U.S. patent application number 14/996553 was filed with the patent office on 2016-07-21 for recombinant proteins and their therapeutic uses.
The applicant listed for this patent is Bioven 3 Limited. Invention is credited to Keith Alan Charlton, Erik D'Hondt.
Application Number | 20160207972 14/996553 |
Document ID | / |
Family ID | 47716105 |
Filed Date | 2016-07-21 |
United States Patent
Application |
20160207972 |
Kind Code |
A1 |
Charlton; Keith Alan ; et
al. |
July 21, 2016 |
RECOMBINANT PROTEINS AND THEIR THERAPEUTIC USES
Abstract
A recombinant protein expressing one or more human growth
factors, tumor antigens, and/or receptors or epitopes thereof on or
within an immunogenic expression creating a recombinant protein in
which one or more epitopes are presented on the surface of the
sequence in their natural configuration. The growth factor, tumor
antigen, and/or receptor, sequence(s) may be expressed within the
encoding sequence at appropriate internal positions or at the
termini as single expressions or as two or more tandem repeats.
Inventors: |
Charlton; Keith Alan;
(Aberdeen, GB) ; D'Hondt; Erik; (Bazel,
BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bioven 3 Limited |
Hamilton |
|
BM |
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|
Family ID: |
47716105 |
Appl. No.: |
14/996553 |
Filed: |
January 15, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13813844 |
Feb 1, 2013 |
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PCT/IB2012/002876 |
Nov 21, 2012 |
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14996553 |
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61654401 |
Jun 1, 2012 |
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61563128 |
Nov 23, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/50 20130101;
A61K 2039/645 20130101; A61K 39/0011 20130101; C07K 14/28 20130101;
A61K 2039/70 20130101; C07K 2319/40 20130101; C07K 14/71 20130101;
A61P 35/00 20180101; C07K 14/485 20130101; A61P 31/04 20180101;
C12N 9/12 20130101; Y02P 20/582 20151101; C07K 14/4753 20130101;
C12N 9/6424 20130101; C07K 14/48 20130101; A61P 37/04 20180101;
C07K 14/4748 20130101; C07K 14/495 20130101; C07K 14/65 20130101;
C07K 14/475 20130101; A61P 43/00 20180101; A61K 39/0005
20130101 |
International
Class: |
C07K 14/485 20060101
C07K014/485; C12N 9/12 20060101 C12N009/12; A61K 39/00 20060101
A61K039/00; C07K 14/65 20060101 C07K014/65; C07K 14/495 20060101
C07K014/495; C07K 14/28 20060101 C07K014/28; C12N 9/64 20060101
C12N009/64 |
Claims
1.-33. (canceled)
34. A recombinant protein, comprising: a polypeptide sequence; and
a first sequence expressing at least a portion of a receptor along
said polypeptide sequence.
35. The recombinant protein according to claim 34, wherein said
polypeptide sequence includes an immunogenic polypeptide
sequence.
36. The recombinant protein according to claim 34, wherein said
polypeptide sequence includes a cholera toxin B (CT-B) protein.
37. The recombinant protein according to claim 34, wherein said at
least said portion of said receptor includes a full length or part
thereof of one or more of the following receptors including but not
limited to Human Epidermal growth factor Receptor 2 (Her2), Human
Epidermal growth factor Receptor 3 (Her3), and other receptors.
38. The recombinant protein according to claim 37, wherein said at
least said portion of said receptor includes a full length or a
portion of two to four different receptors present in said
synthetic protein.
39. The recombinant protein according claim 37, wherein said at
least said portion of said receptor includes a full length or a
portion of one or more receptors in said synthetic protein as
single epitopes or as two or more tandem repeats.
40. The recombinant protein according to claim 34, further
comprising a second sequence expressing at least a portion of a
growth factor along said polypeptide sequence.
41. The recombinant protein according to claim 40, wherein said at
least said portion of said growth factor includes a full length or
part thereof of one or more of the following growth factors
including but not limited to IGF-1, IGF-2, FGF1, FGF2, TGF-.alpha.,
TGF-.beta., VEGF-A, VEGF-B, VEGF-C, VEGF, D, PDGF, NGF, EGF, HGF,
BMP's, and IL's 1-6.
42. The recombinant protein according to claim 41, wherein said at
least said portion of said growth factor includes a full length or
neutralizing portion of two to four different growth factors
present in said synthetic protein.
43. The recombinant protein according claim 41, wherein said at
least said portion of said growth factor includes a full length or
neutralizing portion of one or more growth factors in said
synthetic protein as single epitopes or as two or more tandem
repeats.
44. The recombinant protein according claim 41, wherein said at
least said portion of said growth factor or a neutralizing portion
thereof is separated from the remaining synthetic protein by a
peptide `spacer`.
45. The recombinant protein according claim 44, wherein said
peptide `spacer` comprises in part a growth factor or neutralizing
portion thereof.
46. The recombinant protein according claim 44, wherein said
peptide `spacer` includes one or more T-cell epitopes.
47. The recombinant protein according to claim 40, further
comprising a third sequence expressing at least a portion of a
tumor antigen along said polypeptide sequence.
48. The recombinant protein according to claim 47, wherein said
tumor antigen is a Prostate Specific Antigen (PSA).
49. The recombinant protein according to claim 47, wherein said at
least said portion of said tumor antigen includes a full length or
part thereof of one or more of the following tumor antigens
including but not limited to PSA, and other tumor antigens.
50. The recombinant protein according to claim 49, wherein said at
least said portion of said tumor antigen includes a full length or
a portion of two to four different tumor antigens present in said
synthetic protein.
51. The recombinant protein according claim 49, wherein said at
least said portion of said tumor antigen includes a full length or
a portion of one or more tumor antigens in said synthetic protein
as single epitopes or as two or more tandem repeats.
52.-54. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Patent
Application Ser. No. 61/563,128 filed Nov. 23, 2011 entitled
"IMMUNOGENIC SYNTHETIC RECOMBINANT PROTEINS" and U.S. Patent
Application Ser. No. 61/654,401 filed Jun. 1, 2012 entitled
"IMMUNOGENIC SYNTHETIC RECOMBINANT PROTEINS", both of which are
incorporated by reference herein in their entirety.
FIELD
[0002] The present disclosure relates to the field of recombinant
proteins for use in treating diseases.
BACKGROUND
[0003] Cancer immunology is the study of interactions between an
immune system and cancer cells such as, tumors or malignancies. The
initiation of an immune response, such as recognition of
cancer-specific antigens, which are expressed by human tumors and
not in normal tissues, is of particular interest. Generally,
methods to control the division and proliferation of the malignant
cells have been to isolate these antigens and present them so that
they are recognized by the immune system as non-self antigens and
induce a specific immune response.
[0004] There are a significant number of growth factors identified
at present, and most, if not all, have been shown to be important
mediators of cell proliferation in various cancers in addition to
being implicated in other disease conditions. Generally, growth
factors are soluble serum proteins that recognize, and are bound by
a group of growth factor receptors located on cell surfaces.
Particular growth factors may be specific for a single receptor, or
may bind to more than one closely related receptor with varying
affinities. Similarly, some receptors bind only a single growth
factor ligand while others can bind to multiple related growth
factors, again usually with differing affinities. Upon binding to
its natural receptor, the cytoplasmic domain of the receptor is
phosphorylated, and this initiates an intra cellular signaling
cascade which results in modulation of transcription of one or more
genes and ultimately to progression through the cell cycle and cell
proliferation.
[0005] Growth factors and their receptors are essential components
of the normal processes of growth, development and repair, and
their tissue distribution profiles and expression levels closely
regulate cell growth. Numerous studies have shown that growth
factors can stimulate proliferation of a variety of cell types both
in vitro and in vivo (Cohen S., Carpenter G., PNAS USA 72, 1317,
1975, Witsch E et al: Physiology: 25(2):85-101, (2010)). Moreover,
certain growth factors have been shown to stimulate proliferation
in some cancer cell lines, for example epidermal growth factor
(EGF) can stimulate some non-small cell lung carcinoma cells
(Osborne C. K. et al. Can Res. 40, 2, 361 (1980)). Other growth
factor such as vascular endothelial growth factor (VEGF),
fibroblast growth factor (FGF), and platelet-derived growth factor
(PDGF) are important in several oncology diseases, such as
non-small cell lung cancer (NSCLC) (Ballas M S, Chachoua A., Onco
Targets and Therapy: 4, 43-58 (2011)). Prostate cancer. (Cox M E et
al; Prostate 69 (1):33-40 (2009)), and Breast cancer (Law J et al.
Cancer Res; 68,24:10238-10346 (2008)).
[0006] High levels of various growth factor receptors have been
reported in malignant tissues. For example, the epidermal growth
factor receptor (EGFR) has been detected at unusually high levels
in malignant tumors of epithelial origin, such as lung, breast,
bladder, ovarian, vulva, colonic, pulmonary, brain and oesophagus
cancers. The role played by growth factors and their receptors in
regulating tumor growth is unknown, but there are suggestions that
growth factor receptor expression in tumor cells provides a
mechanism for autocrine growth stimulation which leads to
uncontrolled proliferation (Schlessinger J., Schreiber A. B., Levi
A., Liberman T., Yarden Y. Crit. Rev. Biochem. 1983, 14 (2)
93-111). Further, Liao Y et al; Hum Pathol 36(11); 1186-1196 (2005)
and Cox M E et al; Prostate: 69(1) 33-40 (2009) describe the role
of increased Insular receptor and growth factor on metastatic
prostate cancer.
[0007] One treatment strategy to target growth factor signaling in
cancer therapy has been to use a passive immunotherapy, such as
using monoclonal antibodies against the particular
receptor/receptors involved. Such studies have demonstrated that
the specific recognition by an antibody of the receptor that is
able to inhibit the binding of the ligand can have an inhibitory
effect on the mitogenic stimulation of malignant cells (SATO J. D.
et al. Methods in Enzymology, vol. 146 pp 63-81, 1987). However,
antibodies which are of murine origin will usually produce a human
anti-mouse antibody response (HAMA), thus limiting them to a single
administration.
[0008] Other treatment strategies have been to use an active
immunotherapy with vaccines that contain the growth factor of
interest to induce an immune response against the molecule to
inhibit the proliferation effect of the growth factor on tumors.
U.S. Pat. No. 5,984,018, to Davila et al., entitled Vaccine
Composition Comprising Autologous Epidermal Growth Factor or a
Fragment or a Derivative Thereof having Anti-tumor Activity and use
Thereof in the Therapy of Malignant Diseases, discloses, for
example, the use of a vaccine that contains a mixture of a growth
factor and an immunogenic (i.e. non-human) carrier protein
chemically conjugated together using gluterhaldehyde. However,
without being bound to any particular theory it is thought that
chemical conjugation hinders immune responses against the
vaccine.
[0009] This is a technically challenging approach, as it requires
that the host generates an immune response to a `self antigen`, and
vertebrate immune systems have evolved to prevent such responses
occurring. Where a strong immune response is generated against, a
self antigen, for example, one that includes T-helper cell
activation, an auto-immune disease state usually results. For many
years it has been hypothesized that some auto-immune disorders, for
example, lupus, multiple sclerosis (MS), diabetes etc,, might be
caused by early exposure to an environmental agent that includes
immunogenic epitopes (T-cell epitopes) that closely mimic host
self-epitopes. This could lead to the stimulation of T-helper cells
that are cross reactive with host epitopes. Subsequent exposure to
the environmental agent could then result in an anti-self immune
response (Albert, L. J., and Inman, R.D New England Journal of
Medicine, December 30.sup.th pp 2068-2074, 1999). It has since been
demonstrated that a viral antigen can indeed generate an anti-self
immune response against a nerve cell protein (Levin, M. C. et. al.,
Nature Medicine vol 8 (5) pp 509-513, 2002).
[0010] U.S. Publ. No. 2006/0251654, to Casimiro et al., entitled
Method for Treatment of Malignant and Infectious Chronic Diseases,
(the '654 publication) discloses a method of treating a subject
bearing a malignant or infectious chronic disease comprising the
method of immunizing the subject with a vaccine containing a self
antigen associated with tire malignant or infectious chronic
disease that is coupled to a carrier protein; treating the subject
with an immune modulator agent; and immunizing the subject again
with the vaccine of the step 1, and an appropriate adjuvant
selected from aluminum hydroxide and Montanide ISA 51 (Seppic,
Paris, France). Unfortunately, the preparation of the vaccine by
chemical conjugation is thought to hinder the immune response.
[0011] The majority of the vaccines described above exhibit a
number of limitations, arising primarily from the method of
manufacture and the potential lack of uniformity and homology of
the protein product. The vaccines described above generally
comprise a mixture of a recombinant carrier protein and
polypeptides of human origin that are chemically conjugated using
gluterhaldehyde. Unfortunately, this reactive reagent can
undesirably form covalent cross-linking bonds between varieties of
chemical groups, and generally leads to a highly heterogeneous
product. Thus, the resulting vaccines may comprise not only carrier
protein molecules with varying numbers of the target human
polypeptide attached (for example, 0, 1, 2, 3 etc.), but the human
polypeptides can each be attached to the carrier via different
atoms and so in different positions and in different orientations.
Furthermore, both the target polypeptide and carrier protein
molecules may be conjugated to themselves, resulting in various
homo-multimers that may have no clinical efficacy and may not
contribute to an anti-cancer patient immune response.
SUMMARY
[0012] The present disclosure is directed towards recombinant
proteins and their respective methods of manufacturing: the
characterization of the recombinant proteins and therapeutic
methods of using the recombinant proteins to treat chronic
diseases, such as, for example, lung, breast, bladder, prostate,
ovarian, vulva, colonic, colorectal, intestinal, pulmonary, brain,
esophageal, other cancers, and other diseases.
[0013] In an illustrative embodiment, the recombinant protein is an
immunogenic protein molecule expressing one or more sequences that
fold into a physical structure, for example expressing one or more
sequences of a cholera toxin B (CT-B) protein from Vibrio cholera
or a synthetic equivalent, and expressing one or more sequences of
one or more epitopes from human growth factors. The expressions of
the growth factors or parts thereof can be present at multiple
sites, as a single antigen, in tandem, and/or longer chains of
antigen molecule(s) per site.
[0014] In another illustrative embodiment, the recombinant protein
is an immunogenic protein molecule expressing one or more sequences
that fold into a physical structure, for example expressing one or
more sequences of a cholera toxin B (CT-B) protein from Vibrio
cholera or a synthetic equivalent, and expressing one or more
sequences of one or more tumor antigens or parts thereof. The
recombinant protein may also include one or more sequences of one
or more growth factors or parts thereof, and/or one or more
sequences of one or more receptors or parts thereof.
[0015] In another illustrative embodiment the recombinant protein
is an immunogenic protein molecule expressing one or more sequences
that fold into a physical structure, for example expressing one or
more sequences of a cholera toxin B (CT-B) protein front Vibrio
cholera or a synthetic equivalent, and expressing one or more
sequences of one or more receptors or parts thereof. The
recombinant protein may also include one or more sequences of one
or more growth factors or parts thereof, and/or one or more
sequences of one or more tumor antigens or parts thereof.
[0016] In these illustrative embodiments, the expressions of the
tumor antigen(s) or parts thereof, the receptor(s) or pans thereof
and/or the growth factor(s) or parts thereof can be present at
multiple sites, as a single antigen or receptor, in tandem, and/or
longer drains of antigen or receptor molecule(s) per site.
[0017] In an illustrative embodiment, the sequence of the tumor
antigen may include a sequence of a Prostate Specific Antigen (PSA)
or part thereof. In an illustrative embodiment, the sequence of the
receptor may include a sequence of a Human Epidermal Growth Factor
Receptor 2 (Her2) or part thereof and/or a Human Epidermal Growth
Factor Receptor 3 (Her3) or part thereof.
