U.S. patent application number 14/670271 was filed with the patent office on 2015-07-23 for aberrant cell-restricted immunoglobulins provided with a toxic moiety.
The applicant listed for this patent is APO-T B.V.. Invention is credited to JOHAN RENES, PAULUS J. G. M. STEVERINK, RALPH ALEXANDER WILLEMSEN.
Application Number | 20150202318 14/670271 |
Document ID | / |
Family ID | 47682044 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150202318 |
Kind Code |
A1 |
RENES; JOHAN ; et
al. |
July 23, 2015 |
ABERRANT CELL-RESTRICTED IMMUNOGLOBULINS PROVIDED WITH A TOXIC
MOIETY
Abstract
Described are immunoglobulins provided with a toxic moiety,
comprising at least an immunoglobulin variable region that
specifically binds to an MHC-peptide complex preferentially
associated with aberrant cells. These immunoglobulins provided with
a toxic moiety are preferably used in selectively modulating
biological processes. The provided immunoglobulins provided with a
toxic moiety are of particular use in pharmaceutical compositions
for the treatment of diseases related to cellular aberrancies, such
as cancers and autoimmune diseases.
Inventors: |
RENES; JOHAN; (AMERSFOORT,
NL) ; STEVERINK; PAULUS J. G. M.; (HUIZEN, NL)
; WILLEMSEN; RALPH ALEXANDER; (ROTTERDAM, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APO-T B.V. |
AMERSFOORT |
|
NL |
|
|
Family ID: |
47682044 |
Appl. No.: |
14/670271 |
Filed: |
March 26, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13739974 |
Jan 11, 2013 |
|
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14670271 |
|
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|
61586568 |
Jan 13, 2012 |
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Current U.S.
Class: |
424/134.1 ;
424/178.1; 530/387.3; 530/391.7 |
Current CPC
Class: |
C07K 16/084 20130101;
A61P 35/00 20180101; C07K 2319/33 20130101; A61K 47/6849 20170801;
A61K 47/6809 20170801; C07K 16/3069 20130101; C07K 2317/569
20130101; C07K 2317/34 20130101; A61K 2039/505 20130101; C07K
16/085 20130101; C07K 16/3092 20130101; C07K 16/40 20130101; A61K
47/6883 20170801; C07K 2317/76 20130101; A61K 47/6851 20170801;
C07K 16/32 20130101; C07K 16/2833 20130101; C07K 16/2884 20130101;
C07K 2317/32 20130101; A61K 47/6813 20170801; C07K 16/30
20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C07K 16/28 20060101 C07K016/28 |
Claims
1. An immunoglobulin provided with a toxic moiety, the
immunoglobulin comprising at least an immunoglobulin variable
region that specifically binds to an MHC-peptide complex
preferentially associated with aberrant cells.
2. The immunoglobulin of claim 1, wherein the immunoglobulin
variable region is a Vh or Vhh.
3. The immunoglobulin of claim 2, wherein the immunoglobulin
variable region further comprises a Vl.
4. The immunoglobulin of claim 3, which is a human IgG.
5. The immunoglobulin of claim 1, wherein the MHC-peptide complex
is specific for aberrant cells.
6. The immunoglobulin of claim 1, wherein the toxic moiety is
chemically linked to the immunoglobulin.
7. The immunoglobulin of claim 1, wherein the toxic moiety is a
fusion protein, fused to the immunoglobulin at the DNA level.
8. A pharmaceutical composition comprising the immunoglobulin of
claim 1, and suitable diluents and/or excipients.
9. A method of treatment of a host suffering from a disease
associated with aberrant cells, comprising: utilizing the
immunoglobulin of claim 1 to treat the host.
10. The method according to claim 9, wherein the toxic moiety is
internalized into an aberrant cell.
11. The method according to claim 9 to treat cancer.
12. The method according to claim 10 to treat cancer.
13. An immunoglobulin provided with a toxic moiety according to
FIG. 5, Panel B.
14. The immunoglobulin of claim 1, wherein the MHC-peptide complex
is specific for aberrant cells, through a peptide derived from
MAGE.
15. The immunoglobulin of claim 14, wherein the MHC-peptide complex
is specific for aberrant cells, through a peptide derived from
MAGE-A.
16. The immunoglobulin of claim 7, wherein the fusion protein is
fused to the immunoglobulin at the DNA level through a linking
sequence.
17. A human IgG immunoglobulin chemically linked to a toxic moiety,
wherein the human IgG immunoglobulin comprises at least an
immunoglobulin variable region that specifically binds to an
MHC-peptide complex preferentially associated with aberrant cells,
wherein the MHC-peptide complex is specific for aberrant cells
through a peptide derived from MAGE-A, and wherein the toxic moiety
is a fusion protein.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/739,974, filed Jan. 11, 2013, pending,
which application claims the benefit under 35 U.S.C. .sctn.119(e)
to U.S. Ser. No. 61/586,568, filed on Jan. 12, 2012, the disclosure
of each of which is hereby incorporated herein in its entirety by
this reference.
TECHNICAL FIELD
[0002] The disclosure relates to the field of biotechnology and
biotherapeutics. More specifically, it relates to immunoglobulins
provided with a toxic moiety and human antibodies. It also relates
to the use of these biotherapeutics in the treatment of a host
suffering from a disease associated with aberrant cells, such as
cancers and autoimmune diseases.
BACKGROUND
[0003] The development of immunoglobulin-drug conjugates is a drug
development field that receives great attention nowadays. Humanized
or human antibodies are the largest and most important class of
immunoglobulins under investigation for use in antibody-drug
conjugates (ADCs) and in immunotoxins and antibody-radionuclide
conjugates. These antibodies target binding sites (over)expressed
at aberrant cells, such as those exposed in cancers and immune or
autoimmune diseases, and during infections. Many of the conjugates
have a limited degree of efficacy. For example, the maximum
tolerated dose of immunotoxins is relatively low due to their
toxicity towards healthy tissue. Lowering the dose is one way of
protecting healthy cells for the non-specific toxic activity of the
toxin or the drug in ADCs. Lowering the dose, however, hampers the
delivery of an efficacious amount of conjugate at the site of for
example a tumor. The unwanted side reactions are mainly due to the
targeting of the antibodies to binding sites that are not
exclusively exposed by aberrant cells but also to some extent by
healthy cells. Thus, insufficient specificity for aberrant cells
over healthy cells hampers desired efficacy and hampers obtaining
the desired safety profiles of the nowadays immunoglobulin-drug
conjugates.
[0004] Toxic moieties currently in the clinic or under
investigation are numerous and Diverse..sup.[6] Amongst the first
toxins that were chemically linked to murine antibodies are
plant-derived protein toxins and bacterial toxins such as saporin,
Diphtheria toxin, Pseudomonas exotoxin, gelonin, ricin, ricin A
chain, abrin and pokeweed antiviral protein. Other immunoglobulins
provided with a toxin moiety comprise single chain Fv fused at the
DNA level with toxins. An example is the recombinant protein BL22
consisting of the Fv portion of an anti-human CD22 antibody fused
to a fragment of Pseudomonas exotoxin-A, that targets B-cell
malignancies such as hairy cell leukemia and non-Hodgkin's
lymphoma. Other examples of immunoglobulins conjugated to toxins
are the antibody-radionuclide conjugates. Human CD20 has been
chosen by drug developers as the target for two monoclonal
antibodies, conjugated with 90-Yttrium or with 131-Iodine, for
treatment of non-Hodgkin's lymphomas. In attempts to improve the
tumor selectivity of certain drugs, murine monoclonal antibodies
were conjugated to compounds such as doxorubicin, vinblastine,
methotrexate, providing so-called antibody-drug conjugates.
Insufficient tumor cell specificity however still limited the
therapeutic usefulness. Even when selecting tumor cell surface
antigens that are (highly) over-expressed at aberrant cells, still
the low expression levels at healthy cells gives rise to
insufficient selectivity of the antibody-drug conjugates. Current
cytotoxic anti-tumor drugs under investigation are for example
maytansinoids and dolastatin analogs, that both target
intracellular tubulin, and duocarmycins and calicheamicins, which
target DNA structure. These compounds are potent in their cytotoxic
activity, though not selective for aberrant cells. Antibiotic
calicheamicin conjugated to an anti-human CD33 monoclonal antibody
was approved and used in the clinic, but was withdrawn due to
serious side effects. Additional examples of drugs currently under
investigation for their potential beneficial use in antibody-drug
conjugates meant for the treatment of cellular aberrancies are
ozogamicin, hydrazone-calicheamicin, vedotin, emtansine,
mertansine. These toxic moieties are conjugated to immunoglobulins
targeting cell surface markers expressed at tumor cells, though
also expressed to some extent at healthy cells. Typical examples of
immunoglobulin-drug conjugate-targeted cell surface markers present
at both tumor cells and healthy cells are CD19, CD20, CD22, CD25,
CD30, CD33, CD56, CD70, HER2/neu. All these immunoglobulin-drug
conjugate development programs thus inherently bear the risk for
unacceptable safety profiles and consequent poor efficacy due to
low maximum tolerated doses. Conjugating drugs, radionuclides or
toxins to immunoglobulins specifically and selectively targeting
aberrant cells and not targeting healthy cells would thus provide
for therapies with improved specificity and selectivity for
aberrant cells and with an improved safety profile.
SUMMARY OF THE DISCLOSURE
[0005] Specific and selective delivery of a toxic moiety in target
aberrant cells demands for binding molecules specific for binding
sites preferentially associated with aberrant cells. These binding
molecules then are used as carriers and transporters of the toxic
moieties, specifically and selectively delivering the toxic
moieties at and in the aberrant cells. Herein, we disclose
immunoglobulin-drug conjugates comprising these preferred features.
The immunoglobulins in the immunoglobulin-drug conjugates hereof
comprise immunoglobulin binding regions with improved selectivity
for aberrant cells by specifically binding to binding sites
preferentially associated with these aberrant cells. We disclose as
preferred targets for the antibody hereof, intracellular proteins
that are associated with aberrant cells. These proteins are
available as peptides presented by MHC on the surface of aberrant
cells. The use of MHC-peptide complexes as targets opens us a new
field of tumor targets, because so far typically targets associated
with the surface of aberrant cells have been envisaged. Although it
is preferred that the target is specific for aberrant cells (tumor
cells) in many cases up-regulated intracellular proteins are also
suitable for at least improving the therapeutic window of
immunotoxins. Our most preferred targets are peptides derived from
MAGE presented in the context of MHC-1. In particular MAGE peptides
that are present in more than one MAGE protein (multi-MAGE epitope;
see WO2012/091564 incorporated herein by reference). The toxic
moiety for use herein is preferably a drug compound, a radionuclide
or a toxin. A toxic moiety is a non-proteinaceous molecule or a
proteinaceous molecule. In the immunoglobulin-drug conjugates
hereof, the toxic moiety is preferably conjugated by chemical
conjugation. Also preferred are immunoglobulins hereof fused at the
DNA level to a proteinaceous toxic moiety.
[0006] The immunoglobulins in the immunoglobulin-drug conjugates
hereof are suitable for the specific and selective localization of
a toxic effect inside targeted aberrant cells, leaving healthy
cells essentially unaffected. Immunoglobulins comprise
immunoglobulin binding domains, referred to as immunoglobulin
variable domains, comprising immunoglobulin variable regions.
Maturation of immunoglobulin variable regions results in variable
domains adapted for specific binding to a target binding site.
Immunoglobulins are therefore particularly suitable for providing
the immunoglobulin-drug conjugates hereof with the ability to
specifically and selectively target aberrant cells. At their
surface, aberrant cells present aberrant cell-associated antigen
peptides in the context of major histocompatibility complex (MHC).
Therefore, for the immunoglobulins in the immunoglobulin-drug
conjugates hereof, aberrant cell-associated MHC-1 peptide complexes
are a preferred target on aberrant cells. In addition, aberrant
cell-associated MHC-2 peptide complexes are valuable targets on,
e.g., tumors of hematopoietic origin, for the immunoglobulins in
the immunoglobulin-drug conjugates hereof. Therefore provided are
immunoglobulins in immunoglobulin-drug conjugates, with improved
specificity and selectivity for aberrant cells by targeting
MHC-peptide complexes which are preferentially associated with
aberrant cells. This improved specificity and selectivity for
aberrant cells is accompanied with a reduced level of unintentional
targeting of healthy cells by the immunoglobulins in the
immunoglobulin-drug conjugates hereof. Most preferably, healthy
cells are not targeted by the immunoglobulin-drug conjugates
hereof.
[0007] Thus, in a first embodiment the invention provides an
immunoglobulin provided with a toxic moiety, comprising at least an
immunoglobulin variable region that specifically binds to an
MHC-peptide complex preferentially associated with aberrant cells.
Preferred immunoglobulins hereof are antibodies, but fragments
and/or derivatives such as Fab and/or ScFv can also be used. Even
more preferred immunoglobulins hereof are antibodies of the
immunoglobulin G (IgG) type. Other immunoglobulins hereof are for
example heavy-chain (only) antibodies comprising Vh or Vhh and IgA,
and their fragments such as Fab fragments, and Fab fragments of
IgGs. Immunoglobulins bind via their immunoglobulin variable
regions to binding sites on molecules, such as epitopes, with a
higher binding affinity than background interactions between
molecules. In the context hereof, background interactions are
typically interactions with an affinity lower than a K.sub.D of
10E-4 M. Immunoglobulin variable domains in light chains (Vl) and
immunoglobulin variable domains in heavy chains (Vh) of antibodies
typically comprise the aberrant cell-specific immunoglobulin
variable regions hereof.
[0008] Thus, in one embodiment, provided is an immunoglobulin
provided with a toxic moiety, comprising at least an immunoglobulin
variable region, wherein the immunoglobulin variable region is a
Vh(h) that specifically binds to an MHC-peptide complex
preferentially associated with aberrant cells. Thus, in yet another
embodiment, also provided is an immunoglobulin provided with a
toxic moiety, comprising at least an immunoglobulin variable
region, wherein the immunoglobulin variable region is a Vh that
specifically binds to an MHC-peptide complex preferentially
associated with aberrant cells, and wherein the immunoglobulin
variable region further comprises a Vl.
[0009] As said, immunoglobulins G are particularly suitable binding
molecules for use in therapies specifically and selectively
targeting aberrant cells, for site-specific delivery of a toxic
moiety hereof Because the anticipated predominant use of the
antibodies hereof is in therapeutic treatment regimes meant for the
human body, in a particular embodiment hereof, the immunoglobulins
provided with a toxic moiety have an amino-acid sequence of human
origin. Thus, in one embodiment, provided is a human IgG provided
with a toxic moiety, comprising at least an immunoglobulin variable
region, wherein the immunoglobulin variable region is a Vh that
specifically binds to an MHC-peptide complex preferentially
associated with aberrant cells, and wherein the immunoglobulin
variable region further comprises a Vl. Of course, humanized
antibodies, with the precursor antibodies encompassing amino acid
sequences originating from other species than human, are also part
hereof Also part hereof are chimeric antibodies, comprising (parts
of) an immunoglobulin variable region hereof originating from a
species other than human, and grafted onto a human antibody.
