain Fv 163Asp
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 Gly Glu Gly Thr Ser Thr Gly Ser Gly 115 120 125 Gly Ser
Gly Gly Ser Gly Gly Ala Asp Asp Ile Val Leu Thr Gln Ser 130 135 140
Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys 145
150 155 160 Arg Ala Ser Gln Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln
Lys Pro 165 170 175 Gly Lys Ala Pro Lys Arg Trp Ile Tyr Asp Thr Ser
Lys Val Ala Ser 180 185 190 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Ser 195 200 205 Leu Thr Ile Asn Ser Leu Glu Ala
Glu Asp Ala Ala Thr Tyr Tyr Cys 210 215 220 Gln Gln Trp Ser Ser Asn
Pro Leu Thr Phe Gly Gly Gly Thr Lys Val 225 230 235 240 Glu Ile Lys
1649PRTmus musculus 164Gln Gln Trp Ser Arg Asn Pro Pro Thr 1 5
1657PRTmus musculus 165Asp Ser Ser Lys Leu Ala Ser 1 5 16610PRTmus
musculus 166Ser Ala Ser Ser Ser Val Ser Tyr Met Asn 1 5 10
167327DNAartificial sequenceHuman-like VL VL region 167gagctcgttg
tgactcagga accttcactc accgtatcac ctggtggaac agtcacactc 60acttgtcgct
cgtcgactgg ggctgttaca actagcaact atgccaactg ggtccaacaa
120aaaccaggtc aggcaccccg tggtctaata ggtggtacca acaagcgcgc
accaggtact 180cctgccagat tctcaggctc cctgcttgga ggcaaggctg
ccctcaccct ctcaggggta 240cagccagagg atgaggcaga atattactgt
gctctatggt acagcaacct ctgggtgttc 300ggtggaggaa ccaaactgac tgtccta
327168109PRTartificial sequenceHuman-like VL VL region 168Glu Leu
Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20
25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg
Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro
Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu
Thr Leu Ser Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr
Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu 100 105 169747DNAARTIFICIAL
SEQUENCEHuman-like VH (SEQ ID NO 110) x Human-like VL (SEQ ID NO
168) scFv ; single chain Fv 169gaggtgcagc tgctcgagtc tggaggagga
ttggtgcagc ctggagggtc attgaaactc 60tcatgtgcag cctctggatt caccttcaat
acctacgcca tgaactgggt ccgccaggct 120ccaggaaagg gtttggaatg
ggttgctcgc ataagaagta aatataataa ttatgcaaca 180tattatgccg
attcagtgaa agacaggttc accatctcca gagatgattc aaaaaacact
240gcctatctac aaatgaacaa cttgaaaact gaggacactg ccgtgtacta
ctgtgtgaga 300catgggaact tcggtaatag ctacgtttcc tggtttgctt
actggggcca agggactctg 360gtcaccgtct cctcaggtgg tggtggttct
ggcggcggcg gctccggtgg tggtggttct 420gagctcgttg tgactcagga
accttcactc accgtatcac ctggtggaac agtcacactc 480acttgtcgct
cgtcgactgg ggctgttaca actagcaact atgccaactg ggtccaacaa
540aaaccaggtc aggcaccccg tggtctaata ggtggtacca acaagcgcgc
accaggtact 600cctgccagat tctcaggctc cctgcttgga ggcaaggctg
ccctcaccct ctcaggggta 660cagccagagg atgaggcaga atattactgt
gctctatggt acagcaacct ctgggtgttc 720ggtggaggaa ccaaactgac tgtccta
747170249PRTartificial sequenceHuman-like VH (SEQ ID NO 110) x
Human-like VL (SEQ ID NO 168) scFv ; single chain Fv 170Glu Val Gln
Leu Leu 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 Thr Phe Asn Thr Tyr 20 25
30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr
Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr
Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe
Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Val 130 135 140 Thr Gln
Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu 145 150 155
160 Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn
165 170 175 Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile
Gly Gly 180 185 190 Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe
Ser Gly Ser Leu 195 200 205 Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser
Gly Val Gln Pro Glu Asp 210 215 220 Glu Ala Glu Tyr Tyr Cys Ala Leu
Trp Tyr Ser Asn Leu Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys
Leu Thr Val Leu 245 1711530DNAARTIFICIAL SEQUENCEEGFR HL x SEQ ID
NO 170 ; bispecific single chain antibody 171caggtgcagc tgcagcagtc
tgggcctgat ctggtgaagc ctggggcctc agtgaagatg 60tcctgcaagg cttctggaca
cactttcact gactgtgtta taatctgggt gaaacagaga 120gctggacagg
gccttgagtg gattggacag atttatccag ggactggtcg ttcttactac
180aatgagattt tcaagggcaa ggccacactg actgcagaca aatcctccaa
cacagtccac 240attcaactca gcagcctgac atctgaggac tctgcggtct
atttctgtgc cctatctact 300cttattcacg ggacctggtt ttcttattgg
ggccaaggga ctctggtcac tgtctcttcc 360ggtggtggtg gttctggcgg
cggcggctcc ggtggtggtg gttctgacat tgtactgacc 420cagtctccag
cttccttacc tgtgtctctg gggcagaggg ccaccatctc atgcagggcc
480agccaaagtg tcagttcatc tacttatagt tatatacact ggtaccaaca
gaaaccagga 540cagccaccca aactcctcat cacgtatgca tccaacctag
aatctggggt ccctgccagg 600ttcagtggca gtgggtctgg gacagacttc
accctcgaca tccatcctgt ggaggaggat 660gattcttcaa catattactg
tcagcacagt tgggagattc catttacgtt cggctcgggg 720acaaagttgg
aaataaaatc cggaggtggt ggctccgagg tgcagctggt ggagtctgga
780ggaggattgg tgcagcctgg agggtcattg aaactctcat gtgcagcctc
tggattcacc 840ttcaatacct acgccatgaa ctgggtccgc caggctccag
gaaagggttt ggaatgggtt 900gctcgcataa gaagtaaata taataattat
gcaacatatt atgccgattc agtgaaagac 960aggttcacca tctccagaga
tgattcaaaa aacactgcct atctacaaat gaacaacttg 1020aaaactgagg
acactgccgt gtactactgt gtgagacatg ggaacttcgg taatagctac
1080gtttcctggt ttgcttactg gggccaaggg actctggtca ccgtctcctc
aggtggtggt 1140ggttctggcg gcggcggctc cggtggtggt ggttctcaga
ccgttgtgac tcaggaacct 1200tcactcaccg tatcacctgg tggaacagtc
acactcactt gtcgctcgtc cactggggct 1260gttacaacta gcaactatgc
caactgggtc caacaaaaac caggtcaggc accccgtggt 1320ctaataggtg
gtaccaacaa gcgcgcacca ggtactcctg ccagattctc aggctccctg
1380cttggaggca aggctgccct caccctctca ggggtacagc cagaggatga
ggcagaatat 1440tactgtgctc tatggtacag caacctctgg gtgttcggtg
gaggaaccaa actgactgtc 1500ctacatcatc accatcatca ttaggtcgac
1530172507PRTartificial sequenceEGFR HL x SEQ ID NO 170 ;
bispecific single chain antibody 172Gln Val Gln Leu Gln Gln Ser Gly
Pro Asp Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys
Lys Ala Ser Gly His Thr Phe Thr Asp Cys 20 25 30 Val Ile Ile Trp
Val Lys Gln Arg Ala Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln
Ile Tyr Pro Gly Thr Gly Arg Ser Tyr Tyr Asn Glu Ile Phe 50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
Asn Thr Val His 65 70 75 80 Ile Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Phe Cys 85 90 95 Ala Leu Ser Thr Leu Ile His Gly
Thr Trp Phe Ser 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 Val Leu Thr Gln Ser Pro Ala 130 135 140 Ser Leu
Pro Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala 145 150 155
160 Ser Gln Ser Val Ser Ser Ser Thr Tyr Ser Tyr Ile His Trp Tyr Gln
165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Thr Tyr Ala
Ser Asn 180 185 190 Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Asp Ile His Pro Val Glu
Glu Asp Asp Ser Ser Thr 210 215 220 Tyr Tyr Cys Gln His Ser Trp Glu