[0018] In an illustrative embodiment, the sequence of the growth
factor may include a sequence of an epidermal growth factor (EGF)
or a substantial portion of the appropriate coding region(s) of the
EGF including a neutralizing domain of the EGF at one or more
positions within the recombinant protein. In other illustrative
embodiments, the sequence of the growth factor may include a full
length or part thereof of one or more of the following growth
factors, and/or alternative self-antigens such as, but not limited
to, other growth factors, including, but not limited to, EGF,
IGF-1, IGF-2, FGF, TGF-.beta., TGF-.alpha., VEGF-A, VEGF-B, VEGF-C,
VEGF-D, PDGF, NGF, EGF, HGF, BMP's, and IL's 1-6. It is
contemplated within the scope of the disclosure that growth factors
may be selected from human and non-human origins. It is further
contemplated within the scope of the disclosure that said sequence
of growth factors can substantially similar to either human or
non-human growth factors or said sequence can contain functional
parts thereof. Further, the recombinant protein may include one or
more expressions of other sequences that can be used to
functionally model part or all of the growth factors within a
recombinant immunogenic protein sequence. In one embodiment,
additional flanking residues may also be expressed or added to the
minimum sequence to allow the entire neutralizing domain of the
molecule to be presented in a natural conformation and to be
accessible to cells of the immune system.
[0019] In the context of the present disclosure, "neutralizing
domain" is defined as a region or regions of cither or both
member(s) of a specific binding pair, e.g. a growth factor and its
cognate receptor, wherein the binding of a third molecule that is
not a member of the specific binding pair to the aforementioned
region(s) will prevent the subsequent binding of the two members of
the specific binding pair. The third molecule can be another
protein molecule including but not limited to an antibody, or can
be a small non-protein molecule, and can be either natural or
synthetic in origin. The neutralizing domain will normally include
those regions of the members of the specific binding pair that are
in direct contact during binding, and will also include regions
out-with said regions where upon binding of a third molecule
introduces sufficient stearic hindrance to prevent the members of
the specific binding pair from binding directly.
[0020] It is well established in the field that specific
recognition of a ligand by its cogitate receptor is defined by an
interaction between the binding site of the receptor and a
particular molecular signature (epitope) of the ligand. Thus an
antibody that either binds to or otherwise blocks the receptor
binding site, or binds to or otherwise blocks the recognition
epitope of the ligand, will prevent ligand-receptor interactions.
Such antibodies are described as being "neutralizing". In the
context of the present disclosure it is desirable that neutralizing
antibodies are generated by the host upon administration of the
recombinant protein, and thus the protein sequence may express or
include one or more of all of, or a suitable sequence derived from,
a growth factor or tumor antigen such that epitopes required for
receptor binding are presented in a functional (native)
conformation.
[0021] In addition to expressing multiple copies of a single tumor
antigen, receptor, and/or growth factor, presented as a single
tumor antigen, receptor, and/or growth factor or part thereof per
physical site, and/or as chains of repetitive tumor antigen,
receptor, and/or growth factor sequences (for example, n=1 or
more); the protein according to the disclosure may also include
expressions of one or more epitopes or binding sites from two or
more different tumor antigens, receptors, and/or growth factors
present as single or as chains at different positions within the
sequence of the recombinant protein.
[0022] The resulting protein may be a single polypeptide expressing
a tumor antigen, a receptor, and/or a growth factor or one or more
epitopes or binding sites thereof within the sequence of the
recombinant protein. In an illustrative embodiment, the sequence of
the recombinant protein expresses one or more portions of a CT-B
sequence and presents the tumor antigen, receptor, and/or growth
factor expression(s) or one or more expression(s) of epitopes or
binding sites thereof on a surface of the recombinant protein in a
natural conformation.
[0023] In another illustrative embodiment, a process of preparing a
protein formulation is disclosed. In this illustrative embodiment,
the process includes assembling one or more single monovalent or
multivalent monomers together preparing a multivalent vaccine
including a recombinant protein including one or more tumor
antigens, receptors, and/or a growth factors or parts thereof.
[0024] In yet another illustrative embodiment, a process for
treating a patient is disclosed. In this illustrative embodiment,
the process includes administering separately to the patient one or
more monovalent or multivalent, one tumor antigen, receptor, and/or
growth factor, synthetic proteins in a same day or at alternate
days or times during a vaccination period.
[0025] In a further illustrative embodiment, a process for treating
a patient is disclosed. In this illustrative embodiment, the
process includes administering separately to the patient one or
more monovalent or multivalent vaccine, one tumor antigen,
receptor, and/or growth factor, synthetic proteins in a
pharmaceutically acceptable carrier including an adjuvant to
promote an immune response.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The embodiments described in the present disclosure are
illustrated in the figures of the accompanying drawings which are
meant to be exemplary and not limiting, in which like references
are intended to refer to like or corresponding parts, and in
which:
[0027] FIG. 1 illustrates a table of sequences and structures of
EGF molecules from a range of organisms;
[0028] FIG. 2 illustrates an embodiment of a structure of a human
EGF molecule, including an EGF neutralizing domain;
[0029] FIG. 3 illustrates an embodiment of a simplified line
structure of the EGF molecule's cysteine pairs, including the EGF
neutralizing domain;
[0030] FIG. 4 illustrates an embodiment of a minimum sequence of
the EGF molecule that presents the EGF neutralizing domain in a
correct conformation:
[0031] FIG. 5 illustrates an embodiment of a structure of a
modified synthetic molecule, expressing the EGF neutralizing
domain;
[0032] FIG. 6 illustrates a bar graph of mAb 10825 and mAb 10827
binding to rHuEGF with optical density (OD) measured at 450 nm;
[0033] FIG. 7 illustrates a bar graph of mAb 10825 and mAb 10827
binding to rHuEGF in competition with a free soluble peptide
derived from the neutralizing domain:
[0034] FIG. 8 illustrates a line graph of the binding of anti-EGF
neutralizing domain mAb 10827 to 6 EGF-CT-B synthetic proteins
adsorbed directly onto ELISA plates;
[0035] FIG. 9 illustrates a line graph of the binding of anti-EGF
neutralizing domain mAb 10827 to 6 EGF-CT-B synthetic proteins
captured by a rabbit anti-CT-B antibody;
[0036] FIG. 10 illustrates a Western blot of the 6 monovalent
synthetic EGF- CT-B proteins run on SDS gel under native
(non-boiled) conditions, and detected with an anti-CT-B
antibody;
[0037] FIG. 11 illustrates a line graph of the binding of anti-EGF
neutralizing domain mAb 10827 to synthetic EGF-CT-B proteins
including either 2 full length EGF sequences (E2) or two partial
EGF sequences (B2);
[0038] FIG. 12 illustrates a Western blot of the bivalent synthetic
EGF-CT-B proteins run on non-denaturing SDS-PAGE gels;
[0039] FIG. 13 illustrates a synthetic protein sequence including
two full length EGF sequences (underlined) and a CT-B sequence
(italics);
[0040] FIG. 14 illustrates a synthetic protein sequence including
two EGF neutralizing domain sequences (underlined) and the CT-B
sequence (italics);
[0041] FIG. 15 illustrates a synthetic protein sequence including
two partial sequences of the EGF molecule including the EGF
neutralizing domain, Cys6 to Cys31, (underlined) and the CT-B
sequence (italics);
[0042] FIG. 16 illustrates a Western blot showing the effect of pH
shift, on the multimerisation of native CT-B protein. Samples on
the right side of the gel were incubated for 5 min at the pH
indicated below prior to gel analysis. Samples on the left side
were incubated at the pH indicated below for 5 min., then
neutralized back to pH 7.0 for 1 hour prior to gel analysis;
[0043] FIG. 17 illustrates a table of constructs T1-T6, E2, and B2
including sequences expressing EGF and CT-B;
[0044] FIG. 18 illustrates a Western blot of the E2 and B2
constructs;
[0045] FIG. 19 illustrates constructs E2, E2N, and E2C including
sequences expressing EGF and CT-B;
[0046] FIG. 20 illustrates constructs including sequences
expressing EGF and CT-B and containing extended amino acid
linkers;
[0047] FIG. 21 illustrates a Western blot of the E2, E2N, and E2C
constructs;
[0048] FIG. 22 illustrates a Western blot of a number of N-terminus
constructs including the extended amino acid linkers; and
[0049] FIG. 23 illustrates a Western blot of a number of C-terminus
constructs including the extended amino acid linkers.
[0050] FIG. 24 illustrates a synthetic protein sequence including
IGF1 (Underlined), EGF (underlined and italics) and the CT-B
sequences (italics);
[0051] FIG. 25 illustrates a bar graph of a capture ELISA
demonstrating the simultaneous presence of IGF, EGF and CTB
sequences on a single recombinant protein. Bars A and B were
captured by and-EGF antibody, and bar C by anti-IGF antibody.
Proteins were detected as follows: A anti-CTB, B anti-IGF and C
anti-CTB;
[0052] FIG. 26 illustrates a synthetic protein sequence including
Hu-IFG1 sequence (underlined) and the CT-B sequence (italics);
[0053] FIG. 27 illustrates a bar graph of a capture ELISA in which
hetero-oligomers of IGF-CTB and EGF-CTB are detected. All samples
include IGF C-terminal to CTB. Samples A and B include EGF
C-terminal to CTB, and samples B and D include EGF N-terminal to
CTB. Samples A and B were captured with an anti-EGF antibody, and
IGF was detected, whereas samples C and D were captured with an
anti-IGF antibody and EGF was detected;
[0054] FIG. 28 (a-e) illustrates synthetic protein sequences
including CT-B sequence (italics) and the growth factor sequences
(underlined) of a) TGF-Beta1, b) FGF2, c) HGF (NK1), d) IGF1/2 and
e) VEGF-A/C (VEGF-C sequence underlined and in italics);
[0055] FIG. 29 illustrates a bar graph of a capture ELISA of a
diverse range of chimeric recombinant proteins including sequences
derived from one or more growth factors together with CTB
sequences. In each case, recombinant protein was captured by an
antibody specific for one of the sequences and then detected with a
antibody specific for a different sequence as follows:
[0056] HGF and IGF B1 were captured with .alpha.-HGF and
.alpha.-TGF B1 antibodies, and CTB was detected;
[0057] FGF2 was captured with .alpha.-CTB antibody and FGF2
detected;
[0058] VEGF A/C was captured with (i) .alpha.-VEGF-A antibody and
(ii) .alpha.-VEGF-C antibody, and CTB was detected in both
cases;
[0059] IGF1/2 was captured by .alpha.-IGF1 antibody in both cases,
and detected with (i) .alpha.-CTB antibody and (ii) .alpha.-IGF2
antibody;
[0060] FIG. 30 illustrates a Western blot of a SDS-PAGE gel of
native recombinant TGF B1-CTB protein according to FIG. 28a
demonstrating the presence of primarily pentameric recombinant
protein;
[0061] FIG. 31 illustrates a synthetic protein sequence including
a) a synthetic protein sequence including TGF-B1 sequence
(underlined) and the CT-B sequence (italics) and b) TGF-Beta2
receptor ligand binding domain sequence (underlined) and the (CT-B
sequence (italics);
[0062] FIG. 32 illustrates a bar chart of a capture ELISA of the
recombinant protein containing both TGF-Beta-R2 and CTB sequences.
The graph demonstrates that both sequences can be bound
simultaneously in both orientation without bias;
[0063] FIG. 33 illustrates that recombinant protein containing
sequences derived from TGF-beta and CTB is able to bind to
recombinant protein containing sequences derived from the ligand
binding domain of TGF beta receptor 2 and CTB;
[0064] FIG. 34 illustrates the IgG antibody responses of Group 1
mice sera at 1/100 dilution to r-EGF following immunization;
[0065] FIG. 35 illustrates the IgG antibody responses of Group 2
mice sera at 1/100 dilution to r-EGF following immunization;
[0066] FIG. 36 illustrates the IgG antibody responses of Group 3
mice sera at (a) 1/100 dilution and (b) 1/8 dilution to r-EGF
following immunization;
[0067] FIG. 37 illustrates the IgG antibody responses of Group 3
mice sera at (a) 1/100 and (b) 1/8 dilution to r-IGF following
immunization;
[0068] FIG. 38 illustrates the IgG antibody responses of Group 4
mice sera at (a) 1/100 and (b) 1/8 dilution to r-EGF following
immunization:
[0069] FIG. 39 illustrates the IgG antibody responses of Group 4
mice sera at (a) 1/100 and (b) 1/8 dilution to r-IGF following
immunization;
[0070] FIG. 40 illustrates the IgG antibody responses of Group 5
mice sera at. 1/8 dilution (except sample 178 at 1/100) to r-IGF
following immunization;
[0071] FIG. 41 illustrates the IgG antibody responses of Group 6
mice sera at 1/100 dilution to a) r-EGF and b) rHu-EGF following
immunization;
[0072] FIG. 42 illustrates the structure of mono-ganglioside GM1,
the natural binding partner of cholera toxin sub-unit B;
[0073] FIG. 43 illustrates the structure of commercially available
D-galactose conjugated to a solid support (Pierce); and
[0074] FIG. 44 illustrates a SDS-PAGE gel of the purification of
rCTB from the culture supernatant (media) of three strains of E.
coli cells transformed with a CTB expression vector as follows:,
Lane 1 show size marker. Lanes 2, 5 and 8 show crude culture
supernatant. Lanes 3, 6 and 9 show crude periplasmic fractions.
Lanes 4, 7 and 10 show eluted purified CTB. Lane 11 shows
His-tagged CTB purified by IMAC.
DETAILED DESCRIPTION
[0075] Detailed embodiments of the present recombinant proteins or
vaccines are disclosed herein, however, it is to be understood that
the disclosed embodiments are merely exemplary, which may be
embodied in various forms. Therefore, specific functional details
disclosed herein are not to be interpreted as limiting, but merely
as a basis for the claims and as a representative basis for
teaching one skilled in the art to variously employ the recombinant
protein disclosed herein.
[0076] The present disclosure provides a homogeneous recombinant
protein for improving the presentation of the maximum number of
growth factor epitopes, tumor antigen epitopes, and/or receptor
binding sites as elements of an immunogenic recombinant protein. In
one illustrative embodiment, a recombinant protein expressing all
or portions of a cholera toxin B (CT-B), and a human epidermal
growth factor (EGF), a tumor antigen, and/or a receptor is
described. In alternative illustrative embodiments, the protein may
express other immunogenic recombinant proteins that are modeled
based upon known immunogenic proteins. It is contemplated within
the scope of the disclosure that such recombinant proteins will be
expressions of polypeptides that are highly immunogenic to the
human immune system. Preferably, the recombinant proteins confer
additional properties to the chimeric protein, for example, high
expression yield and ease of manufacture, oral stability and the
ability to cross from gut to blood stream, and/or previous safe use
in humans.
[0077] In an illustrative embodiment, the recombinant proteins
disclosed herein may include or express a high proportion of a
protein sequence derived from target self antigens, as a function
of total molecular weight. This can be achieved, for example, by
using a large protein model containing multiple growth factor
epitopes. These growth factor epitopes can be multiple copies of
whole or part of a single growth factor, or copies of whole or part
of more than one different growth factor.
[0078] According to the disclosure, the expressions of the growth
factor epitopes should be folded allowing their natural
conformation to be substantially retained and presented to
components of the host immune system in such a way as to elicit a
robust host immune response to said epitopes. Examples of suitable
natural protein models to model an epitope supporting domain of a
recombinant protein include, but are not limited to, cholera toxin
B sub-unit. E. coli heat-labile LT and LT-II enterotoxin B
subunits. veratoxin, pertussis toxin, C. jejuni enterotoxin, Shiga
toxin, listeria toxin, tetanus toxoid, diphtheria toxoid, N.
meningitidisI outer membrane protein, bacteriophage coat protein,
adenovirus and other viral coat proteins. Alternatively, a non-self
component of the protein can be small. As a minimum, the non-self
sequenced) should comprise about 9, 10, 11 or more amino acids in
length, and include either entirely or in-part at least one human
T-cell epitope. Alternatively, non-natural `synthetic` polypeptides
may be used that fulfill the requirements of conferring
immunogenicity to the whole protein and allowing appropriate
presentation of growth factor(s), receptors, tumor antigens or
epitopes thereof to the host immune system.
[0079] In an illustrative embodiment, the epitope supporting domain
of the recombinant protein, whether derived from a natural or
synthetic polypeptide sequence, should have the capacity to
self-assemble into oligomeric multimers under appropriate
chemical/environmental conditions, or to be reduced to monomers
under alternative conditions. Ideally, multimerisation domains will
assemble into stable multimers with a discreet number of sub-units,
for example dimers, trimers, tetramers, pentamers, etc., such that
a product of homogeneous size is generated. Examples of natural
polypeptides include, but are not limited to, leucine zippers, lac
repressor protein, streptavidin/avidin. cholera toxin B sub-unit, B
sub-units of other AB.sub.5 toxins, Pseudomonas trimerization
domain, and viral capsid proteins.