[0010] An aberrant cell is defined as a cell that deviates from its
healthy normal counterparts. Aberrant cells are for example tumor
cells, cells invaded by a pathogen such as a virus, and autoimmune
cells.
[0011] Thus, in one embodiment, provided is an immunoglobulin
according to any of the aforementioned embodiments wherein the
MHC-peptide complex is specific for aberrant cells.
[0012] In the molecules hereof, the toxic moieties are preferably
chemically linked to the immunoglobulins via any linker chemistry
know in the art, and optionally via an additional spacer. Hereof,
one or several, preferably two to six toxic moiety molecules are
chemically linked to an immunoglobulin molecule hereof. The number
of conjugated toxic moiety molecules per single immunoglobulin
molecule is restricted by boundaries such as the number of
available sites for conjugation on the immunoglobulin, the
stability of the conjugate, the preservation of the ability of the
immunoglobulin to specifically bind to an aberrant cell, etc. Of
course, also two, three, etc., different toxic moieties can be
linked to an immunoglobulin, depending amongst others on available
binding sites and the applied linker chemistry. Chemical linking of
the toxic moieties has several advantages when working with
immunoglobulins. This way, toxic moieties cannot interfere with
expression, folding, assembly and secretion of the immunoglobulin
molecules.
[0013] Thus, in one embodiment, provided is an immunoglobulin
according to any of the aforementioned embodiments wherein the
toxic moiety is chemically linked to the immunoglobulin. It is then
also part of the current invention that toxic moieties are
covalently bound via peptide bonds, and preferably via a peptide
linker, to the immunoglobulins hereof The toxic moiety and the
immunoglobulin are then fused at the DNA level.
[0014] Thus, in one embodiment, provided is an immunoglobulin
according to any of the aforementioned embodiments wherein the
toxic moiety is a protein, preferably fused to the immunoglobulin
at the DNA level, preferably through a linker sequence. In many
instances, a simple Gly--Ser linker of 4-15 amino-acid residues may
suffice, but if greater flexibility between the immunoglobulin and
the toxic moiety is desired, longer or more complex linkers may be
used. Preferred linkers are (Gly.sub.4Ser).sub.n (SEQ ID NO:109),
(GlySerThrSerGlySer).sub.n (SEQ ID NO:110),
GlySerThrSerGlySerGlyLysProGlySerGlyGluGlySerThrLysGly (SEQ ID
NO:105),
GlyPheAlaLysThrThrAlaProSerValTyrProLeuAlaProValLeuGluSerSerGlyS-
erGly (SEQ ID NO:111) or any other linker that provides flexibility
allowing protein folding, stability against undesired proteolytic
activity and flexibility for the immunoglobulins hereof to exert
their activity.
[0015] Another group of preferred linkers are linkers based on
hinge regions of immunoglobulins. These linkers tend to be quite
flexible and quite resistant to proteases. The most preferred
linkers based on hinge regions are GluProLysSerCysAspLysThrHisThr
(linking Ch1 and Ch2 in IgG1) (SEQ ID NO:106),
GluLeuLysThrProLeuGlyAspThrThrHisThr (IgG3) (SEQ ID NO:107), and
GluSerLysTyrGlyProPro (IgG4) (SEQ ID NO:108). Thus, the role of any
applied chemical linker in conjugates hereof or the role of any
applied peptide linker in fused molecules hereof is aiding the dual
activity of the antibodies hereof, i.e., specific and selective
binding of the immunoglobulin to aberrant cells, and subsequent
delivery of at least the toxic moiety in the targeted aberrant
cells. Thus, in one embodiment, provided is the use of an
immunoglobulin provided with a toxic moiety according to any of the
aforementioned embodiments, for the treatment of a host suffering
from a disease associated with aberrant cells. In a further
embodiment, provided is the use of an immunoglobulin provided with
a toxic moiety according to any of the aforementioned embodiments,
for the treatment of a host suffering from a disease associated
with aberrant cells wherein at least the toxic moiety is
internalized into the aberrant cell. The immunoglobulins provided
with a toxic moiety are for example used for the treatment of
cancer. Thus, in one embodiment, provided is an immunoglobulin
provided with a toxic moiety according to any of the aforementioned
embodiments for use in the treatment of cancer.
[0016] Preferred toxic moieties are numerous. Several examples of
preferred toxic moieties hereof are drugs such as doxorubicin,
cisplatin, carboplatin, vinblastine, methotrexate, chelated
radioactive metal ions, (synthetic) antineoplastic agents such as
monomethyl auristatin E, radioactive iodine, radionuclides such as
90-Yttrium, 131-Iodine, to name a few, which are chemically
conjugated to the immunoglobulins hereof. Also preferred toxic
moieties are proteinaceous toxins such as a fragment of Pseudomonas
exotoxin-A, statins, ricin A, gelonin, saporin, interleukin-2,
interleukin-12, viral proteins E4 or f4, apoptin and NS1, and
non-viral proteins HAMLET, TRAIL and mda-7. Thus, in one embodiment
hereof, antibodies are provided for the specific targeting of
aberrant cells, wherein the toxic moiety is selected from the list
of available toxic moieties comprising toxins such as a fragment of
Pseudomonas exotoxin-A, statins, chelated radioactive metal ions,
radioactive iodine, ricin A, gelonin, saporin, interleukin-2,
interleukin-12, radionuclides such as 90-Yttrium, 131-Iodine, drugs
such as doxorubicin, taxol or derivatives, 5-FU, anthracyclines,
vinca alkaloids, calicheamicins, cisplatin, carboplatin,
vinblastine, methotrexate, (synthetic) antineoplastic agents such
as monomethyl auristatin E, apoptin, parvovirus-H1 NS1 protein, E4
or f4, TRAIL, mda-7, HAMLET.
[0017] Proteinaceous molecules are molecules comprising at least a
string of amino acid residues. In addition, hereof the
proteinaceous molecules may comprise carbohydrates, disulphide
bonds, phosphorylations, sulphatations, etc.
[0018] When antibodies hereof are designed to first bind to a
target aberrant cell, followed by internalization, the toxic moiety
can then subsequently have its intracellular (cytotoxic) function,
i.e., inducing apoptosis.
[0019] For administration to subjects the antibodies hereof must be
formulated. Typically these antibodies will be given parenterally.
For formulation simply water (saline) for injection may suffice.
For stability reasons more complex formulations may be necessary.
The invention contemplates lyophilized compositions as well as
liquid compositions, provided with the usual additives. Thus, in
one embodiment, provided is a pharmaceutical composition comprising
an immunoglobulin provided with a toxic moiety according to any of
the aforementioned embodiments and suitable diluents and/or
excipients.
[0020] The dosage of the antibodies hereof are established through
animal studies, (cell-based) in vitro studies, and clinical studies
in so-called rising-dose experiments. Typically, the doses will be
comparable with present day antibody dosages (at the molar level).
Typically, such dosages are 3-15 mg/kg body weight, or 25-1000 mg
per dose.
[0021] In addition, especially in the more difficult to treat
cellular aberrancies the first applications of the antibodies
hereof will (at least initially) probably take place in combination
with other treatments (standard care). Of course, also provided are
antibodies for use in novel or first treatments of any malignancy
accompanied by the occurrence of aberrant cells, for which current
treatments are not efficient enough or for which currently no
treatment options are available. Thus, for example, also provided
is a pharmaceutical composition comprising an invented
immunoglobulin provided with a toxic moiety and a conventional
cytostatic and/or tumoricidal agent. Moreover, also provided is a
pharmaceutical composition comprising an invented immunoglobulin
provided with a toxic moiety for use in an adjuvant treatment of
cancer. Thus, in one embodiment hereof, an invented immunoglobulin
provided with a toxic moiety for use in an adjuvant treatment of
cancer is provided. Additionally, also provided is a pharmaceutical
composition comprising an invented immunoglobulin provided with a
toxic moiety for use in a combination chemotherapy treatment of
cancer. Examples of chemotherapeutical treatments that are combined
with the pharmaceutical composition of the current invention are
etoposide, paclitaxel, cisplatin, doxorubicin and methotrexate.
[0022] The pharmaceutical compositions hereof will typically find
their use in the treatment of cancer, particularly in forms of
cancer where the targets of the preferred antibodies hereof
(complexes of MHC and tumor-specific antigen peptides) are
presented by the tumors. Table 1, for example, gives a list of
tumors on which complexes of MHC and MAGE-A peptides have been
found. It is easy using an antibody hereof to identify tumors that
present these target MHC-peptide complexes. This can be done in
vitro or in vivo (imaging).
[0023] It is preferred that the cell-surface molecules comprising
the binding sites for the antibodies hereof are internalized into
the targeted aberrant cell, together with the antibodies hereof, or
together with at least the toxic moiety of the antibodies hereof.
In a particularly preferred embodiment hereof the targeted aberrant
cells go into apoptosis as a result of the internalization. Thus,
in one embodiment, provided is the use of an immunoglobulin
provided with a toxic moiety according to any of the aforementioned
embodiments, for the treatment of a host suffering from cancer,
wherein at least the toxic moiety is internalized into the aberrant
cell.
[0024] Also comprised herein is a nucleic acid molecule encoding
the immunoglobulin part of an antibody according to any of the
embodiments hereof, when the toxic moiety is chemically linked to
the immunoglobulin in the antibody hereof. Thus, also comprised
herein is a nucleic acid molecule encoding an immunoglobulin and a
toxic moiety according to any of the embodiments hereof, when the
toxic moiety is fused to the immunoglobulin at the DNA level. These
molecules hereof can be produced in prokaryotes or eukaryotes. The
codon usage of prokaryotes may be different from that in
eukaryotes. The nucleic acids hereof can be adapted in these
respects. Also, elements that are necessary for secretion may be
added, as well as promoters, terminators, enhancers, etc. Also,
elements that are necessary and/or beneficial for the isolation
and/or purification of the immunoglobulins hereof or of the
antibodies hereof may be added. Typically, the nucleic acids hereof
are provided in an expression vector suitable for the host in which
they are to be produced. Choice of a production platform will
depend on the size of the molecule, the expected issues around
protein folding, whether amino acid sequences are present in the
immunoglobulin or in the antibody that require glycosylation,
expected issues around isolation and/or purification, etc. For
example, the presence of disulfide bonds in immunoglobulins or
proteinaceous toxins hereof will typically guide the selection of
the preferred production platform. Thus, typically nucleic acids
hereof are adapted to the production and purification platform in
which the immunoglobulins optionally with their fused proteinaceous
toxins hereof are to be produced. Thus, provided is a vector
comprising a nucleic acid molecule encoding an immunoglobulin or an
antibody hereof For stable expression in a eukaryote it is
preferred that the nucleic acid encoding the immunoglobulin or the
antibody hereof is integrated in the host cell genome (at a
suitable site that is not silenced). In one embodiment, also
provided is a vector comprising means for integrating the nucleic
acid in the genome of a host cell. The disclosure further comprises
the host cell or the organism in which the nucleic acid molecule
encoding for the immunoglobulin hereof optionally with their fused
proteinaceous toxins, is present and which is thus capable of
producing the immunoglobulin optionally with their fused
proteinaceous toxins hereof. Thus, in a preferred embodiment, also
provided is a cell comprising a nucleic acid molecule hereof,
preferably integrated in its genome and/or a vector hereof,
comprising a nucleic acid molecule encoding an immunoglobulin
optionally with their fused proteinaceous toxins hereof.
[0025] Included herein invention is also a method for producing an
immunoglobulin optionally with their fused proteinaceous toxins
hereof, comprising culturing a cell hereof, comprising a nucleic
acid molecule encoding an immunoglobulin optionally with their
fused proteinaceous toxins hereof, preferably integrated in the
cell's genome and/or a vector hereof, comprising a nucleic acid
molecule encoding an immunoglobulin optionally with their fused
proteinaceous toxins hereof, allowing for expression of the
immunoglobulin optionally with their fused proteinaceous toxins and
separating the immunoglobulin optionally with their fused
proteinaceous toxins from the culture.
[0026] In one embodiment hereof, the immunoglobulin variable
domains in the molecules hereof target one binding site. Also
bi-specific immunoglobulins provided with a toxic moiety are
provided that are specifically binding to two different binding
sites associated with the cell surface of aberrant cells. By
targeting with a single antibody hereof two different binding sites
on an aberrant cell such as a tumor cell, the risk that both
targets are also jointly present on a healthy cell is significantly
further diminished. The affinity of the antibodies hereof for the
two different target binding sites separately, preferably is
designed such that K.sub.on and K.sub.off are very much skewed
towards binding to both different binding sites simultaneously.
Thus, the specificity of the bi-specific antibodies hereof is
increased by increasing their specificity for binding to two
different binding sites associated with aberrant cells. Thus, in
one embodiment hereof, the antibody according to any of the
previous embodiments is a hetero-dimeric bi-specific immunoglobulin
G or heavy-chain only antibody comprising two different but
complementary heavy chains. The two different but complementary
heavy chains may then be dimerized through their respective Fc
regions. Upon applying preferred pairing biochemistry,
hetero-dimers are preferentially formed over homo-dimers. For
example, two different but complementary heavy chains are subject
to forced pairing upon applying the "knobs-into-holes" CH3 domain
engineering technology as described (Ridgway et al., Protein
Engineering, 1996 (ref. 14)). In a preferred embodiment hereof the
two different immunoglobulin variable regions in the bi-specific
immunoglobulins hereof specifically bind to an MHC-peptide complex
preferentially associated with aberrant cells.
[0027] Typical preferred antibodies hereof are exemplified by the
antibodies outlined in this section, in FIG. 5B, and by the
examples provided below and in the Examples section. Thus the
invention provides an immunoglobulin provided with a toxic moiety
according to FIG. 5B.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1: Specific binding of HLA-A0201/multi-MAGE-A-specific
phage clones isolated from a large human non-immune antibody Fab
phage library. Individual antibody Fab expressing phages that were
selected against biotinylated HLA-A0201/multi-MAGE-A were analyzed
by ELISA for their capacity to bind the relevant peptide/MHC
complex only. Streptavidin coated 96-well plates were incubated
with soluble HLA-A0201/multi-MAGE-A (A2/multiMage) or HLA-A0201/JCV
(A2/JC) peptide/MHC complexes (10 .mu.g/ml, washed to remove
non-bound complexes and incubated with individual phage clones.
Non-binding phages were first removed by three washes with
PBS/TWEEN.RTM., followed by incubation with anti-M13 antibody (1
.mu.g/ml, Amersham) for one hour by room temperature. Finally the
wells were incubated with an HRP-labeled secondary antibody and
bound phages detected.
[0029] FIG. 2: Phages AH5, CB1 and CG1 specifically bind cells
presenting the multi-MAGE-Apeptide. Phages AH5, CB1, CG1, BD5 and
BC7 that had shown specific binding in ELISA using the relevant
HLA-A201/multi-MAGE-A complex and an irrelevant HLA-A201 complex
loaded with a JCV peptide were analyzed for their capacity to bind
cells presenting the multi-MAGE-A peptide in HLA-A0201 molecules.