Ile Pro Phe Thr Phe Gly Ser Gly 225 230 235 240 Thr Lys Leu Glu Ile
Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 245 250 255 Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu 260 265 270 Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp 275 280
285 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg
290 295 300 Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val
Lys Asp 305 310 315 320 Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Thr Ala Tyr Leu Gln 325 330 335 Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Val Tyr Tyr Cys Val Arg 340 345 350 His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe Ala Tyr Trp Gly 355 360 365 Gln Gly Thr Leu Val
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 370 375 380 Gly Gly Ser
Gly Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu Pro 385 390 395 400
Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser 405
410 415 Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln
Gln 420 425 430 Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr
Asn Lys Arg 435 440 445 Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser
Leu Leu Gly Gly Lys 450 455 460 Ala Ala Leu Thr Leu Ser Gly Val Gln
Pro Glu Asp Glu Ala Glu Tyr 465 470 475 480 Tyr Cys Ala Leu Trp Tyr
Ser Asn Leu Trp Val Phe Gly Gly Gly Thr 485 490 495 Lys Leu Thr Val
Leu His His His His His His 500 505 1731527DNAartificial
sequenceEGFR LH x SEQ ID NO 170 ; bispecific single chain antibody
173gacattgtgc tgacacagtc tcctgcttcc ttacctgtgt ctctggggca
gagggccacc 60atctcatgca gggccagcca aagtgtcagt tcatctactt atagttatat
acactggtac 120caacagaaac caggacagcc acccaaactc ctcatcacgt
atgcatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg
tctgggacag acttcaccct cgacatccat 240cctgtggagg aggatgattc
ttcaacatat tactgtcagc acagttggga gattccattt 300acgttcggct
cggggacaaa gttggaaata aaaggtggtg gtggttctgg cggcggcggc
360tccggtggtg gtggttctca ggttcagctg cagcagtctg gacctgatct
ggtgaagcct 420ggggcctcag tgaagatgtc ctgcaaggct tctggacaca
ctttcactga ctgtgttata 480atctgggtga aacagagagc tggacagggc
cttgagtgga ttggacagat ttatccaggg 540actggtcgtt cttactacaa
tgagattttc aagggcaagg ccacactgac tgcagacaaa 600tcctccaaca
cagtccacat tcaactcagc agcctgacat ctgaggactc tgcggtctat
660ttctgtgccc tatctactct tattcacggg acctggtttt cttattgggg
ccaagggact 720ctggtcactg tctcttccgg aggtggtggc tccgaggtgc
agctggtgga gtctggagga 780ggattggtgc agcctggagg gtcattgaaa
ctctcatgtg cagcctctgg attcaccttc 840aatacctacg ccatgaactg
ggtccgccag gctccaggaa agggtttgga atgggttgct 900cgcataagaa
gtaaatataa taattatgca acatattatg ccgattcagt gaaagacagg
960ttcaccatct ccagagatga ttcaaaaaac actgcctatc tacaaatgaa
caacttgaaa 1020actgaggaca ctgccgtgta ctactgtgtg agacatggga
acttcggtaa tagctacgtt 1080tcctggtttg cttactgggg ccaagggact
ctggtcaccg tctcctcagg tggtggtggt 1140tctggcggcg gcggctccgg
tggtggtggt tctcagaccg ttgtgactca ggaaccttca 1200ctcaccgtat
cacctggtgg aacagtcaca ctcacttgtc gctcgtccac tggggctgtt
1260acaactagca actatgccaa ctgggtccaa caaaaaccag gtcaggcacc
ccgtggtcta 1320ataggtggta ccaacaagcg cgcaccaggt actcctgcca
gattctcagg ctccctgctt 1380ggaggcaagg ctgccctcac cctctcaggg
gtacagccag aggatgaggc agaatattac 1440tgtgctctat ggtacagcaa
cctctgggtg ttcggtggag gaaccaaact gactgtccta 1500catcatcacc
atcatcatta ggtcgac 1527174506PRTartificial sequenceEGFR LH x SEQ ID
NO 170 ; bispecific single chain antibody 174Asp Ile Val Leu Thr
Gln Ser Pro Ala Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Gln Arg Ala
Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Thr
Tyr Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40
45 Lys Leu Leu Ile Thr Tyr 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 Asp
Ile His 65 70 75 80 Pro Val Glu Glu Asp Asp Ser Ser Thr Tyr Tyr Cys
Gln His Ser Trp 85 90 95 Glu Ile Pro Phe Thr Phe Gly Ser Gly Thr
Lys Leu Glu Ile Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gln Val 115 120 125 Gln Leu Gln Gln Ser Gly
Pro Asp Leu Val Lys Pro Gly Ala Ser Val 130 135 140 Lys Met Ser Cys
Lys Ala Ser Gly His Thr Phe Thr Asp Cys Val Ile 145 150 155 160 Ile
Trp Val Lys Gln Arg Ala Gly Gln Gly Leu Glu Trp Ile Gly Gln 165 170
175 Ile Tyr Pro Gly Thr Gly Arg Ser Tyr Tyr Asn Glu Ile Phe Lys Gly
180 185 190 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Val His
Ile Gln 195 200 205 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Phe Cys Ala Leu 210 215 220 Ser Thr Leu Ile His Gly Thr Trp Phe Ser
Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Glu Val Gln Leu Val 245 250 255 Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser 260 265 270 Cys Ala Ala
Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val 275 280 285 Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 290 295
300 Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg
305 310 315 320 Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr
Leu Gln Met 325 330 335 Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr
Tyr Cys Val Arg His 340 345 350 Gly Asn Phe Gly Asn Ser Tyr Val Ser
Trp Phe Ala Tyr Trp Gly Gln 355 360 365 Gly Thr Leu Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly 370 375 380 Gly Ser Gly Gly Gly
Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser 385 390 395 400 Leu Thr
Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser 405 410 415
Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys 420
425 430 Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg
Ala 435 440 445 Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly
Gly Lys Ala 450 455 460 Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp
Glu Ala Glu Tyr Tyr 465 470 475 480 Cys Ala Leu Trp Tyr Ser Asn Leu
Trp Val Phe Gly Gly Gly Thr Lys 485 490 495 Leu Thr Val Leu His His
His His His His 500 505 1751530DNAartificial sequenceEGFR HL x SEQ
ID NO 194 ; bispecific single chain antibody 175caggtgcagc
tgcagcagtc tgggcctgat ctggtgaagc ctggggcctc agtgaagatg 60tcctgcaagg
cttctggaca cactttcact gactgtgtta taatctgggt gaaacagaga
120gctggacagg gccttgagtg gattggacag atttatccag ggactggtcg
ttcttactac 180aatgagattt tcaagggcaa ggccacactg actgcagaca
aatcctccaa cacagtccac 240attcaactca gcagcctgac atctgaggac
tctgcggtct atttctgtgc cctatctact 300cttattcacg ggacctggtt
ttcttattgg ggccaaggga ctctggtcac tgtctcttcc 360ggtggtggtg
gttctggcgg cggcggctcc ggtggtggtg gttctgacat tgtactgacc
420cagtctccag cttccttacc tgtgtctctg gggcagaggg ccaccatctc
atgcagggcc 480agccaaagtg tcagttcatc tacttatagt tatatacact
ggtaccaaca gaaaccagga 540cagccaccca aactcctcat cacgtatgca
tccaacctag aatctggggt ccctgccagg 600ttcagtggca gtgggtctgg
gacagacttc accctcgaca tccatcctgt ggaggaggat 660gattcttcaa
catattactg tcagcacagt tgggagattc catttacgtt cggctcgggg