[0080] According to the disclosure the recombinant proteins,
whether either growth factors or parts thereof, cellular receptors
or parts thereof, tumor antigens or parts thereof, are related to
broad range of either cellular pathways involved in chronic disease
or cancers for growth factors and receptors and to broadest
possible range of solid tumors for use of tumor antigens within the
said synthetic proteins. The proteins are in the form of a
recombinant protein and may be use fill in treating chronic
diseases, for example, breast, lung, bladder, ovarian, vulva,
colonic, pulmonary, brain, colorectal intestinal, head and neck,
and esophagus cancers. As different tumor antigens can be expressed
and multiple cellular receptors and growth factors over expressed
in the said diseases, the proteins described hereunder can contain
one or more different tumor antigens, one or more different
receptors or growth factors of one or multiple cellular pathways
associated with the disease. These proteins are called
"multivalent".
[0081] In an illustrative embodiment, a protein comprised of a
homogeneous recombinant protein expressing one or more epidermal
growth factor (EGF) neutralizing domains is disclosed. The protein
is in the form of a recombinant protein and may be useful in
treating chronic diseases, for example, breast, lung, bladder,
ovarian, vulva, colonic, pulmonary, brain, colorectal, head and
neck, and esophagus cancers. In an illustrative embodiment, the
protein is a recombinant protein expressing or including EGF
sequences and CT-B sequences.
[0082] In another illustrative embodiment, a protein comprised of a
homogeneous recombinant protein expressing one fibroblast growth
factor (FGF) is disclosed. In an illustrative embodiment the
protein is a recombinant protein expressing or including FGF
sequences and CT-B sequences.
[0083] In a further illustrative embodiment, a protein comprised of
a homogeneous recombinant protein expressing one transforming
growth factor-Beta 1 (TGF-.beta.1) is disclosed. In an illustrative
embodiment, the protein is a recombinant protein expressing or
including TGF-.beta.1 sequences and CT-B sequences.
[0084] In yet another illustrative embodiment, a protein comprised
of a homogeneous recombinant protein expressing one transforming
growth factor- Beta 1 (TGF-.beta.1) is disclosed. In an
illustrative embodiment, the protein is a recombinant protein
expressing or including TGF-.beta.1 sequences and CT-B
sequences.
[0085] In one illustrative embodiment, a protein comprised of a
homogeneous recombinant protein expressing one insulin-like growth
factor-1 (IGF-1) is disclosed. In an illustrative embodiment, the
protein is a recombinant protein expressing or including IGF-1
sequences and CT-B sequences.
[0086] In another illustrative embodiment, a protein comprised of a
homogeneous recombinant protein expressing one hepatocyte growth
factor (HGF) is disclosed. In an illustrative embodiment, the
protein is a recombinant protein expressing or including HGF
sequences and CT-B sequences.
[0087] In a further illustrative embodiment, a protein comprised of
a homogeneous recombinant protein expressing one Insulin-like
growth factor-1 (IGF-1) and one insulin-like growth factor-2 is
disclosed. In an illustrative embodiment, the protein is a
recombinant protein expressing or including IGF-1 sequences. IGF-2
sequences and CT-B sequences.
[0088] In yet another illustrative embodiment, a protein comprised
of a homogeneous recombinant protein expressing one vascular
endothelial growth factor-A (VEGF-A) and one vascular endothelial
growth factor-C (VEGF-C) is disclosed. In an illustrative
embodiment, the protein is a recombinant protein expressing or
including VEGF-A neutralizing domain sequences, VEGF-C sequences
and CT-B sequences.
[0089] To determine the appropriate coding region(s) of the HuEGF
to express or include, the sequences and structures of EGF
molecules from a range of organisms are analyzed. A table
illustrating sequences and structures of EGF molecules from a range
of organisms is described with reference to FIG. 1. As illustrated
in FIG. 1, a box 100 encloses a portion of the sequence of the EGF
molecules from the range of organisms, which represents the
neutralizing domain epitope of the EGF molecules. While there is a
significant amount of conservation between the neutralizing domain
epitopes of the EGF molecules from different species, there is also
a great deal of variation between species. Notably for in vivo
studies, one neutralizing domain (boxed sequence 100) is fully
conserved amongst primates, but is different in rodents and other
species. Similarly, the different sequences of the EGF molecules
equate to differences in tertiary structure.
[0090] A structure of the human EGF molecule, including the EGF
neutralizing domain, according to an illustrative embodiment is
described with reference to FIG. 2. The EGF molecule contains six
cysteine residues including Cys6, Cys14, Cys20, Cys31, Cys33, and
Cys42. The six cysteine residues are important in determining the
folding of the EGF molecule. The EGF neutralizing domain 200
(illustrated as an anti-parallel .beta.-sheet) is constrained by
two separate disulphide linked cysteine pairs, Cys6-Cys20 and
Cys14-Cys31. The two disulphide linked cysteine pairs, Cys6 Cys20
and Cys14-Cys31 are important because these two pairs define the
minimum sequence or minimum peptide of the EGF molecule that
presents the EGF neutralizing domain 200 in the correct
conformation.
[0091] A simplified line structure of the EGF molecule's cysteine
pairs, including the EGF B-loop 200, according to an illustrative
embodiment is described with reference to FIG. 3. As illustrated in
FIG. 3, Cys6 is linked to Cys20, Cys14 is linked to Cys31, and
Cys33 is linked to Cys42. The EGF B-loop 200 is located between
Cys20 and Cys31, Thus, the minimum sequence or minimum peptide 400
of the EGF molecule that presents the EGF neutralizing domain 200
in the correct conformation is the sequence from Cys6 to Cys31, as
illustrated in FIG. 4.
[0092] A structure of a modified recombinant protein molecule
according to the disclosure expressing at least a portion of the
EGF molecule, including the EGF neutralizing domain according to an
illustrative embodiment is described with reference to FIG. 5. A
single mutation or change is made to Cys33 of the EGF molecule to
produce the modified synthetic molecule changing Cys33 to Ala33 to
remove the Cys33 to prevent any possible mis-folding problems.
[0093] Alanine is used because alanine is fairly `neutral` in terms
of functional characteristics and has the smallest side chain apart
from glycine. Alanine is therefore considered the least likely
residue to impart any non-native characteristics to the modified
recombinant protein. It is contemplated within tire scope of the
disclosure that potentially any other residue could be used, or
even no change made at all.
[0094] In an illustrative embodiment, any part, of the EGF molecule
could be used from the region defined by residues Met21-Ala30 up to
the entire EGF sequence. The sequences selected for expression in
the recombinant EGF-CT-B proteins in the examples include all of
the EGF sequence, and separately a region that is thought required
for correct presentation of the neutralizing domain defined as a
neutralizing domain in the context used, and doesn't include any
other part of the EGF that is not considered necessary to achieve
this.
[0095] In another illustrative embodiment, a protein comprised of a
homogeneous recombinant protein expressing a neutralizing domain of
vascular endothelial growth factor-A (VEGF-A) is disclosed. In an
illustrative embodiment, the protein is a recombinant protein
expressing or including VEGF-A sequences and CT-B sequences. In an
illustrative embodiment, the VEGF-A sequence will include the
neutralizing domain comprising the sequence from Cys57 to Cys104 of
the mature protein. In another illustrative embodiment, the
sequence of VEGF-A will include one or more flanking residues
extending up to Val14 and Lys108.
[0096] In another illustrative embodiment, a protein comprised of a
homogeneous recombinant protein expressing the ligand binding
domain of TGF-Beta receptor II is disclosed. In an illustrative
embodiment, the protein is a recombinant protein expressing or
including TGFB-RII sequences and CT-B sequences. The TGFB-RII
sequence will include any sequence of the extra-cellular domain
between Thr23 and Gln166.
[0097] In another illustrative embodiment, a protein comprised of a
homogeneous recombinant protein expressing the ligand binding
domain of the HGF receptor (c-Met) is disclosed. In an illustrative
embodiment, the protein is a recombinant protein expressing or
including HGF receptor sequences and CT-B sequences. Preferably,
the HGF receptor sequence will include any sequence of the
extra-cellular SEMA domain between Lys27 and Leu515.
EXAMPLE I
ELISA Protocols
[0098] In order to determine whether recombinant proteins, such as
the synthetic EGF-CT-B proteins according to the disclosure, can
display the EGF B-loop in the correct conformation, two commercial
monoclonal antibodies (Santa Cruz Antibodies, Cat No's 10825 and
10827) that were known to block binding of EGF to the EGF receptor
were obtained. Without being bound to any particular theory, it is
postulated from a number of sources that binding to the EGF
receptor is achieved in part via the region defined by residues
Met21-Ala30.
[0099] In an illustrative embodiment. 1 ug/ml and 2 ug/ml
concentrations of mAb 10825 and mAb 10827 were used to bind a
recombinant EGF (rEGF) protein in ELISA, and optical density (OD)
was measured at 450 nm. The results are illustrated in a bar graph
with reference to FIG. 6. As illustrated in FIG. 6, the rEGF
retains its natural conformation when adsorbed onto an ELISA plate
and 1 ug/ml of either mAb 10825 or mAb 10827 is sufficient to
obtain a good signal.
[0100] To assess recognition of residues Met21-Ala30, a plate was
coated with about 100 ul/well protein (rEGF) at about 1 ug/ml and
incubated at about 37.degree. C. for about 1 h. The plate was
washed twice with about 200 ul/well PBS-0.5% Tween (PBST), then
twice with about 200 ul PBS. The plate was blocked with about 200
ul/well PBS-2% milk powder (MPBS) and incubated for about 1 hour at
about. 37.degree. C. The plate was then washed twice with PBST and
twice with PBS, as above. About 100 ul of the test antibodies were
added at either about 1 ug/ml or about 2 ug/ml and incubated for
about 1 hour at about room temp (RT). The plate was washed again as
described above. Secondary, an antibody (HRP-Iabeled anti-mouse
Fc-specific, Sigma product code A0168) was added at about 1/1000
dilution, about 100 ul/well and incubated for about 1 h at about
RT. The plate was washed again as above, and developed with about
100 ul/well Sureblue TMB substrate until color developed (usually
about 5-10 min). The reaction was stopped with about 50 ul/well 1M
H2SO4, and the plate was read at about 450 nm.
[0101] Additionally, a competitive binding ELISA was carried out.
In the second ELISA the binding of each of the mAb 10825 and mAb
10827 antibodies to rEGF was assessed in the presence of either
free soluble peptide corresponding to the epitope of interest
(peptide sequence MYIEALDKYA) or a control irrelevant peptide
(peptide sequence SLAGSSGALSK). ELISAs with about 100 ul/well at
about 1 ug/ml of mAb 10825 plus about 1 ug/ml of the free soluble
peptide corresponding to the target epitope, about 1 ug/ml of mAb
10827 plus about 1 ug/ml of the free soluble peptide Met21-Ala30,
about 1 ug/ml of mAb 10825 plus about 1 ug/ml of the control
irrelevant peptide, and about 1 ug/ml of mAb 10827 plus about 1
ug/ml of the control irrelevant peptide were conducted.
[0102] The optical density (OD) was measured at 450 nm. The results
are illustrated in a bar graph with reference to FIG. 7. As
illustrated in FIG. 7, of the two antibodies, mAb 10825 and mAb
10827, it is clear that the mAb 10827 antibody binds to the
Met21-Ala30 neutralizing epitope and the mAb 10825 antibody docs
not. The mAb 10825 antibody is probably neutralizing by virtue of
stearically hindering receptor binding by blocking a region of EGF
conformationally proximal to the region defined by residues
Met21-Ala30. Thus, the mAb 10827 antibody binds to the rEGF
neutralizing epitope Met21-Ala30 in its native state, and was used
in the following analysis of the synthetic EGF-CT-B vaccine
precursors.
EXAMPLE II
EGF Neutralizing Epitope Presentation
[0103] To determine whether or not the recombinant protein EGF-CT-B
vaccine expressing the EGF on a termini of the CT-B sequence
interferes with or otherwise influences any of the desired inherent
characteristics of the EGF domain(s), specifically the correct
conformational presentation of the EGF Met21-Ala30 epitope, and the
ability of CT-B monomers to assemble into multimers (pentamer
rings) under appropriate physico-chemical conditions, six
recombinant proteins were created expressing the entire EGF coding
region on the CT-B sequence at either the N (Test 1-Test 3) or
C-terminus (Test 4-Test 6).
[0104] Test 1 and Test 4 include the recombinant protein EGF-CT-B
vaccine expressing the full length EGF sequence directly on the
CT-B domain. Test 2 and Test 5 include the synthetic EGF-CT-B
vaccine expressing the full length EGF sequence separated from the
CT-B domain by a short 3 amino acid peptide sequence. The
recombinant protein EGF-CT-B vaccine expressing the EGF sequence on
the N-terminal, includes SerGlyGly as the 3 amino acid peptide
sequence, and includes a KpnI restriction site. The recombinant
protein EGF-CT-B vacccineexpressing the EGF sequence on the
C-terminal, includes ScrScrGly as the 3 amino acid peptide
sequence, and includes a Xhol restriction site.
[0105] Test 3 and Test 6 include the recombinant protein EGF-CT-B
expressing the full length EGF sequence separated from the CT-B
domain by a short 5 amino acid peptide sequence. The recombinant
protein EGF-CT-B expressing the EGF sequence on the N-terminal,
includes GlyGlySerGlyGly as the 5 amino acid peptide sequence, and
includes a KpnI restriction site. The synthetic EGF-CT-B expressing
the EGF sequence on the C-terminal. includes SerSerGlyGlyGly as the
5 amino acid peptide sequence, and includes a Xhol restriction
site. The short 3 and 5 amino acid peptide sequences serve both to
distance the growth factor domain from the CT-B sequence, and also
to allow a degree of freedom of movement of one domain relative to
the other, thus reducing any potential steric hindrance.
[0106] Each of the six recombinant protein EGF-CT-B were cloned
into a bacterial expression vector (pIMS147), such that the
synthetic recombinant EGF-CT-B proteins could be expressed in E.
coli periplasm, and purified by the inclusion of a C-terminal 6xHis
tag. Each recombinant EGF-CT-B sequence was expressed, purified,
and quantified by means of protein get/Bradford assay.
[0107] The presentation of the EGF neutralizing epitope Met21-Ala30
in each of the six recombinant EGF-CT-B proteins was determined by
ELISA. The recombinant EGF-CT-B proteins, including one terminal
EGF domain were immobilized onto an ELISA plate. The EGF
Met21-Ala30 epitopes were detected with the mAb 10827 antibody
(Santa Cruz).
[0108] The ELISA plate was coated with serial 2-fold dilutions of
synthetic EGF- CT-B 6-His purified proteins and incubated at about
37.degree. C. for about 1 hour. The plate was washed and blocked
with about 2% MPBS, as described above. Washing involved pipetting
about 200 ul PBS or PBST into each well, inverting the plate and
flicking to empty the wells, and repeating. The mAb 10827 antibody
was then added to all the wells at about 1 .mu.g/ml and incubated
at about room temperature for about 1 hour. The plate was washed
once more and an anti-mouse Horse-Raddish Peroxidase (HRP) was
added to the wells and incubated for about a further 1 hour. The
plate was washed again and developed using SureBlue TMB.
[0109] Upon adding the SureBlue TMB substrate, the HRP conjugated
to the secondary antibody enzymaticrally processes the substrate to
yield a blue product. The reaction was observed and monitored until
it was decided that the color intensity has reached a sufficient
level. (If color begins to appear in the control wells, which
contain no primary antibody, then the reaction is stopped at this
point). The reaction is stopped by addition of about 50 ul H2SO4
which destroys HRP activity. It also changes the color of the
reaction product from blue to yellow. This can then be measured in
a plate reader at about 450 nm absorbance.