To this end, human B-LCL (BSM) were loaded with multi-MAGE-A
peptide (10 .mu.g in 100 .mu.PBS) for 30 minutes at 37.degree. C.,
followed by incubation with the Fab phages AH5, CB1, CG1, BD5 and
BC7 and analyzed by flow-cytometry using anti-phage antibodies and
a fluorescently labeled secondary antibody.
[0030] FIG. 3: Phages expressing HLA-A2/multi-MAGE-A-specific Fab
bind tumor cells of distinct histologic origin. Phages AH5, CB1 and
CG1 specific for HLA-A0201/multi-MAGE-A and a positive control
phage specific for HA-0101/MAGE-A1 were used for staining of
distinct tumor cell lines. To this end the prostate cancer cell
line LNCaP, the multiple myeloma cell line MDN, the melanoma cell
lines MZ2-MEL43 and G43, and the breast cancer cell line MDA-MD157
were incubated with the different phages (30 minutes at 4.degree.
C.), bound phages were then detected by flow cytometry using
anti-phage antibodies and fluorescently labeled secondary
antibodies.
[0031] FIG. 4: Phage AH5 specifically binds HLA-A0201/multi-MAGE-A
complexes only. To determine specificity of the phage AH5 an ELISA
was performed using relevant and irrelevant peptide/MHC complexes.
HLA-A0201 with multi-MAGE-A, gp100, JCV and MAGE-C2 peptides, as
well as HLA-A1 with MAGE-A1 peptide were coated on streptavidin
96-well plates and incubated with phage AH5.
[0032] FIG. 5: Cartoon displaying examples of preferred
immunoglobulins provided with a toxic moiety, hereof.
[0033] Panel A: Cartoon displaying the topology of the twelve
immunoglobulin domains assembled in an immunoglobulin G.
[0034] Panel B: Examples are provided of preferred immunoglobulins
provided with a toxic moiety, hereof. Shown are immunoglobulins
provided with a single toxic moiety such as for example a
cytostatic agent, linked to the immunoglobulin with a chemical
linker (exemplified by I. and II.; immunoglobulin-toxic moiety
conjugates), or immunoglobulins provided with a single toxic
moiety, linked to the immunoglobulin with a peptide linker
(exemplified by III.; fused immunoglobulin-toxic moiety molecule).
In IV., an immunoglobulin provided with a toxic moiety, hereof, is
shown, comprising one immunoglobulin heavy chain comprising a fused
proteinaceous toxic moiety, comprising immunoglobulin variable
regions specific for a certain binding site, and comprising a
second immunoglobulin heavy chain comprising immunoglobulin
variable regions specific for a different binding site. Of course,
also part hereof are bi-specific immunoglobulins provided with a
toxic moiety, hereof, comprising two heavy chains comprising
different immunoglobulin variable regions specific for different
binding sites and further comprising the same or different
proteinaceous toxic moieties fused two the heavy chains. Of course,
as part hereof, more than one, and typically two to six toxic
moiety molecules can be fused or conjugated to an immunoglobulin
molecule.
[0035] FIG. 6: Human Fab phage F9 specifically binds HLA-A2/
FLWGPRALV (SEQ ID NO:23) positive CMT64 mouse lung tumor cells.
Human Fab clone F9 was analyzed for its capacity to bind mouse lung
tumor cells (CMT64) stably expressing the HLA-A2/FLWGPRALV (SEQ ID
NO:23) complex. Purified Clone F9 Fab fragments (3 .mu.g total)
were incubated with 0.5.times.10.sup.6 CMT64 cells that do not
express human HLA, that express HLA-A2/YLEYRQVPG (SEQ ID NO:3) or
that express HLA-A2/FLWGPRALV (SEQ ID NO:23). After one hour
incubation on ice CMT64 cells were incubated with a fluorescently
labeled secondary antibody and analyzed by flow cytometry.
[0036] FIG. 7: Llama VHH specifically binds CMT64 mouse lung tumor
cells expressing human HLA-A2/multi-MAGE-A. Llama VHH specific for
A2/FLW or A2/YLE were analyzed by flow cytometry for their binding
capacity to CMT64 cells expressing these human
HLA-A0201/multi-MAGE-A complexes. Purified VHH fragments (3 .mu.g
total) were incubated with 0.5.times.10.sup.6 CMT64 cells, which do
not express human HLA, that express HLA-A2/YLEYRQVPG (SEQ ID NO:3)
or that express HLA-A2/FLWGPRALV (SEQ ID NO:23). After one hour
incubation on ice CMT64 cells were incubated with a fluorescently
labeled secondary antibody and analyzed by flow cytometry.
DETAILED DESCRIPTION
[0037] One aspect hereof relates to a method for providing the
antibodies hereof. As described hereinabove, it typically involves
providing a nucleic acid construct encoding the desired
immunoglobulin part of antibodies hereof, or encoding the desired
immunoglobulin fused to a proteinaceous toxic moiety. The nucleic
acid construct can be introduced, preferably via a plasmid or
expression vector, into a prokaryotic host cell and/or in a plant
cell and/or in a eukaryotic host cell capable of expressing the
construct. In one embodiment, a method hereof to provide an
immunoglobulin or to provide an immunoglobulin fused to a
proteinaceous toxic moiety comprises the steps of providing a host
cell with the nucleic acid(s) encoding the immunoglobulin or the
immunoglobulin fused to a proteinaceous toxic moiety, and allowing
the expression of the nucleic acid(s) by the host cell.
[0038] It is part hereof that nucleic acids coding for selected
(human) immunoglobulin Vh(h) domains according to any of the above
embodiments are combined with nucleic acids coding for human
immunoglobulin heavy chain constant domains, providing nucleic acid
molecules hereof encoding for a heavy chain of a human antibody.
The human antibody heavy chain protein product of such a nucleic
acid molecule hereof, then may be hetero-dimerized with a universal
human antibody light chain. It is also part hereof that nucleic
acids coding for (jointly) selected human immunoglobulin Vl domains
and Vh domains according to any of the above embodiments are
combined with nucleic acids coding for a human immunoglobulin light
chain constant domain and are combined with nucleic acids coding
for human immunoglobulin heavy chain constant domains,
respectively, providing nucleic acid molecules hereof encoding for
a light chain and for a heavy chain of a human antibody. In yet
another embodiment hereof, the nucleic acids coding for the
complementarity determining regions 1, 2 and 3 (CDR1, CDR2, CDR3),
forming together the immunoglobulin variable region of a selected
immunoglobulin Vh domain and/or a selected immunoglobulin Vl domain
according to any of the above embodiments are combined with nucleic
acids coding for human immunoglobulin Vh domain frame work regions
and/or human immunoglobulin Vl domain frame work regions,
respectively, providing nucleic acid molecules hereof encoding for
a heavy chain variable domain (Vh) of a human antibody and/or
encoding for a light chain variable domain (Vl) of a human antibody
(A method known in the art as "grafting"). These nucleic acid
molecules encoding for variable domains Vh and/or Vl are, as part
hereof, then combined with nucleic acids coding for human
immunoglobulin constant domains, providing a nucleic acid molecule
encoding for a human antibody heavy chain and/or providing a
nucleic acid molecule encoding for a human antibody light
chain.
[0039] Hereof, immunoglobulins or immunoglobulins fused to a
proteinaceous toxic moiety are for example expressed in plant
cells, eukaryotic cells or in prokaryotic cells. Non-limited
examples of suitable expression systems are tobacco plants, Pichia
pastoris, Saccharomyces cerevisiae. Also cell-free recombinant
protein production platforms are suitable. Preferred host cells are
bacteria, like for example bacterial strain BL21 or strain SE1, or
mammalian host cells, more preferably human host cells. Suitable
mammalian host cells include human embryonic kidney (HEK-293)
cells, PERC6.RTM. cells or preferably Chinese hamster ovary (CHO)
cells, which can be commercially obtained. Insect cells, such as S2
or S9 cells, may also be used using baculovirus or insect cell
expression vectors, although they are less suitable when the
immunoglobulins or the fused immunoglobulins-toxic moiety molecules
hereof include elements that involve glycosylation. The produced
immunoglobulins or fused immunoglobulin-toxic moiety molecules
hereof can be extracted or isolated from the host cell or, if they
are secreted, from the culture medium of the host cell. Thus, in
one embodiment a method hereof comprises providing a host cell with
one or more nucleic acid(s) encoding the immunoglobulin or the
fused immunoglobulin-toxic moiety molecule, allowing the expression
of the nucleic acids by the host cell. In another preferred
embodiment a method hereof comprises providing a host cell with one
or more nucleic acid(s) encoding two or more different
immunoglobulins or two or more different fused immunoglobulin-toxic
moiety molecules, allowing the expression of the nucleic acids by
the host cell. For example, in one embodiment, nucleic acids
encoding for a so-called universal immunoglobulin light chain and
nucleic acids encoding for two or more different immunoglobulin
heavy chains are provided, enabling isolation of mono-specific
immunoglobulins or mono-specific fused immunoglobulin-toxic moiety
molecules comprising homo-dimers of heavy chains and/or enabling
isolation of bi-specific immunoglobulins or bi-specific fused
immunoglobulin-toxic moiety molecules comprising hetero-dimers of
heavy chains, with all different heavy chains complexed with a
universal light chain. Methods for the recombinant expression of
(mammalian) proteins in a (mammalian) host cell are well known in
the art.
[0040] As said, it is preferred that the immunoglobulins hereof are
linked with the toxic moieties via bonds and/or binding
interactions other than peptide bonds. Methods for linking
proteinaceous molecules such as immunoglobulins to other
proteinaceous molecules or non-proteinaceous molecules are numerous
and well known to those skilled in the art of protein linkage
chemistry. Protein linkage chemistry not based on peptide bonds can
be based on covalent interactions and/or on non-covalent
interactions. A typical example of linkage chemistries applicable
for linking toxic moieties to immunoglobulins hereof are the
various applications of the Universal Linkage System disclosed in
patent applications WO92/01699, WO96/35696, WO98/45304, WO03040722,
the contents of each of which are incorporated herein by this
reference.
[0041] As will be clear, an antibody hereof finds its use in many
therapeutic applications and non-therapeutic applications, e.g.,
diagnostics, or scientific applications. Antibodies hereof, or more
preferably the immunoglobulin part of the antibodies hereof,
suitable for diagnostic purposes are of particular use for
monitoring the expression levels of molecules exposing binding
sites on aberrant cells that are targeted by antibodies hereof. In
this way, it is monitored whether the therapy remains efficacious
or whether other antibodies hereof targeting one or two different
binding sites on the aberrant cells should be applied instead. This
is beneficial when the expression levels of the first or the first
two targeted binding site(s) are below a certain threshold, whereas
another or new binding sites (still) can serve as newly targeted
binding sites for antibodies hereof comprising the appropriate
specific immunoglobulin variable regions for these alternative
binding site(s). Antibodies hereof may also be used for the
detection of (circulating) tumor cells, and for the target-cell
specific delivery of immune-stimulatory molecules. For these later
two uses, the sole immunoglobulins hereof without the fused or
conjugated toxic moiety may also be used.
[0042] Provided herein is a method for inducing ex vivo or in vivo
a modulating effect on a biological process in a target cell,
comprising contacting the cell with an antibody hereof in an amount
that is effective to induce the modulating effect. Preferably, the
antibody hereof is used for a modulating effect on a biological
process of aberrant cells in a subject, more preferably a human
subject. For therapeutic applications in humans it is of course
preferred that an antibody hereof does not contain amino acid
sequences of non-human origin. More preferred are antibodies
hereof, which only contain human amino acid sequences. Therefore, a
therapeutically effective amount of an antibody hereof capable of
recognizing and binding to one or two disease-specific binding
sites and subsequently inducing a modulating effect on a biological
process in the cell, can be administered to a patient to stimulate
eradication of aberrant cells expressing the binding site(s)
without affecting the viability of (normal) cells not expressing
the disease-specific binding site(s). The specific killing of
aberrant cells while minimizing or even avoiding the deterioration
or even death of healthy cells will generally improve the
therapeutic outcome of a patient after administration of the
antibodies hereof.
[0043] Accordingly, also provided is the use of an antibody hereof
as medicament. In another aspect, provided is the use of an
antibody hereof for the manufacture of a medicament for the
treatment of cancer, autoimmune disease, infection or any other
disease of which the symptoms are reduced upon targeting aberrant
cells expressing disease-specific binding sites with antibodies
hereof. For example, an antibody hereof is advantageously used for
the manufacture of a medicament for the treatment of various
cancers (e.g., solid tumors, hematologic malignancies).
[0044] An example of a preferred antibody hereof is an antibody
comprising at least an immunoglobulin variable region specifically
binding to the complex between MHC-1 HLA-0201 and a multi-MAGE-A
epitope, conjugated with a toxic moiety, using for example
Universal Linkage System linker chemistry for conjugation. A second
example of a preferred antibody hereof is an antibody comprising at
least an immunoglobulin variable region specifically binding to the
complex between MHC-1 HLA-CW7 and a multi-MAGE-A epitope,
conjugated with a toxic moiety, using for example Universal Linkage
System linker chemistry for conjugation. With the bi-specific
antibodies hereof, difficult to target and/or difficult to reach
aberrant cells have a higher chance of being "hit" by at least one
of the two different immunoglobulin variable regions in the
bi-specific antibodies hereof, thereby providing at least in part
the therapeutic activity. An example of a preferred bi-specific
antibody hereof is an immunoglobulin comprising an immunoglobulin
variable region specific for the complex between MHC-1 HLA-0201 and
a multi-MAGE-A epitope and comprising a second immunoglobulin
variable region specific for the complex between MHC-1 HLA-CW7 and
a second multi-MAGE-A epitope, conjugated with a toxic moiety.
[0045] Antibody fragments of human origin can be isolated from
large antibody repertoires displayed by phages. One aspect hereof,
known by the art, is the use of human antibody phage display
libraries for the selection of human antibody fragments specific
for a selected binding site, e.g., an epitope. Examples of such
libraries are phage libraries comprising human Vh repertoires,
human Vh-Vl repertoires, human Vh-Chl or human antibody Fab
fragment repertoires.
[0046] Although the disclosure contemplates many different
combinations of MHC and antigenic peptides the most preferred is
the combination of MHC-1 and an antigenic peptide from a tumor
related antigen presented by the MHC-1, exclusively expressed by
aberrant cells and not by healthy cells. Because of HLA
restrictions, there are many combinations of MHC-1-peptide
complexes as well as of MHC-2-peptide complexes that can be
designed based on the rules for presentation of peptides in MHC.