720acaaagttgg aaataaaatc cggaggtggt ggctcccaga ccgttgtgac
tcaggaacct 780tcactcaccg tatcacctgg tggaacagtc acactcactt
gtcgctcgtc cactggggct 840gttacaacta gcaactatgc caactgggtc
caacaaaaac caggtcaggc accccgtggt 900ctaataggtg gtaccaacaa
gcgcgcacca ggtactcctg ccagattctc aggctccctg 960cttggaggca
aggctgccct caccctctca ggggtacagc cagaggatga ggcagaatat
1020tactgtgctc tatggtacag caacctctgg gtgttcggtg gaggaaccaa
actgactgtc 1080ctaggtggtg gtggttctgg cggcggcggc tccggtggtg
gtggttctga ggtgcagctg 1140gtggagtctg gaggaggatt ggtgcagcct
ggagggtcat tgaaactctc atgtgcagcc 1200tctggattca ccttcaatac
ctacgccatg aactgggtcc gccaggctcc aggaaagggt 1260ttggaatggg
ttgctcgcat aagaagtaaa tataataatt atgcaacata ttatgccgat
1320tcagtgaaag acaggttcac catctccaga gatgattcaa aaaacactgc
ctatctacaa 1380atgaacaact tgaaaactga ggacactgcc gtgtactact
gtgtgagaca tgggaacttc 1440ggtaatagct acgtttcctg gtttgcttac
tggggccaag ggactctggt caccgtctcc 1500tcacatcatc accatcatca
ttaggtcgac 1530176507PRTartificial sequenceEGFR HL x SEQ ID NO 194
; bispecific single chain antibody 176Gln Val Gln Leu Gln Gln Ser
Gly Pro Asp Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser
Cys Lys Ala Ser Gly His Thr Phe Thr Asp Cys 20 25 30 Val Ile Ile
Trp Val Lys Gln Arg Ala Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly
Gln Ile Tyr Pro Gly Thr Gly Arg Ser Tyr Tyr Asn Glu Ile Phe 50 55
60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Val His
65 70 75 80 Ile Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Phe Cys 85 90 95 Ala Leu Ser Thr Leu Ile His Gly Thr Trp Phe Ser
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 Val Leu Thr Gln Ser Pro Ala 130 135 140 Ser Leu Pro Val Ser Leu
Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala 145 150 155 160 Ser Gln Ser
Val Ser Ser Ser Thr Tyr Ser Tyr Ile His Trp Tyr Gln 165 170 175 Gln
Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Thr Tyr Ala Ser Asn 180 185
190 Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205 Asp Phe Thr Leu Asp Ile His Pro Val Glu Glu Asp Asp Ser
Ser Thr 210 215 220 Tyr Tyr Cys Gln His Ser Trp Glu Ile Pro Phe Thr
Phe Gly Ser Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Ser Gly Gly
Gly Gly Ser Gln Thr Val Val 245 250 255 Thr Gln Glu Pro Ser Leu Thr
Val Ser Pro Gly Gly Thr Val Thr Leu 260 265 270 Thr Cys Arg Ser Ser
Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 275 280 285 Trp Val Gln
Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly 290 295 300 Thr
Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu 305 310
315 320 Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu
Asp 325 330 335 Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu
Trp Val Phe 340 345 350 Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly
Gly Gly Ser Gly Gly 355 360 365 Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val Glu Ser Gly 370 375 380 Gly Gly Leu Val Gln Pro Gly
Gly Ser Leu Lys Leu Ser Cys Ala Ala 385 390 395 400 Ser Gly Phe Thr
Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala 405 410 415 Pro Gly
Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn 420 425 430
Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile 435
440 445 Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn
Leu 450 455 460 Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His
Gly Asn Phe 465 470 475 480 Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr
Trp Gly Gln Gly Thr Leu 485 490 495 Val Thr Val Ser Ser His His His
His His His 500 505 1771530DNAartificial sequenceEGFR LH x SEQ ID
NO 194 ; bispecific single chain antibody 177gacattgtgc tgacacagtc
tcctgcttcc ttacctgtgt ctctggggca gagggccacc 60atctcatgca gggccagcca
aagtgtcagt tcatctactt atagttatat acactggtac 120caacagaaac
caggacagcc acccaaactc ctcatcacgt atgcatccaa cctagaatct
180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct
cgacatccat 240cctgtggagg aggatgattc ttcaacatat tactgtcagc
acagttggga gattccattt 300acgttcggct cggggacaaa gttggaaata
aaaggtggtg gtggttctgg cggcggcggc 360tccggtggtg gtggttctca
ggttcagctg cagcagtctg gacctgatct ggtgaagcct 420ggggcctcag
tgaagatgtc ctgcaaggct tctggacaca ctttcactga ctgtgttata
480atctgggtga aacagagagc tggacagggc cttgagtgga ttggacagat
ttatccaggg 540actggtcgtt cttactacaa tgagattttc aagggcaagg
ccacactgac tgcagacaaa 600tcctccaaca cagtccacat tcaactcagc
agcctgacat ctgaggactc tgcggtctat 660ttctgtgccc tatctactct
tattcacggg acctggtttt cttattgggg ccaagggact 720ctggtcactg
tctcttcctc cggaggtggt ggctcccaga ccgttgtgac tcaggaacct
780tcactcaccg tatcacctgg tggaacagtc acactcactt gtcgctcgtc
cactggggct 840gttacaacta gcaactatgc caactgggtc caacaaaaac
caggtcaggc accccgtggt 900ctaataggtg gtaccaacaa gcgcgcacca
ggtactcctg ccagattctc aggctccctg 960cttggaggca aggctgccct
caccctctca ggggtacagc cagaggatga ggcagaatat 1020tactgtgctc
tatggtacag caacctctgg gtgttcggtg gaggaaccaa actgactgtc
1080ctaggtggtg gtggttctgg cggcggcggc tccggtggtg gtggttctga
ggtgcagctg 1140gtggagtctg gaggaggatt ggtgcagcct ggagggtcat
tgaaactctc atgtgcagcc 1200tctggattca ccttcaatac ctacgccatg
aactgggtcc gccaggctcc aggaaagggt 1260ttggaatggg ttgctcgcat
aagaagtaaa tataataatt atgcaacata ttatgccgat 1320tcagtgaaag
acaggttcac catctccaga gatgattcaa aaaacactgc ctatctacaa
1380atgaacaact tgaaaactga ggacactgcc gtgtactact gtgtgagaca
tgggaacttc 1440ggtaatagct acgtttcctg gtttgcttac tggggccaag
ggactctggt caccgtctcc 1500tcacatcatc accatcatca ttaggtcgac
1530178507PRTartificial sequenceEGFR LH x SEQ ID NO 194 ;
bispecific single chain antibody 178Asp Ile Val Leu Thr Gln Ser Pro
Ala Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser
Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Thr Tyr Ser Tyr
Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu
Leu Ile Thr Tyr 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 Asp Ile His 65 70 75 80 Pro Val Glu Glu
Asp Asp Ser Ser Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile
Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 115
120 125 Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala Ser
Val 130 135 140 Lys Met Ser Cys Lys Ala Ser Gly His Thr Phe Thr Asp
Cys Val Ile 145 150 155 160 Ile Trp Val Lys Gln Arg Ala Gly Gln Gly
Leu Glu Trp Ile Gly Gln 165 170 175 Ile Tyr Pro Gly Thr Gly Arg Ser
Tyr Tyr Asn Glu Ile Phe Lys Gly 180 185 190 Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Asn Thr Val His Ile Gln 195 200 205 Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Leu 210 215 220 Ser Thr
Leu Ile His Gly Thr Trp Phe Ser Tyr Trp Gly Gln Gly Thr 225 230 235
240 Leu Val Thr Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Thr Val Val
245 250 255 Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val
Thr Leu 260 265 270 Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