[0110] The results of the binding ELISAs are illustrated in a line
graph with reference to FIG. 8. As illustrated in FIG. 8, the mAb
10827 antibody was able to bind to all six recombinant EGF-CT-B
6-His purified proteins, demonstrating that in each formulation the
EGF-Met21-Ala30 epitope is presented in its native conformation and
is accessible to components of the immune system.
[0111] In order to confirm that the synthetic recombinant EGF-CF-B
protein included expressions of the EGF domain and the CT-B
sequence, a second ELISA was performed whereby rather than
adsorbing the recombinant protein directly onto the plates, the
recombinant protein was instead captured using a rabbit anti-CT-B
antibody (Antibodies On-Line), as shown in FIG. 9. As this
`capture` antibody is specific to native CT-B, the assay
demonstrates that the detected EGF neutralizing domains are
components of a larger recombinant protein that includes a
correctly folded CT-B domain.
EXAMPLE III
EGF-CT-B Protein Multimer Assembly
[0112] In order to examine the effect of expressing a structural
domain comprising a growth factor on the termini of the CT-B
derived recombinant protein on assembly of multimers from monomeric
sub-units, synthetic proteins Test 1-Test 6 were run on an SDS-PAGE
gel under native conditions (non-reduced, non-boiled). The
synthetic recombinant EGF-CT-B proteins were then transferred onto
a nitrocellulose membrane by electro-blotting, and were probed
using a rabbit anti-CT-B antibody (as described above in example
II). Binding of a secondary HRP-labeled anti-rabbit antibody was
detected via the light emitted using ECL substrate on
autoradiograph film. As illustrated in FIG. 10, the Western blot
confirms the presence of high molecular weight CT-B, indicating
that the synthetic EGF-CT-B monomer proteins are able to assemble
into multimers via the CT-B domain.
[0113] In a separate experiment, duplicate samples of native
(non-boiled or reduced) CT-B protein were incubated for 5 min. at a
range of different pH values from pH 1.0 to 7.0. Following
incubation, one of each duplicate sample was neutralized back to pH
7.0 for one hour. All samples were then run on an SDS-PAGE gel,
Western blotted, and protein detected with anti-CTB antibody (FIG.
16). This demonstrates that i) CTB pentamers can be reduced to
monomers at pH 3.0 or below in 5 min., and ii) that returning to
neutral pH restores the formation of pentamers. It has previously
been demonstrated that a chimeric protein comprising a CT-B protein
fused to a camelid antibody binding site and tags via a suitable
linker (molecular weight of 16 kDa) can be made to form
functionally active pentamers (Li et. al., 2009 Molecular
Immunology 46; 1718-1726).
EXAMPLE IV
Bivalent Synthetic EGF-CT-B Proteins
[0114] In an illustrative embodiment, two additional synthetic
recombinant EGF-CT-B proteins were created. in which i) a full
length EGF gene is expressed at both the N- and C-termini,
separated from the CT-B gene by the three amino acid sequence as
described for Test-2 and Test-5 above, and designated `E2` or ii) a
truncated EGF including the Met21-Ala30 neutralizing epitope is
expressed at both termini of the CT-B gene as above, and designated
`B2`. Both recombinant proteins were cloned into the E. coli
expression vector pIMS147 as described above. Both recombinant
EGF-CT-B proteins were expressed and purified as described
previously, and assayed for the presence of correctly folded CT-B
domain and presentation of EGF neutralizing epitope Met21-Ala30 in
the correct conformation. The results are illustrated in a line
graph with reference to FIG. 11. As illustrated in FIG. 11, both of
the E2 and B2 recombinant EGF-CT-B proteins comprise both a CT-B
domain and at least one functionally correct EOF Met21-Ala30
epitope displayed so as to be accessible to an antibody.
[0115] Further analysis involved running samples of purified E2 and
B2 recombinant EGF-CT-B proteins on non-denaturing SDS-PAGE gels at
pH 7.0 without first boiling the samples, and transferring to
nitrocellulose membranes via electro-transfer. The transferred
proteins were detected using the AbOL (Antibodies On-line)
anti-CT-B rabbit polyclonal antibody and an MRP-labeled anti-rabbit
antibody. As illustrated in FIG. 12, the Western blot indicates
that the CT-B domain-containing recombinant proteins exist both as
monomers, and have also formed into a series of oligomeric
multimers comprising dimers, trimers, tetramers and pentamers.
EXAMPLE V
EGF-CT-B Protein Sequence
[0116] One example of a sequence of a synthetic recombinant
EGF-CT-B protein is illustrated in FIG. 13. As illustrated in FIG.
13. the sample sequence illustrates the synthetic protein sequence
including two full length EGF sequences, which are underlined, and
a CT-B sequence, which is italicized.
EXAMPLE VI
EGF-CT-B Protein Sequence
[0117] Another example of a sequence of a synthetic recombinant
EGF-CT-B protein is illustrated in FIG. 14. As illustrated in FIG.
14, the sample sequence illustrates the protein sequence including
two EGF neutralizing domain sequences, which are underlined, and a
CT-B sequence, which is italicized.
EXAMPLE VII
EGF-CT-B Protein Sequence
[0118] Yet another example of a sequence of a recombinant EGF-CT-B
protein is illustrated in FIG. 15. As illustrated in FIG. 15, the
sample sequence illustrates the protein sequence including partial
sequences of the EGF molecule including the EGF neutralizing domain
(Cys6 to Cys31), which are underlined, and a CT-B sequence, which
is italicized.
EXAMPLE VIII
EGF-CT-B Protein Sequences Including Linkers
[0119] In other illustrative embodiments, additional recombinant
EGF-CT-B proteins including one or more linkers or space is are
disclosed herein. One or more of the embodiments described above
include EGF fused to CT-B at one or both termini of the CT-B such
that one gene ran directly into the next. These resulting
recombinant or chimeric proteins essentially included EGF fused
directly to CT-B. In other illustrative embodiments, the EGF and
CF-B components of the chimeric protein are effectively separated
by 3 or 5 amino acids, which form a flexible spacer or linker
between the two domains. The following amino acids that can be used
as linkers included but are not limited to the following: SSG,
SSGGG, SGG, GGSGG, and GGGGS
[0120] The addition of the linkers can reduce interferences, for
example, from steric hindrance, and aid in the formation of
pentamers by the CT-B domain. The linkers also enabled unique
restriction sites to be introduced within the linkers to allow
subsequent manipulation of the genetic constructs. In this example,
eight constructs (T1-T6, E2, and B2) are described, having the
sequences listed in the Table illustrated in FIG. 17. In one
illustrative embodiment the restriction sites include but are not
limited to the following: Xho1, Kpn1, BspE1, and Spe1.
[0121] Western blot analysis of the constructs T1-T6, E2, and B2
was performed and are described below in connection with FIG. 18.
As illustrated in FIG. 18, the Western blot of the constructs E2
and B2, there appears to be some interference, for example, steric
hindrance and/or other interference, that caused the proteins
produced to be comprised of a variety of oligomers, for example,
monomer, dimer, trimer, etc. Alternatively, the concentration of
protein present in samples may have influences oligomerization, as
it is a dependent factor for native CTB pentamerization.
[0122] The lowest bands correspond to monomers, the next up to
dimers etc. As B2 includes truncated EGFs, it appears to be smaller
than E2, which is illustrated by B2 being lower on the Western
blot.
[0123] A similar result is found for the constructs T1-T6, although
the numbers and proportions of oligomers vary from construct to
construct. Initially, it appeared that proteins with EGF on the
N-terminus including the amino acid linkers might give a higher
proportion of pentamer, However, subsequently it was found that the
proportion of pentamer varied from batch to batch.
[0124] Since it was initially postulated that fusion at one or
other terminus favors pentamerization, two tandem fusions in
addition to the E2 construct were constructed and are illustrated
in FIG. 19. The first tandem fusion, designated E2N. includes two
consecutive EGF's at the N-terminus of the CT-B. Wherein L-3 is
SGG, L-4 is GSSG The second fusion, designated E2C, includes two
consecutive EGFs at the C-terminus of the CT-B Wherein 1.-3 is SSG,
L-5 is GGSGG
[0125] In an illustrative embodiment, the amino acid linker lengths
at the N-terminus and the C-terminus were extended to determine
whether or not the amino acid linker length at each end yields
pentamer only, or perhaps that one end, the N-terminus or the
C-terminus, yields a higher proportion of pentamer. Referring to
FIG. 20, the N-terminus and C-terminus amino acid linkers were
extended using the constructs T2/3 and T4/5, respectively. The
illustration (FIG. 20) refers to the c-terminal fusion E2C. In this
illustrative example, 1.,3 is SSG, L5 is SSGGG, L8 is SSGGGSGG and
L10 is SSGGGGSGGG. In the N-terminal version, the inserted linker
spacers were about 7 and 9 residues in length. In that example the
4 linkers would be: L3 SGG, L5 GGSGG, L7 TSGGGSG and L9 TSGGGGSGG.
Each of the linker-spacers can be inserted into each of the shorter
L3 and L5 linkers. As a result, inserting L7 into L5 or L9 into L3
both yield linkers of 12 residues, HOWEVER they would have
different sequences, termed `a` and `b` below. The N-terminus
linkers were also extended to 10, 12 and 14 amino acids, and the
C-terminus were extended to 11, 13 and 15 amino acids, as
illustrated in FIG. 20. In this illustrative example L10 is
SSGGGSGGSSG. L12a is GGSGGTSGGGSG, L12b is SGGTSGGGGSGG, and L14 is
GGSGGTSGGGGSGG. Similarly. L11 is SSGGGSGGSSG, L13a SSGGGGSGGGSSG,
L13b SSGGGSGGSSGGG, and L15 SSGGGGSGGGSSGGG.
[0126] Referring to FIG. 21. Western blot analysis of the tandem
EGF fusions, E2N and E2C. compared to the original bivalent
construct with the original E2 demonstrate that both E2 and E2C
produce many oligomers. E2N also produces oligomers, however there
is a strong indication that the first EGF domain is being either
expressed as a truncated protein, or is being cleaved off at some
stage during expression/purification.
[0127] A comparative Western blot analysis was also performed on
the monovalent `T` constructs with the extended linkers, and is
illustrated in FIG. 22. When the above linker extensions were
introduced to the constructs already named T2 and T3 (N-terminal, 3
and 5 aa linkers respectively), we get T2SL (Short extended Linker,
L10). T2LL (Long Linker, L12a) T3SL (Short linker L12b), and T3LL
(Long Linker L14). Similarly the N-terminal T5 and T6 constructs
become T5SL (with L11), T5LL (with L13a), T6SL (with L13b) and T6LL
(with L15).
[0128] When the linker spacers are inserted, they can actually be
cloned in either of two directions, giving quite different
sequences. Wherever possible, sufficient clones were sequenced to
find one with the insertion in the desired direction. In the case
of T3LL-Rev, initially we only had a clone with the desired linker
length (i.e. 14 aa's) but with the insert in the `wrong`
orientation. It does serve to illustrate how the precise sequences
of these linkers isn't necessarily critical at least as far as
acting as a physical spacer. The actual linker sequence of T3LL-Rev
would be GGSGGTRPSTAATS. (underlined=inverted section).
[0129] As illustrated in the Western blot illustrated in FIG. 22, N
and R refer to native and reduced/denatured protein, respectively.
The first two lanes illustrate wild type CT-B as a pentamer
(native) and a monomer (reduced). As illustrated in the other
lanes, it can be seen that T3 (including the 5 amino acid linker)
produced some oligomers of various sizes, however all N-terminus
constructs with longer linkers produce primarily pentamer when run
under native conditions.
[0130] In contrast as illustrated in FIG. 23. the Western blot of
the C-terminus constructs produced multiple bands under native
conditions even with extended linkers.
[0131] Based on this data, the tandem N-terminus fusion of EGF to
CT-B appears to be of significant interest. Additionally, the first
linker (between the two EGF domains) may be extended to attempt to
prevent the truncation/proteolysis described above with the E2N
construct and to allow flexibility when introducing alternative
growth factors. The Sequence for the N-terminus FUSION of EGF to
CT-B with the extended first linker) is as follows:
TABLE-US-00001 H H H H H H I E G R N S D S E C P L S H D G Y C L H
D G V C M Y I E A L D K Y A C N C V V G Y I G E R C Q Y R D L K W W
E L R G G S G G T S G G G G S G G T P Q N I T D L C A E Y H N T Q I
H T L N D K I F S Y T E S L A G K R E M A I I T F K N G A T F Q V E
V P G S Q H I D S Q K K A I E R M K D T L R I A Y L T E A K V E K L
C V W N N K T P H A I A A I S M A N
[0132] While the homogeneous recombinant proteins expressing or
incorporating EGF B-loop epitopes have been described and
illustrated in connection with certain embodiments, many variations
and modifications will be evident (o those skilled in the art and
may be made without departing from the spirit and scope of the
disclosure.
EXAMPLE IX
Bi-specific IGF1 -EGF-CTB Protein (a)
[0133] In order to establish the feasibility of targeting more
titan one growth factor with a single synthetic recombinant protein
a gene encoding the human insulin-like growth factor 1 (IGF1) was
synthesized including short flanking regions to enable cloning into
the construct E2N described in example VIII. Briefly, the
N-terminal EOF gene was excised from the vector by digesting the
DNA with the restriction endonucleases Nco1 and Xho1. It was then
replaced with the similarly digested human IGF I gene using methods
familiar to those skilled in the art. The resulting DNA vector was
sequenced to confirm that it encoded the required recombinant gene
in such a way as to allow the recombinant protein to be expressed
as designed, The sequence of the novel recombinant protein is
illustrated in FIG. 24.
[0134] Subsequently, the protein generated by the expression of the
aforementioned vector was analyzed by ELISA to demonstrate that
both growth factors can be simultaneously displayed to components
(i.e. antibodies) of the mammalian immune system. Briefly, wells of
an ELISA plate were coated with an appropriate dilution of anti-CTB
antibody and then blocked with PBS containing 2% milk powder as
described previously. Samples of the recombinant protein were
applied to the plate and incubated for 1 hour at room temperature.
After washing, different wells prepared as described were then
incubated with 1/1000 (or as per supplier's recommendations) of
either i) mouse anti-EGF antibody AbOL 10827 or ii) rabbit
anti-human IGF1 2o antibody. After washing, the wells were
incubated with an appropriate dilution of i) HRP-labeled anti-
mouse antibody or ii) HRP-labeled anti-rabbit antibody and then
developed as described previously. As illustrated in FIG. 25, the
signals generated confirmed that both IGF and EGF are displayed in
their native configurations. The signal generated by the anti-IGF
antibody also confirms that IGF, EGF and CTB sequences are present
in the same molecule due to the relative positions of the encoding
DNA sequences in the expression vector,
EXAMPLE X
Bi-Specific IGF1-EGF-CTB Protein (b)
[0135] In order to demonstrate that bi-specific recombinant
proteins can be generated using the natural characteristic of CTB
to form oligomers, the IGF gene described in example IX was
modified by PCR using techniques familiar to those skilled in the
art to enable it to be cloned into the T5 construct, replacing the
EGF gene, The resulting recombinant protein included IGF sequences
C-terminal to CTB sequences, and separated by a 3 amino acid linker
(FIG. 26).
[0136] Samples of the above recombinant protein were combined
separately with equal (molar) amounts of i) T2 protein and ii) T5
protein. Each of the mixtures was adjusted to pH 3.0 by the
addition of buffered 10 mM Tris-HCL as required and incubated at
4.degree. C. for 15 min to dissociate any oligomers present. The
protein mixtures were then neutralized, and incubation continued
for 60 min in order to encourage oligomerization. To detect the
presence of hetero-oligomers, wells of an ELISA plate were coated
with either mouse anti-EGF antibody or rabbit anti-IGF antibody,
and blocked. After washing. IGF-CTB/T2 mix and IGF-CTB/T5 mix were
applied separately to either wells coated with anti-EGF antibody or
with anti-IGF antibody, and incubated for 60 min at room
temperature.