These rules include size limits on peptides that can be presented
in the context of MHC, restriction sites that need to be present
for processing of the antigen in the cell, anchor sites that need
to be present on the peptide to be presented, etc. The exact rules
differ for the different HLA classes and for the different MHC
classes. We have found that MAGE-derived peptides are very suitable
for presentation in an MHC context. An MHC-1 presentable antigenic
peptide with the sequence Y-L-E-Y-R-Q-V-P-G in MAGE-A (SEQ ID NO:3)
was identified, that is present in almost every MAGE-A variant
(multi MAGE peptide) and that will be presented by one of the most
prevalent MHC-1 alleles in the Caucasian population (namely
HLA-A0201). A second MAGE peptide that is presented by another
MHC-1 allele (namely HLA-CW7) and that is present in many MAGE
variants, like, for example, MAGE-A2, -A3, -A6 and -Al12, is
E-G-D-C-A-P-E-E-K (SEQ ID NO:4). These two combinations of MHC-1
and MAGE peptides together would cover 80% of the Caucasian
population. The same approach can be followed for other MHC
molecules, other HLA restrictions and other antigenic peptides
derived from tumor-associated antigens. Relevant is that the chosen
antigenic peptide to elicit the response to must be presented in
the context of an MHC molecule and recognized in that context only.
Furthermore, the antigenic peptide must be derived from a
sufficiently tumor-specific antigen and the HLA restriction must
occur in a relevant part of the population. One of the important
advantages of the invention is that tumors that down regulate their
targeted MHC-peptide complex, can be treated with a second
immunoglobulin comprising at least one variable region binding to a
different MHC-peptide complex based on the same antigen. If this
one is down regulated a third one will be available. For
heterozygotes six different targets on MHC-1 may be available.
Since cells need to be "inspected" by the immune system from time
to time, escape through down regulation of all MHC molecules does
not seem a viable escape route. In the case that MAGE is the
antigen from which the peptide is derived escape through down
regulation of the antigen is also not possible, because MAGE seems
important for survival of the tumor..sup.[8]Thus the invention, in
an important aspect reduces or even prevents escape of the tumor
from the therapy. Thus, provided is in a preferred embodiment an
antibody hereof whereby the immunoglobulin variable region is
capable of binding to an MHC-I--peptide complex. In a further
preferred embodiment the invention provides an immunoglobulin
whereby the immunoglobulin variable region is capable of binding to
MHC-I--peptide complexes comprising an antigenic peptide derived
from a tumor related antigen, in particular MHC-I--peptide
complexes comprising an antigenic peptide present in a variety of
MAGE antigens, whereby the immunoglobulin is provided with a toxic
moiety.
[0047] Because in one embodiment the invention uses MHC molecules
as a target, and individuals differ in the availability of MHC
targets, the invention also provides a so-called companion
diagnostic to determine the HLA composition of an individual.
Although the invention preferably uses a more or less universal
(MAGE) peptide, the invention also provides a diagnostic for
determining the expression of the particular antigen by the tumor.
In this manner the therapy can be geared to the patient
(personalized medicine, patient stratification), particularly also
in the set-up to prevent escape as described herein before. It is
known that the HLA restriction patterns of the Asian population and
the black population are different from the Caucasian population.
For different populations different MHC-peptide complexes can be
targeted.
[0048] Although the present specification presents more specific
disclosure on tumors, it must be understood that other aberrant
cells can also be targeted by the antibodies of the invention.
These other aberrant cells are typically cells that also
proliferate without sufficient control. This occurs in autoimmune
diseases. It is typical that these cells start to show expression
of tumor antigens. In particular MAGE polypeptides have been
identified in rheumatoid arthritis..sup.[7]
[0049] In literature, it is shown that a single nine amino-acid
(A.A.) peptide present in MAGE-A2, -A3, -A4, -A6, -A10, and -Al2 is
presented by HLA-A0201 on tumor cells, and can be recognized by
cytotoxic T-lymphocytes..sup.[1 ] This nine amino acid residues
peptide with sequence Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:3) is almost
identical to the HLA-A0201 presented MAGE-A1 peptide
Y-L-E-Y-R-Q-V-P-D (SEQ ID NO:5), except for the anchor residue at
position 9. Replacement of the anchor residue with Valine results
in a nine-amino-acid-residue peptide with enhanced binding capacity
to HLA-A0201 molecules..sup.[1 ] Human and mouse T-lymphocytes
recognizing the Y-L-E-Y-R-Q-V-P-V (SEQ ID NO:6) peptide presented
by HLA-0201 also recognize the original MAGE-A Y-L-E-Y-R-Q-V-P-G
(SEQ ID NO:3) and Y-L-E-Y-R-Q-V-P-D (SEQ ID NO:5) peptides
presented on tumors of distinct origin. As diverse tumors may each
express at least one MAGE-A gene, targeting of this so-called
multi-MAGE-A epitope includes the vast majority of tumors. As an
example, MAGE-A expression in human prostate tumor cell lines and
in human xenographs was analyzed and shown to be highly diverse,
but in each individual sample tested at least one MAGE-A gene was
expressed (Table 2), confirming that targeting this multi-MAGE-A
epitope serves as a universal HLA-A0201 restricted target for
therapy.
[0050] Of course several other multi-MAGE or multi-target epitopes
may be designed. In principle the invention contemplates
combinations of tumor-specific antigen-derived MHC presented
epitopes in different HLA restrictions of both MHC-I and MHC-II,
targeted by immunoglobulins linked to a toxic moiety, to induce
apoptosis in aberrant cells. Examples of MHC-MAGE peptide
combinations that can be targeted by antibodies hereof are peptide
IMPKAGLLI (MAGE-A3) (SEQ ID NO:8) and HLA-DP4 or peptide
243-KKLLTQHFVQENYLEY-258 (MAGE-A3) (SEQ ID NO:9) and HLA-DQ6. Other
non-limiting examples of tumor-specific complexes of HLA and
antigen peptide are: HLA A1--MAGE-A1 peptide EADPTGHSY (SEQ ID
NO:10), HLA A3--MAGE-A1 SLFRAVITK (SEQ ID NO:11), HLA A24--MAGE-A1
NYKHCFPEI (SEQ ID NO:12), HLA A28--MAGE-A1 EVYDGREHSA (SEQ ID
NO:13), HLA B37--MAGE-A1/A2/A3/A6 REPVTKAEML (SEQ ID NO:14),
expressed at aberrant cells related to melanoma, breast carcinoma,
SCLC, sarcoma, NSCLC, colon carcinoma (N. Renkvist et al., Cancer
Immunol. Immunother. (2001) V50:3-15 (ref. 13)). Further examples
are HLA B53--MAGE-A1 DPARYEFLW (SEQ ID NO:15), HLA Cw2--MAGE-A1
SAFPTTINF (SEQ ID NO:16), HLA Cw3-MAGE-A1SAYGEPRKL (SEQ ID NO:17),
HLA Cw16--MAGE-A1 SAYGEPRKL (SEQ ID NO:18), HLA A2--MAGE
A2KMVELVHFL (SEQ ID NO:19), HLA A2--MAGE-A2 YLQLVFGIEV (SEQ ID
NO:20), HLA A24--MAGE-A2 EYLQLVFGI (SEQ ID NO:21), HLA-A1--MAGE-A3
EADPIGHLY (SEQ ID NO:22), HLA A2--MAGE-A3 FLWGPRALV (SEQ ID NO:23),
HLA B44--MAGE-A3 MEVDPIGHLY (SEQ ID NO:24), HLA B52--MAGE-A3
WQYFFPVIF (SEQ ID NO:25), HLA A2--MAGE-A4 GVYDGREHTV (SEQ ID
NO:26), HLA A34--MAGE-A6 MVKISGGPR (SEQ ID NO:27), HLA A2--MAGE-A10
GLYDGMEHL (SEQ ID NO:28), HLA Cw7--MAGE-A12 VRIGHLYIL (SEQ ID
NO:29), HLA Cw16--BAGE AARAVFLAL (SEQ ID NO:30), expressed by for
example melanoma, bladder carcinoma, NSCLC, sarcoma, HLA
A2--DAM-6/-10 FLWGPRAYA (SEQ ID NO:31), expressed by for example
skin tumors, lung carcinoma, ovarian carcinoma, mammary carcinoma,
HLA Cw6--GAGE-1/-2/-8 YRPRPRRY (SEQ ID NO:32), HLA
A29--GAGE-3/-4/-5/-6/-7B YYWPRPRRY (SEQ ID NO:33), both expressed
by for example melanoma, leukemia cells, bladder carcinoma, HLA B13
NA88-A MTQGQHFLQKV (SEQ ID NO:34), expressed by melanoma, HLA
A2--NY-ESO-1 SLLMWITQCFL (SEQ ID NO:35), HLA A2--NY-ESO-la
SLLMWITQC (SEQ ID NO:36), HLA A2--NY-ESO-1a QLSLLMWIT (SEQ ID
NO:37), HLA A31NY-ESO-1a ASGPGGGAPR (SEQ ID NO:38), the latter four
expressed by for example melanoma, sarcoma, B-lymphomas, prostate
carcinoma, ovarian carcinoma, bladder carcinoma.
[0051] The disclosure is further described by the following
non-limiting Examples.
Abbreviations used
[0052] A.A., amino acid; Ab, antibody; .beta.2-M, CDR,
complementarity determining region; CHO, Chinese hamster ovary; CT,
cancer testis antigens; CTL, cytotoxic T-lymphocyte; E4orf4,
adenovirus early region 4 open reading frame; EBV, Epstein-Barr
virus; ELISA, enzyme linked immunosorbent assay; HAMLET, human
.alpha.lactalbumin made lethal to tumor cells; HEK, human embryonic
kidney; HLA, human leukocyte antigen; Ig, immunoglobulin; i.v.,
intravenously; kDa, kilo Dalton; MAGE, melanoma-associated antigen;
Mda-7, melanoma differentiation-associated gene-7; MHC, major
histocompatibility complex; MHC-p, MHC-peptide; NS1,
parvovirus-H1-derived non-structural protein 1; PBSM, PBS
containing 2% non-fat dry milk; TCR, T-cell receptor; VH, Vh or
V.sub.H, amino-acid sequence of an immunoglobulin variable heavy
domain; Vl, amino-acid sequence of an immunoglobulin variable light
domain; TRAIL, tumor necrosis factor-related apoptosis-inducing
ligand.
EXAMPLES
Example 1
[0053] Non-exhaustive examples of immunoglobulins hereof comprising
at least an immunoglobulin variable region that specifically binds
to an MHC-peptide complex preferentially associated with aberrant
cells or to an aberrant cell surface marker preferentially
associated with aberrant cells, with domain topologies as outlined
for example in FIG. 5B, are:
[0054] Antibodies hereof comprising immunoglobulin variable regions
that specifically bind to: [0055] (a) a complex comprising a T-cell
epitope selected from 146-KLQCVDLHV-154 (SEQ ID NO:74),
141-FLTPKKLQCV-150 (SEQ ID NO:75), 154-VISNDVCAQV-163 (SEQ ID
NO:76), 154-YISNDVCAQV-163 (SEQ ID NO:77) of PSA, presented by
HLA-A2 and/or 162-QVHPQKVTK-170 (SEQ ID NO:78) of PSA, presented by
HLA-A3, and/or 152-CYASGWGSI-160 (SEQ ID NO:79), 248-HYRKWIKDTI-257
(SEQ ID NO:80) of PSA, presented by HLA-A24, and/or 4-LLHETDSAV-12
(SEQ ID NO:81), 711-ALFDIESKV-719 (SEQ ID NO:82), 27-VLAGGFFLL-35
(SEQ ID NO:83) of PSMA, presented by HLA-A2, and/or
178-NYARTEDFF-186 (SEQ ID NO:84), 227-LYSDPADYF-235 (SEQ ID NO:85),
624-TYSVSFDSL-632 (SEQ ID NO:86) of PSMA, presented by HLA-A24,
and/or 299-ALDVYNGLL-307 (SEQ ID NO:87) of PAP, presented by HLA-A2
and/or 213-LYCESVHNF-221 (SEQ ID NO:88) of PAP, presented by
HLA-A24 and/or 199-GQDLFGIWSKVYDPL-213 (SEQ ID NO:89),
228-TEDTMTKLRELSELS-242 (SEQ ID NO:90) of PAP, presented by MHC-2
and/or 14-ALQPGTALL-22 (SEQ ID NO:91), 105-AILALLPAL-113 (SEQ ID
NO:92), 7-ALLMAGLAL-15 (SEQ ID NO:93), 21-LLCYSCKAQV-30 (SEQ ID
NO:94) of PSCA, presented by HLA-A2 and/or 155-LLANGRMPTVLQCVN-169
(SEQ ID NO:95) of Kallikrein 4, presented by DRB1*0404 and/or
160-RMPTVLQCVNVSVVS-174 (SEQ ID NO:96) of Kallikrein 4, presented
by DRB1*0701 and/or 125-SVSESDTIRSISIAS-139 (SEQ ID NO:97) of
Kallikrein 4, presented by DPB1*0401, for the treatment of prostate
cancer;
[0056] (b) the HLA B8 restricted epitope from EBV nuclear antigen
3, FLRGRAYGL (SEQ ID NO:98), complexed with MHC I, for the
clearance of EBV infected cells;
[0057] (c) the MAGE-A peptide YLEYRQVPG (SEQ ID NO:3) presented by
MHC 1 HLA-A0201, for treatment of cancers accompanied by tumor
cells expressing these MHC-peptide complexes (see Table 1);
[0058] (d) the MAGE-A peptide EGDCAPEEK (SEQ ID NO:4) presented by
MHC-1 HLA-CW7, for treatment of cancers accompanied by tumor cells
expressing these MHC-peptide complexes (see Table 1);
[0059] (e) complexes of HLA-A2 and HLA-A2 restricted
CD8.sup.+T-cell epitopes, e.g., nonamer peptides FLFLLFFWL (SEQ ID
NO:99) (from prostatic acid phosphatase (PAP, also
prostatic-specific acid phosphatase (PSAP))), TLMSAMTNL (SEQ ID
NO:100) (from PAP), ALDVYNGLL (SEQ ID NO:101) (from PAP), human
HLA-A2.1-restricted CTL epitope ILLWQPIPV (SEQ ID NO:102) (from
PAP-3), six-transmembrane epithelial antigen of prostate (STEAP),
or complexes of HLA-A2.1 and HLA-A2.1-restricted CTL epitope
LLLGTIHAL (SEQ ID NO:103) (from STEAP-3), epitopes from mucin
(MUC-1 and MUC-2), MUC-1-32mer (CHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPA
(SEQ ID NO:104)), epitopes from Globo H, Lewis.sup.y, Tn(c), TF(c)
clusters, GM2, prostate-specific membrane antigen (PSMA),
kallikrein 4, prostein, or complexes of HLA-A2.1 and
HLA-A2.1-restricted epitopes from BA46, PTH-rP, HER-2/neu, hTERT,
and MAGE-A8, for the treatment of prostate cancer;
[0060] (f) an aberrant cell-specific epitope in aberrant
cell-specific altered MUC-1 complexed with MHC, or to an aberrant
cell-specific epitope in aberrant cell-specific altered MUC-1 for,
the targeting of aberrant cells in for example breast cancer or for
the treatment of colorectal cancer;
[0061] (g) an aberrant cell-specific epitope of the aberrant
cell-specific epidermal growth factor receptor mutant form vIII
complexed with MHC, or to an aberrant cell-specific epitope of the
epidermal growth factor receptor mutant form vIII, for the
treatment of the brain neoplasm glioblastoma multiforme;
[0062] (h) the complex of MHC with T-cell epitope peptide 369-376
from human Her-2/neu, for the treatment of malignancies related to
Her-2 and/or Her-1 over-expression;
[0063] (i) an epitope of the aberrant cell-specific surface marker
CD44 splice variants known as CD44-v6, CD44-v9, CD44-v10, complexed
with MHC, or to an aberrant cell-specific epitope of an aberrant
cell-specific CD44 splice variant, for the treatment of multiple
myeloma;
[0064] Target binding sites suitable for specific and selective
targeting of infected aberrant cells by antibodies hereof are
pathogen-derived antigen peptides complexed with MHC molecules.