Asn Tyr Ala Asn 275 280 285 Trp Val Gln Gln Lys Pro Gly Gln Ala Pro
Arg Gly Leu Ile Gly Gly 290 295 300 Thr Asn Lys Arg Ala Pro Gly Thr
Pro Ala Arg Phe Ser Gly Ser Leu 305 310 315 320 Leu Gly Gly Lys Ala
Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp 325 330 335 Glu Ala Glu
Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe 340 345 350 Gly
Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly 355 360
365 Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly
370 375 380 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys
Ala Ala 385 390 395 400 Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn
Trp Val Arg Gln Ala 405 410 415 Pro Gly Lys Gly Leu Glu Trp Val Ala
Arg Ile Arg Ser Lys Tyr Asn 420 425 430 Asn Tyr Ala Thr Tyr Tyr Ala
Asp Ser Val Lys Asp Arg Phe Thr Ile 435 440 445 Ser Arg Asp Asp Ser
Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu 450 455 460 Lys Thr Glu
Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe 465 470 475 480
Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 485
490 495 Val Thr Val Ser Ser His His His His His His 500 505
1791530DNAartificial sequenceSEQ ID NO 170 x EGFR HL ; bispecific
single chain antibody 179gaggtgcagc tggtggagtc tggaggagga
ttggtgcagc ctggagggtc attgaaactc 60tcatgtgcag cctctggatt caccttcaat
acctacgcca tgaactgggt ccgccaggct 120ccaggaaagg gtttggaatg
ggttgctcgc ataagaagta aatataataa ttatgcaaca 180tattatgccg
attcagtgaa agacaggttc accatctcca gagatgattc aaaaaacact
240gcctatctac aaatgaacaa cttgaaaact gaggacactg ccgtgtacta
ctgtgtgaga 300catgggaact tcggtaatag ctacgtttcc tggtttgctt
actggggcca agggactctg 360gtcaccgtct cctcaggtgg tggtggttct
ggcggcggcg gctccggtgg tggtggttct 420cagaccgttg tgactcagga
accttcactc accgtatcac ctggtggaac agtcacactc 480acttgtcgct
cgtccactgg ggctgttaca actagcaact atgccaactg ggtccaacaa
540aaaccaggtc aggcaccccg tggtctaata ggtggtacca acaagcgcgc
accaggtact 600cctgccagat tctcaggctc cctgcttgga ggcaaggctg
ccctcaccct ctcaggggta 660cagccagagg atgaggcaga atattactgt
gctctatggt acagcaacct ctgggtgttc 720ggtggaggaa ccaaactgac
tgtcctatcc ggaggtggtg gctcccaggt gcagctgcag 780cagtctgggc
ctgatctggt gaagcctggg gcctcagtga agatgtcctg caaggcttct
840ggacacactt tcactgactg tgttataatc tgggtgaaac agagagctgg
acagggcctt 900gagtggattg gacagattta tccagggact ggtcgttctt
actacaatga gattttcaag 960ggcaaggcca cactgactgc agacaaatcc
tccaacacag tccacattca actcagcagc 1020ctgacatctg aggactctgc
ggtctatttc tgtgccctat ctactcttat tcacgggacc 1080tggttttctt
attggggcca agggactctg gtcactgtct cttccggtgg tggtggttct
1140ggcggcggcg gctccggtgg tggtggttct gacattgtac tgacccagtc
tccagcttcc 1200ttacctgtgt ctctggggca gagggccacc atctcatgca
gggccagcca aagtgtcagt 1260tcatctactt atagttatat acactggtac
caacagaaac caggacagcc acccaaactc 1320ctcatcacgt atgcatccaa
cctagaatct ggggtccctg ccaggttcag tggcagtggg 1380tctgggacag
acttcaccct cgacatccat cctgtggagg aggatgattc ttcaacatat
1440tactgtcagc acagttggga gattccattt acgttcggct cggggacaaa
gttggaaata 1500aaacatcatc accatcatca ttaggtcgac
1530180507PRTartificial sequenceSEQ ID NO 170 x EGFR HL ,
bispecific single chain antibody 180Glu 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 Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65
70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gln Thr Val Val 130 135 140 Thr Gln Glu Pro Ser Leu
Thr Val Ser Pro Gly Gly Thr Val Thr Leu 145 150 155 160 Thr Cys Arg
Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 165 170 175 Trp
Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly 180 185
190 Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu
195 200 205 Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro
Glu Asp 210 215 220 Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
Leu Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Thr Val Leu
Ser Gly Gly Gly Gly Ser Gln 245 250 255 Val Gln Leu Gln Gln Ser Gly
Pro Asp Leu Val Lys Pro Gly Ala Ser 260 265 270 Val Lys Met Ser Cys
Lys Ala Ser Gly His Thr Phe Thr Asp Cys Val 275 280 285 Ile Ile Trp
Val Lys Gln Arg Ala Gly Gln Gly Leu Glu Trp Ile Gly 290 295 300 Gln
Ile Tyr Pro Gly Thr Gly Arg Ser Tyr Tyr Asn Glu Ile Phe Lys 305 310
315 320 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Val His
Ile 325 330 335 Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Phe Cys Ala 340 345 350 Leu Ser Thr Leu Ile His Gly Thr Trp Phe Ser
Tyr Trp Gly Gln Gly 355 360 365 Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly 370 375 380 Ser Gly Gly Gly Gly Ser Asp
Ile Val Leu Thr Gln Ser Pro Ala Ser 385 390 395 400 Leu Pro Val Ser
Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser 405 410 415 Gln Ser
Val Ser Ser Ser Thr Tyr Ser Tyr Ile His Trp Tyr Gln Gln 420 425 430
Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Thr Tyr Ala Ser Asn Leu 435
440 445 Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp 450 455 460 Phe Thr Leu Asp Ile His Pro Val Glu Glu Asp Asp Ser
Ser Thr Tyr 465 470 475 480 Tyr Cys Gln His Ser Trp Glu Ile Pro Phe
Thr Phe Gly Ser Gly Thr 485 490 495 Lys Leu Glu Ile Lys His His His
His His His 500 505 1811530DNAartificial sequenceSEQ ID NO 194 x
EGFR HL ; bispecific single chain antibody 181cagaccgttg tgactcagga
accttcactc accgtatcac ctggtggaac agtcacactc 60acttgtcgct cgtccactgg
ggctgttaca actagcaact atgccaactg ggtccaacaa 120aaaccaggtc
aggcaccccg tggtctaata ggtggtacca acaagcgcgc accaggtact
180cctgccagat tctcaggctc cctgcttgga ggcaaggctg ccctcaccct
ctcaggggta 240cagccagagg atgaggcaga atattactgt gctctatggt
acagcaacct ctgggtgttc 300ggtggaggaa ccaaactgac tgtcctaggt
ggtggtggtt ctggcggcgg cggctccggt 360ggtggtggtt ctgaggtgca
gctggtggag tctggaggag gattggtgca gcctggaggg 420tcattgaaac
tctcatgtgc agcctctgga ttcaccttca atacctacgc catgaactgg
480gtccgccagg ctccaggaaa gggtttggaa tgggttgctc gcataagaag
taaatataat 540aattatgcaa catattatgc cgattcagtg aaagacaggt
tcaccatctc cagagatgat 600tcaaaaaaca ctgcctatct acaaatgaac
aacttgaaaa ctgaggacac tgccgtgtac 660tactgtgtga gacatgggaa
cttcggtaat agctacgttt cctggtttgc ttactggggc 720caagggactc
tggtcaccgt ctcctcatcc ggaggtggtg gctcccaggt gcagctgcag
780cagtctgggc ctgatctggt gaagcctggg gcctcagtga agatgtcctg
caaggcttct 840ggacacactt tcactgactg tgttataatc tgggtgaaac
agagagctgg acagggcctt 900gagtggattg gacagattta tccagggact
ggtcgttctt actacaatga gattttcaag 960ggcaaggcca cactgactgc
agacaaatcc tccaacacag tccacattca actcagcagc 1020ctgacatctg
aggactctgc ggtctatttc tgtgccctat ctactcttat tcacgggacc
1080tggttttctt attggggcca agggactctg gtcactgtct cttccggtgg
tggtggttct 1140ggcggcggcg gctccggtgg tggtggttct gacattgtac
tgacccagtc tccagcttcc 1200ttacctgtgt ctctggggca gagggccacc
atctcatgca gggccagcca aagtgtcagt 1260tcatctactt atagttatat