[0137] After washing, antibody specific to the growth factor not
targeted by the coating antibody was added and incubated for 60
min. Thus, rabbit anti-IGF antibody was applied to wells coated
with mouse anti-EGF antibody, and vice-versa. After washing to
remove unbound 2o antibody. HRP-labeled anti-mouse or HRP-labeled
anti rabbit antibody was applied as appropriate to target the 2o
antibody. The results are illustrated in FIG. 27. and demonstrate
that anti-EGF coating antibody can capture and immobilize protein
containing IGF sequences. Similarly. anti-IGF antibody can capture
and immobilize protein that includes EGF sequences. In both cases,
this is caused by oligomerization of IGF and EGF-containing
monomers such that both are present. Moreover, the hetero-oligomers
are able to form when both growth factors are located at opposite
termini of the CTB component (i.e. IGF-CTB and T2) and when both
growth factors are on the same (C) terminus (i.e. IGF-CTB and T5).
The assay also works in either orientation.
EXAMPLE XI
Diverse Growth Factor Presentation
[0138] In order to further demonstrate the flexibility of the
present invention, a panel of recombinant proteins were generated
that included sequence derived from CTB together with additional
sequence derived from one or more of a range of growth factors and
representing a range of domains of varying size according to FIG.
28 using standard techniques familiar to those practiced in the
art. Samples of each of the proteins was prepared by expression of
the genetic construct in E. coli and purified using IMAC via the
hexa-histidine tag N-terminal to each protein. Purified recombinant
proteins were assayed by ELISA to demonstrate that the each of the
different sequences was present and displayed correctly using
antibodies specific for each sequence (FIG. 29), A native protein
was run with samples of the recombinant protein including sequences
derived from mTGF B1 and CTB and a Western blot prepared (FIG. 30).
Protein was detected with .alpha.-CTB antibody and showed that
under the conditions used the recombinant chimeric protein wax able
to form stable pentamers. retaining this characteristic of CTB.
EXAMPLE XII
Growth Factor Receptor Presentation
[0139] In order to demonstrate that the technology described in the
present disclosure is applicable to the functional display of
proteins other than growth factors, recombinant proteins including
sequences derived from growth factor receptor and CTB were
generated, and shown to present such sequences in a natural
conformation in conjunction with CTB sequences. DNA encoding the
protein sequence of human TGF-beta1 was cloned upstream of the CTB
gene, by replacing the EGF coding DNA from the T3LL clone using
standard techniques familiar to those practiced in the art. This
construct was used to generate a recombinant protein including both
human TGF-beta1 and CTB sequences (FIG. 31a). Likewise, a second
recombinant protein was generated that included sequences of the
extra cellular ligand-binding domain of the human TGF Beta receptor
2 and CTB (FIG. 31b).
[0140] The simultaneous presentation of both TGF-Beta R2 and CTB
sequences on a single recombinant protein was established by
capture ELISA. Briefly, wells of an ELISA plate were coated with i)
mouse anti-CTB antibody or ii) goat anti-TGF Beta R2 antibody and
blocked with PBS containing milk powder. Samples of the recombinant
protein according to FIG. 31b were then contacted to the wells and
incubated for about 1 hour. Following washing, the wells were
contacted with i) goat anti-TGF Beta R2 antibody or ii) mouse
anti-CTB antibody respectively and incubated for 1 hour. Following
washing, the wells were contacted with i) HRP-labeled anti-sheep
(goat) antibody and ii) HRP-labeled anti-mouse antibody
respectively and incubated for about 1 hour. The plate was
developed with TMB substrate and color intensity measured at 450
nm. The assay demonstrated that both TGF-beta R2 and CTB sequences
were present on the same chimeric recombinant protein (FIG.
32).
[0141] In order to demonstrate that both TGF-Beta 1 and TGF Beta R2
were both presented separately with CTB sequences in a native
configuration, the interaction between TGF beta1 and Hs natural
receptor was determined by ELISA. Briefly, wells of an ELISA plate
were coated with mouse anti-CTB antibody as blocked. The wells were
then contacted with the recombinant protein containing human
TGF-beta1 and CTB sequences as described in FIG. 31a and incubated
for about 1 hour. After washing, the wells were contacted with the
recombinant protein containing human TGF-betaR2 and CTB sequences
as described in FIG. 31b and incubated for about 1 hour. The wells
were washed and then contacted with goat anti-TGF Beta R2 antibody
for 1 hour. Finally, the wells were washed and contacted with
HRP-labeled anti-sheep (goat) antibody for about 1 hour. The plate
was developed with TMB substrate and read at 450 nm. FIG. 33
illustrates that the two recombinant proteins are able to reproduce
the natural receptor-ligand binding interaction, and that this is
not disturbed by the anti-receptor antibody used in the assay.
EXAMPLE XIII
Immune Responses of Mice to Recombinant Protein Formulations
[0142] In another experiment groups of mice were immunized with
recombinant proteins including sequences from CTB and one or more
growth factors according to the present disclosure in order to
assess the effects of various formulations on immune responses of
said mice. Six groups of mice, each comprising six mice were
immunized, with a different recombinant protein formulation
according to the schedule described below.
[0143] Unless otherwise stated, mice were immunized with 25 .mu.g
recombinant protein in 75 .mu.l buffer, emulsified in 75 .mu.l
montanide adjuvant Immunogens were administered via i.m. injection
at day 0 and day 14, Serum samples were taken at day 0
(pre-immunization) and day 28 and were analyzed for the presence of
IgG antibodies against the growth factor sequences contained within
the immunizing recombinant protein. The groups of mice were
immunized with the following antigens:
[0144] Group 1: SB1, 75 .mu.l (25 .mu.g) recombinant protein
including human IGF and CTB sequences according to FIG. 26
emulsified with 75 .mu.l montanide;
[0145] Group 2: SB2, 75 .mu.l (25 .mu.g) recombinant protein
including human EGF and CTB sequences as described in example VIII
and referred to as T3LL, emulsified with 75 .mu.l montanide;
[0146] Group 3: SB3, 75 .mu.l (25 .mu.g) recombinant protein
including human IGF, human EGF and CTB sequences according to FIG.
24 and as described in example IX, emulsified with 75 .mu.l
montanide;
[0147] Group 4: SB4. 37.5 .mu.l (12.5 .mu.g) SB1 and 37.5 .mu.l
(12.5 .mu.g) SB2 combined by the method as described in example X
and including oligomers containing both IGF-CTB and EGF-CTB,
emulsified with 75 .mu.l montanide;
[0148] Group 5: SB5, 75 .mu.l (25 .mu.g) SB1, as for Group 1,
except emulsified with 20 .mu.l Matrix-M adjuvant; and
[0149] Group 6: SB6, 37.5 .mu.l (12.5 .mu.g) SB1 emulsified with
37.5 .mu.l montanide, followed after 5 min by 37.5 .mu.l (12.5
.mu.g) SB2 emulsified with 37.5 .mu.l montanide and administered
via a different location.
[0150] Immediately prior to, and 14 days after immunization, blood
samples were taken and scrum analyzed by ELISA. for the presence
and relative titres of IgG antibodies against the growth factor
component of the recombinant protein immunizing antigens. ELISA
plates were coated with commercially available recombinant human
IGF or EGF at 1 .mu.g/ml concentration. After blocking and washing,
serum from subject mice at various dilutions was applied to wells
and incubated for 1 hour at room temperature. Un-bound antibody and
other proteins were removed by washing, and bound mouse IgG
detected with HRP-labeled anti- mouse antibody.
[0151] All six groups included animals that raised a specific
immune response to the growth factor component of the immunogenic
recombinant chimeric proteins. It is evident that stronger
responses are seen to EGF than to IGF throughout, including groups
where sequences from only one growth factor was included (Groups 1
and 2, FIGS. 34 and 35). Without being bound to any particular
theory, this is probably a reflection of the degrees of homology
between the mouse and human proteins, whereby the EGF's differ by
15/53 residues and the IGF's only differ at 4 of 70 residues. It is
also notable that differences between the responses of individual
animals within a group are often greater than differences between
groups to the same antigen.
[0152] The use of Matrix-M rather than Montanide as adjuvant (Group
5 compared to Group 1, FIGS. 40 and 34) resulted in a poorer
response, with one of the mice not responding at all, and four
other samples needing to be screened at much higher concentrations
than with Montanide.
[0153] Groups 3, 4 and 6 received proteins that included sequences
from both EGF and IGF, the difference being the formulation or
administration. Group 3 mice, receiving recombinant protein that
included both EGF and IGF sequences on each protein molecule, all
responded to EGF though two of the six did not show an .alpha.-IGF
response (FIGS. 36 and 37), Groups 4 and 6 mice also all generated
antibodies to EGF (FIGS. 38,39 and 41). In Group 4 one animal did
not respond to IGF and another gave only a very weak response. Only
in Group 6, where EGF and IGF-containing proteins were administered
separately and at. different locations, did all 6 animals mount a
response to IGF.
EXAMPLE XIV
Generic Single-Step Purification
[0154] A simple first-stage purification process is desired that
can be applied to any and all of the immunogenic recombinant
proteins detailed in the present disclosure. Ideally, the
purification will not require the inclusion of an affinity tag such
as hexa-histidine, MBP, FLAG etc. The recombinant proteins of the
present disclosure are related in that they all include at least
some sequence derived from the Vibrio cholera CT-B toxin sub-unit,
or a synthetic functional equivalent, lit is envisaged that
purification could be achieved by the use of monoclonal or
polyclonal antibodies, however monoclonal antibodies are expensive
to produce. Polyclonal antibodies are less expensive, however it is
likely that variations in performance will be seen between batches
from the same animal, and between individual animals.
Immuno-affinity purification also requires harsh conditions such as
low pH to elute target protein that can adversely affect the target
protein, and will limit the re-use of the affinity matrix. It also
involves the introduction of additional protein into the production
process, which is preferably avoided.
[0155] In the native CT holotoxin, the toxin binds to
mono-ganglioside Gm1 (FIG. 42) found on the surface of most
mammalian cells, including epithelial cells of the respiratory
tract and gut. Binding is effected by the CT-B sub-unit, and only
CT-B oligomers bind of Gm1. It is therefore envisaged that CTB
immobilized onto a suitable support could be used for the
purification of the immunogenic recombinant proteins of the present
disclosure. The use of CTB is not thought to be a preferred method
however for several reasons, notably that CTB Is only available
commercially as material purified from bovine brain. The use of
animal material, and the use of bovine brain tissue in particular
is not suitable for use in production of therapeutic products.
[0156] The binding of CTB to Gm1 is known to involve a terminal
galactose moiety on the branched glyco-molecule Gm1 binding to two
adjacent CTB sub-units. It is therefore envisaged that galactose
immobilized to a suitable solid support would provide a generic
means to purifying the recombinant proteins of the present
disclosure. To assess the applicability of this approach, the gene
encoding CTB was cloned into a bacterial protein expression vector
designed for periplasms protein recovery using techniques familiar
to those practiced in the art and transformed into various strains
of E. coli bacteria. Galactose-sepharose resin (FIG. 43) was
sourced from Pierce (Pierce Cat No. 20372). Fifty milliliter
cultures of the CTB-expressing clones in XL1-Blue, BL21 and TG1 E.
coli strains were grown and induced to express recombinant CTB
overnight at 37.degree. C. The cells were harvested by
centrifugation and the clarified media retained for extraction of
CTB. The periplasmic contents of the cell pellets were released by
osmotic shock using standard methods familiar to those practiced in
the art yielding 10 ml per culture.
[0157] The galactose sepharose resin was washed with 200 mM NaCl,
50 mM Tris HCl 5 mM EDTA pH 7.5 (TEN buffer) according to
manufacturer s instructions. NaCl, Tris-HCl pH 7.5 and EDTA were
added to the conditioned media and periplasmic fractions to a final
concentration of 200 mM NaCl, 50 mM Tris-HCl and 5 mM EDTA, 0.5 ml
washed galactose sepharose was added to each conditioned media and
periplasmic fraction, and incubated with agitation at 4.degree. C.
for 2-3 h. The resin was recovered into BioRad columns and washed
with 30 bed volumes of ice-cold TEN buffer. The bound protein was
eluted by re-suspending the resin in 0.5 ml 1 M galactose in PBS
and incubating for 10 min. The column was drained and the elate
retained few analysis. The elution step was repeated several times,
and fractions analyzed for the presence of CTB. Almost all of the
expressed CTB protein was found in the culture media Samples of
pre-purification conditioned media and periplasmic fraction,
together with pooled column eluates containing purified CTB (from
the media) were analysed by SDS- PAGE and compared with His-tagged
CTB purified by IMAC (FIG. 44). It can be seen that highly purified
CTB was obtained from the culture supernatants of all three
strains, with XL1-Blue cells giving the highest yields (Lanes 4, 7
and 10). The purity compares well with that seen from IMAC
purification (Lane 11), and includes significant pentameric
protein.
Additional Embodiments
[0158] In another illustrative embodiment, a vaccine comprised of a
homogeneous recombinant protein for improving the presentation of
and increasing the number of tumor antigen epitopes as elements of
a synthetic immunogenic recombinant protein is disclosed herein. In
one illustrative embodiment, a vaccine formed from a recombinant
protein expressing all or portions of a polypeptide sequence and a
tumor antigen is described herein.
[0159] In an illustrative embodiment, the recombinant proteins
disclosed herein may include or express a high proportion of a
protein sequence derived from rumor antigens and/or epitopes
thereof, as a function of total molecular weight. These tumor
antigen epitopes can be multiple copies of whole or part of a
single tumor antigen, or copies of whole or part of more than one
different tumor antigen.
[0160] In an illustrative embodiment, the recombinant protein is an
immunogenic protein molecule expressing one or more sequences that
fold into a physical structure, for example expressing one or more
sequences of a cholera toxin B (CT-B) protein from Vibrio cholera
or a synthetic equivalent, and expressing one or more sequences of
one or more tumor antigens or parts thereof.
[0161] In an illustrative embodiment, the sequence of the tumor
antigen may- include a sequence of a Prostate Specific Antigen
(PSA) or part thereof. In other illustrative embodiments, the tumor
antigen may include a full length or part thereof of one or more of
the following tumor antigens, including, but not limited to, PSA,
and other tumor antigens.
[0162] In another illustrative embodiment, a protein comprised of a
homogeneous recombinant protein for improving the presentation of
and increasing the number of receptor binding sites as elements of
a immunogenic recombinant protein is disclosed herein. In one
illustrative embodiment, a recombinant protein expressing all or
portions of a polypeptide sequence and a receptor is described
herein.
[0163] In an illustrative embodiment, the recombinant proteins
disclosed herein may include or express a high proportion of a
protein sequence derived from receptors and/or binding sites
thereof, as a function of total molecular weight. These binding
sites can be multiple copies of whole or part of a single receptor,
or copies of whole or part of more than one different receptor.
[0164] In an illustrative embodiment, the recombinant protein Is an
immunogenic protein molecule expressing one or more sequences that
fold into a physical structure, for example expressing one or more
sequences of a cholera toxin B (CT-B) protein from Vibrio cholera
or a synthetic equivalent, and expressing one or more sequences of
one or more receptors or parts thereof.
[0165] In an illustrative embodiment, the sequence of the receptor
may include a sequence of a Human Epidermal growth factor Receptor
2 (Her2) or part thereof and/or a Human Epidermal growth factor
Receptor 3 (Her3) or part thereof In other illustrative
embodiments, the receptor may include a full length or part thereof
of one or more of the following receptors, including, but not
limited to, Her2, Her3, and other receptors,
[0166] In other illustrative embodiments, the recombinant protein
is an immunogenic protein molecule expressing one or more sequences
that fold into a physical structure, for example expressing one or
more sequences of a CT-B or a synthetic modified variant, and
expressing various combinations of one or more sequences of one or
more growth factors or parts thereof, one or more sequences of one
or more tumor antigens or parts thereof, and one or more sequences
of one or more receptors or parts thereof.
[0167] In an illustrative embodiment, the recombinant protein
includes expressions or sequences of one or more growth factors or
parts thereof and one or more sequences of one or more tumor
antigens or parts thereof. In one embodiment, the recombinant
protein includes one or more sequences of a CT-B or a synthetic
modified variant, a PSA or part thereof, and an IGF-1 or part
thereof.
[0168] In another illustrative embodiment, the recombinant protein
includes expressions or sequences of one or more growth factors or
parts thereof and one or more sequences of one or more receptors or
parts thereof. In one embodiment, the recombinant protein includes
one or more sequences of a CT-B or a synthetic modified variant, a
Her2 or part thereof, and an IGF-1 or part thereof. In another
embodiment, the recombinant protein includes one or more sequences
of a CT-B or a synthetic modified variant, a Her2 or pan thereof, a
Her2 or part thereof, and a PDGF or part thereof.