Examples of T-cell epitopes of the E6 and E7 protein of human
papilloma virus, complexed with indicated HLA molecules, are
provided below. Any combination of an HLA molecule complexed with a
pathogen-derived T-cell epitope provides a specific target on
infected aberrant cells for antibodies hereof. An example of an
infected aberrant cell is a keratinocyte in the cervix infected by
human papilloma virus (HPV), presenting T-cell epitopes derived
from for example E6 or E7 protein, in the context of MHC. Examples
of suitable target HPV 16 E6 T-cell epitopes are peptides FQDPQERPR
(SEQ ID NO:39), TTLEQQYNK (SEQ ID NO:40), ISEYRHYCYS (SEQ ID NO:41)
and GTTLEQQYNK (SEQ ID NO:42) binding to HLA A1, KISEYRHYC (SEQ ID
NO:43) and YCYSIYGTTL (SEQ ID NO:44) binding to HLA A2, LLRREVYDF
(SEQ ID NO:45) and IVYRDGNPY (SEQ ID NO:46) binding to HLA A3,
TTLEQQYNK (SEQ ID NO:47) binding to HLA A11, CYSLYGTTL (SEQ ID
NO:48), KLPQLCTEL (SEQ ID NO:49), HYCYSLYGT (SEQ ID NO:50),
LYGTTLEQQY (SEQ ID
[0065] NO:51), EVYDFAFRDL (SEQ ID NO:52) and VYDFAFRDLC (SEQ ID
NO:53) binding to HLA A24, 29-TIHDIILECV-38 (SEQ ID NO:54) binding
to HLA A*0201. Equally suitable are HPV 16 E7 T-cell epitopes such
as 86-TLGIVCPI-93 (SEQ ID NO:55), 82-LLMGTLGIV-90 (SEQ ID NO:56),
85-GTLGIVCPI-93 (SEQ ID NO:57) and 86-TLGIVCPIC-94 (SEQ ID NO:58)
binding to HLA A*0201, HPV 18 E6 T-cell epitopes and HPV 18 E7
T-cell epitopes, binding to HLA A1, A2, A3, A11 or A24. Yet
additional examples of T-cell epitopes related to HPV infected
cells are HPV E7-derived peptides 1-MHGDTPTLHEYD-12 (SEQ ID NO:59),
48-DRAHYNIVTFCCKCD-62 (SEQ ID NO:60) and 62-DSTLRLCVQSTHVD-75 (SEQ
ID NO:61) binding to HLA DR, 7-TLHEYMLDL-15 (SEQ ID NO:62),
11-YMLDLQPETT-20 (SEQ ID NO:63), 11-YMLDLQPET-19 (SEQ ID NO:64) and
12-MLDLQPETT-20 (SEQ ID NO:65) binding to HLA A*201,
16-QPETTDLYCY-25 (SEQ ID NO:66), 44-QAEPDRAHY-52 (SEQ ID NO:67) and
46-EPDRAHYNIV-55 (SEQ ID NO:68) binding to HLA B18,
35-EDEIDGPAGQAEPDRA-50 (SEQ ID NO:69) binding to HLA DQ2,
43-GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR-77 (SEQ ID NO:70) binding to
HLA DR3, 50-AHYNIVTFCCKCD-62 (SEQ ID NO:71) binding to HLA DR15,
58-CCKCDSTLRLC-68 (SEQ ID NO:72) binding to HLA DR17 and
61-CDSTLRLCVQSTHVDIRTLE-80 (SEQ ID NO:73) binding to
HLA-DRB1*0901.
[0066] A good source for selecting binding sites suitable for
specific and selective targeting of aberrant cells by antibodies
hereof, is the Peptide Database listing T-cell defined tumor
antigens and the HLAs binding the T-cell epitopes.sup.[9-12](on the
WorldWideWeb at
cancerimmunity.org/peptidedatabase/Tcellepitopes.htm). The database
provides combinations of antigen peptides complexed with MHC
molecules comprising the indicated class of HLA, unique to tumor
cells or over-expressed by tumor cells.
Example 2
Selection of Human Antibody Fragments Specific for
HLA-A0201/Multi-MAGE-A.
[0067] To obtain human antibody fragments comprising immunoglobulin
variable regions specific for the HLA-A0201 presented multi-MAGE-A
epitope Y-L-E-Y-R-Q-V-P-V (SEQ ID NO:6) and FLWGPRALV (SEQ ID
NO:23) a Human Fab phage display library was constructed according
to the procedure previously described by de Haard et al..sup.[2]
and used for selections 1) essentially as described by Chames et
al. using biotinylated MHC/p complexes,.sup.[2] or 2) on cells
expressing the relevant antigen.
[0068] 2.1: Selection of human antibody fragments specific for
HLA-A0201/YLEYRQVPV (SEQ ID NO:6) using biotinylated MHC-peptide
complexes:
[0069] Human Fab phages (10.sup.13 colony forming units) were first
pre-incubated for one hour at room temperature in PBS containing 2%
non-fat dry milk (PBSM). In parallel, 200 .mu.l Streptavidin-coated
beads (Dynal.TM.) were equilibrated for one hour in PBSM. For
subsequent rounds, 100 .mu.l beads were used. To deplete for
pan-MHC binders, each selection round, 200 nM of biotinylated MHC
class I-peptide (MHC-p) complexes containing an irrelevant peptide
(Sanquin, the Netherlands) were added to the phages and incubated
for 30 minutes under rotation. Equilibrated beads were added, and
the mixture was incubated for 15 minutes under rotation. Beads were
drawn to the side of the tube using magnetic force. To the depleted
phage fraction, subsequently decreasing amounts of biotinylated
MHC-p complexes (200 nM for the first round, and 20 nM for the
second and third round) were added and incubated for one hour at
room temperature, with continuous rotation. Simultaneously, a
pan-MHC class I binding soluble Fab (D3) was added to the
phage-MHC-p complex mixture (50, 10, and 5 .mu.g for rounds 1-3,
respectively). Equilibrated streptavidin-coated beads were added,
and the mixture was incubated for 15 minutes under rotation. Phages
were selected by magnetic force. Non-bound phages were removed by
five washing steps with PBSM, five steps with PBS containing 0.1%
TWEEN.RTM., and five steps with PBS. Phages were eluted from the
beads by ten minutes incubation with 500 .mu.l freshly prepared
tri-ethylamine (100 mM). The pH of the solution was neutralized by
the addition of 500 .mu.l 1 M Tris (pH 7.5). The eluted phages were
incubated with logarithmic growing E. Coli TG1 cells (OD.sub.600nm
of 0.5) for 30 minutes at 37.degree. C. Bacteria were grown
overnight on 2.times.TYAG plates. Next day, colonies were
harvested, and a 10 0 inoculum was used in 50 ml 2x TYAG. Cells
were grown until an .sub.OD600nm of 0.5, and 5 ml of this
suspension was infected with Ml3k07 helper phage (5 x 10.sup.11
colony forming units). After 30 minutes incubation at 37.degree.
C., the cells were centrifuged, resuspended in 25 ml 2x TYAK, and
grown overnight at 30.degree. C. Phages were collected from the
culture supernatant as described previously, and were used for the
next round panning. After three selection rounds a 261-fold
enrichment was obtained, and 46 out of 282 analyzed clones were
shown to be specific for the HLA-A2-multi-MAGE-A complex (FIG. 1).
ELISA using the HLA-A0201/multi-MAGE-A complexes as well as
HLA-A0201 complexes with a peptide derived from JC virus was used
to determine the specificity of the selected Fab.
[0070] 2.2: Selection of human Fab specific for HLA-A0201/FLWGPRALV
(SEQ ID NO:23) using cells.
[0071] Selections of Fab-phages specifically binding to
HLA-A0201/FLWGPRALV (SEQ ID NO:23) were performed using mouse CMT64
lung tumor cells. To obtain CMT64 cells stably expressing
HLA-A0201/FLWGPRALV (SEQ ID NO:23) (A2/FLW) complexes, the CMT64
cells were retroviral infected with a vector encoding a single
chain peptide-.beta.2M-HLA-A0201 heavy chain construct (SEQ ID
NO:2). Human Fab phages (10.sup.13 colony forming units) were first
pre-incubated for one hour at room temperature in PBS containing 2%
FCS (PBSF). In parallel, 1.0.times.10.sup.6 CMT64-A2/FLW cells were
equilibrated for one hour in PBSF. The phages were first incubated
for one hour with 10.times.10.sup.6 CMT 64 cells expressing
HLA-A0210/YLEYRQVPG (SEQ ID NO:3) to deplete non-specifically
binding phages. The non-bound fraction was then incubated (one hour
at 4.degree. C.) with HLA-A0201/FLWGPRALV (SEQ ID NO:23) expressing
CMT64 cells. After extensive washing, bound phages were eluted by
adding 500 .mu.l freshly prepared tri-ethylamine (100 mM). The pH
of the solution was neutralized by the addition of 500 .mu.l 1 M
Tris (pH 7.5). The eluted phages were incubated with logarithmic
growing E. Coli TG1 cells (OD.sub.600nm of 0.5) for 30 minutes at
37.degree. C. Bacteria were grown overnight on 2.times. TYAG
plates. Next day, colonies were harvested. After four rounds of
selection, individual clones were selected and tested for
specificity of binding.
[0072] 2.3: Human Fab specific for HLA-A0201/multi-MAGE-A epitopes
bind antigen-positive cells.
TABLE-US-00001 Multi-MAGE-A; Y-L-E-Y-R-Q-V-P-V
[0073] Fab phages were analyzed for their capacity to bind
HLA-A0201-positive EBV-transformed B-LCL loaded with the
multi-MAGE-A peptide Y-L-E-Y-R-Q-V-P-V (SEQ ID NO:6). The B-LCL
line BSM (0.5.times.10.sup.6) was loaded with multi-MAGE-A peptide
(10 .mu.g in 100.mu.l PBS) for 30 minutes at 37.degree. C.,
followed by incubation with the Fab phages AH5, CB1, CG1, BD5 and
BC7 and analyzed by flow-cytometry. As shown in FIG. 2, Fab AH5,
CB1 and CG1, specifically bound to the peptide loaded cells only,
whereas Fab BD5 and BC7 displayed non-specific binding to BSM that
was not loaded with the multi-MAGE-A peptide. No binding was
observed by AH5, CB1 and CG1 to non-peptide loaded cells.
[0074] Phages presenting AH5, CB1 and CG1, as well as the
HLA-A0101/MAGE-A1-specific Fab phage G8 (4) were then used to stain
tumor cell lines of distinct histologic origin. To this end
prostate cancer cells (LNCaP), multiple myeloma cells (MDN),
melanoma cells (MZ2-MEL43 and G43), and breast cancer cells
(MDA-MB157) were stained and analyzed by flow cytometry (FIG. 3).
The Fab AH5 specifically bound multiple myeloma cells MDN, and not
the HLA-A0201-negative melanoma and breast cancer cells. Both CB1
and CG1 displayed non-specific binding on the melanoma cell line
G43. The positive control Fab G8 demonstrated binding to all cell
lines tested.
TABLE-US-00002 Multi-MAGE-A: (SEQ ID NO: 23) F-L-W-G-P-R-A-L-V
[0075] To determine the cell-binding capacity of the
HLA-A0201/FLWGPRALV (SEQ ID NO:23) selected Fab clone F9 soluble
Fab fragments were made by induction of TG-1 bacteria. TG-1
containing pCes-F9 were grown until OD=0.8 and Fab production was
induced by addition of 1 mM IPTG. After 13 hours induction the
bacterial periplasmic fraction was isolated and dialyzed overnight.
Next day soluble Fab F9 fragments were purified by IMAC.
[0076] Purified Fab F9 was added to 0.5.times.10.sup.6 CMT 64 cells
expressing either HLA-A0210/YLEYRQVPG (SEQ ID NO:3),
HLA-A0201/FLWGPRALV (SEQ ID NO:23), or CMT 64 cells that do not
express human HLA. As shown in FIG. 6 the Fab clone F9 specifically
binds HLA-A0201/FLWGPRALV (SEQ ID NO:23) expressing CMT64 cells and
not CMT 64 cells that do not express human HLA or that do express
the irrelevant HLA-A0201/YLEYRQVPG (SEQ ID NO:3) molecules.
[0077] 2.4: Fab AH5 binds HLA-A0201/multi-MAGE-A complexes
only.
[0078] ELISA using multiple peptide/MHC complexes then confirmed
the specificity of Fab-AH5. To this end HLA-A0201 complexes
presenting peptides multi-MAGE-A, gp100, JCV and MAGE-C2, as well
as a HLA-A1/MAGE-A1 complex were immobilized on 96-well plates and
incubated with phages displaying Fab AH5 and control Fab G8. As
shown in FIG. 4, AH5 only binds HLA-A0201/multi-MAGE-A and not the
irrelevant complexes HLA-A0201/gp100, HLA-A0201/MAGE-C2,
HLA-A0201/JCV and HLA-A0101/MAGE-A1. The positive control Fab G8
only binds to its relevant target HLA-A0101/MAGE-A1.
[0079] The nucleic acids encoding for the HLA-A0201--multi-MAGE-A
complex binding Fab AH5 will be combined with nucleic acids
encoding for human antibody Ch2-Ch3 domains, providing nucleic acid
molecules encoding for a human antibody light chain encompassing
the selected Cl-Vl encoding nucleic acids and encoding for a human
antibody heavy chain encompassing the selected Ch-Vh encoding
nucleic acids. These nucleic acid molecules encoding the desired
immunoglobulin will be introduced, via a plasmid or via an
expression vector, into a eukaryotic host cell such as a CHO cell.
After expression of the immunoglobulin, it will be isolated from
the cell culture and purified. Then, a selected toxic moiety will
be linked to the immunoglobulin, for example using Universal
Linkage System linker chemistry.