acactggtac caacagaaac caggacagcc acccaaactc 1320ctcatcacgt
atgcatccaa cctagaatct ggggtccctg ccaggttcag tggcagtggg
1380tctgggacag acttcaccct cgacatccat cctgtggagg aggatgattc
ttcaacatat 1440tactgtcagc acagttggga gattccattt acgttcggct
cggggacaaa gttggaaata 1500aaacatcatc accatcatca ttaggtcgac
1530182507PRTartificial sequenceSEQ ID NO 194 x EGFR HL ;
bispecific single chain antibody 182Gln Thr Val Val Thr Gln Glu Pro
Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys
Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn
Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60
Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65
70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr
Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu Lys Leu 130 135 140 Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp 145 150 155 160 Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg 165 170 175 Ser
Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp 180 185
190 Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln
195 200 205 Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
Val Arg 210 215 220 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
Ala Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser
Ser Gly Gly Gly Gly Ser Gln 245 250 255 Val Gln Leu Gln Gln Ser Gly
Pro Asp Leu Val Lys Pro Gly Ala Ser 260 265 270 Val Lys Met Ser Cys
Lys Ala Ser Gly His Thr Phe Thr Asp Cys Val 275 280 285 Ile Ile Trp
Val Lys Gln Arg Ala Gly Gln Gly Leu Glu Trp Ile Gly 290 295 300 Gln
Ile Tyr Pro Gly Thr Gly Arg Ser Tyr Tyr Asn Glu Ile Phe Lys 305 310
315 320 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Val His
Ile 325 330 335 Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Phe Cys Ala 340 345 350 Leu Ser Thr Leu Ile His Gly Thr Trp Phe Ser
Tyr Trp Gly Gln Gly 355 360 365 Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly 370 375 380 Ser Gly Gly Gly Gly Ser Asp
Ile Val Leu Thr Gln Ser Pro Ala Ser 385 390 395 400 Leu Pro Val Ser
Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser 405 410 415 Gln Ser
Val Ser Ser Ser Thr Tyr Ser Tyr Ile His Trp Tyr Gln Gln 420 425 430
Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Thr Tyr Ala Ser Asn Leu 435
440 445 Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp 450 455 460 Phe Thr Leu Asp Ile His Pro Val Glu Glu Asp Asp Ser
Ser Thr Tyr 465 470 475 480 Tyr Cys Gln His Ser Trp Glu Ile Pro Phe
Thr Phe Gly Ser Gly Thr 485 490 495 Lys Leu Glu Ile Lys His His His
His His His 500 505 1831530DNAartificial sequenceSEQ ID NO 170 x
EGFR LH ; bispecific single chain antibody 183gaggtgcagc tggtggagtc
tggaggagga ttggtgcagc ctggagggtc attgaaactc 60tcatgtgcag cctctggatt
caccttcaat acctacgcca tgaactgggt ccgccaggct 120ccaggaaagg
gtttggaatg ggttgctcgc ataagaagta aatataataa ttatgcaaca
180tattatgccg attcagtgaa agacaggttc accatctcca gagatgattc
aaaaaacact 240gcctatctac aaatgaacaa cttgaaaact gaggacactg
ccgtgtacta ctgtgtgaga 300catgggaact tcggtaatag ctacgtttcc
tggtttgctt actggggcca agggactctg 360gtcaccgtct cctcaggtgg
tggtggttct ggcggcggcg gctccggtgg tggtggttct 420cagaccgttg
tgactcagga accttcactc accgtatcac ctggtggaac agtcacactc
480acttgtcgct cgtccactgg ggctgttaca actagcaact atgccaactg
ggtccaacaa 540aaaccaggtc aggcaccccg tggtctaata ggtggtacca
acaagcgcgc accaggtact 600cctgccagat tctcaggctc cctgcttgga
ggcaaggctg ccctcaccct ctcaggggta 660cagccagagg atgaggcaga
atattactgt gctctatggt acagcaacct ctgggtgttc 720ggtggaggaa
ccaaactgac tgtcctatcc ggaggtggtg gctccgacat tgtgctgaca
780cagtctcctg cttccttacc tgtgtctctg gggcagaggg ccaccatctc
atgcagggcc 840agccaaagtg tcagttcatc tacttatagt tatatacact
ggtaccaaca gaaaccagga 900cagccaccca aactcctcat cacgtatgca
tccaacctag aatctggggt ccctgccagg 960ttcagtggca gtgggtctgg
gacagacttc accctcgaca tccatcctgt ggaggaggat 1020gattcttcaa
catattactg tcagcacagt tgggagattc catttacgtt cggctcgggg
1080acaaagttgg aaataaaagg tggtggtggt tctggcggcg gcggctccgg
tggtggtggt 1140tctcaggttc agctgcagca gtctggacct gatctggtga
agcctggggc ctcagtgaag 1200atgtcctgca aggcttctgg acacactttc
actgactgtg ttataatctg ggtgaaacag 1260agagctggac agggccttga
gtggattgga cagatttatc cagggactgg tcgttcttac 1320tacaatgaga
ttttcaaggg caaggccaca ctgactgcag acaaatcctc caacacagtc
1380cacattcaac tcagcagcct gacatctgag gactctgcgg tctatttctg
tgccctatct 1440actcttattc acgggacctg gttttcttat tggggccaag
ggactctggt cactgtctct 1500tcccatcatc accatcatca ttaggtcgac
1530184507PRTartificial sequenceSEQ ID NO 170 x EGFR LH ;
bispecific single chain antibody 184Glu 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 Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50
55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gln Thr Val Val 130 135 140 Thr Gln Glu Pro Ser
Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu 145 150 155 160 Thr Cys
Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 165 170 175
Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly 180
185 190 Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser
Leu 195 200 205 Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln
Pro Glu Asp 210 215 220 Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser
Asn Leu Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Thr Val
Leu Ser Gly Gly Gly Gly Ser Asp 245 250 255 Ile Val Leu Thr Gln Ser
Pro Ala Ser Leu Pro Val Ser Leu Gly Gln 260 265 270 Arg Ala Thr Ile
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser Thr 275 280 285 Tyr Ser
Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys 290 295 300
Leu Leu Ile Thr Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg 305
310 315 320 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asp Ile
His Pro 325 330 335 Val Glu Glu Asp Asp Ser Ser Thr Tyr Tyr Cys Gln
His Ser Trp Glu 340 345 350 Ile Pro Phe Thr Phe Gly Ser Gly Thr Lys
Leu Glu Ile Lys Gly Gly 355 360 365 Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gln Val Gln 370 375 380 Leu Gln Gln Ser Gly Pro
Asp Leu Val Lys Pro Gly Ala Ser Val Lys 385 390 395 400 Met Ser Cys
Lys Ala Ser Gly His Thr Phe Thr Asp Cys Val Ile Ile 405 410 415 Trp
Val Lys Gln Arg Ala Gly Gln Gly Leu Glu Trp Ile Gly Gln Ile 420 425
430 Tyr Pro Gly Thr Gly Arg Ser Tyr Tyr Asn Glu Ile Phe Lys Gly Lys
435 440 445 Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Val His Ile
Gln Leu 450 455 460 Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe
Cys Ala Leu Ser 465 470 475 480 Thr Leu Ile His Gly Thr Trp Phe Ser
Tyr Trp Gly Gln Gly Thr Leu 485 490 495 Val Thr Val Ser Ser His His
His His His His 500 505 1851530DNAartificial sequenceSEQ ID NO 194
x EGFR LH ; bispecific single chain antibody 185cagaccgttg
tgactcagga accttcactc accgtatcac ctggtggaac agtcacactc 60acttgtcgct