[0169] In another illustrative embodiment, the recombinant protein
includes expressions or sequences of one or more tumor antigens or
parts thereof and one or more sequences of one or more receptors or
parts thereof.
[0170] In yet another illustrative embodiment, the recombinant
protein includes expressions or sequences of one or more growth
factors or pans thereof, one or more sequences of one or more tumor
antigens or parts thereof, and one or more sequences of one or more
receptors or parts thereof.
[0171] In any of the embodiments described above, in addition to
expressing one or more copies of a single tumor antigen, receptor,
and/or growth factor, presented as a single tumor antigen,
receptor, and/or growth factor or part thereof per physical site,
and/or as chains of repetitive tumor antigen, receptor, and/or
growth factor sequences (for example, n=1 to 10). The recombinant
proteins according to the disclosure may also include expressions
of one or more neutralizing domains or binding sites from two or
more different tumor antigens, receptors, and/or growth factors
present as single or as chains at different positions within the
sequences of the recombinant proteins. For example. Ac recombinant
proteins may include expressions or sequences of a full length or a
portion of two to four different tumor antigens, receptors, and/or
growth factors, and/or a full length or a portion of one or more
tumor antigens, receptors, and/or growth factors as single epitopes
or binding sites or as two or more tandem repeats.
[0172] The resulting proteins are single polypeptides expressing a
tumor antigen, receptor, and/or growth factor or one or more
epitopes or binding sites thereof within the sequence of the
recombinant proteins. In an illustrative embodiment, the sequences
of the recombinant proteins expresses one or more portions of a
CT-B sequence and presents the tumor antigen, receptor, and/or
growth factor expression(s) including at least one or more
expression(s) of epitopes or binding sites thereof on a surface of
the immunogenic recombinant proteins in a natural conformation.
[0173] According to the disclosure, the expressions of the tumor
antigen epitopes, receptor binding sites, and/or growth factor
epitopes should be folded allowing their natural conformation to be
substantially retained and presented to components of the host
immune system in such a way as to elicit a robust host immune
response. Examples of suitable natural protein models include, but
are not limited to, cholera toxin B sub-unit, listeria, tetanus
toxoid, diphtheria toxoid, bacteriophage coat protein, adenovirus
and other viral coat proteins. Alternatively, non-natural
`synthetic` polypeptides may be used that fulfill the requirements
of conferring immunogenicity to the whole protein and allowing
appropriate presentation of tumor antigen epitopes, receptor
binding sites, and/or growth factor epitopes to the host immune
system.
Adjuvant
[0174] Certain illustrative embodiments as provided herein include
recombinant proteins according to the disclosure within vaccine
compositions and immunological adjuvant compositions, including
pharmaceutical compositions, that contain, in addition to
recombinant proteins at least one adjuvant, which refers to a
component of such compositions that has adjuvant activity. An
adjuvant having such adjuvant activity includes a composition that,
when administered to a subject such as a human (e.g., a human
patient), a non-human primate, a mammal or another higher
eukaryotic organism having a recognized immune system, is capable
of altering (i.e., increasing or decreasing in a statistically
significant manner, and in certain preferred embodiments, enhancing
or increasing) the potency and/or longevity of an immune response.
In certain illustrative embodiments disclosed herein a desired
antigen and or antigens contain within a protein carrier, and
optionally one or more adjuvant, may so alter, , elicit or enhance,
an immune response that is directed against the desired antigen and
or antigens which may be administered at the same time or may be
separated in time and/or space (e.g., at a different anatomic site)
in its administration, but certain illustrative embodiments are not
intended to be so limited and thus also contemplate administration
of recombinant protein in a composition that does not include a
specified antigen but which may also include but is not limited to
one or more co-adjuvant, an imidazoquinline immune response
modifier.
[0175] Accordingly and as noted above, adjuvant include
compositions that have adjuvant effects, such as saponins and
saponin mimetics, including QS21 and QS21 mimetics (see, e.g., U.S.
Pat. No. 5,057,540: EP 0 362 279 B1; WO 95717210), alum, plant
alkaloids such as tomatine, detergents such as (but not limited to)
saponin, polysorbate 80, Span 85 and stearyl tyrosine, one or more
cytokines (e.g., GM-CSF, IL,-2, IL-7, IL-12, TNF-alpha, IFN-gamma),
an imidazoquinoline immune response modifier, and a double stem
loop immune modifier (dSLIM, e.g., Weeratna et al., 2005 Vaccine
23:5263).
[0176] Detergents including saponins are taught in, e.g., U.S. Pat.
No. 6,544,518; Lacaille-Dubois, M and Wagner H. (1996 Phytomedicine
2:363-386), U.S. Pat. No. 5,057,540, Kensil, Crit. Rev Ther Drug
Carrier Syst, 1996,12 (1-2):1-55, and EP 0 362 279 B1. Particulate
structures, termed Immune Stimulating Complexes (ISCOMS),
comprising fractions of Qui A (saponin) are haemolytic and have
been used in the manufacture of vaccines (Morein, B., EP 0 109 942
B1). These structures have been reported to have adjuvant activity
(EP 0 109 942 B1: WO 96/11711). The haemolytic saponins QS21 and
QS17 (HPLC purified fractions of Quil A) have been described as
potent systemic adjuvant, and the method of their production is
disclosed in U.S. Pat. No. 5,057,540 and EP 0 362 279 B1. Also
described in these references is the use of QS7 (a non-haemolytic
fraction of Quil-A) which acts as a potent adjuvant for systemic
vaccines. Use of QS21 is further described in Kensil et al. (1991.
J. Immunology 146:431-437;. Combinations of QS21 and polysorbate or
cyclodextrin are also known (WO 99/10008). Particulate adjuvant
systems comprising fractions of QuilA, such as QS21 and QS7 are
described in WO 96/33739 and WO 96/11711. Other saponins which have
been used in systemic vaccination studies include those derived
from other plant species such as Gypsophila and Saponaria (Bomford
et al., Vaccine, 10(9):572-577, 1992).
[0177] Escin is another detergent related to the saponins for use
in the adjuvant compositions of the embodiments herein disclosed.
Escin is described in the Merck index (12.sup.th Ed.: entry 3737)
as a mixture of saponin occurring in the seed of the horse chestnut
tree, Aesculus hippocastanum. Its isolation is described by
chromatography and purification (Fiedler, Arzneimittel-Forsch, 4,
213 (1953)), and by ion-exchange resins (Erbring et al., U.S. Pat.
No. 3,238,190). Fractions of escin (also known as aescin) have been
purified and shown to be biologically active (Yoshikawa M, et al
(Chem Pharm Bull (Tokyo) 1996 August; 44(8): 14.54-1464)).
Digitonin is another detergent, also being described in the Merck
index (12th Ed., entry 3204) as a saponin, being derived from the
seeds of Digitalis purpurea and purified according to the procedure
described by Gisvold et al, J. Am. Pharm. Assoc., 1934.23. 664: and
Rubenstroth-Bauer, Physiol Chem., 1955, 301, 621.
[0178] Other adjuvant or co-adjuvant for use according to certain
herein disclosed embodiments include a block co-polymer or
biodegradable polymer, which refers to a class of polymeric
compounds with which those in the relevant art will be familiar.
Examples of a block co-polymer or biodegradable polymer that may be
included in a vaccine composition or a immunological adjuvant
include Pluronic.RTM. L121 (BASF Corp., Mount Olive, N.J.; see,
e.g., Yeh et al, 1996 Pharm. Res. 13:1693).
[0179] Certain further illustrative embodiments contemplate
immunological adjuvant that include but are not limited to an oil,
which in some such embodiments may contribute co-adjuvant activity
and in other such embodiments may additionally or alternatively
provide a pharmaceutically acceptable carrier or excipient Any
number of suitable oils are known and may be selected tor inclusion
in vaccine compositions and immunological adjuvant compositions
based on the present disclosure. Examples of such oils, by way of
illustration and not limitation, include squalene, squalane,
mineral oil, olive oil, cholesterol and a mannide monooleate.
[0180] Immune response modifiers such as imidazoquinoline immune
response modifiers are also known in the art and may also be
included as adjuvant or co-adjuvant in certain presently disclosed
embodiments.
[0181] As also noted above, one type of adjuvant or co-adjuvant for
use in a vaccine composition according to the disclosure as
described herein may be the aluminum, co-adjuvant, which are
generally referred to as "alum." Alum co-adjuvant are based on the
following: aluminum oxy-hydroxidc; aluminum hydroxyphosphoate; or
various proprietary salts. Alum co-adjuvant are be advantageous
because they have a good safety record, augment antibody responses,
stabilize antigens, and are relatively simple for large-scale
production. (Edelman 2002 Mol Biotechnol 21:129-148; Edelman, R.
1980 Rev. Infect. Dis. 2:370-383.)
Pharmaceutical Compositions
[0182] In certain illustrative embodiments, the pharmaceutical
composition is a vaccine composition that comprises both the
recombinant protein according to the disclosure and may further
comprise one or more components, as provided herein, that are
selected from TLR agonist, co-adjuvant (including, , a cytokine, an
imidazoquinoline immune response modifier and/or a dSLIM) and the
like and/or a recombinant expression construct, in combination with
a pharmaceutically acceptable carrier, excipient or diluent.
[0183] Illustrative carriers will be nontoxic to recipients at the
dosages and concentrations employed. For vaccines comprising
recombinant protein, about 0.01 .mu.g/kg to about 100 mg/kg body
weight will be administered, typically by the intradermal,
subcutaneous, intramuscular or intravenous route, or by other
routes.
[0184] It will be evident to those skilled in the art that the
number and frequency of administration will be dependent upon the
response of the host. "Pharmaceutically acceptable carriers" for
therapeutic use are well known in the pharmaceutical an, and are
described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co. (A. R. Gennaro edit. 1985). For example,
sterile saline and phosphate-buffered saline at physiological pH
may be used. Preservatives, stabilizers, dyes and even flavoring
agents may be provided in the pharmaceutical composition. For
example, sodium benzoate, ascorbic acid and esters of
p-hydroxybenzoic acid may be added as preservatives. In addition,
antioxidants and suspending agents may be used.
[0185] The pharmaceutical compositions may be in any form which
allows for the composition to be administered to a patient. For
example, the composition may be in the form of a solid, liquid or
gas (aerosol). Typical routes of administration include, without
limitation, oral, topical, parenteral (e.g., sublingually or
buccally), sublingual, rectal, vaginal, and intranasal (e.g., as a
spray). The term parenteral as used herein includes iontophoretic
sonophoretic, passive transdermal, microneedle administration and
also subcutaneous injections, intravenous, intramuscular,
intrasternal, intracavernous, intrathecal, intrameatal,
intraurethral injection or infusion techniques. In a particular
embodiment, a composition as described herein (including vaccine
and pharmaceutical compositions) is administered intradermally by a
technique selected from iontophoresis, microcavitation,
sonophoresis or microneedles.
[0186] The pharmaceutical composition is formulated so as to allow
the active ingredients contained therein to be bioavailable upon
administration of the composition to a patient Compositions that
will be administered to a patient take the form of one or more
dosage units, where for example, u tablet may be a single dosage
unit, and a container of one or mows compounds of the invention in
aerosol form may hold a plurality of dosage units.
[0187] For oral administration, an excipient and/or binder may be
present Examples are sucrose, kaolin, glycerin, starch dextrins,
sodium alginate, carboxymethylcellulose and ethyl cellulose.
Coloring and/or flavoring agents may be present. A coating shell
may be employed.
[0188] The composition may be in the form of a liquid, e.g., an
elixir, syrup, solution, emulsion or suspension. The liquid may be
for oral administration or for delivery by injection, as two
examples. When intended for oral administration, preferred
compositions contain one or more of a sweetening agent,
preservatives, dye/colorant and flavor enhancer. In a composition
intended to be administered by injection, one or more of a
surfactant, preservative, wetting agent, dispersing agent,
suspending agent, buffer, stabilizer and isotonic agent may be
included.
[0189] A liquid pharmaceutical composition as used herein, whether
in the form of a solution, suspension or other like form, may
include one or more of the following carriers or excipients:
sterile diluents such as water for injection, saline solution,
preferably physiological saline, Ringer's solution, isotonic sodium
chloride, fixed oils such as squalene, squalane, mineral oil, a
mannide monooleate, cholesterol, and/or synthetic mono or
digylcerides which may serve as the solvent or suspending medium,
polyethylene glycols, glycerin, propylene glycol or other solvents;
antibacterial agents such as benzyl alcohol or methyl paraben;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. The parenteral
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic. An injectable
pharmaceutical composition is preferably sterile.
[0190] In a particular embodiment a pharmaceutical or vaccine
composition of the invention comprises a stable aqueous suspension
of less than 0.2 um and further comprises at least one component
selected from the group consisting of phospholipids, fatty acids,
surfactants, detergents, saponins, fluorodated lipids, and the
like.
[0191] It may also be desirable to include other components in a
vaccine or pharmaceutical composition, such as delivery vehicles
including but not limited to aluminum salts, water-in-oil
emulsions, biodegradable oil vehicles, oil-in-water emulsions,
biodegradable microcapsules, and liposomes. Examples of additional
irmnunostimulatory substances (co-adjuvant) for use in such
vehicles are also described above and may include
N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucan, IL-12,
OM-CSF, gamma interferon and IL-12.
[0192] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention, the type of carrier will vary depending on the mode
of administration and whether a sustained release is desired. For
parenteral administration, such as subcutaneous injection, the
carrier preferably comprises water, saline, alcohol, a fat, a wax
or a buffer. For oral administration, any of the above carriers or
a solid carrier, such as mannitol, lactose, starch, magnesium
stearate, sodium saccharine, talcum, cellulose, glucose, sucrose,
and magnesium carbonate, may be employed. Biodegradable
microspheres (e.g., polylactic galactide) may also be employed as
carriers for the pharmaceutical compositions of this invention.
[0193] Pharmaceutical compositions may also contain diluents such
as buffers, antioxidants such as ascorbic acid, low molecular
weight (less than about 10 residues) polypeptides, proteins, amino
acids, carbohydrates including glucose, sucrose or dextrins.
chelating agents such as EDTA, glutathione and other stabilizers
and excipients. Neutral buffered saline or saline mixed with
nonspecific serum albumin are exemplary appropriate diluents.
Preferably, product may be formulated as a lyophilizate using
appropriate excipient solutions (e.g., sucrose) as diluents.
[0194] In an illustrative embodiment, the epitope or receptor
supporting domain of the recombinant protein, whether derived from
a natural or synthetic polypeptide sequence, should have the
capacity to self-assemble into oligomers multimers under
appropriate chemical/environmental conditions, or to be reduced to
monomers under alternative conditions. Ideally, multimerisation
domains will assemble into stable multimers with a discreet number
of sub-units, for example dimers, trimers, tetramers, pentamers,
etc., such that a product of homogeneous size is generated.
Examples of natural polypeptides include, but are not limited to,
leucine zippers, lac repressor protein, streptavidin/avidin.
cholera toxin B sub-unit, Pseudomonas triroerization domain, and
viral capsid proteins.
[0195] In an illustrative embodiment, a process of preparing a
multivalent molecule is disclosed. In this illustrative embodiment,
the process includes assembling multimers from monomeric sub-units
to form a synthetic protein including one or more tumor antigens,
receptors, and/or a growth factors or parts thereof.
[0196] In another illustrative embodiment, a process of preparing a
vaccine formulation is disclosed. In this illustrative embodiment,
the process includes mixing one or more single monovalent multimers
together preparing a multivalent vaccine including a recombinant
protein including one or more tumor antigens, receptors, and/or a
growth factors or parts thereof.
[0197] In yet another illustrative embodiment, a process for
treating a patient is disclosed. In this illustrative embodiment,
the process includes administering separately to the patient one or
more monovalent, one tumor antigen, receptor, and/or growth factor,
recombinant proteins in a same day or at alternate days or times
during a vaccination period.