Example 3
Cell Binding and Internalization of an Immunoglobulin Provided with
a Toxic Moiety
[0080] Binding capacity of an antibody hereof is analyzed by
flow-cytometry. For example, an antibody comprising immunoglobulin
variable regions specific for complexes of HLA-A0201 and the
multi-MAGE-A peptide is analyzed. HLA-A0201/multi-MAGE-A-positive
tumor cells (Daju, MDN and mel 624) and
HLA-A0201/multi-MAGE-A-negative cells (BSM, G43 and 293) are
incubated on ice with purified antibody and detected by addition of
fluorescently labeled antibodies. Cells bound by the antibody are
quantified and visualized by flow-cytometry. Internalization of
antibody is analyzed by confocal microscopy. To this end, cells are
incubated with the antibody, kept on ice for 30 minutes to allow
binding but no internalization. Next, fluorescently labeled
antibodies specific for the antibody are added. To induce
internalization cells are transferred to 37.degree. C. and fixed
with 1% PFA after 5, 10 and 15 minutes.
Example 4
Apoptosis Induction by Antibodies Hereof in Diverse Tumor Cells
[0081] 4.1: Killing of diverse tumor cells by immunoglobulin
provided with a toxic moiety.
[0082] Antibodies hereof are analyzed for their capacity to induce
apoptosis by incubation with diverse tumor cells, known to express
the antigens comprising the binding sites for the immunoglobulin
variable regions. For example, an antibody comprising
immunoglobulin variable region VH specific for complexes of
HLA-A0201 and the multi-MAGE-A peptide, AH5-BTX, is coupled to a
synthetic HPMA polymer containing the BTX peptide and Doxorubicin
(as we described in WO2009131435, the contents of which are
incorporated herein by this reference) and analyzed. To this end,
antibodies hereof coupled to doxorubicin are analyzed for their
capacity to induce apoptosis by incubation with diverse tumor cells
known to express both HLA-A0201 and MAGE-A genes. The cell-lines
Daju, Mel 624 (melanoma), PC346C (prostate cancer), and MDN
(multiple myeloma) as well as MAGE-A-negative cells (911 and
HEK293T) are incubated with different concentrations of the
antibodies hereof (in DMEM medium, supplemented with pen/strep,
Glutamine and non-essential amino acids). Several hours later,
cells are visually inspected for classical signs of apoptosis such
as detachment of the cells from tissue culture plates and membrane
blebbing. In addition, cells are stained for active caspase-3 to
demonstrate apoptosis. It is excepted that the antibodies hereof
induce apoptosis in the Daju Mel 624, PC346C and MDN cells. Cells
that are not treated with the antibodies hereof are not affected,
as well as cells that do not express HLA-A0201 (HEK293T) and MAGE-A
genes (911 and HEK293T).
[0083] Another antibody, comprising Vh and Vl domains (scFv) with
specificity for complexes of HLA-A01, presenting a MAGE-A1 peptide
was also analyzed. The scFv-BTX construct was coupled to the HPMA
polymer containing doxorubicin and incubated with MAGE-A1-positive
and MAGE-A1-negative cells. Apoptosis is shown by staining for
active caspase-3.
[0084] 4.2: Detection of Active Caspase-3
[0085] A classical intra-cellular hallmark for apoptosis is the
presence of active caspase-3. To determine whether or not the
antibodies hereof induce active caspase-3, Daju, Mel624 and MDN
cells are incubated with various concentrations of antibodies
hereof. After four and 13 hours FAM-DEVD-FMK, a fluorescently
caspase-3/7 inhibitor, is added and positively stained cells are
visualized by fluorescent microscopy and flow-cytometry. Caspase-3
activity is shown in antigen-positive cells and not in
antigen-negative cells, with the (fragment of the) antigen
providing the specific target-binding site for the antibodies
hereof.
[0086] 4.3: Treatment of Tumor Bearing Mice with Immunoglobulins
Provided with a Toxic Moiety.
[0087] Nude mice (NOD-scid, eight per group) with a palpable
subcutaneous transplantable human tumor (Daju or MDN) are injected
with different doses of immunoglobulins provided with a toxic
moiety. As a control mice are treated with standard chemotherapy or
receive an injection with PBS. Mice receiving an optimal dose of
the immunoglobulins provided with a toxic moiety survive
significantly longer that those mice receiving chemotherapy or PBS,
when the aberrant cells expose the target binding sites for the
antibodies hereof.
Example 5
[0088] 5.1: Selection of Llama VHH with Specificity for
HLA-A0201/FLWGPRALV (SEQ ID NO:23) and HLA-A0201/YLEYRQVPG (SEQ ID
NO:3)
[0089] Selection of Llama VHH fragments with specificity for
HLA-A0201/FLWGPRALV (SEQ ID NO:23) (A2/FLW) and HLA-A0201/YLEYRQVPG
(SEQ ID NO:3) (A2/YLE) were performed on CMT64 cells stably
expressing these HLA/peptide complexes. Llama VHH phages (10.sup.11
colony forming units) were first pre-incubated for one hour at room
temperature in PBS containing 2% FCS (PBSF). In parallel,
1.0.times.10.sup.6 CMT64-A2/FLW and 1.0.times.10.sup.6 CMT64 A2/YLE
cells were equilibrated for one hour in PBSF. To deplete for
non-specific binding phages 10.times.10.sup.6 CMT 64 cells
expressing either A2/FLW or A2/YLE were incubated for one hour with
the llama VHH. The non-bound fractions were then incubated (one
hour at 4.degree. C.) with A2/FLW or A2/YLE expressing CMT64 cells.
After extensive washing, bound phages were eluted by adding 500
.mu.l freshly prepared tri-ethylamine (100 mM). The pH of the
solution was neutralized by the addition of 500 .mu.l 1 M Tris (pH
7.5). The eluted phages were incubated with logarithmic growing E.
Coli TG1 cells (OD.sub.600nm of 0.5) for 30 minutes at 37.degree.
C. Bacteria were grown overnight on 2.times. TYAG plates. Next day,
colonies were harvested. After four rounds of selection individual
clones were selected and tested for specificity of binding.
[0090] 5.2: Llama VHH Specific for HLA-A0201/Multi-MAGE-A Epitopes
Bind Antigen-Positive Cells
[0091] To determine the cell-binding capacity of the A2/FLW and
A2/YLE selected VHH soluble VHH fragments were made by induction of
TG-1 bacteria. TG-1 containing pHen-VHH were grown until OD=0.8 and
Fab production was induced by addition of 1 mM IPTG. After 13 hours
induction, the bacterial periplasmic fraction was isolated and
dialyzed overnight. Next day, soluble VHH fragments were purified
by IMAC.
[0092] CMT 64 cells (0.5.times.10.sup.6) expressing either
HLA-A0210/YLEYRQVPG (SEQ ID NO:3), HLA-A0201/FLWGPRALV (SEQ ID
NO:23), or CMT 64 cells that do not express human HLA were
incubated with purified VHH fragments for one hour at 4.degree. C.
As shown in FIG. 7, the A2/FLW-specific VHH bind
HLA-A0201/FLWGPRALV (SEQ ID NO:23) expressing CMT64 cells and not
CMT 64 cells that do not express human HLA or that do express the
irrelevant HLA-A0201/YLEYRQVPG (SEQ ID NO:3) molecules. The
A2/YLE-specific VHH only bind HLA-A2/YLEYRQVPG (SEQ ID NO:3)
expressing CMT64 cells and not A2/FLW-positive CMT64 cells and
CMT64 cells that do not express human HLA.
References (the Contents of Each of which are Incorporated Herein
by this Reference)
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TABLE-US-00003 TABLE 1 Examples of the frequency of MAGE-A
expression by human cancers. Frequency of expression (%) Cancer
MAGE-A1 MAGE-A2 MAGE-A3 MAGE-A4 MAGE-A6 MAGE-A10 MAGE-A11 Melanoma
16 E 36 E 64 E 74 Head and neck 25 42 33 8 N N N Bladder 21 30 35
33 15 N 9 Breast 6 19 10 13 5 N N Colorectal N 5 5 N 5 N N Lung 21
30 46 11 8 N N Gastric 30 22 57 N N N N Ovarian 55 32 20 E 20 N N
Osteosarcoma 62 75 62 12 62 N N hepatocarcinoma 68 30 68 N 30 30 30
Renal cell 22 16 76 30 N N N carcinoma E, expressed but the
frequency is not known; N, expression by tumors has never been
observed
TABLE-US-00004 TABLE 2 MAGE-A expression in human prostate cancer
cell lines and prostate cancer xenografts. Cell line/ MAGE-
Xenograft A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 LNCaP + ++ ++ ++ +
PC346C + ++ ++ + ++ + + ++ OVCAR + + + + JON ++ ++ ++ + + PNT 2 C2
+ + + + + SD48 + + + + PC-3 + + + PC 374 + PC 346p + ++ ++ ++ + ++
+ PC 82 + + PC 133 ++ + + PC 135 + PC 295 + PC 324 + + + PC 310 +
++ + ++ + PC 339 ++ ++ + ++ + + + Expression of the MAGE-A1, A2,
A3, A4, A5, A6, A7, A8, A9, A10, A11 and A12 genes in diverse
prostate tumor cell lines and prostate xenografts was analyzed by
RT-PCR. Shown are expression levels in individual samples tested.
Blank = no expression, + = low expression, ++ = high expression.
All cell lines/xenografts express at least one MAGE-A gene.
SEQUENCE IDENTIFIERS
TABLE-US-00005 [0107] Amino acid sequence Vh AH5 SEQ ID NO: 1
QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNK
YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTV SSGSTSGS.
single chain HLA-A0201/FLWGPRALV construct. SEQ ID NO: 2
MAVMAPRTLVLLLSGALALTQTWAFLWGPRALVGGGGSGGGGSGGGGSGGGSGIQRT
PKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYL
LYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGSHSMRY
FFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRK
VKAHSQTHRVDLGTLRGYYNQSESHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYI
ALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQ
RTDSPKAHVTHHPRSKGEVTLRCWALGFYPADITLTWQLNGEELTQDMELVETRPAGD
GTFQKWASVVVPLGKEQNYTCRVYHEGLPEPLTLRWEPPPSTDSYMVIVAVLGVLGAM
AIIGAVVAFVMKRRRNTGGGDYALAPGSQSSEMSLRDCKA, Amino acid sequence MHC-1
HLA-A0201 presentable peptide in MAGE-A SEQ ID NO: 3 YLEYRQVPG.
Amino acid sequence MHC-1 HLA-CW7 presentable peptide in MAGE-A SEQ
ID NO: 4 EGDCAPEEK. Amino acid sequence MHC-1 HLA-A0201 presentable
peptide in MAGE-A1 SEQ ID NO: 5 YLEYRQVPD. Amino acid sequence
MHC-1 HLA-A0201 presentable peptide in MAGE-A1 with enhanced
binding capacity for HLA-A0201 SEQ ID NO: 6 YLEYRQVPV. Amino acid
sequence Vh binding domain 11H SEQ ID NO: 7
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWLSYISSDGSTIY
YADSVKGRFTVSRDNAKNSLSLQMNSLRADDTAVYYCAVSPRGYYYYGLDLWGQGTT VTVSS.