cgtccactgg ggctgttaca actagcaact atgccaactg ggtccaacaa
120aaaccaggtc aggcaccccg tggtctaata ggtggtacca acaagcgcgc
accaggtact 180cctgccagat tctcaggctc cctgcttgga ggcaaggctg
ccctcaccct ctcaggggta 240cagccagagg atgaggcaga atattactgt
gctctatggt acagcaacct ctgggtgttc 300ggtggaggaa ccaaactgac
tgtcctaggt ggtggtggtt ctggcggcgg cggctccggt 360ggtggtggtt
ctgaggtgca gctggtggag tctggaggag gattggtgca gcctggaggg
420tcattgaaac tctcatgtgc agcctctgga ttcaccttca atacctacgc
catgaactgg 480gtccgccagg ctccaggaaa gggtttggaa tgggttgctc
gcataagaag taaatataat 540aattatgcaa catattatgc cgattcagtg
aaagacaggt tcaccatctc cagagatgat 600tcaaaaaaca ctgcctatct
acaaatgaac aacttgaaaa ctgaggacac tgccgtgtac 660tactgtgtga
gacatgggaa cttcggtaat agctacgttt cctggtttgc ttactggggc
720caagggactc tggtcaccgt ctcctcatcc ggaggtggtg gctccgacat
tgtgctgaca 780cagtctcctg cttccttacc tgtgtctctg gggcagaggg
ccaccatctc atgcagggcc 840agccaaagtg tcagttcatc tacttatagt
tatatacact ggtaccaaca gaaaccagga 900cagccaccca aactcctcat
cacgtatgca tccaacctag aatctggggt ccctgccagg 960ttcagtggca
gtgggtctgg gacagacttc accctcgaca tccatcctgt ggaggaggat
1020gattcttcaa catattactg tcagcacagt tgggagattc catttacgtt
cggctcgggg 1080acaaagttgg aaataaaagg tggtggtggt tctggcggcg
gcggctccgg tggtggtggt 1140tctcaggttc agctgcagca gtctggacct
gatctggtga agcctggggc ctcagtgaag 1200atgtcctgca aggcttctgg
acacactttc actgactgtg ttataatctg ggtgaaacag 1260agagctggac
agggccttga gtggattgga cagatttatc cagggactgg tcgttcttac
1320tacaatgaga ttttcaaggg caaggccaca ctgactgcag acaaatcctc
caacacagtc 1380cacattcaac tcagcagcct gacatctgag gactctgcgg
tctatttctg tgccctatct 1440actcttattc acgggacctg gttttcttat
tggggccaag ggactctggt cactgtctct 1500tcccatcatc accatcatca
ttaggtcgac 1530186507PRTartificial sequenceSEQ ID NO 194 x EGFR LH
; bispecific single chain antibody 186Gln Thr Val Val Thr Gln Glu
Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr
Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala
Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu
Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55
60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val
65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr
Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu Lys Leu 130 135 140 Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp 145 150 155 160 Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg 165 170 175 Ser
Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp 180 185
190 Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln
195 200 205 Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
Val Arg 210 215 220 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
Ala Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser
Ser Gly Gly Gly Gly Ser Asp 245 250 255 Ile Val Leu Thr Gln Ser Pro
Ala Ser Leu Pro Val Ser Leu Gly Gln 260 265 270 Arg Ala Thr Ile Ser
Cys Arg Ala Ser Gln Ser Val Ser Ser Ser Thr 275 280 285 Tyr Ser Tyr
Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys 290 295 300 Leu
Leu Ile Thr Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg 305 310
315 320 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asp Ile His
Pro 325 330 335 Val Glu Glu Asp Asp Ser Ser Thr Tyr Tyr Cys Gln His
Ser Trp Glu 340 345 350 Ile Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu
Glu Ile Lys Gly Gly 355 360 365 Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gln Val Gln 370 375 380 Leu Gln Gln Ser Gly Pro Asp
Leu Val Lys Pro Gly Ala Ser Val Lys 385 390 395 400 Met Ser Cys Lys
Ala Ser Gly His Thr Phe Thr Asp Cys Val Ile Ile 405 410 415 Trp Val
Lys Gln Arg Ala Gly Gln Gly Leu Glu Trp Ile Gly Gln Ile 420 425 430
Tyr Pro Gly Thr Gly Arg Ser Tyr Tyr Asn Glu Ile Phe Lys Gly Lys 435
440 445 Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Val His Ile Gln
Leu 450 455 460 Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
Ala Leu Ser 465 470 475 480 Thr Leu Ile His Gly Thr Trp Phe Ser Tyr
Trp Gly Gln Gly Thr Leu 485 490 495 Val Thr Val Ser Ser His His His
His His His 500 505 1871515DNAartificial sequenceCAIX HL x SEQ ID
NO 194 ; bispecific single chain antibody 187gacgtgaagc tcgtggagtc
tgggggaggc ttagtgaagc ttggagggtc cctgaaactc 60tcctgtgcag cctctggatt
cactttcagt aactattaca tgtcttgggt tcgccagact 120ccagagaaga
ggctggagtt ggtcgcagcc attaatagtg atggtggtat cacctactat
180ctagacactg tgaagggccg attcaccatt tcaagagaca atgccaagaa
caccctgtac 240ctgcaaatga gcagtctgaa gtctgaggac acagccttgt
tttactgtgc aagacaccgc 300tcgggctact tttctatgga ctactggggt
caaggaacct cagtcaccgt ctcctcaggt 360ggtggtggtt ctggcggcgg
cggctccggt ggtggtggtt ctgacattgt gatgacccag 420tctcaaagat
tcatgtccac aacagtagga gacagggtca gcatcacctg caaggccagt
480cagaatgtgg tttctgctgt tgcctggtat caacagaaac caggacaatc
tcctaaacta 540ctgatttact cagcatccaa tcggtacact ggagtccctg
atcgcttcac aggcagtgga 600tctgggacag atttcactct caccattagc
aatatgcagt ctgaagacct ggctgatttt 660ttctgtcaac aatatagcaa
ctatccgtgg acgttcggtg gaggcaccaa gctggaaatc 720aaatccggag
gtggtggctc ccagaccgtt gtgactcagg aaccttcact caccgtatca
780cctggtggaa cagtcacact cacttgtcgc tcgtccactg gggctgttac
aactagcaac 840tatgccaact gggtccaaca aaaaccaggt caggcacccc
gtggtctaat aggtggtacc 900aacaagcgcg caccaggtac tcctgccaga
ttctcaggct ccctgcttgg aggcaaggct 960gccctcaccc tctcaggggt
acagccagag gatgaggcag aatattactg tgctctatgg 1020tacagcaacc
tctgggtgtt cggtggagga accaaactga ctgtcctagg tggtggtggt
1080tctggcggcg gcggctccgg tggtggtggt tctgaggtgc agctggtgga
gtctggagga 1140ggattggtgc agcctggagg gtcattgaaa ctctcatgtg
cagcctctgg attcaccttc 1200aatacctacg ccatgaactg ggtccgccag
gctccaggaa agggtttgga atgggttgct 1260cgcataagaa gtaaatataa
taattatgca acatattatg ccgattcagt gaaagacagg 1320ttcaccatct
ccagagatga ttcaaaaaac actgcctatc tacaaatgaa caacttgaaa
1380actgaggaca ctgccgtgta ctactgtgtg agacatggga acttcggtaa
tagctacgtt 1440tcctggtttg cttactgggg ccaagggact ctggtcaccg
tctcctcaca tcatcaccat 1500catcattagg tcgac 1515188502PRTartificial
sequenceCAIX HL x SEQ ID NO 194 ; bispecific single chain antibody
188Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Leu Gly Gly
1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg
Leu Glu Leu Val 35 40 45 Ala Ala Ile Asn Ser Asp Gly Gly Ile Thr
Tyr Tyr Leu Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu
Lys Ser Glu Asp Thr Ala Leu Phe Tyr Cys 85 90 95 Ala Arg His Arg
Ser Gly Tyr Phe Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser
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 Val Met Thr Gln Ser Gln Arg Phe 130
135 140 Met Ser Thr Thr Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala
Ser 145 150 155 160 Gln Asn Val Val Ser Ala Val Ala Trp Tyr Gln Gln
Lys Pro Gly Gln 165 170 175 Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser
Asn Arg Tyr Thr Gly Val 180 185 190 Pro Asp Arg Phe Thr Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Asn Met Gln Ser
Glu Asp Leu Ala Asp Phe Phe Cys Gln Gln 210 215 220 Tyr Ser Asn Tyr
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys
Ser Gly Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser 245 250
255 Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser
260 265 270 Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln
Gln Lys 275 280 285 Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr
Asn Lys Arg Ala 290 295 300 Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser
Leu Leu Gly Gly Lys Ala 305 310 315 320 Ala Leu Thr Leu Ser Gly Val
Gln Pro Glu Asp Glu Ala Glu Tyr Tyr 325 330 335 Cys Ala Leu Trp Tyr
Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys 340 345 350 Leu Thr Val
Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 355 360 365 Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 370 375
380 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
385 390 395 400 Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 405 410 415 Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr 420 425 430 Tyr Ala Asp Ser Val Lys Asp Arg Phe
Thr Ile Ser Arg Asp Asp Ser 435 440 445 Lys Asn Thr Ala Tyr Leu Gln
Met Asn Asn Leu Lys Thr Glu Asp Thr 450 455 460 Ala Val Tyr Tyr Cys
Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val 465 470 475 480 Ser Trp
Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 485 490 495
His His His His His His 500 1891515DNAartificial sequenceCAIX HL x
SEQ ID NO 170 ; bispecific single chain antibody 189gacgtgaagc
tcgtggagtc tgggggaggc ttagtgaagc ttggagggtc cctgaaactc 60tcctgtgcag
cctctggatt cactttcagt aactattaca tgtcttgggt tcgccagact
120ccagagaaga ggctggagtt ggtcgcagcc attaatagtg atggtggtat
cacctactat 180ctagacactg tgaagggccg attcaccatt tcaagagaca
atgccaagaa caccctgtac 240ctgcaaatga gcagtctgaa gtctgaggac
acagccttgt tttactgtgc aagacaccgc 300tcgggctact tttctatgga
ctactggggt caaggaacct cagtcaccgt ctcctcaggt 360ggtggtggtt
ctggcggcgg cggctccggt ggtggtggtt ctgacattgt gatgacccag
420tctcaaagat tcatgtccac aacagtagga gacagggtca gcatcacctg
caaggccagt 480cagaatgtgg tttctgctgt tgcctggtat caacagaaac
caggacaatc tcctaaacta 540ctgatttact cagcatccaa tcggtacact
ggagtccctg atcgcttcac aggcagtgga 600tctgggacag atttcactct
caccattagc aatatgcagt ctgaagacct ggctgatttt 660ttctgtcaac
aatatagcaa ctatccgtgg acgttcggtg gaggcaccaa gctggaaatc
720aaatccggag gtggtggctc cgaggtgcag ctggtggagt ctggaggagg
attggtgcag 780cctggagggt cattgaaact ctcatgtgca gcctctggat
tcaccttcaa tacctacgcc 840atgaactggg tccgccaggc tccaggaaag
ggtttggaat gggttgctcg cataagaagt 900aaatataata attatgcaac
atattatgcc gattcagtga aagacaggtt caccatctcc 960agagatgatt
caaaaaacac tgcctatcta caaatgaaca acttgaaaac tgaggacact
1020gccgtgtact actgtgtgag acatgggaac ttcggtaata gctacgtttc
ctggtttgct 1080tactggggcc aagggactct ggtcaccgtc tcctcaggtg
gtggtggttc tggcggcggc 1140ggctccggtg gtggtggttc tcagaccgtt
gtgactcagg aaccttcact caccgtatca 1200cctggtggaa cagtcacact
cacttgtcgc tcgtccactg gggctgttac aactagcaac 1260tatgccaact
gggtccaaca aaaaccaggt caggcacccc gtggtctaat aggtggtacc
1320aacaagcgcg caccaggtac tcctgccaga ttctcaggct ccctgcttgg
aggcaaggct 1380gccctcaccc tctcaggggt acagccagag gatgaggcag
aatattactg tgctctatgg 1440tacagcaacc tctgggtgtt cggtggagga
accaaactga ctgtcctaca tcatcaccat 1500catcattagg tcgac
1515190502PRTartificial sequenceCAIX HL x SEQ ID NO 170 ;
bispecific single chain antibody 190Asp Val Lys Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Leu Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ser Trp
Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Leu Val 35 40 45 Ala Ala
Ile Asn Ser Asp Gly Gly Ile Thr Tyr Tyr Leu Asp Thr Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu
Phe Tyr Cys 85 90 95 Ala Arg His Arg Ser Gly Tyr Phe Ser Met Asp
Tyr Trp Gly Gln Gly 100 105 110 Thr Ser 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 Val Met Thr Gln Ser Gln Arg Phe 130 135 140 Met Ser Thr Thr Val
Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser 145 150 155 160 Gln Asn
Val Val Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 165 170 175
Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val 180
185 190 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr 195 200 205 Ile Ser Asn Met Gln Ser Glu Asp Leu Ala Asp Phe Phe
Cys Gln Gln 210 215 220 Tyr Ser Asn Tyr Pro Trp Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile 225 230 235 240 Lys Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val Glu Ser Gly Gly 245 250 255 Gly Leu Val Gln Pro Gly
Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser 260 265 270 Gly Phe Thr Phe
Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro 275 280 285 Gly Lys
Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn 290 295 300
Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser 305
310 315 320 Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn
Leu Lys 325 330 335 Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His
Gly Asn Phe Gly 340 345 350 Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp
Gly Gln Gly Thr Leu Val 355 360 365 Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 370 375 380 Gly Gly Ser Gln Thr Val
Val Thr Gln Glu Pro Ser Leu Thr Val Ser 385 390 395 400 Pro Gly Gly
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val 405 410 415 Thr
Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala 420 425
430 Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro
435 440 445 Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu
Thr Leu 450 455 460 Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr
Cys Ala Leu Trp 465 470 475 480 Tyr Ser Asn Leu Trp Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 485 490 495 His His His His His His 500
1911515DNAartificial sequenceCAIX LH x SEQ ID NO 170 ; bispecific
single chain antibody 191gacattgtga tgacccagtc tcaaagattc
atgtccacaa cagtaggaga cagggtcagc 60atcacctgca aggccagtca gaatgtggtt
tctgctgttg cctggtatca acagaaacca 120ggacaatctc ctaaactact
gatttactca gcatccaatc ggtacactgg agtccctgat 180cgcttcacag
gcagtggatc tgggacagat ttcactctca ccattagcaa tatgcagtct
240gaagacctgg ctgatttttt ctgtcaacaa tatagcaact atccgtggac
gttcggtgga 300ggcaccaagc tggaaatcaa aggtggtggt ggttctggcg
gcggcggctc cggtggtggt 360ggttctgacg tgaagctcgt ggagtctggg
ggaggcttag tgaagcttgg agggtccctg 420aaactctcct gtgcagcctc
tggattcact ttcagtaact attacatgtc ttgggttcgc 480cagactccag
agaagaggct ggagttggtc gcagccatta atagtgatgg tggtatcacc
540tactatctag acactgtgaa gggccgattc accatttcaa gagacaatgc
caagaacacc 600ctgtacctgc aaatgagcag tctgaagtct gaggacacag