[0198] While the recombinant protein is described as including or
expressing one or more of all or a portion of at least one sequence
of the tumor antigens, the growth factors, and/or the receptors,
and the CT-B sequence, the recombinant protein may include the
natural CT-B sequence or a sequence substantially similar to the
natural CT-B sequence and/or a synthetic sequence.
[0199] While the recombinant protein is described as including or
expressing the CT-B sequence, the recombinant protein may include
or express a derivation of the CT-B sequence or a sequence that is
substantially similar to the CT-B sequence.
[0200] While the homogeneous recombinant proteins expressing or
incorporating one or more tumor antigens, growth factors, and/or
receptors have been described and illustrated in connection with
certain embodiments, many variations and modifications will be
evident to those skilled in the an and may be made without
departing from the spirit and scope of the disclosure, the
disclosure is thus not to be limited to the precise details of
methodology or construction set forth above as such variations and
modification are intended to be included within the scope of the
disclosure.
TABLE-US-00002 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS:
47 <210> SEQ ID NO 1 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<221> NAME/KEY: source <223> OTHER INFORMATION:
/note="Description of Artificial Sequence: Synthetic peptide"
<400> SEQUENCE: 1 Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala 1 5
10 <210> SEQ ID NO 2 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<221> NAME/KEY: source <223> OTHER INFORMATION:
/note="Description of Artificial Sequence: Synthetic peptide"
<400> SEQUENCE: 2 Ser Leu Ala Gly Ser Ser Gly Ala Leu Ser Lys
1 5 10 <210> SEQ ID NO 3 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <221> NAME/KEY: source <223> OTHER
INFORMATION: /note="Description of Artificial Sequence: Synthetic
peptide" <400> SEQUENCE: 3 Gly Gly Ser Gly Gly 1 5
<210> SEQ ID NO 4 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<221> NAME/KEY: source <223> OTHER INFORMATION:
/note="Description of Artificial Sequence: Synthetic peptide"
<400> SEQUENCE: 4 Ser Ser Gly Gly Gly 1 5 <210> SEQ ID
NO 5 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <221>
NAME/KEY: source <223> OTHER INFORMATION: /note="Description
of Artificial Sequence: Synthetic 6xHis tag" <400> SEQUENCE:
5 His His His His His His 1 5 <210> SEQ ID NO 6 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <221> NAME/KEY: source
<223> OTHER INFORMATION: /note="Description of Artificial
Sequence: Synthetic peptide" <400> SEQUENCE: 6 Gly Gly Gly
Gly Ser 1 5 <210> SEQ ID NO 7 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <221> NAME/KEY: source <223> OTHER
INFORMATION: /note="Description of Artificial Sequence: Synthetic
peptide" <400> SEQUENCE: 7 Gly Ser Ser Gly 1 <210> SEQ
ID NO 8 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <221>
NAME/KEY: source <223> OTHER INFORMATION: /note="Description
of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 8
Ser Ser Gly Gly Gly Ser Gly Gly 1 5 <210> SEQ ID NO 9
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <221> NAME/KEY:
source <223> OTHER INFORMATION: /note="Description of
Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 9 Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 <210> SEQ ID NO 10
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <221> NAME/KEY:
source <223> OTHER INFORMATION: /note="Description of
Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 10
Thr Ser Gly Gly Gly Ser Gly 1 5 <210> SEQ ID NO 11
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <221> NAME/KEY:
source <223> OTHER INFORMATION: /note="Description of
Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 11
Thr Ser Gly Gly Gly Gly Ser Gly Gly 1 5 <210> SEQ ID NO 12
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <221> NAME/KEY:
source <223> OTHER INFORMATION: /note="Description of
Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 12
Ser Ser Gly Gly Gly Ser Gly Gly Ser Ser Gly 1 5 10 <210> SEQ
ID NO 13 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <221>
NAME/KEY: source <223> OTHER INFORMATION: /note="Description
of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 13
Gly Gly Ser Gly Gly Thr Ser Gly Gly Gly Ser Gly 1 5 10 <210>
SEQ ID NO 14 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<221> NAME/KEY: source <223> OTHER INFORMATION:
/note="Description of Artificial Sequence: Synthetic peptide"
<400> SEQUENCE: 14 Ser Gly Gly Thr Ser Gly Gly Gly Gly Ser
Gly Gly 1 5 10 <210> SEQ ID NO 15 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <221> NAME/KEY: source <223> OTHER
INFORMATION: /note="Description of Artificial Sequence: Synthetic
peptide" <400> SEQUENCE: 15 Gly Gly Ser Gly Gly Thr Ser Gly
Gly Gly Gly Ser Gly Gly 1 5 10 <210> SEQ ID NO 16 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <221> NAME/KEY: source
<223> OTHER INFORMATION: /note="Description of Artificial
Sequence: Synthetic peptide" <400> SEQUENCE: 16 Ser Ser Gly
Gly Gly Gly Ser Gly Gly Gly Ser Ser Gly 1 5 10 <210> SEQ ID
NO 17 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence
<220> FEATURE: <221> NAME/KEY: source <223> OTHER
INFORMATION: /note="Description of Artificial Sequence: Synthetic
peptide" <400> SEQUENCE: 17 Ser Ser Gly Gly Gly Ser Gly Gly
Ser Ser Gly Gly Gly 1 5 10 <210> SEQ ID NO 18 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <221> NAME/KEY: source
<223> OTHER INFORMATION: /note="Description of Artificial
Sequence: Synthetic peptide" <400> SEQUENCE: 18 Ser Ser Gly
Gly Gly Gly Ser Gly Gly Gly Ser Ser Gly Gly Gly 1 5 10 15
<210> SEQ ID NO 19 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<221> NAME/KEY: source <223> OTHER INFORMATION:
/note="Description of Artificial Sequence: Synthetic peptide"
<400> SEQUENCE: 19 Gly Gly Ser Gly Gly Thr Arg Pro Ser Thr
Ala Ala Thr Ser 1 5 10 <210> SEQ ID NO 20 <211> LENGTH:
180 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <221> NAME/KEY: source <223> OTHER
INFORMATION: /note="Description of Artificial Sequence: Synthetic
polypeptide" <400> SEQUENCE: 20 His His His His His His Ile
Glu Gly Arg Asn Ser Asp Ser Glu Cys 1 5 10 15 Pro Leu Ser His Asp
Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr 20 25 30 Ile Glu Ala
Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile 35 40 45 Gly
Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg Gly 50 55
60 Gly Ser Gly Gly Thr Ser Gly Gly Gly Gly Ser Gly Gly Thr Pro Gln
65 70 75 80 Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr Gln Ile
His Thr 85 90 95 Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu
Ala Gly Lys Arg 100 105 110 Glu Met Ala Ile Ile Thr Phe Lys Asn Gly
Ala Thr Phe Gln Val Glu 115 120 125 Val Pro Gly Ser Gln His Ile Asp
Ser Gln Lys Lys Ala Ile Glu Arg 130 135 140 Met Lys Asp Thr Leu Arg
Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu 145 150 155 160 Lys Leu Cys
Val Trp Asn Asn Lys Thr Pro His Ala Ile Ala Ala Ile 165 170 175 Ser
Met Ala Asn 180 <210> SEQ ID NO 21 <211> LENGTH: 53
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 21 Asn Ser Asp Ser Glu Cys Pro Leu Ser His
Asp Gly Tyr Cys Leu His 1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu
Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30 Cys Val Val Gly Tyr Ile
Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45 Trp Trp Glu Leu
Arg 50 <210> SEQ ID NO 22 <211> LENGTH: 53 <212>
TYPE: PRT <213> ORGANISM: Pan troglodytes <400>
SEQUENCE: 22 Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr
Cys Leu His 1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp
Lys Tyr Ala Cys Asn 20 25 30 Cys Val Val Gly Tyr Ile Gly Glu Arg
Cys Gln Tyr Arg Asp Leu Lys 35 40 45 Trp Trp Glu Leu Arg 50
<210> SEQ ID NO 23 <211> LENGTH: 53 <212> TYPE:
PRT <213> ORGANISM: Macaca sp. <400> SEQUENCE: 23 Asn
Ser Asp Ser Gly Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His 1 5 10
15 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn
20 25 30 Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp
Leu Lys 35 40 45 Trp Trp Glu Leu Arg 50 <210> SEQ ID NO 24
<211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM:
Rattus norvegicus <400> SEQUENCE: 24 Asn Ser Asn Thr Gly Cys
Pro Pro Ser Tyr Asp Gly Tyr Cys Leu Asn 1 5 10 15 Gly Gly Val Cys
Met Tyr Val Glu Ser Val Asp Arg Tyr Val Cys Asn 20 25 30 Cys Val
Ile Gly Tyr Ile Gly Glu Arg Cys Gln His Arg Asp Leu Arg 35 40 45
Trp Trp Lys Leu Arg 50 <210> SEQ ID NO 25 <211> LENGTH:
33 <212> TYPE: PRT <213> ORGANISM: Rattus rattus
<400> SEQUENCE: 25 Met Tyr Val Glu Ser Val Asp Arg Tyr Val
Cys Asn Cys Val Ile Gly 1 5 10 15 Tyr Ile Gly Glu Arg Cys Gln His
Arg Asp Leu Arg Trp Trp Asn Trp 20 25 30 Arg <210> SEQ ID NO
26 <211> LENGTH: 53 <212> TYPE: PRT <213>
ORGANISM: Mus musculus <400> SEQUENCE: 26 Asn Ser Tyr Pro Gly
Cys Pro Ser Ser Tyr Asp Gly Tyr Cys Leu Asn 1 5 10 15 Gly Gly Val
Cys Met His Ile Glu Ser Leu Asp Ser Tyr Thr Cys Asn 20 25 30 Cys
Val Ile Gly Tyr Ser Gly Asp Arg Cys Gln Thr Arg Asp Leu Arg 35 40
45 Trp Trp Glu Leu Arg 50 <210> SEQ ID NO 27 <211>
LENGTH: 53 <212> TYPE: PRT <213> ORGANISM: Sus scrofa
<400> SEQUENCE: 27 Asn Ser Tyr Ser Glu Cys Pro Pro Ser His
Asp Gly Tyr Cys Leu His 1 5 10 15 Gly Gly Val Cys Met Tyr Ile Glu
Ala Val Asp Ser Tyr Ala Cys Asn 20 25 30 Cys Val Phe Gly Tyr Val
Gly Glu Arg Cys Gln His Arg Asp Leu Lys 35 40 45 Trp Trp Glu Leu
Arg 50 <210> SEQ ID NO 28 <211> LENGTH: 52 <212>
TYPE: PRT <213> ORGANISM: Felis catus <400> SEQUENCE:
28 Asn Ser Tyr Gln Glu Cys Pro Pro Ser Tyr Asp Gly Tyr Cys Leu Tyr
1 5 10 15 Asn Gly Val Cys Met Tyr Ile Glu Ala Val Asp Arg Tyr Ala
Cys Asn 20 25 30 Cys Val Phe Gly Tyr Val Gly Glu Arg Cys Gln His
Arg Asp Leu Lys 35 40 45 Trp Glu Leu Arg 50 <210> SEQ ID NO
29 <211> LENGTH: 52 <212> TYPE: PRT <213>
ORGANISM: Canis lupus <400> SEQUENCE: 29
Asn Gly Tyr Arg Glu Cys Pro Ser Ser Tyr Asp Gly Tyr Cys Leu Tyr 1 5
10 15 Asn Gly Val Cys Met Tyr Ile Glu Ala Val Asp Arg Tyr Ala Cys
Asn 20 25 30 Cys Val Phe Gly Tyr Val Gly Glu Arg Cys Gln His Arg
Asp Leu Lys 35 40 45 Trp Glu Leu Arg 50 <210> SEQ ID NO 30
<211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM:
Equus caballus <400> SEQUENCE: 30 Asn Ser Tyr Gln Glu Cys Ser
Gln Ser Tyr Asp Gly Tyr Cys Leu His 1 5 10 15 Gly Gly Lys Cys Val
Tyr Leu Val Gln Val Asp Thr His Ala Cys Asn 20 25 30 Cys Val Val
Gly Tyr Val Gly Glu Arg Cys Gln His Gln Asp Leu Arg 35 40 45 Trp
Trp Glu Leu Arg 50 <210> SEQ ID NO 31 <211> LENGTH: 48
<212> TYPE: PRT <213> ORGANISM: Taeniopygia guttata
<400> SEQUENCE: 31 Cys Pro Pro Ser Tyr Glu Ser Tyr Cys Leu
His Gly Gly Val Cys Asn 1 5 10 15 Tyr Val Ser Asp Leu Gln Asp Tyr
Ala Cys Asn Cys Val Thr Gly Tyr 20 25 30 Val Gly Glu Arg Cys Gln
Phe Ser Asp Leu Glu Trp Trp Glu Gln Arg 35 40 45 <210> SEQ ID
NO 32 <211> LENGTH: 46 <212> TYPE: PRT <213>
ORGANISM: Gallus gallus <400> SEQUENCE: 32 Cys Pro Pro Ala
Tyr Asp Ser Tyr Cys Leu His Gly Gly Val Cys Asn 1 5 10 15 Tyr Val
Ser Asp Leu Gln Asp Tyr Ala Cys Asn Cys Val Thr Gly Tyr 20 25 30
Val Gly Glu Arg Cys Gln Phe Ser Asp Leu Glu Trp Trp Glu 35 40 45
<210> SEQ ID NO 33 <211> LENGTH: 47 <212> TYPE:
PRT <213> ORGANISM: Xenopus sp. <400> SEQUENCE: 33 Glu
Cys Pro Leu Ala Tyr Asp Gly Tyr Cys Leu Asn Gly Gly Val Cys 1 5 10
15 Ile His Phe Pro Glu Leu Lys Asp Tyr Gly Cys Arg Cys Val Ala Gly
20 25 30 Tyr Val Gly Glu Arg Cys Gln Phe Asp Asp Leu Lys Ser Trp
Glu 35 40 45 <210> SEQ ID NO 34 <211> LENGTH: 53
<212> TYPE: PRT <213> ORGANISM: Danio rerio <400>
SEQUENCE: 34 Asn Gly Val Gln Ser Cys Pro Ser Thr His Asp Ser Tyr
Cys Leu Tyr 1 5 10 15 Asp Gly Val Cys Phe Tyr Phe Pro Glu Met Glu
Ser Tyr Ala Cys Asn 20 25 30 Cys Val Leu Gly Tyr Met Gly Glu Arg
Cys Gln Phe Ser Asp Leu Glu 35 40 45 Trp Trp Glu Leu Gln 50
<210> SEQ ID NO 35 <211> LENGTH: 38 <212> TYPE:
PRT <213> ORGANISM: Branchiostoma sp. <400> SEQUENCE:
35 Cys Pro Pro Arg Tyr Glu Gly Phe Cys Leu His Gly Gly Ile Cys Phe
1 5 10 15 Tyr Val Asp Arg Leu Gly Val Gly Cys Ser Cys Pro Val Met
Tyr Glu 20 25 30 Gly Glu Arg Cys Gln Tyr 35 <210> SEQ ID NO
36 <211> LENGTH: 225 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <221>
NAME/KEY: source <223> OTHER INFORMATION: /note="Description
of Artificial Sequence: Synthetic polypeptide" <400>
SEQUENCE: 36 His His His His His His Ile Glu Gly Arg Asn Ser Asp
Ser Glu Cys 1 5 10 15 Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp
Gly Val Cys Met Tyr 20 25 30 Ile Glu Ala Leu Asp Lys Tyr Ala Cys
Asn Cys Val Val Gly Tyr Ile 35 40 45 Gly Glu Arg Cys Gln Tyr Arg
Asp Leu Lys Trp Trp Glu Leu Arg Ser 50 55 60 Gly Gly Thr Pro Gln
Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn 65 70 75 80 Thr Gln Ile
His Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser 85 90 95 Leu
Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala 100 105
110 Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys
115 120 125 Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr
Leu Thr 130 135 140 Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn
Lys Thr Pro His 145 150 155 160 Ala Ile Ala Ala Ile Ser Met Ala Asn
Ser Ser Gly Asn Ser Asp Ser 165 170 175 Glu Cys Pro Leu Ser His Asp
Gly Tyr Cys Leu His Asp Gly Val Cys 180 185 190 Met Tyr Ile Glu Ala
Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly 195 200 205 Tyr Ile Gly
Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu 210 215 220 Arg
225 <210> SEQ ID NO 37 <211> LENGTH: 139 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <221> NAME/KEY: source <223> OTHER
INFORMATION: /note="Description of Artificial Sequence: Synthetic
polypeptide" <400> SEQUENCE: 37 His His His His His His Ile
Glu Gly Arg Cys Met Tyr Ile Glu Ala 1 5 10 15 Leu Asp Lys Tyr Ser
Gly Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys 20 25 30 Ala Glu Tyr
His Asn Thr Gln Ile His Thr Leu Asn Asp Lys Ile Phe 35 40 45 Ser
Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr 50 55
60 Phe Lys Asn Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser Gln His
65 70 75 80 Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr
Leu Arg 85 90 95 Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu
Cys Val Trp Asn 100 105 110 Asn Lys Thr Pro His Ala Ile Ala Ala Ile