amino acid sequence of MAGE-A3 peptide epitope binding to HLA SEQ
ID NO: 8 IMPKAGLLI, amino acid sequence of MAGE-A3 peptide epitope
binding to HLA SEQ ID NO: 9 KKLLTQHFVQENYLEY, amino acid sequence
of MAGE peptide epitope binding to HLA SEQ ID NO: 10 EADPTGHSY,
amino acid sequence of MAGE peptide epitope binding to HLA SEQ ID
NO: 11 SLFRAVITK, amino acid sequence of MAGE peptide epitope
binding to HLA SEQ ID NO: 12 NYKHCFPEI, amino acid sequence of MAGE
peptide epitope binding to HLA SEQ ID NO: 13 EVYDGREHSA, amino acid
sequence of MAGE peptide epitope binding to HLA SEQ ID NO: 14
REPVTKAEML, amino acid sequence of MAGE peptide epitope binding to
HLA SEQ ID NO: 15 DPARYEFLW, amino acid sequence of MAGE peptide
epitope binding to HLA SEQ ID NO: 16 SAFPTTINF amino acid sequence
of MAGE peptide epitope binding to HLA SEQ ID NO: 17 SAYGEPRKL,
amino acid sequence of MAGE peptide epitope binding to HLA SEQ ID
NO: 18 SAYGEPRKL, amino acid sequence of MAGE peptide epitope
binding to HLA SEQ ID NO: 19 KMVELVHFL, amino acid sequence of MAGE
peptide epitope binding to HLA SEQ ID NO: 20 YLQLVFGIEV, amino acid
sequence of MAGE peptide epitope binding to HLA SEQ ID NO: 21
EYLQLVFGI, amino acid sequence of MAGE peptide epitope binding to
HLA SEQ ID NO: 22 EADPIGHLY, amino acid sequence of MAGE peptide
epitope binding to HLA SEQ ID NO: 23 FLWGPRALV, amino acid sequence
of MAGE peptide epitope binding to HLA SEQ ID NO: 24 MEVDPIGHLY,
amino acid sequence of MAGE peptide epitope binding to HLA SEQ ID
NO: 25 WQYFFPVIF, amino acid sequence of MAGE peptide epitope
binding to HLA SEQ ID NO: 26 GVYDGREHTV, amino acid sequence of
MAGE peptide epitope binding to HLA SEQ ID NO: 27 MVKISGGPR, amino
acid sequence of MAGE peptide epitope binding to HLA SEQ ID NO: 28
GLYDGMEHL, amino acid sequence of MAGE peptide epitope binding to
HLA SEQ ID NO: 29 VRIGHLYIL, amino acid sequence of BAGE peptide
epitope binding to HLA SEQ ID NO: 30 AARAVFLAL, amino acid sequence
of DAM-6 and DAM-10 peptide epitope binding to HLA SEQ ID NO: 31
FLWGPRAYA, amino acid sequence of GAGE-1/-2/-8 peptide epitope
binding to HLA SEQ ID NO: 32 YRPRPRRY, amino acid sequence of
GAGE-3/-4/-5/-6/-7B peptide epitope binding to HLA SEQ ID NO: 33
YYWPRPRRY, amino acid sequence of NA88-A peptide epitope binding to
HLA SEQ ID NO: 34 MTQGQHFLQKV, amino acid sequence of NY-ESO-1
peptide epitope binding to HLA SEQ ID NO: 35 SLLMWITQCFL, amino
acid sequence of NY-ESO-1a peptide epitope binding to HLA SEQ ID
NO: 36 SLLMWITQC, amino acid sequence of NY-ESO-1a peptide epitope
binding to HLA SEQ ID NO: 37 QLSLLMWIT, amino acid sequence of
NY-ESO-1a peptide epitope binding to HLA SEQ ID NO: 38 ASGPGGGAPR,
HPV 16 E6 T-cell epitope binding to HLA A1 SEQ ID NO: 39 FQDPQERPR,
HPV 16 E6 T-cell epitope binding to HLA A1 SEQ ID NO: 40 TTLEQQYNK,
HPV 16 E6 T-cell epitope binding to HLA A1 SEQ ID NO: 41
ISEYRHYCYS, HPV 16 E6 T-cell epitope binding to HLA A1 SEQ ID NO:
42 GTTLEQQYNK, HPV 16 E6 T-cell epitope binding to HLA A2 SEQ ID
NO: 43 KISEYRHYC, HPV 16 E6 T-cell epitope binding to HLA A2 SEQ ID
NO: 44 YCYSIYGTTL, HPV 16 E6 T-cell epitope binding to HLA A3 SEQ
ID NO: 45 LLRREVYDF, HPV 16 E6 T-cell epitope binding to HLA A3 SEQ
ID NO: 46 IVYRDGNPY, HPV 16 E6 T-cell epitope binding to HLA A11
SEQ ID NO: 47 TTLEQQYNK, HPV 16 E6 T-cell epitope binding to HLA
A24 SEQ ID NO: 48 CYSLYGTTL, HPV 16 E6 T-cell epitope binding to
HLA A24 SEQ ID NO: 49 KLPQLCTEL, HPV 16 E6 T-cell epitope binding
to HLA A24 SEQ ID NO: 50 HYCYSLYGT, HPV 16 E6 T-cell epitope
binding to HLA A24 SEQ ID NO: 51 LYGTTLEQQY, HPV 16 E6 T-cell
epitope binding to HLA A24 SEQ ID NO: 52 EVYDFAFRDL, HPV 16 E6
T-cell epitope binding to HLA A24 SEQ ID NO: 53 VYDFAFRDLC, HPV 16
E6 T-cell epitope binding to HLA A*0201 SEQ ID NO: 54
29-TIHDIILECV-38, HPV 16 E7 T-cell epitope binding to HLA A*0201
SEQ ID NO: 55 86-TLGIVCPI-93, HPV 16 E7 T-cell epitope binding to
HLA A*0201 SEQ ID NO: 56 82-LLMGTLGIV-90, HPV 16 E7 T-cell epitope
binding to HLA A*0201 SEQ ID NO: 57 85-GTLGIVCPI-93, HPV 16 E7
T-cell epitope binding to HLA A*0201
SEQ ID NO: 58 86-TLGIVCPIC-94, HPV E7 T-cell epitope binding to HLA
DR SEQ ID NO: 59 1-MHGDTPTLHEYD-12, HPV E7 T-cell epitope binding
to HLA DR SEQ ID NO: 60 48-DRAHYNIVTFCCKCD-62, HPV E7 T-cell
epitope binding to HLA DR SEQ ID NO: 61 62-DSTLRLCVQSTHVD-75, HPV
E7 T-cell epitope binding to HLA A*201 SEQ ID NO: 62
7-TLHEYMLDL-15, HPV E7 T-cell epitope binding to HLA A*201 SEQ ID
NO: 63 11-YMLDLQPETT-20, HPV E7 T-cell epitope binding to HLA A*201
SEQ ID NO: 64 11-YMLDLQPET-19, HPV E7 T-cell epitope binding to HLA
A*201 SEQ ID NO: 65 12-MLDLQPETT-20, HPV E7 T-cell epitope binding
to HLA B18 SEQ ID NO: 66 16-QPETTDLYCY-25, HPV E7 T-cell epitope
binding to HLA B18 SEQ ID NO: 67 44-QAEPDRAHY-52, HPV E7 T-cell
epitope binding to HLA B18 SEQ ID NO: 68 46-EPDRAHYNIV-55, HPV E7
T-cell epitope binding to HLA DQ2 SEQ ID NO: 69
35-EDEIDGPAGQAEPDRA-50, HPV E7 T-cell epitope binding to HLA DR3
SEQ ID NO: 70 43-GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR-77, HPV E7
T-cell epitope binding to HLA DR15 SEQ ID NO: 71
50-AHYNIVTFCCKCD-62, HPV E7 T-cell epitope binding to HLA DR17 SEQ
ID NO: 72 58-CCKCDSTLRLC-68, HPV E7 T-cell epitope binding to
HLA-DRB1*0901 SEQ ID NO: 73 61-CDSTLRLCVQSTHVDIRTLE-80, PSA T-cell
epitope binding to HLA-A2 SEQ ID NO: 74 146-KLQCVDLHV-154, PSA
T-cell epitope binding to HLA-A2 SEQ ID NO: 75 141-FLTPKKLQCV-150,
PSA T-cell epitope binding to HLA-A2 SEQ ID NO: 76
154-VISNDVCAQV-163, PSA T-cell epitope binding to HLA-A2 SEQ ID NO:
77 154-YISNDVCAQV-163, PSA T-cell epitope binding to HLA-A3 SEQ ID
NO: 78 162-QVHPQKVTK-170, PSA T-cell epitope binding to HLA-A24 SEQ
ID NO: 79 152-CYASGWGSI-160, PSA T-cell epitope binding to HLA-A24
SEQ ID NO: 80 248-HYRKWIKDTI-257, PSMA T-cell epitope binding to
HLA-A2 SEQ ID NO: 81 4-LLHETDSAV-12, PSMA T-cell epitope binding to
HLA-A2 SEQ ID NO: 82 711-ALFDIESKV-719, PSMA T-cell epitope binding
to HLA-A2 SEQ ID NO: 83 27-VLAGGFFLL-35, PSMA T-cell epitope
binding to HLA-A24 SEQ ID NO: 84 178-NYARTEDFF-186, PMSA T-cell
epitope binding to HLA-A24 SEQ ID NO: 85 227-LYSDPADYF-235, T-cell
epitope binding to HLA-A24 SEQ ID NO: 86 624-TYSVSFDSL-632, PAP
T-cell epitope binding to HLA-A2 SEQ ID NO: 87 299-ALDVYNGLL-307,
PAP T-cell epitope binding to HLA-A24 SEQ ID NO: 88
213-LYCESVHNF-221, PAP T-cell epitope binding to MHC-2 SEQ ID NO:
89 199-GQDLFGIWSKVYDPL-213, PAP T-cell epitope binding to MHC-2 SEQ
ID NO: 90 228-TEDTMTKLRELSELS-242, PSCA T-cell epitope binding to
HLA-A2 SEQ ID NO: 91 14-ALQPGTALL-22, PSCA T-cell epitope binding
to HLA-A2 SEQ ID NO: 92 105-AILALLPAL-113, PSCA T-cell epitope
binding to HLA-A2 SEQ ID NO: 93 7-ALLMAGLAL-15, PSCA T-cell epitope
binding to HLA-A2 SEQ ID NO: 94 21-LLCYSCKAQV-30, Kallikrein 4
T-cell epitope binding to DRB1*0404 SEQ ID NO: 95
155-LLANGRMPTVLQCVN-169, Kallikrein 4 T-cell epitope binding to
DRB1*0701 SEQ ID NO: 96 160-RMPTVLQCVNVSVVS-174, Kallikrein 4
T-cell epitope binding to DPB1*0401 SEQ ID NO: 97
125-SVSESDTIRSISIAS-139, EBV nuclear antigen 3 T-cell epitope
binding to MHC I HLA B8 SEQ ID NO: 98 FLRGRAYGL, HLA-A2 restricted
CD8.sup.+ T-cell epitope of PAP binding to HLA-A2 SEQ ID NO: 99
FLFLLFFWL, HLA-A2 restricted CD8.sup.+ T-cell epitope of PAP
binding to HLA-A2 SEQ ID NO: 100 TLMSAMTNL, HLA-A2 restricted
CD8.sup.+ T-cell epitope of PAP binding to HLA-A2 SEQ ID NO: 101
ALDVYNGLL, human HLA-A2.1-restricted CTL epitope of PAP-3 binding
to HLA A2.1 SEQ ID NO: 102 ILLWQPIPV, HLA-A2.1-restricted CTL
epitope of STEAP-3 binding to HLA-A2.1 SEQ ID NO: 103 LLLGTIHAL,
HLA-A2.1-restricted CTL epitope of MUC-1 and MUC-2 binding to
HLA-A2.1 SEQ ID NO: 104 CHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPA,
(GlySerThrSerGlySer)n Ig linker SEQ ID NO: 105
GSTSGSGKPGSGEGASTKGGFAKTTAPSVYPLAPVLESSGSG, IgG1 Ch1-Ch2 hinge
region linker SEQ ID NO: 106 EPKSCDKTHT, IgG3 hinge region linker
SEQ ID NO: 107 ELKTPLGDTTHT, IgG4 hinge region linker SEQ ID NO:
108 ESKYGPP,
Sequence CWU 1
1
1111123PRTArtificialVhAH5 1Gln Leu Gln Leu Gln Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Gly Gly Ser Tyr Tyr Val Pro Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser 115 120
2501PRTArtificialsingle chain HLA-A0201/FLWGPRALV construct 2Met
Ala Val Met Ala Pro Arg Thr Leu Val Leu Leu Leu Ser Gly Ala 1 5 10
15 Leu Ala Leu Thr Gln Thr Trp Ala Phe Leu Trp Gly Pro Arg Ala Leu
20 25 30 Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 35 40 45 Gly Gly Gly Ser Gly Ile Gln Arg Thr Pro Lys Ile
Gln Val Tyr Ser 50 55 60 Arg His Pro Ala Glu Asn Gly Lys Ser Asn
Phe Leu Asn Cys Tyr Val 65 70 75 80 Ser Gly Phe His Pro Ser Asp Ile
Glu Val Asp Leu Leu Lys Asn Gly 85 90 95 Glu Arg Ile Glu Lys Val
Glu His Ser Asp Leu Ser Phe Ser Lys Asp 100 105 110 Trp Ser Phe Tyr
Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys 115 120 125 Asp Glu
Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys 130 135 140
Ile Val Lys Trp Asp Arg Asp Met Gly Gly Gly Gly Ser Gly Gly Gly 145
150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Ser His Ser Met Arg Tyr
Phe Phe 165 170 175 Thr Ser Val Ser Arg Pro Gly Arg Gly Glu Pro Arg
Phe Ile Ala Val 180 185 190 Gly Tyr Val Asp Asp Thr Gln Phe Val Arg
Phe Asp Ser Asp Ala Ala 195 200 205 Ser Gln Arg Met Glu Pro Arg Ala
Pro Trp Ile Glu Gln Glu Gly Pro 210 215 220 Glu Tyr Trp Asp Gly Glu
Thr Arg Lys Val Lys Ala His Ser Gln Thr 225 230 235 240 His Arg Val
Asp Leu Gly Thr Leu Arg Gly Tyr Tyr Asn Gln Ser Glu 245 250 255 Ser
His Thr Val Gln Arg Met Tyr Gly Cys Asp Val Gly Ser Asp Trp 260 265
270 Arg Phe Leu Arg Gly Tyr His Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr
275 280 285 Ile Ala Leu Lys Glu Asp Leu Arg Ser Trp Thr Ala Ala Asp
Met Ala 290 295 300 Ala Gln Thr Thr Lys His Lys Trp Glu Ala Ala His
Val Ala Glu Gln 305 310 315 320 Leu Arg Ala Tyr Leu Glu Gly Thr Cys
Val Glu Trp Leu Arg Arg Tyr 325 330 335 Leu Glu Asn Gly Lys Glu Thr
Leu Gln Arg Thr Asp Ser Pro Lys Ala 340 345 350 His Val Thr His His
Pro Arg Ser Lys Gly Glu Val Thr Leu Arg Cys 355 360 365 Trp Ala Leu
Gly Phe Tyr Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu 370 375 380 Asn
Gly Glu Glu Leu Thr Gln Asp Met Glu Leu Val Glu Thr Arg Pro 385 390
395 400 Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ser Val Val Val Pro
Leu 405 410 415 Gly Lys Glu Gln Asn Tyr Thr Cys Arg Val Tyr His Glu
Gly Leu Pro 420 425 430 Glu Pro Leu Thr Leu Arg Trp Glu Pro Pro Pro
Ser Thr Asp Ser Tyr 435 440 445 Met Val Ile Val Ala Val Leu Gly Val
Leu Gly Ala Met Ala Ile Ile 450 455 460 Gly Ala Val Val Ala Phe Val
Met Lys Arg Arg Arg Asn Thr Gly Gly 465 470 475 480 Gly Asp Tyr Ala
Leu Ala Pro Gly Ser Gln Ser Ser Glu Met Ser Leu 485 490 495 Arg Asp
Cys Lys Ala 500 39PRTArtificialMHC-1 HLA-A0201 presentable peptide
in MAGE-A 3Tyr Leu Glu Tyr Arg Gln Val Pro Gly 1 5
49PRTArtificialMHC-1 HLA-CW7 presentable peptide in MAGE-A 4Glu Gly
Asp Cys Ala Pro Glu Glu Lys 1 5 59PRTArtificialMHC-1 HLA-A0201
presentable peptide in MAGE-A1 5Tyr Leu Glu Tyr Arg Gln Val Pro Asp
1 5 69PRTArtificialMHC-1 HLA-A0201 presentable peptide in MAGE-A1,
with enhanced binding capacity for HLA-A0201 6Tyr Leu Glu Tyr Arg
Gln Val Pro Val 1 5 7121PRTArtificialVh binding domain 11H 7Glu Val
Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Leu 35 40 45 Ser Tyr Ile Ser Ser Asp Gly Ser Thr Ile Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala
Lys Asn Ser Leu Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Asp