ccttgtttta ctgtgcaaga 660caccgctcgg gctacttttc tatggactac
tggggtcaag gaacctcagt caccgtctcc 720tcctccggag gtggtggctc
cgaggtgcag ctggtggagt ctggaggagg attggtgcag 780cctggagggt
cattgaaact ctcatgtgca gcctctggat tcaccttcaa tacctacgcc
840atgaactggg tccgccaggc tccaggaaag ggtttggaat gggttgctcg
cataagaagt 900aaatataata attatgcaac atattatgcc gattcagtga
aagacaggtt caccatctcc 960agagatgatt caaaaaacac tgcctatcta
caaatgaaca acttgaaaac tgaggacact 1020gccgtgtact actgtgtgag
acatgggaac ttcggtaata gctacgtttc ctggtttgct 1080tactggggcc
aagggactct ggtcaccgtc tcctcaggtg gtggtggttc tggcggcggc
1140ggctccggtg gtggtggttc tcagaccgtt gtgactcagg aaccttcact
caccgtatca 1200cctggtggaa cagtcacact cacttgtcgc tcgtccactg
gggctgttac aactagcaac 1260tatgccaact gggtccaaca aaaaccaggt
caggcacccc gtggtctaat aggtggtacc 1320aacaagcgcg caccaggtac
tcctgccaga ttctcaggct ccctgcttgg aggcaaggct 1380gccctcaccc
tctcaggggt acagccagag gatgaggcag aatattactg tgctctatgg
1440tacagcaacc tctgggtgtt cggtggagga accaaactga ctgtcctaca
tcatcaccat 1500catcattagg tcgac 1515192502PRTartificial
sequenceCAIX LH x SEQ ID NO 170 ; bispecific single chain antibody
192Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Ser Thr Thr Val Gly
1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Val
Ser Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val
Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Asn Met Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Phe
Phe Cys Gln Gln Tyr Ser Asn Tyr Pro Trp 85 90 95 Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Lys Leu Val Glu 115 120 125
Ser Gly Gly Gly Leu Val Lys Leu Gly Gly Ser Leu Lys Leu Ser Cys 130
135 140 Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Tyr Met Ser Trp Val
Arg 145 150 155 160 Gln Thr Pro Glu Lys Arg Leu Glu Leu Val Ala Ala
Ile Asn Ser Asp 165 170 175 Gly Gly Ile Thr Tyr Tyr Leu Asp Thr Val
Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr Leu Gln Met Ser Ser Leu 195 200 205 Lys Ser Glu Asp Thr Ala
Leu Phe Tyr Cys Ala Arg His Arg Ser Gly 210 215 220 Tyr Phe Ser Met
Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 225 230 235 240 Ser
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly 245 250
255 Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser
260 265 270 Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln
Ala Pro 275 280 285 Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser
Lys Tyr Asn Asn 290 295 300 Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys
Asp Arg Phe Thr Ile Ser 305 310 315 320 Arg Asp Asp Ser Lys Asn Thr
Ala Tyr Leu Gln Met Asn Asn Leu Lys 325 330 335 Thr Glu Asp Thr Ala
Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly 340 345 350 Asn Ser Tyr
Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 355 360 365 Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 370 375
380 Gly Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser
385 390 395 400 Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr
Gly Ala Val 405 410 415 Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln
Lys Pro Gly Gln Ala 420 425 430 Pro Arg Gly Leu Ile Gly Gly Thr Asn
Lys Arg Ala Pro Gly Thr Pro 435 440 445 Ala Arg Phe Ser Gly Ser Leu
Leu Gly Gly Lys Ala Ala Leu Thr Leu 450 455 460 Ser Gly Val Gln Pro
Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp 465 470 475 480 Tyr Ser
Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 485 490 495
His His His His His His 500 193747DNAartificial sequenceHuman-like
VL (SEQ ID NO 168) x Human-like VH (SEQ ID NO 110) scFv ; single
chain Fv 193cagaccgttg tgactcagga accttcactc accgtatcac ctggtggaac
agtcacactc 60acttgtcgct cgtccactgg ggctgttaca actagcaact atgccaactg
ggtccaacaa 120aaaccaggtc aggcaccccg tggtctaata ggtggtacca
acaagcgcgc accaggtact 180cctgccagat tctcaggctc cctgcttgga
ggcaaggctg ccctcaccct ctcaggggta 240cagccagagg atgaggcaga
atattactgt gctctatggt acagcaacct ctgggtgttc 300ggtggaggaa
ccaaactgac tgtcctaggt ggtggtggtt ctggcggcgg cggctccggt
360ggtggtggtt ctgaggtgca gctggtggag tctggaggag gattggtgca
gcctggaggg 420tcattgaaac tctcatgtgc agcctctgga ttcaccttca
atacctacgc catgaactgg 480gtccgccagg ctccaggaaa gggtttggaa
tgggttgctc gcataagaag taaatataat 540aattatgcaa catattatgc
cgattcagtg aaagacaggt tcaccatctc cagagatgat 600tcaaaaaaca
ctgcctatct acaaatgaac aacttgaaaa ctgaggacac tgccgtgtac
660tactgtgtga gacatgggaa cttcggtaat agctacgttt cctggtttgc
ttactggggc 720caagggactc tggtcaccgt ctcctca 747194249PRTartificial
sequenceHuman-like VL (SEQ ID NO 168) x Human-like VH (SEQ ID NO
110) scFv ; single chain Fv 194Gln Thr Val Val Thr Gln Glu Pro Ser
Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg
Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp
Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly
Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser
Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70
75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser
Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Lys Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe
Thr Phe Asn Thr Tyr Ala Met Asn Trp 145 150 155 160 Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg 165 170 175 Ser Lys
Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp 180 185 190
Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln 195
200 205 Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val
Arg 210 215 220 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala
Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser 245
19513PRTartificial sequenceepitope 195Glu Phe Ser Glu Leu Glu Gln
Ser Gly Tyr Tyr Val Cys 1 5 10 19613PRTartificial sequenceepitope
196Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Lys 1 5 10
19734DNAartificial sequence5' EGFR XbaI ; oligonucleotide
197ggtctagagc atgcgaccct ccgggacggc cggg 3419836DNAARTIFICIAL
SEQUENCE3' EGFR SalI ; oligonucleotide 198ttttaagtcg actcatgctc
caataaattc actgct 3619913PRTartificial sequenceepitope 199Gln Asp
Gly Asn Glu Glu Met Gly Ser Ile Thr Gln Thr 1 5 10
20013PRTartificial sequenceepitope 200Tyr Tyr Val Ser Tyr Pro Arg
Gly Ser Asn Pro Glu Asp 1 5 10 20113PRTartificial sequenceepitope
201Glu Phe Ser Glu Met Glu Gln Ser Gly Tyr Tyr Val Cys 1 5 10
2025PRTartificial sequenceepitope 202Phe Ser Glu Leu Glu 1 5
20313PRTartificial sequenceepitope 203Gln Tyr Pro Gly Ser Glu Ile
Leu Trp Gln His Asn Asp 1 5 10 2045PRTartificial sequenceepitope
204Phe Ser Glu Leu Glu 1 5 2055PRTartificial sequenceepitope 205Phe
Ser Glu Leu Glu 1 5 2065PRTartificial sequenceepitope 206Phe Ser
Glu Met Glu 1 5 20713PRTartificial sequenceepitope 207Glu Phe Ser
Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys 1 5 10
* * * * *
References