Ser Met Ala Asn Ser Ser 115 120 125 Gly Cys Met Tyr Ile Glu Ala Leu
Asp Lys Tyr 130 135 <210> SEQ ID NO 38 <211> LENGTH:
171 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <221> NAME/KEY: source <223> OTHER
INFORMATION: /note="Description of Artificial Sequence: Synthetic
polypeptide" <400> SEQUENCE: 38 His His His His His His Ile
Glu Gly Arg Cys Pro Leu Ser His Asp 1 5 10 15 Gly Tyr Cys Leu His
Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp 20 25 30 Lys Tyr Ala
Cys Ser Gly Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys 35 40 45 Ala
Glu Tyr His Asn Thr Gln Ile His Thr Leu Asn Asp Lys Ile Phe 50 55
60 Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr
65 70 75 80 Phe Lys Asn Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser
Gln His 85 90 95 Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys
Asp Thr Leu Arg 100 105 110 Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu
Lys Leu Cys Val Trp Asn
115 120 125 Asn Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn
Ser Ser 130 135 140 Gly Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His
Asp Gly Val Cys 145 150 155 160 Met Tyr Ile Glu Ala Leu Asp Lys Tyr
Ala Cys 165 170 <210> SEQ ID NO 39 <211> LENGTH: 253
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <221> NAME/KEY: source <223> OTHER
INFORMATION: /note="Description of Artificial Sequence: Synthetic
polypeptide" <400> SEQUENCE: 39 His His His His His His Ile
Glu Gly Arg Gly Pro Glu Thr Leu Cys 1 5 10 15 Gly Ala Glu Leu Val
Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly 20 25 30 Phe Tyr Phe
Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala 35 40 45 Pro
Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu 50 55
60 Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala
65 70 75 80 Gly Ser Ser Gly Asn Ser Asp Ser Glu Cys Pro Leu Ser His
Asp Gly 85 90 95 Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu
Ala Leu Asp Lys 100 105 110 Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile
Gly Glu Arg Cys Gln Tyr 115 120 125 Arg Asp Leu Lys Trp Trp Glu Leu
Arg Gly Gly Ser Gly Gly Thr Ser 130 135 140 Gly Gly Gly Gly Gly Ser
Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys 145 150 155 160 Ala Glu Tyr
His Asn Thr Gln Ile His Thr Leu Asn Asp Lys Ile Phe 165 170 175 Ser
Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr 180 185
190 Phe Lys Asn Gly Ala Thr Phe Gln Val Glu Val Pro Ser Gln His Ile
195 200 205 Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu
Arg Ile 210 215 220 Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys
Val Trp Asn Asn 225 230 235 240 Lys Thr Pro His Ala Ile Ala Ala Ile
Ser Met Ala Asn 245 250 <210> SEQ ID NO 40 <211>
LENGTH: 185 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <221> NAME/KEY: source
<223> OTHER INFORMATION: /note="Description of Artificial
Sequence: Synthetic polypeptide" <400> SEQUENCE: 40 His His
His His His His Ile Glu Gly Arg Thr Pro Gln Asn Ile Thr 1 5 10 15
Asp Leu Cys Ala Glu Tyr His Asn Thr Gln Ile His Thr Leu Asn Asp 20
25 30 Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met
Ala 35 40 45 Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe Gln Val Glu
Val Pro Ser 50 55 60 Gln His Ile Asp Ser Gln Lys Lys Ala Ile Glu
Arg Met Lys Asp Thr 65 70 75 80 Leu Arg Ile Ala Tyr Leu Thr Glu Ala
Lys Val Glu Lys Leu Cys Val 85 90 95 Trp Asn Asn Lys Thr Pro His
Ala Ile Ala Ala Ile Ser Met Ala Asn 100 105 110 Ser Ser Gly Gly Pro
Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala 115 120 125 Leu Gln Phe
Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr 130 135 140 Gly
Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp 145 150
155 160 Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr
Cys 165 170 175 Ala Pro Leu Lys Pro Ala Lys Ser Ala 180 185
<210> SEQ ID NO 41 <211> LENGTH: 228 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<221> NAME/KEY: source <223> OTHER INFORMATION:
/note="Description of Artificial Sequence: Synthetic polypeptide"
<400> SEQUENCE: 41 His His His His His His Ile Glu Gly Arg
Thr Pro Gln Asn Ile Thr 1 5 10 15 Asp Leu Cys Ala Glu Tyr His Asn
Thr Gln Ile His Thr Leu Asn Asp 20 25 30 Lys Ile Phe Ser Tyr Thr
Glu Ser Leu Ala Gly Lys Arg Glu Met Ala 35 40 45 Ile Ile Thr Phe
Lys Asn Gly Ala Thr Phe Gln Val Glu Val Pro Gly 50 55 60 Ser Gln
His Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp 65 70 75 80
Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys 85
90 95 Val Trp Asn Asn Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met
Ala 100 105 110 Asn Ser Ser Gly Ala Leu Asp Thr Asn Tyr Cys Phe Ser
Ser Thr Glu 115 120 125 Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp
Phe Arg Lys Asp Leu 130 135 140 Gly Trp Lys Trp Ile His Glu Pro Lys
Gly Tyr His Ala Asn Phe Cys 145 150 155 160 Leu Gly Pro Cys Pro Tyr
Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys 165 170 175 Val Leu Ala Leu
Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ser Pro 180 185 190 Cys Cys
Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val 195 200 205
Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser 210
215 220 Cys Lys Cys Ser 225 <210> SEQ ID NO 42 <211>
LENGTH: 261 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <221> NAME/KEY: source
<223> OTHER INFORMATION: /note="Description of Artificial
Sequence: Synthetic polypeptide" <400> SEQUENCE: 42 His His
His His His His Ile Glu Gly Arg Thr Pro Gln Asn Ile Thr 1 5 10 15
Asp Leu Cys Ala Glu Tyr His Asn Thr Gln Ile His Thr Leu Asn Asp 20
25 30 Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met
Ala 35 40 45 Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe Gln Val Glu
Val Pro Gly 50 55 60 Ser Gln His Ile Asp Ser Gln Lys Lys Ala Ile
Glu Arg Met Lys Asp 65 70 75 80 Thr Leu Arg Ile Ala Tyr Leu Thr Glu
Ala Lys Val Glu Lys Leu Cys 85 90 95 Val Trp Asn Asn Lys Thr Pro
His Ala Ile Ala Ala Ile Ser Met Ala 100 105 110 Asn Ser Ser Gly Pro
Ala Leu Pro Glu Asp Gly Gly Ala Ala Phe Pro 115 120 125 Pro Gly His
Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly 130 135 140 Phe
Phe Leu Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu 145 150
155 160 Lys Ser Asp Pro His Val Lys Leu Gln Leu Gln Ala Glu Glu Arg
Gly 165 170 175 Val Val Ser Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu
Ala Met Lys 180 185 190 Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val
Thr Glu Glu Cys Phe 195 200 205 Phe Phe Glu Arg Leu Glu Ser Asn Asn
Tyr Asn Thr Tyr Arg Ser Arg 210 215 220 Lys Tyr Ser Ser Trp Tyr Val
Ala Leu Lys Arg Thr Gly Gln Tyr Lys 225 230 235 240 Leu Gly Ser Lys
Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro 245 250 255 Met Ser
Ala Lys Ser 260 <210> SEQ ID NO 43 <211> LENGTH: 299
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <221> NAME/KEY: source <223> OTHER
INFORMATION: /note="Description of Artificial Sequence: Synthetic
polypeptide" <400> SEQUENCE: 43
His His His His His His Gln Lys Lys Arg Arg Asn Thr Leu His Glu 1 5
10 15 Phe Lys Lys Ser Ala Lys Thr Thr Leu Thr Lys Glu Asp Pro Leu
Leu 20 25 30 Lys Ile Lys Thr Lys Lys Val Asn Ser Ala Asp Glu Cys
Ala Asn Arg 35 40 45 Cys Ile Arg Asn Arg Gly Phe Thr Phe Thr Cys
Lys Ala Phe Val Phe 50 55 60 Asp Lys Ser Arg Lys Arg Cys Tyr Trp
Tyr Pro Phe Asn Ser Met Ser 65 70 75 80 Ser Gly Val Lys Lys Gly Phe
Gly His Glu Phe Asp Leu Tyr Glu Asn 85 90 95 Lys Asp Tyr Ile Arg
Asn Cys Ile Ile Gly Lys Gly Gly Ser Tyr Lys 100 105 110 Gly Thr Val
Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro Trp Asn 115 120 125 Ser
Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr Arg Gly 130 135
140 Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly
145 150 155 160 Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr
Glu Val Cys 165 170 175 Asp Ile Pro Gln Cys Ser Gly Gly Ser Gly Gly
Thr Ser Gly Gly Gly 180 185 190 Gly Ser Gly Gly Thr Pro Gln Asn Ile
Thr Asp Leu Cys Ala Glu Tyr 195 200 205 His Asn Thr Gln Ile His Thr
Leu Asn Asp Lys Ile Phe Ser Tyr Thr 210 215 220 Glu Ser Leu Ala Gly
Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn 225 230 235 240 Gly Ala
Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser 245 250 255
Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr 260
265 270 Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys
Thr 275 280 285 Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn 290 295
<210> SEQ ID NO 44 <211> LENGTH: 256 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<221> NAME/KEY: source <223> OTHER INFORMATION:
/note="Description of Artificial Sequence: Synthetic polypeptide"
<400> SEQUENCE: 44 His His His His His His Ile Glu Gly Arg
Thr Pro Gln Asn Ile Thr 1 5 10 15 Asp Leu Cys Ala Glu Tyr His Asn
Thr Gln Ile His Thr Leu Asn Asp 20 25 30 Lys Ile Phe Ser Tyr Thr
Glu Ser Leu Ala Gly Lys Arg Glu Met Ala 35 40 45 Ile Ile Thr Phe
Lys Asn Gly Ala Thr Phe Gln Val Glu Val Pro Gly 50 55 60 Ser Gln
His Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp 65 70 75 80
Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys 85
90 95 Val Trp Asn Asn Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met
Ala 100 105 110 Asn Ser Ser Gly Gly Pro Glu Thr Leu Cys Gly Ala Glu
Leu Val Asp 115 120 125 Ala Leu Gln Phe Val Cys Gly Pro Arg Gly Phe
Tyr Phe Asn Lys Pro 130 135 140 Thr Gly Tyr Gly Ser Ser Ile Arg Arg
Ala Pro Gln Thr Gly Ile Val 145 150 155 160 Asp Glu Cys Cys Phe Arg
Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr 165 170 175 Cys Ala Pro Leu
Lys Pro Thr Lys Ala Ala Gly Gly Ser Ala Tyr Gly 180 185 190 Pro Gly
Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe 195 200 205
Val Cys Ser Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ser Ser Arg Ala 210
215 220 Asn Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe Arg Ser
Cys 225 230 235 240 Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro
Ala Lys Ser Glu 245 250 255 <210> SEQ ID NO 45 <211>
LENGTH: 309 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <221> NAME/KEY: source
<223> OTHER INFORMATION: /note="Description of Artificial
Sequence: Synthetic polypeptide" <400> SEQUENCE: 45 His His
His His His His Ile Glu Gly Arg Thr Pro Gln Asn Ile Thr 1 5 10 15
Asp Leu Cys Ala Glu Tyr His Asn Thr Gln Ile His Thr Leu Asn Asp 20
25 30 Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met
Ala 35 40 45 Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe Gln Val Glu
Val Pro Gly 50 55 60 Ser Gln His Ile Asp Ser Gln Lys Lys Ala Ile
Glu Arg Met Lys Asp 65 70 75 80 Thr Leu Arg Ile Ala Tyr Leu Thr Glu
Ala Lys Val Glu Lys Leu Cys 85 90 95 Val Trp Asn Asn Lys Thr Pro
His Ala Ile Ala Ala Ile Ser Met Ala 100 105 110 Asn Ser Ser Gly Val
Ile Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr 115 120 125 Cys Arg Pro
Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp 130 135 140 Glu
Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys 145 150
155 160 Ala Gly Cys Cys Asn Asp Glu Ala Leu Glu Cys Val Pro Thr Ser
Glu 165 170 175 Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His
Gln Ser Gln 180 185 190 His Ile Gly Glu Met Ser Phe Leu Gln His Ser
Arg Cys Glu Cys Arg 195 200 205 Pro Lys Lys Thr Glu Ile Leu Lys Ser
Ile Asp Asn Glu Trp Arg Lys 210 215 220 Thr Gln Cys Met Pro Arg Glu
Val Cys Ile Asp Val Gly Lys Glu Phe 225 230 235 240 Gly Ala Ala Thr
Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr 245 250 255 Arg Cys
Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr 260 265 270
Ser Thr Gly Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu 275
280 285 Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr
Ser 290 295 300 Cys Arg Cys Met Ser 305 <210> SEQ ID NO 46
<211> LENGTH: 234 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <221> NAME/KEY:
source <223> OTHER INFORMATION: /note="Description of
Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE:
46 His His His His His His Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser
1 5 10 15 Thr Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe
Arg Lys 20 25 30 Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly
Tyr His Ala Asn 35 40 45 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp
Ser Leu Asp Thr Gln Tyr 50 55 60 Ser Lys Val Leu Ala Leu Tyr Asn
Gln His Asn Pro Gly Ala Ser Ala 65 70 75 80 Ala Pro Cys Cys Val Pro
Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr 85 90 95 Tyr Val Gly Arg
Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val 100 105 110 Arg Ser
Cys Lys Cys Ser Gly Gly Ser Gly Gly Thr Ser Gly Gly Gly 115 120 125
Gly Gly Ser Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr 130
135 140 His Asn Thr Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr
Thr 145 150 155 160 Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile
Thr Phe Lys Asn 165 170 175 Gly Ala Thr Phe Gln Val Glu Val Pro Ser
Gln His Ile Asp Ser Gln 180 185 190 Lys Lys Ala Ile Glu Arg Met Lys
Asp Thr Leu Arg Ile Ala Tyr Leu 195 200 205 Thr Glu Ala Lys Val Glu
Lys Leu Cys Val Trp Asn Asn Lys Thr Pro 210 215 220 His Ala Ile Ala
Ala Ile Ser Met Ala Asn 225 230 <210> SEQ ID NO 47
<211> LENGTH: 234 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence
<220> FEATURE: <221> NAME/KEY: source <223> OTHER
INFORMATION: /note="Description of Artificial Sequence: Synthetic
polypeptide" <400> SEQUENCE: 47 His His His His His His Ile
Glu Gly Arg Ala Val Lys Phe Pro Gln 1 5 10 15 Leu Cys Lys Phe Cys
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys 20 25 30 Ser Cys Met
Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln 35 40 45 Glu
Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu 50 55
60 Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu
65 70 75 80 Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys
Lys Pro 85 90 95 Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp
Glu Cys Asn Asp 100 105 110 Asn Ile Ile Phe Ser Glu Gly Gly Ser Gly
Gly Thr Ser Gly Gly Gly 115 120 125 Gly Gly Ser Gly Thr Pro Gln Asn
Ile Thr Asp Leu Cys Ala Glu Tyr 130 135 140 His Asn Thr Gln Ile His
Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr 145 150 155 160 Glu Ser Leu
Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn 165 170 175 Gly
Ala Thr Phe Gln Val Glu Val Pro Ser Gln His Ile Asp Ser Gln 180 185
190 Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu
195 200 205 Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys
Thr Pro 210 215 220 His Ala Ile Ala Ala Ile Ser Met Ala Asn 225
230
* * * * *