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Ser Pro Arg Gly Tyr
Tyr Tyr Tyr Gly Leu Asp Leu Trp Gly 100 105 110 Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120 89PRTArtificialMAGE-A3 peptide epitope
binding to HLA 8Ile Met Pro Lys Ala Gly Leu Leu Ile 1 5
916PRTArtificialMAGE-A3 peptide epitope binding to HLA 9Lys Lys Leu
Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu Tyr 1 5 10 15
109PRTArtificialMAGE peptide epitope binding to HLA 10Glu Ala Asp
Pro Thr Gly His Ser Tyr 1 5 119PRTArtificialMAGE peptide epitope
binding to HLA 11Ser Leu Phe Arg Ala Val Ile Thr Lys 1 5
129PRTArtificialMAGE peptide epitope binding to HLA 12Asn Tyr Lys
His Cys Phe Pro Glu Ile 1 5 1310PRTArtificialMAGE peptide epitope
binding to HLA 13Glu Val Tyr Asp Gly Arg Glu His Ser Ala 1 5 10
1410PRTArtificialMAGE peptide epitope binding to HLA 14Arg Glu Pro
Val Thr Lys Ala Glu Met Leu 1 5 10 159PRTArtificialMAGE peptide
epitope binding to HLA 15Asp Pro Ala Arg Tyr Glu Phe Leu Trp 1 5
169PRTArtificialMAGE peptide epitope binding to HLA 16Ser Ala Phe
Pro Thr Thr Ile Asn Phe 1 5 179PRTArtificialMAGE peptide epitope
binding to HLA 17Ser Ala Tyr Gly Glu Pro Arg Lys Leu 1 5
189PRTArtificialMAGE peptide epitope binding to HLA 18Ser Ala Tyr
Gly Glu Pro Arg Lys Leu 1 5 199PRTArtificialMAGE peptide epitope
binding to HLA 19Lys Met Val Glu Leu Val His Phe Leu 1 5
2010PRTArtificialMAGE peptide epitope binding to HLA 20Tyr Leu Gln
Leu Val Phe Gly Ile Glu Val 1 5 10 219PRTArtificialMAGE peptide
epitope binding to HLA 21Glu Tyr Leu Gln Leu Val Phe Gly Ile 1 5
229PRTArtificialMAGE peptide epitope binding to HLA 22Glu Ala Asp
Pro Ile Gly His Leu Tyr 1 5 239PRTArtificialMAGE peptide epitope
binding to HLA 23Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5
2410PRTArtificialMAGE peptide epitope binding to HLA 24Met Glu Val
Asp Pro Ile Gly His Leu Tyr 1 5 10 259PRTArtificialMAGE peptide
epitope binding to HLA 25Trp Gln Tyr Phe Phe Pro Val Ile Phe 1 5
2610PRTArtificialMAGE peptide epitope binding to HLA 26Gly Val Tyr
Asp Gly Arg Glu His Thr Val 1 5 10 279PRTArtificialMAGE peptide
epitope binding to HLA 27Met Val Lys Ile Ser Gly Gly Pro Arg 1 5
289PRTArtificialMAGE peptide epitope binding to HLA 28Gly Leu Tyr
Asp Gly Met Glu His Leu 1 5 299PRTArtificialMAGE peptide epitope
binding to HLA 29Val Arg Ile Gly His Leu Tyr Ile Leu 1 5
309PRTArtificialBAGE peptide epitope binding to HLA 30Ala Ala Arg
Ala Val Phe Leu Ala Leu 1 5 319PRTArtificialDAM-6 and DAM-10
peptide epitope binding to HLA 31Phe Leu Trp Gly Pro Arg Ala Tyr
Ala 1 5 328PRTArtificialGAGE-1/-2/-8 peptide epitope binding to HLA
32Tyr Arg Pro Arg Pro Arg Arg Tyr 1 5
339PRTArtificialGAGE-3/-4/-5/-6/-7B peptide epitope binding to HLA
33Tyr Tyr Trp Pro Arg Pro Arg Arg Tyr 1 5 3411PRTArtificialNA88-A
peptide epitope binding to HLA 34Met Thr Gln Gly Gln His Phe Leu
Gln Lys Val 1 5 10 3511PRTArtificialNY-ESO-1 peptide epitope
binding to HLA 35Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu 1 5 10
369PRTArtificialNY-ESO-1a peptide epitope binding to HLA 36Ser Leu
Leu Met Trp Ile Thr Gln Cys 1 5 379PRTArtificialNY-ESO-1a peptide
epitope binding to HLA 37Gln Leu Ser Leu Leu Met Trp Ile Thr 1 5
3810PRTArtificialNY-ESO-1a peptide epitope binding to HLA 38Ala Ser
Gly Pro Gly Gly Gly Ala Pro Arg 1 5 10 399PRTArtificialHPV 16 E6
T-cell epitope binding to HLA A1 39Phe Gln Asp Pro Gln Glu Arg Pro
Arg 1 5 409PRTArtificialHPV 16 E6 T-cell epitope binding to HLA A1
40Thr Thr Leu Glu Gln Gln Tyr Asn Lys 1 5 4110PRTArtificialHPV 16
E6 T-cell epitope binding to HLA A1 41Ile Ser Glu Tyr Arg His Tyr
Cys Tyr Ser 1 5 10 4210PRTArtificialHPV 16 E6 T-cell epitope
binding to HLA A1 42Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys 1 5 10
439PRTArtificialHPV 16 E6 T-cell epitope binding to HLA A2 43Lys
Ile Ser Glu Tyr Arg His Tyr Cys 1 5 4410PRTArtificialHPV 16 E6
T-cell epitope binding to HLA A2 44Tyr Cys Tyr Ser Ile Tyr Gly Thr
Thr Leu 1 5 10 459PRTArtificialHPV 16 E6 T-cell epitope binding to
HLA A3 45Leu Leu Arg Arg Glu Val Tyr Asp Phe 1 5
469PRTArtificialHPV 16 E6 T-cell epitope binding to HLA A3 46Ile
Val Tyr Arg Asp Gly Asn Pro Tyr 1 5 479PRTArtificialHPV 16 E6
T-cell epitope binding to HLA A11 47Thr Thr Leu Glu Gln Gln Tyr Asn
Lys 1 5 489PRTArtificialHPV 16 E6 T-cell epitope binding to HLA A24
48Cys Tyr Ser Leu Tyr Gly Thr Thr Leu 1 5 499PRTArtificialHPV 16 E6
T-cell epitope binding to HLA A24 49Lys Leu Pro Gln Leu Cys Thr Glu
Leu 1 5 509PRTArtificialHPV 16 E6 T-cell epitope binding to HLA A24
50His Tyr Cys Tyr Ser Leu Tyr Gly Thr 1 5 5110PRTArtificialHPV 16
E6 T-cell epitope binding to HLA A24 51Leu Tyr Gly Thr Thr Leu Glu
Gln Gln Tyr 1 5 10 5210PRTArtificialHPV 16 E6 T-cell epitope
binding to HLA A24 52Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu 1 5 10
5310PRTArtificialHPV 16 E6 T-cell epitope binding to HLA A24 53Val
Tyr Asp Phe Ala Phe Arg Asp Leu Cys 1 5 10 5410PRTArtificialHPV 16
E6 T-cell epitope binding to HLA A*0201 54Thr Ile His Asp Ile Ile
Leu Glu Cys Val 1 5 10 558PRTArtificialHPV 16 E7 T-cell epitope
binding to HLA A*0201 55Thr Leu Gly Ile Val Cys Pro Ile 1 5
569PRTArtificialHPV 16 E7 T-cell epitope binding to HLA A*0201
56Leu Leu Met Gly Thr Leu Gly Ile Val 1 5 579PRTArtificialHPV 16 E7
T-cell epitope binding to HLA A*0201 57Gly Thr Leu Gly Ile Val Cys
Pro Ile 1 5 589PRTArtificialHPV 16 E7 T-cell epitope binding to HLA
A*0201 58Thr Leu Gly Ile Val Cys Pro Ile Cys 1 5
5912PRTArtificialHPV E7 T-cell epitope binding to HLA DR 59Met His
Gly Asp Thr Pro Thr Leu His Glu Tyr Asp 1 5 10 6015PRTArtificialHPV
E7 T-cell epitope binding to HLA DR 60Asp Arg Ala His Tyr Asn Ile
Val Thr Phe Cys Cys Lys Cys Asp 1 5 10 15 6114PRTArtificialHPV E7
T-cell epitope binding to HLA DR 61Asp Ser Thr Leu Arg Leu Cys Val
Gln Ser Thr His Val Asp 1 5 10 629PRTArtificialHPV E7 T-cell
epitope binding to HLA A*201 62Thr Leu His Glu Tyr Met Leu Asp Leu
1 5 6310PRTArtificialHPV E7 T-cell epitope binding to HLA A*201
63Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr 1 5 10
649PRTArtificialHPV E7 T-cell epitope binding to HLA A*201 64Tyr
Met Leu Asp Leu Gln Pro Glu Thr 1 5 659PRTArtificialHPV E7 T-cell
epitope binding to HLA A*201 65Met Leu Asp Leu Gln Pro Glu Thr Thr
1 5 6610PRTArtificialHPV E7 T-cell epitope binding to HLA B18 66Gln
Pro Glu Thr Thr Asp Leu Tyr Cys Tyr 1 5 10 679PRTArtificialHPV E7
T-cell epitope binding to HLA B18 67Gln Ala Glu Pro Asp Arg Ala His
Tyr 1 5 6810PRTArtificialHPV E7 T-cell epitope binding to HLA B18
68Glu Pro Asp Arg Ala His Tyr Asn Ile Val 1 5 10
6916PRTArtificialHPV E7 T-cell epitope binding to HLA DQ2 69Glu Asp
Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala 1 5 10 15
7035PRTArtificialHPV E7 T-cell epitope binding to HLA DR3 70Gly Gln
Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys 1 5 10 15
Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val 20
25 30 Asp Ile Arg 35 7113PRTArtificialHPV E7 T-cell epitope binding
to HLA DR15 71Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp 1
5 10 7211PRTArtificialHPV E7 T-cell epitope binding to HLA DR17
72Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys 1 5 10
7320PRTArtificialHPV E7 T-cell epitope binding to HLA DRB1*0901
73Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile 1
5 10 15 Arg Thr Leu Glu 20 749PRTArtificialPSA T-cell epitope
binding to HLA-A2 74Lys Leu Gln Cys Val Asp Leu His Val 1 5
7510PRTArtificialPSA T-cell epitope binding to HLA-A2 75Phe Leu Thr
Pro Lys Lys Leu Gln Cys Val 1 5 10 7610PRTArtificialPSA T-cell
epitope binding to HLA-A2 76Val Ile Ser Asn Asp Val Cys Ala Gln Val
1 5 10 7710PRTArtificialPSA T-cell epitope binding to HLA-A2 77Tyr
Ile Ser Asn Asp Val Cys Ala Gln Val 1 5 10 789PRTArtificialPSA
T-cell epitope binding to HLA-A3 78Gln Val His Pro Gln Lys Val Thr
Lys 1 5 799PRTArtificialPSA T-cell epitope binding to HLA-A24 79Cys
Tyr Ala Ser Gly Trp Gly Ser Ile 1 5 8010PRTArtificialPSA T-cell
epitope binding to HLA-A24 80His Tyr Arg Lys Trp Ile Lys Asp Thr
Ile 1 5 10 819PRTArtificialPSMA T-cell epitope binding to HLA-A2
81Leu Leu His Glu Thr Asp Ser Ala Val 1 5 829PRTArtificialPSMA
T-cell epitope binding to HLA-A2 82Ala Leu Phe Asp Ile Glu Ser Lys
Val 1 5 839PRTArtificialPSMA T-cell epitope binding to HLA-A2 83Val
Leu Ala Gly Gly Phe Phe Leu Leu 1 5 849PRTArtificialPSMA T-cell
epitope binding to HLA-A24 84Asn Tyr Ala Arg Thr Glu Asp Phe Phe 1
5 859PRTArtificialPSMA T-cell epitope binding to HLA-A24 85Leu Tyr
Ser Asp Pro Ala Asp Tyr Phe 1 5 869PRTArtificialPSMA T-cell epitope
binding to HLA-A24 86Thr Tyr Ser Val Ser Phe Asp Ser Leu 1 5
879PRTArtificialPAP T-cell epitope binding to HLA-A2 87Ala Leu Asp
Val
Tyr Asn Gly Leu Leu 1 5 889PRTArtificialPAP T-cell epitope binding
to HLA-A24 88Leu Tyr Cys Glu Ser Val His Asn Phe 1 5
8915PRTArtificialPAP T-cell epitope binding to MHC-2 89Gly Gln Asp
Leu Phe Gly Ile Trp Ser Lys Val Tyr Asp Pro Leu 1 5 10 15
9015PRTArtificialPAP T-cell epitope binding to MHC-2 90Thr Glu Asp
Thr Met Thr Lys Leu Arg Glu Leu Ser Glu Leu Ser 1 5 10 15
919PRTArtificialPSCA T-cell epitope binding to HLA-A2 91Ala Leu Gln
Pro Gly Thr Ala Leu Leu 1 5 929PRTArtificialPSCA T-cell epitope
binding to HLA-A2 92Ala Ile Leu Ala Leu Leu Pro Ala Leu 1 5
939PRTArtificialPSCA T-cell epitope binding to HLA-A2 93Ala Leu Leu
Met Ala Gly Leu Ala Leu 1 5 9410PRTArtificialPSCA T-cell epitope
binding to HLA-A2 94Leu Leu Cys Tyr Ser Cys Lys Ala Gln Val 1 5 10
9515PRTArtificialKallikrein 4 T-cell epitope binding to DRB1*0404
95Leu Leu Ala Asn Gly Arg Met Pro Thr Val Leu Gln Cys Val Asn 1 5
10 15 9615PRTArtificialKallikrein 4 T-cell epitope binding to
DRB1*0701 96Arg Met Pro Thr Val Leu Gln Cys Val Asn Val Ser Val Val
Ser 1 5 10 15 9715PRTArtificialKallikrein 4 T-cell epitope binding
to DPB1*0401 97Ser Val Ser Glu Ser Asp Thr Ile Arg Ser Ile Ser Ile
Ala Ser 1 5 10 15 989PRTArtificialEBV nuclear antigen 3 T-cell
epitope binding to MHC I HLA B8 98Phe Leu Arg Gly Arg Ala Tyr Gly
Leu 1 5 999PRTArtificialHLA-A2 restricted CD8+ T-cell epitope of
PAP binding to HLA-A2 99Phe Leu Phe Leu Leu Phe Phe Trp Leu 1 5
1009PRTArtificialHLA-A2 restricted CD8+ T-cell epitope of PAP
binding to HLA-A2 100Thr Leu Met Ser Ala Met Thr Asn Leu 1 5
1019PRTArtificialHLA-A2 restricted CD8+ T-cell epitope of PAP
binding to HLA-A2 101Ala Leu Asp Val Tyr Asn Gly Leu Leu 1 5
1029PRTArtificialhuman HLA-A2.1-restricted CTL epitope of PAP-3
binding to HLA A2.1 102Ile Leu Leu Trp Gln Pro Ile Pro Val 1 5
1039PRTArtificialHLA-A2.1-restricted CTL epitope of STEAP-3 binding
to HLA-A2.1 103Leu Leu Leu Gly Thr Ile His Ala Leu 1 5
10433PRTArtificialHLA-A2.1-restricted CTL epitope of MUC-1 and
MUC-2 binding to HLA-A2.1 104Cys His Gly Val Thr Ser Ala Pro Asp
Thr Arg Pro Ala Pro Gly Ser 1 5 10 15 Thr Ala Pro Pro Ala His Gly
Val Thr Ser Ala Pro Asp Thr Arg Pro 20 25 30 Ala
10518PRTArtificialIg linker 105Gly Ser Thr Ser Gly Ser Gly Lys Pro
Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly
10610PRTArtificialIgG1 Ch1-Ch2 hinge region linker 106Glu Pro Lys
Ser Cys Asp Lys Thr His Thr 1 5 10 10712PRTArtificialIgG3 hinge
region linker 107Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr 1
5 10 1087PRTArtificialIgG4 hinge region linker 108Glu Ser Lys Tyr
Gly Pro Pro 1 5 1095PRTArtificialIg linker 109Gly Gly Gly Gly Ser 1
5 1106PRTArtificialIg linker 110Gly Ser Thr Ser Gly Ser 1 5
11123PRTArtificialIg linker 111Gly Phe Ala Lys Thr Thr Ala Pro Ser
Val Tyr Pro Leu Ala Pro Val 1 5 10 15 Leu Glu Ser Ser Gly Ser Gly
20
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